U.S. patent application number 14/000819 was filed with the patent office on 2015-03-12 for compounds and methods for conjugation of biomolecules.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. The applicant listed for this patent is Scott Clarke, Kyle Gee, Scott Grecian, Upinder Singh. Invention is credited to Scott Clarke, Kyle Gee, Scott Grecian, Upinder Singh.
Application Number | 20150072396 14/000819 |
Document ID | / |
Family ID | 45879028 |
Filed Date | 2015-03-12 |
United States Patent
Application |
20150072396 |
Kind Code |
A1 |
Gee; Kyle ; et al. |
March 12, 2015 |
Compounds and Methods for Conjugation of Biomolecules
Abstract
Low-copper click chemistry, 1.3-dipolar cycloadditions, and
Staudinger ligations for modifying biomolecules is provided.
Compositions, methods, and kits relating to low-copper click
chemistry, 1.3-dipolar cycloadditions, and Staudinger ligations are
also provided.
Inventors: |
Gee; Kyle; (Springfield,
OR) ; Singh; Upinder; (Eugene, OR) ; Grecian;
Scott; (Jasper, OR) ; Clarke; Scott; (Eugene,
OR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gee; Kyle
Singh; Upinder
Grecian; Scott
Clarke; Scott |
Springfield
Eugene
Jasper
Eugene |
OR
OR
OR
OR |
US
US
US
US |
|
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
45879028 |
Appl. No.: |
14/000819 |
Filed: |
March 1, 2012 |
PCT Filed: |
March 1, 2012 |
PCT NO: |
PCT/US2012/027285 |
371 Date: |
March 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61449396 |
Mar 4, 2011 |
|
|
|
61558148 |
Nov 10, 2011 |
|
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|
Current U.S.
Class: |
435/188 ;
435/196; 435/201; 435/231; 435/325; 514/281; 525/329.4; 525/330.3;
525/331.5; 525/333.6; 525/333.7; 525/420; 525/50; 530/303; 530/310;
530/317; 530/351; 530/370; 530/377; 530/391.5; 530/399; 530/409;
536/102; 536/123.1; 536/18.1; 536/2; 536/21; 536/27.14; 536/56;
536/6.4; 546/194; 546/246; 546/257; 546/273.7; 546/277.4;
546/281.7; 546/282.7; 546/300; 546/309; 546/310; 546/48; 548/202;
548/365.4; 549/510 |
Current CPC
Class: |
C07D 215/12 20130101;
C07D 213/79 20130101; C07D 401/14 20130101; C09B 11/24 20130101;
G01N 33/533 20130101; C07D 213/53 20130101; C09B 23/06 20130101;
G01N 33/582 20130101; C07D 213/68 20130101; C07K 14/575 20130101;
C07D 213/78 20130101; C09B 23/083 20130101; C07D 401/04 20130101;
C07D 235/14 20130101; C07D 213/74 20130101; C07D 213/81
20130101 |
Class at
Publication: |
435/188 ;
435/325; 530/391.5; 530/303; 536/21; 549/510; 530/310; 435/196;
435/201; 435/231; 530/409; 536/2; 536/123.1; 536/56; 536/102;
546/281.7; 536/18.1; 536/6.4; 514/281; 530/317; 536/27.14; 530/351;
530/399; 530/370; 530/377; 546/309; 546/310; 546/194; 546/273.7;
546/282.7; 546/277.4; 546/48; 546/246; 548/202; 548/365.4; 546/300;
546/257; 525/50; 525/330.3; 525/329.4; 525/333.6; 525/331.5;
525/333.7; 525/420 |
International
Class: |
G01N 33/58 20060101
G01N033/58; C07K 14/575 20060101 C07K014/575; C07D 401/14 20060101
C07D401/14 |
Claims
1. A compound of the formula: ##STR00085## wherein: A is a carbon,
or A, R.sub.5, and R.sub.6 are absent; R.sub.1, R.sub.2, R.sub.3,
and R.sub.4, are independently selected from hydrogen, halogen,
--SO.sub.3X, a carboxylic acid, a salt of carboxylic acid, CN,
nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl,
aryl, heteroaryl, substituted aryl, arylalkyl, substituted
arylalkyl, and substituted heteroaryl, arylcarboxamido, alkyl and
aryl portions are optionally substituted one or more times by
halogen, --SO.sub.3X, a carboxylic acid, a salt of carboxylic acid,
CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, alkylthio, alkanoylamino,
alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, arylcarboxamido;
or two substituents selected from R.sub.1, R.sub.2, R.sub.3, and
R.sub.4, wherein each of the at least two substituents are on
different carbon atoms together form a fused moiety selected from
cycloalkyl, heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, and all of the
remaining substituents are independently selected from hydrogen,
halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl; or at least two of the remaining
substituents together form a fused moiety selected from cycloalkyl,
heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, and any
remaining substituents are independently selected from hydrogen,
halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl; R.sub.5, and R.sub.6, are independently
selected from hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl,
aryl, heteroaryl, substituted aryl, arylalkyl, substituted
arylalkyl, and substituted heteroaryl, arylcarboxamido, alkyl and
aryl portions are optionally substituted one or more times by
halogen, --SO.sub.3X, a carboxylic acid, a salt of carboxylic acid,
CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, alkylthio, alkanoylamino,
alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, arylcarboxamido;
at least one substituent selected from R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and R.sub.6 comprises X-L-, wherein: X is selected
from a reporter molecule, a carrier molecule, a solid phase, a
therapeutic molecule such as peptide, a protein, an antibody, a
polysaccharide, a nucleic acid polymer, an ion complexing moiety, a
lipid or a non-biological organic polymer or polymeric micro or
nano particle, that are optionally bound to one or more additional
fluorophores; or X is a reactive group such as carboxylic acid, an
activated ester of carboxylic acid, an amine, a hydrazine, a
haloacetamide, an alkyl halide, an isothiocynate or a maleimide
group; and L is an independently a single covalent bond or L is
covalent linkage having 1-24 non-hydrogen atoms selected from the
group consisting of C, N, O, P and S and composed of any
combinations of single, double, triple or aromatic carbon-carbon
bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds,
carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds
and phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl; Z is
an independently a single covalent bond or Z is covalent linkage
having 1-10 non-hydrogen atoms selected from the group consisting
of C, N, O, P and S and composed of any combinations of single,
double, triple or aromatic carbon-carbon bonds, carbon-nitrogen
bonds, carbon-oxygen bonds, and carbon-sulfur bonds in the form of
a straight- or branched-chain alkyl or heteroalkyl chain; and G is
a chemical handle selected from an azide-reactive group, an
alkyne-reactive group, and a phosphine-reactive group.
2. The compound of claim 1, wherein the compound is of the formula:
##STR00086##
3. The compound of claim 2, wherein the compound is of the formula:
##STR00087##
4. The compound of claim 2, wherein the compound is of the formula:
##STR00088## wherein R.sub.2, R.sub.3, R.sub.4, and R.sub.7 to
R.sub.12 are independently selected from hydrogen, halogen,
--SO.sub.3X, a carboxylic acid, a salt of carboxylic acid, CN,
nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl,
aryl, heteroaryl, substituted aryl, arylalkyl, substituted
arylalkyl, and substituted heteroaryl, arylcarboxamido, alkyl and
aryl portions are optionally substituted one or more times by
halogen, --SO.sub.3X, a carboxylic acid, a salt of carboxylic acid,
CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, alkylthio, alkanoylamino,
alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, arylcarboxamido;
or two substituents selected from R.sub.2, R.sub.3, R.sub.4, and
R.sub.7 to R.sub.12, wherein the two substituents are on different
carbon atoms together form a fused moiety selected from cycloalkyl,
heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, and all of the
remaining substituents are independently selected from hydrogen,
halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl; or two of the remaining substituents also
together form a fused moiety selected from cycloalkyl,
heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, and the
remaining substituents are independently selected from hydrogen,
halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl; or R.sub.7 to R.sub.12, wherein the two
substituents are on same carbon atom together form a spirocyclic
moiety selected from alkyl or heteroalkyl, portions of which are
further optionally substituted with halogen, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl; at
least one substituent selected from R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and R.sub.6 comprises X-L-, wherein: X is selected
from a reporter molecule, a carrier molecule, a solid phase, a
therapeutic molecule such as peptide, a protein, an antibody, a
polysaccharide, a nucleic acid polymer, an ion complexing moiety, a
lipid or a non-biological organic polymer or polymeric micro or
nano particle, that are optionally bound to one or more additional
fluorophores; or X is a reactive group such as carboxylic acid, an
activated ester of carboxylic acid, an amine, a hydrazine, a
haloacetamide, an alkyl halide, an isothiocynate or a maleimide
group; and L is an independently a single covalent bond or L is
covalent linkage having 1-24 non-hydrogen atoms selected from the
group consisting of C, N, O, P and S and composed of any
combinations of single, double, triple or aromatic carbon-carbon
bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds,
carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds
and phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl; and G
is a chemical handle selected from an azide-reactive group, an
alkyne-reactive group, and a phosphine-reactive group.
5-10. (canceled)
11. The compound of claim 1, wherein all but one of the R
substituents are H.
12. The compound of claim 1, wherein L is an alkyl group having a
chain length of 0 to 15 atoms.
13. The compound of claim 12, wherein L is an alkyl group having a
chain length of 0 to 5 atoms.
14. The compound of claim 12, wherein L is
--NH--(CH.sub.2).sub.n--NH--C(O)--, wherein n is 1 to 12.
15. The compound of claim 1, wherein the reporter molecule
comprises a chromophore, fluorophore, fluorescent protein,
phosphorescent dye, tandem dye, particle, hapten, enzyme, or
radioisotope.
16. The compound of claim 15, wherein the fluorophore is a
xanthene, coumarin, cyanine, pyrene, oxazine, borapolyazaindacene,
or carbopyranine.
17. The compound of claim 15, wherein the enzyme is horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
beta-lactamase.
18. The compound of claim 15, wherein the particle is a
semiconductor nanocrystal.
19. The compound of claim 1, wherein the carrier molecule is an
amino acid, peptide, protein, polysaccharide, nucleoside,
nucleotide, oligonucleotide, nucleic acid, hapten, psoralen, drug,
hormone, lipid, lipid assembly, tyramine, synthetic polymer,
polymeric microparticle, biological cell, cellular component, ion
chelating moiety, enzymatic substrate, or virus.
20. The compound of claim 1, wherein the carrier molecule is an
antibody, antibody fragment, antigen, avidin, streptavidin, biotin,
dextran, IgG binding protein, fluorescent protein, agarose, or
non-biological microparticle.
21. The compound of claim 1, wherein the solid support is an
aerogel, hydrogel, resin, bead, biochip, microfluidic chip, silicon
chip, multi-well plate, membrane, conducting metal, nonconducting
metal, glass, or magnetic support.
22. The compound of claim 1, wherein the solid support is a silica
gel, polymeric membrane, particle, derivatized plastic film, glass
bead, cotton, plastic bead, alumina gel, polysaccharide,
poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose,
agar, cellulose, dextran, starch, FICOLL, heparin, glycogen,
amylopectin, mannan, inulin, nitrocellulose, diazocellulose,
polyvinylchloride, polypropylene, polyethylene, nylon, latex bead,
magnetic bead, paramagnetic bead, superparamagnetic bead, or
starch.
23. The compound of claim 1, wherein the therapeutic molecule is
taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,
mitomycin, etoposide, tenoposide, vincristine, vinblastine,
colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,
mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,
glucocorticoid, procaine, tetracaine, lidocaine, propranolol,
puromycin, or analogs or homologs thereof.
24. The compound of claim 1, wherein the therapeutic molecule is an
antimetabolite, alkylating agent, anthracycline, antibiotic, or
anti-mitotic agent.
25. The compound of claim 1, wherein the therapeutic molecule is
abrin, ricin A, pseudomonas exotoxin, diphtheria toxin, tumor
necrosis factor, .gamma.-interferon, .alpha.-interferon, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator, interleukin-1, interleukin-2, interleukin-6, granulocyte
macrophage colony stimulating factor, or granulocyte colony
stimulating factor.
26. The compound of claim 1, wherein G is an alkyne-reactive
group.
27. The compound of claim 26, wherein the alkyne-reactive group is
an azide.
28. The compound of claim 1, wherein G is an azide-reactive
group.
29. The compound of claim 28, wherein the azide-reactive group is a
terminal alkyne.
30-61. (canceled)
62. The compound of claim 1 selected from the group consisting of:
##STR00089## ##STR00090## ##STR00091##
63. (canceled)
64. The compound of claim 12, which is ##STR00092##
65. A compound selected from the group consisting of:
##STR00093##
66. A composition comprising: a compound of the formula:
##STR00094## wherein: A is a carbon, or A, R.sub.5, and R.sub.6 are
absent; R.sub.1, R.sub.2, R.sub.3, and R.sub.4, are independently
selected from hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido, alkyl and aryl portions are optionally substituted
one or more times by halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido; or two substituents selected from R.sub.1,
R.sub.2, R.sub.3, and R.sub.4, wherein each of the at least two
substituents are on different carbon atoms together form a fused
moiety selected from cycloalkyl, heterocycloalkyl, substituted
cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, and all of the remaining substituents are independently
selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl; or at least two
of the remaining substituents together form a fused moiety selected
from cycloalkyl, heterocycloalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl, and
any remaining substituents are independently selected from
hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl; R.sub.5, and
R.sub.6, are independently selected from hydrogen, halogen,
--SO.sub.3X, a carboxylic acid, a salt of carboxylic acid, CN,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido, alkyl and aryl portions are optionally substituted
one or more times by halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido; at least one substituent selected from R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 comprises X-L-,
wherein: X is selected from a reporter molecule, a carrier
molecule, a solid phase, a therapeutic molecule such as peptide, a
protein, an antibody, a polysaccharide, a nucleic acid polymer, an
ion complexing moiety, a lipid or a non-biological organic polymer
or polymeric micro or nano particle, that are optionally bound to
one or more additional fluorophores; or X is a reactive group such
as carboxylic acid, an activated ester of carboxylic acid, an
amine, a hydrazine, a haloacetamide, an alkyl halide, an
isothiocynate or a maleimide group; and L is an independently a
single covalent bond or L is covalent linkage having 1-24
non-hydrogen atoms selected from the group consisting of C, N, O, P
and S and composed of any combinations of single, double, triple or
aromatic carbon-carbon bonds, carbon-nitrogen bonds,
nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds,
phosphorus-oxygen bonds and phosphorus-nitrogen bonds in the form
of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy,
substituted alkoxy, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl; Z is an independently a single covalent
bond or Z is covalent linkage having 1-10 non-hydrogen atoms
selected from the group consisting of C, N, O, P and S and composed
of any combinations of single, double, triple or aromatic
carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygen bonds,
and carbon-sulfur bonds in the form of a straight- or
branched-chain alkyl or heteroalkyl chain; and G is a chemical
handle selected from an azide-reactive group, an alkyne-reactive
group, and a phosphine-reactive group; and a compound of the
formula: ##STR00095## wherein X, Y, and Z each independently have
the formula: ##STR00096## wherein: R.sub.1, R.sub.2, R.sub.3,
R.sub.4 and R.sub.5 are independently selected from hydrogen,
halogen, --SO.sub.3X, a carboxylic acid, a salt of carboxylic acid,
CN, nitro, hydroxyl, amino, alkylthio, alkanoylamino,
alkylaminocarbonyl, arylcarboxamido, alkyl aryl portions are
optionally substituted one or more times by halogen, --SO.sub.3X, a
carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl,
amino, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido; and B, C, D, and E are C or N; and
L.sub.1, L.sub.2, and L.sub.3 are covalent linkage having 1-5
non-hydrogen atoms selected from the group consisting of C, N, O, P
and S and composed of any combinations of single, double, triple or
aromatic carbon-carbon bonds, carbon-nitrogen bonds,
nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds,
and phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
and substituted heterocycloalkyl.
Description
RELATED APPLICATIONS
[0001] This application is a 371 application of PCT application No.
PCT/US2012/027285, filed Mar. 1, 2012 and claims priority to U.S.
provisional application No. 61/449,396, filed Mar. 4, 2011 and U.S.
provisional application No. 61/558,148, filed Nov. 10, 2011, which
disclosures are herein incorporated by reference in their
entirety.
FIELD
[0002] This invention relates to click chemistry, 1,3-dipolar
cycloadditions, and Staudinger ligations for conjugating
biomolecules.
BACKGROUND
[0003] Conjugation of biomolecules, such as polynucleotides,
proteins, lipids, etc., can be useful for detection, isolation,
and/or identification of biomolecules. Click chemistry was
developed by K. Barry Sharpless as a robust and specific method of
ligating two molecules together. See, e.g., Kolb et al. Angew.
Chemie Intern. 40(11): 2004-21 (2001). Classic click reactions
typically require Cu(I) ions in order to proceed efficiently.
However, Cu(I) ions can have deleterious effects on cells and
biomolecules. Reducing the amount and/or accessibility of Cu(I)
ions used in click reactions could therefore be beneficial for
conjugating biomolecules.
SUMMARY
[0004] In one aspect, the invention provides a compound of the
formula:
##STR00001##
wherein:
[0005] A is a carbon, or A, R.sub.5, and R.sub.6 are absent;
[0006] R.sub.1, R.sub.2, R.sub.3, and R.sub.4, are independently
selected from hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido, alkyl and aryl portions are optionally substituted
one or more times by halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido; or two substituents selected from R.sub.1,
R.sub.2, R.sub.3, and R.sub.4, wherein each of the at least two
substituents are on different carbon atoms together form a fused
moiety selected from cycloalkyl, heterocycloalkyl, substituted
cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, and all of the remaining substituents are independently
selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl; or at least two
of the remaining substituents together form a fused moiety selected
from cycloalkyl, heterocycloalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl, and
any remaining substituents are independently selected from
hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl;
[0007] R.sub.5, and R.sub.6, are independently selected from
hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a salt of
carboxylic acid, CN, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl,
aryl, heteroaryl, substituted aryl, arylalkyl, substituted
arylalkyl, and substituted heteroaryl, arylcarboxamido, alkyl and
aryl portions are optionally substituted one or more times by
halogen, --SO.sub.3X, a carboxylic acid, a salt of carboxylic acid,
CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, alkylthio, alkanoylamino,
alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido;
[0008] at least one substituent selected from R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5 and R.sub.6 comprises X-L-, wherein:
[0009] X is selected from a reporter molecule, a carrier molecule,
a solid phase, a therapeutic molecule such as peptide, a protein,
an antibody, a polysaccharide, a nucleic acid polymer, an ion
complexing moiety, a lipid or a non-biological organic polymer or
polymeric micro or nano particle, that are optionally bound to one
or more additional fluorophores; or [0010] X is a reactive group
such as carboxylic acid, an activated ester of carboxylic acid, an
amine, a hydrazine, a haloacetamide, an alkyl halide, an
isothiocynate or a maleimide group; and [0011] L is an
independently a single covalent bond or L is covalent linkage
having 1-24 non-hydrogen atoms selected from the group consisting
of C, N, O, P and S and composed of any combinations of single,
double, triple or aromatic carbon-carbon bonds, carbon-nitrogen
bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur
bonds, phosphorus-oxygen bonds and phosphorus-nitrogen bonds in the
form of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy,
substituted alkoxy, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl;
[0012] Z is an independently a single covalent bond or Z is
covalent linkage having 1-10 non-hydrogen atoms selected from the
group consisting of C, N, O, P and S and composed of any
combinations of single, double, triple or aromatic carbon-carbon
bonds, carbon-nitrogen bonds, carbon-oxygen bonds, and
carbon-sulfur bonds in the form of a straight- or branched-chain
alkyl or heteroalkyl chain; and
[0013] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0014] In some embodiments, the compound is of the formula (I). In
some of these, the compound is of the formula:
##STR00002##
[0015] In others, the compound is of the formula:
##STR00003##
wherein
[0016] R.sub.2, R.sub.3, R.sub.4, and R.sub.7 to R.sub.12 are
independently selected from hydrogen, halogen, --SO.sub.3X, a
carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl,
amino, hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido, alkyl and aryl portions are optionally
substituted one or more times by halogen, --SO.sub.3X, a carboxylic
acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino,
hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido; or two substituents selected from
R.sub.2, R.sub.3, R.sub.4, and R.sub.7 to R.sub.12, wherein the two
substituents are on different carbon atoms together form a fused
moiety selected from cycloalkyl, heterocycloalkyl, substituted
cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, and all of the remaining substituents are independently
selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl; or two of the
remaining substituents also together form a fused moiety selected
from cycloalkyl, heterocycloalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl, and
the remaining substituents are independently selected from
hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl; or R.sub.7 to
R.sub.12, wherein the two substituents are on same carbon atom
together form a spirocyclic moiety selected from alkyl or
heteroalkyl, portions of which are further optionally substituted
with halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl;
[0017] at least one substituent selected from R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5 and R.sub.6 comprises X-L-, wherein:
[0018] X is selected from a reporter molecule, a carrier molecule,
a solid phase, a therapeutic molecule such as peptide, a protein,
an antibody, a polysaccharide, a nucleic acid polymer, an ion
complexing moiety, a lipid or a non-biological organic polymer or
polymeric micro or nano particle, that are optionally bound to one
or more additional fluorophores; or [0019] X is a reactive group
such as carboxylic acid, an activated ester of carboxylic acid, an
amine, a hydrazine, a haloacetamide, an alkyl halide, an
isothiocynate or a maleimide group; and [0020] L is an
independently a single covalent bond or L is covalent linkage
having 1-24 non-hydrogen atoms selected from the group consisting
of C, N, O, P and S and composed of any combinations of single,
double, triple or aromatic carbon-carbon bonds, carbon-nitrogen
bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur
bonds, phosphorus-oxygen bonds and phosphorus-nitrogen bonds in the
form of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy,
substituted alkoxy, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl; and
[0021] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0022] In some embodiments, the compound of the formula (I), (II)
or (III) is selected from the group consisting of:
##STR00004## ##STR00005## ##STR00006##
[0023] In another aspect, the invention provides a compound of the
formula:
##STR00007##
wherein:
[0024] R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently
selected from hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido, alkyl and aryl portions are optionally substituted
one or more times by halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido; or two substituents selected from R.sub.1,
R.sub.2, R.sub.3, and R.sub.4 together form a fused moiety selected
from cycloalkyl, heterocycloalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl, and
the remaining two substituents also together form a fused moiety
selected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl, or
the remaining two substituents are independently selected from
hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl;
[0025] R.sub.5, and R.sub.6, are independently selected from
hydrogen, --SO.sub.3X, a carboxylic acid, a salt of carboxylic
acid, CN, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido, alkyl and aryl portions are optionally
substituted one or more times by halogen, --SO.sub.3X, a carboxylic
acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino,
hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido; or
[0026] at least one substituent selected from R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, and R.sub.6 comprises X-L-, wherein:
[0027] X is selected from a reporter molecule, a carrier molecule,
a solid phase, or a therapeutic molecule such as peptide, a
protein, an antibody, a polysaccharide, a nucleic acid polymer, an
ion complexing moiety, a lipid or a non-biological organic polymer
or polymeric micro or nano particle, that are optionally bound to
one or more additional fluorophores; or [0028] X is a reactive
group such as carboxylic acid, an activated ester of carboxylic
acid, an amine, a hydrazine, a haloacetamide, an alkyl halide, an
isothiocynate or a maleimide group; and [0029] L is an
independently a single covalent bond or L is covalent linkage
having 1-24 non-hydrogen atoms selected from the group consisting
of C, N, O, P and S and composed of any combinations of single,
double, triple or aromatic carbon-carbon bonds, carbon-nitrogen
bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur
bonds, phosphorus-oxygen bonds and phosphorus-nitrogen bonds in the
form of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy,
substituted alkoxy, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl;
[0030] A is a carbon, and R.sub.5 and R.sub.6 are independently
selected from hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido, alkyl and aryl portions are optionally substituted
one or more times by halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido or A, R.sub.5, and R.sub.6 are absent;
[0031] B is selected from O, S, and NR.sub.7, wherein R.sub.7 is
selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl and X-L wherein:
[0032] X is selected from a reporter molecule, a carrier molecule,
a solid phase, a therapeutic molecule such as peptide, a protein,
an antibody, a polysaccharide, a nucleic acid polymer, an ion
complexing moiety, a lipid or a non-biological organic polymer or
polymeric micro or nano particle, that are optionally bound to one
or more additional fluorophores; or [0033] X is a reactive group
such as carboxylic acid, an activated ester of carboxylic acid, an
amine, a hydrazine, a haloacetamide, an alkyl halide, an
isothiocynate or a maleimide group; and [0034] L is an
independently a single covalent bond or L is covalent linkage
having 1-24 non-hydrogen atoms selected from the group consisting
of C, N, O, P and S and composed of any combinations of single,
double, triple or aromatic carbon-carbon bonds, carbon-nitrogen
bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur
bonds, phosphorus-oxygen bonds and phosphorus-nitrogen bonds in the
form of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy,
substituted alkoxy, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl;
[0035] Z is an independently a single covalent bond or Z is
covalent linkage having 1-10 non-hydrogen atoms selected from the
group consisting of C, N, O, P and S and composed of any
combinations of single, double, triple or aromatic carbon-carbon
bonds, carbon-nitrogen bonds, carbon-oxygen bonds, and
carbon-sulfur bonds in the form of a straight- or branched-chain
alkyl or heteroalkyl chain; and
[0036] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0037] In another aspect, the invention provides a compound of the
formula:
##STR00008##
wherein:
[0038] A is a carbon, or A, R.sub.5, and R.sub.6 are absent;
[0039] R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are independently
selected from hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl
arylcarboxamido, alkyl and aryl portions are optionally substituted
one or more times by halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, alkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,
substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido;
[0040] R.sub.5, and R.sub.6, are independently selected from
hydrogen, --SO.sub.3X, a carboxylic acid, a salt of carboxylic
acid, CN, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido, alkyl and aryl portions are optionally
substituted one or more times by halogen, --SO.sub.3X, a carboxylic
acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino,
hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido; or two substituents selected from
R.sub.1, R.sub.2, R.sub.3, and R.sub.4, wherein the two
substituents are on different carbon atoms together form a fused
moiety selected from cycloalkyl, heterocycloalkyl, substituted
cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, and the remaining substituents are independently
selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl; or
[0041] at least one substituent selected from R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, and R.sub.6 comprises X-L-, wherein:
[0042] X is selected from a reporter molecule, a carrier molecule,
a solid phase, or a therapeutic molecule such as peptide, a
protein, an antibody, a polysaccharide, a nucleic acid polymer, an
ion complexing moiety, a lipid or a non-biological organic polymer
or polymeric micro or nano particle, that are optionally bound to
one or more additional fluorophores; or [0043] X is a reactive
group such as carboxylic acid, an activated ester of carboxylic
acid, an amine, a hydrazine, a haloacetamide, an alkyl halide, an
isothiocynate or a maleimide group; and [0044] L is an
independently a single covalent bond or L is covalent linkage
having 1-24 non-hydrogen atoms selected from the group consisting
of C, N, O, P and S and composed of any combinations of single,
double, triple or aromatic carbon-carbon bonds, carbon-nitrogen
bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur
bonds, phosphorus-oxygen bonds and phosphorus-nitrogen bonds in the
form of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl;
[0045] B, C, D and E are selected from O, S, and N where N is
further substituted with either R.sub.7, R.sub.8 or R.sub.9,
wherein R.sub.7, R.sub.8 or R.sub.9 are selected from hydrogen,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, substituted
heteroaryl and X-L wherein: [0046] X is selected from a reporter
molecule, a carrier molecule, a solid phase, or a therapeutic
molecule such as peptide, a protein, an antibody, a polysaccharide,
a nucleic acid polymer, an ion complexing moiety, a lipid or a
non-biological organic polymer or polymeric micro or nano particle,
that are optionally bound to one or more additional fluorophores;
or [0047] X is a reactive group such as carboxylic acid, an
activated ester of carboxylic acid, an amine, a hydrazine, a
haloacetamide, an alkyl halide, an isothiocynate or a maleimide
group; and [0048] L is an independently a single covalent bond or L
is covalent linkage having 1-24 non-hydrogen atoms selected from
the group consisting of C, N, O, P and S and composed of any
combinations of single, double, triple or aromatic carbon-carbon
bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds,
carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds
and phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl;
[0049] Z is an independently a single covalent bond or Z is
covalent linkage having 1-10 non-hydrogen atoms selected from the
group consisting of C, N, O, P and S and composed of any
combinations of single, double, triple or aromatic carbon-carbon
bonds, carbon-nitrogen bonds, carbon-oxygen bonds, and
carbon-sulfur bonds in the form of a straight- or branched-chain
alkyl or heteroalkyl having a chain length of 1-10 atoms, or is
absent; and
[0050] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0051] In some embodiments, the compound is of formula (VI), and
R.sub.1, R.sub.2, R.sub.3, and R.sub.4, are independently selected
from hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a salt of
carboxylic acid, CN, nitro, hydroxyl, amino, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, alkylthio, alkanoylamino,
alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, arylcarboxamido,
alkyl and aryl portions are optionally substituted one or more
times by halogen, --SO.sub.3X, a carboxylic acid, a salt of
carboxylic acid, CN, nitro, hydroxyl, amino, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, alkylthio, alkanoylamino,
alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, arylcarboxamido;
or R.sub.1 and R.sub.2, R.sub.2 and R.sub.3, R.sub.3 and R.sub.4
together form a fused moiety selected from cycloalkyl,
heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, and R.sub.5 and
R.sub.6 are independently selected from hydrogen, halogen, alkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,
substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl.
[0052] One embodiment is the compound:
##STR00009##
[0053] In another aspect, the invention provides a compound of the
formula:
##STR00010##
wherein:
[0054] A is a carbon, or A, R.sub.5, and R.sub.6 are absent;
[0055] R.sub.1, R.sub.2, and R.sub.3 are independently selected
from hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a salt of
carboxylic acid, CN, nitro, hydroxyl, amino, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, alkylthio, alkanoylamino,
alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, arylcarboxamido,
alkyl and aryl portions are optionally substituted one or more
times by halogen, --SO.sub.3X, a carboxylic acid, a salt of
carboxylic acid, CN, nitro, hydroxyl, amino, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, alkylthio, alkanoylamino,
alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido;
[0056] R.sub.5, and R.sub.6, are independently selected from
hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a salt of
carboxylic acid, CN, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl,
aryl, heteroaryl, substituted aryl, arylalkyl, substituted
arylalkyl, and substituted heteroaryl, arylcarboxamido, alkyl and
aryl portions are optionally substituted one or more times by
halogen, --SO.sub.3X, a carboxylic acid, a salt of carboxylic acid,
CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, alkylthio, alkanoylamino,
alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, arylcarboxamido;
or
two substituents selected from R.sub.1, R.sub.2, R.sub.3, R.sub.5,
and R.sub.6, wherein the two substituents are on different carbon
atoms, together form a fused moiety selected from cycloalkyl,
heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, and the
remaining substituents are selected from hydrogen, halogen, alkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,
substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl;
[0057] at least one substituent selected from R.sub.1, R.sub.2,
R.sub.3, R.sub.5, and R.sub.6 comprises X-L-, wherein: [0058] X is
selected from a reporter molecule, a carrier molecule, a solid
phase, or a therapeutic molecule such as peptide, a protein, an
antibody, a polysaccharide, a nucleic acid polymer, an ion
complexing moiety, a lipid or a non-biological organic polymer or
polymeric micro or nano particle, that are optionally bound to one
or more additional fluorophores; or [0059] X is a reactive group
such as carboxylic acid, an activated ester of carboxylic acid, an
amine, a hydrazine, a haloacetamide, an alkyl halide, an
isothiocynate or a maleimide group; and [0060] L is an
independently a single covalent bond or L is covalent linkage
having 1-24 non-hydrogen atoms selected from the group consisting
of C, N, O, P and S and composed of any combinations of single,
double, triple or aromatic carbon-carbon bonds, carbon-nitrogen
bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur
bonds, phosphorus-oxygen bonds and phosphorus-nitrogen bonds in the
form of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy,
substituted alkoxy, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl;
[0061] Z is an independently a single covalent bond or Z is
covalent linkage having 1-10 non-hydrogen atoms selected from the
group consisting of C, N, O, P and S and composed of any
combinations of single, double, triple or aromatic carbon-carbon
bonds, carbon-nitrogen bonds, carbon-oxygen bonds, and
carbon-sulfur bonds in the form of a straight- or branched-chain
alkyl or heteroalkyl having a chain length of 1-10 atoms, or is
absent; and
[0062] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0063] In another aspect, the invention provides a compound of the
formula:
##STR00011##
wherein:
[0064] A is a carbon, or A, R.sub.5, and R.sub.6 are absent;
[0065] R.sub.1, and R.sub.2 are independently selected from
hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a salt of
carboxylic acid, CN, nitro, hydroxyl, amino, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl, substituted
alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl, substituted
arylalkyl, and substituted heteroaryl, arylcarboxamido, alkyl and
aryl portions are optionally substituted one or more times by
halogen, --SO.sub.3X, a carboxylic acid, a salt of carboxylic acid,
CN, nitro, hydroxyl, amino, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl,
aryl, heteroaryl, substituted aryl, arylalkyl, substituted
arylalkyl, and substituted heteroaryl, arylcarboxamido;
[0066] R.sub.5, and R.sub.6, are independently selected from
hydrogen, --SO.sub.3X, a carboxylic acid, a salt of carboxylic
acid, CN, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido, alkyl and aryl portions are optionally
substituted one or more times by halogen, --SO.sub.3X, a carboxylic
acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino,
hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido; or
[0067] at least one substituent selected from R.sub.1, R.sub.2,
R.sub.5, and R.sub.6 comprises X-L-, wherein: [0068] X is selected
from a reporter molecule, a carrier molecule, a solid phase, or a
therapeutic molecule such as peptide, a protein, an antibody, a
polysaccharide, a nucleic acid polymer, an ion complexing moiety, a
lipid or a non-biological organic polymer or polymeric micro or
nano particle that are optionally bound to one or more additional
fluorophores; or [0069] X is a reactive group such as carboxylic
acid, an activated ester of carboxylic acid, an amine, a hydrazine,
a haloacetamide, an alkyl halide, an isothiocynate or a maleimide
group; and [0070] L is an independently a single covalent bond or L
is covalent linkage having 1-24 non-hydrogen atoms selected from
the group consisting of C, N, O, P and S and composed of any
combinations of single, double, triple or aromatic carbon-carbon
bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds,
carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds
and phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl;
[0071] Z is an independently a single covalent bond or Z is
covalent linkage having 1-10 non-hydrogen atoms selected from the
group consisting of C, N, O, P and S and composed of any
combinations of single, double, triple or aromatic carbon-carbon
bonds, carbon-nitrogen bonds, carbon-oxygen bonds, and
carbon-sulfur bonds in the form of a straight- or branched-chain
alkyl or heteroalkyl having a chain length of 1-10 atoms, or is
absent; and
[0072] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0073] In another aspect, the invention provides a compound of the
formula:
##STR00012##
wherein:
[0074] A is a carbon, or A, R.sub.5, and R.sub.6 are absent;
[0075] m and n is an integer between 1 and 4;
[0076] B is O or S and R.sub.3 and R.sub.4 are absent or N and
R.sub.3 or R.sub.4 is absent;
[0077] R.sub.7 is selected from hydrogen, alkyl, heteroalkyl,
substituted alkyl, and substituted heteroalkyl;
[0078] R.sub.1, R.sub.2, R.sub.3, R.sub.4, each R', and each R''
are independently selected from hydrogen, halogen, --SO.sub.3X, a
carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl,
amino, hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido, alkyl and aryl portions are optionally
substituted one or more times by halogen, --SO.sub.3X, a carboxylic
acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino,
hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido; or two substituents selected from
R.sub.1, R.sub.2, R.sub.3, R.sub.4, an R', and an R'', wherein the
two substituents are on different carbon atoms together form a
fused moiety selected from cycloalkyl, heterocycloalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl, and all of the remaining substituents are
independently selected from hydrogen, halogen, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl; or
two of the remaining substituents also together form a fused moiety
selected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl, and
the remaining substituents are independently selected from
hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl;
[0079] wherein, R.sub.5, and R.sub.6, are independently selected
from hydrogen, --SO.sub.3X, a carboxylic acid, a salt of carboxylic
acid, CN, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido, alkyl and aryl portions are optionally
substituted one or more times by halogen, --SO.sub.3X, a carboxylic
acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino,
hydrazine, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido; or
[0080] at least one substituent selected from R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, an R', and an R'' comprises
X-L-, wherein: [0081] X is selected from a reporter molecule, a
carrier molecule, a solid phase, or a therapeutic molecule such as
peptide, a protein, an antibody, a polysaccharide, a nucleic acid
polymer, an ion complexing moiety, a lipid or a non-biological
organic polymer or polymeric micro or nano particle, that are
optionally bound to one or more additional fluorophores; or [0082]
X is a reactive group such as carboxylic acid, an activated ester
of carboxylic acid, an amine, a hydrazine, a haloacetamide, an
alkyl halide, an isothiocynate or a maleimide group; and [0083] L
is an independently a single covalent bond or L is covalent linkage
having 1-24 non-hydrogen atoms selected from the group consisting
of C, N, O, P and S and composed of any combinations of single,
double, triple or aromatic carbon-carbon bonds, carbon-nitrogen
bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur
bonds, phosphorus-oxygen bonds and phosphorus-nitrogen bonds in the
form of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy,
substituted alkoxy, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl;
[0084] Z is an independently a single covalent bond or Z is
covalent linkage having 1-10 non-hydrogen atoms selected from the
group consisting of C, N, O, P and S and composed of any
combinations of single, double, triple or aromatic carbon-carbon
bonds, carbon-nitrogen bonds, carbon-oxygen bonds, and
carbon-sulfur bonds in the form of a straight- or branched-chain
alkyl or heteroalkyl having a chain length of 1-10 atoms, or is
absent; and
[0085] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0086] In some embodiments of the compound of the formula (I),
(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX),
(X), (XI) or (XII), all but one of the R substituents are H.
[0087] In some embodiments of the compound of the formula (I),
(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX),
(X), (XI) or (XII), L is an alkyl group having a chain length of 0
to 15 atoms. In some of these, L is an alkyl group having a chain
length of 0 to 5 atoms. In others, L is
--NH--(CH.sub.2).sub.n--NH--C(O)--, wherein n is 1 to 12. In
others, the compound is:
##STR00013##
[0088] In some embodiments of the compound of the formula (I),
(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX),
(X), (XI) or (XII), the reporter molecule comprises a chromophore,
fluorophore, fluorescent protein, phosphorescent dye, tandem dye,
particle, hapten, enzyme, or radioisotope. In some of these, the
fluorophore is a xanthene, coumarin, cyanine, pyrene, oxazine,
borapolyazaindacene, or carbopyranine. In others, the enzyme is
horseradish peroxidase, alkaline phosphatase, beta-galactosidase,
or beta-lactamase. In others, the particle is a semiconductor
nanocrystal.
[0089] In some embodiments of the compound of the formula (I),
(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX),
(X), (XI) or (XII), the carrier molecule is an amino acid, peptide,
protein, polysaccharide, nucleoside, nucleotide, oligonucleotide,
nucleic acid, hapten, psoralen, drug, hormone, lipid, lipid
assembly, tyramine, synthetic polymer, polymeric microparticle,
biological cell, cellular component, ion chelating moiety,
enzymatic substrate, or virus.
[0090] In some embodiments of the compound of the formula (I),
(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX),
(X), (XI) or (XII), the carrier molecule is an antibody, antibody
fragment, antigen, avidin, streptavidin, biotin, dextran, IgG
binding protein, fluorescent protein, agarose, or non-biological
microparticle.
[0091] In some embodiments of the compound of the formula (I),
(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX),
(X), (XI) or (XII), the solid support is an aerogel, hydrogel,
resin, bead, biochip, microfluidic chip, silicon chip, multi-well
plate, membrane, conducting metal, nonconducting metal, glass, or
magnetic support.
[0092] In some embodiments of the compound of the formula (I),
(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX),
(X), (XI) or (XII), the solid support is a silica gel, polymeric
membrane, particle, derivatized plastic film, glass bead, cotton,
plastic bead, alumina gel, polysaccharide, poly(acrylate),
polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose,
dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan,
inulin, nitrocellulose, diazocellulose, polyvinylchloride,
polypropylene, polyethylene, nylon, latex bead, magnetic bead,
paramagnetic bead, superparamagnetic bead, or starch.
[0093] In some embodiments of the compound of the formula (I),
(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX),
(X), (XI) or (XII), the therapeutic molecule is taxol, cytochalasin
B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine,
tetracaine, lidocaine, propranolol, puromycin, or analogs or
homologs thereof.
[0094] In some embodiments of the compound of the formula (I),
(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX),
(X), (XI) or (XII), the therapeutic molecule is an antimetabolite,
alkylating agent, anthracycline, antibiotic, or anti-mitotic
agent.
[0095] In some embodiments of the compound of the formula (I),
(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX),
(X), (XI) or (XII), the therapeutic molecule is abrin, ricin A,
pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor,
.gamma.-interferon, .alpha.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator,
interleukin-1, interleukin-2, interleukin-6, granulocyte macrophage
colony stimulating factor, or granulocyte colony stimulating
factor.
[0096] In some embodiments of the compound of the formula (I),
(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX),
(X), (XI) or (XII), G is an alkyne-reactive group. In some of
these, the alkyne-reactive group is an azide.
[0097] In some embodiments of the compound of the formula (I),
(II), (III), (IV), (XIII), (V), (VI), (VII), (VIII), (XIV), (IX),
(X), (XI) or (XII), G is an azide-reactive group. In some of these,
the azide-reactive group is a terminal alkyne.
[0098] In another aspect, the invention provides a method of
modifying a biomolecule comprising the step of reacting in a
solution a biomolecule comprising an azide reactive moiety with a
compound of the formula (I), (II), (III), (IV), (XIII), (V), (VI),
(VII), (VIII), (XIV), (IX), (X), (XI) or (XII), wherein the
therapeutic molecule is an antimetabolite, alkylating agent,
anthracycline, antibiotic, or anti-mitotic agent to provide a
modified biomolecule.
[0099] In some embodiments, the azide reactive moiety comprises a
terminal alkyne, an activated alkyne, or triarylphosphine.
[0100] In another aspect, the invention provides a method of
modifying a biomolecule comprising the step of reacting in a
solution a biomolecule comprising an alkyne reactive moiety with a
compound of the formula (I), (II), (III), (IV), (XIII), (V), (VI),
(VII), (VIII), (XIV), (IX), (X), (XI) or (XII), wherein G is an
alkyne reactive moiety to provide a modified biomolecule.
[0101] In some embodiments, the alkyne reactive moiety comprises an
azide.
[0102] In some embodiments, the biomolecule is a nucleic acid,
oligonucleotide, protein, peptide, carbohydrate, polysaccharide,
glycoprotein, lipid, hormone, drug, or prodrug.
[0103] In some embodiments, the solution further comprises copper
ions. In some of these, the solution further comprises at least one
reducing agent. In some of these, the at least one reducing agent
is ascorbate, Tris(2-Carboxyethyl)Phosphine (TCEP), TCP
(2,4,6-trichlorophenol), NADH, NADPH, thiosulfate,
2-mercaptoethanol, dithiothreitol, glutathione, cysteine, metallic
copper, quinone, hydroquinone, vitamin K1, Fe2+, Co2+, or an
applied electric potential. In some of these, the at least one
reducing agent is ascorbate.
[0104] In some of these, the solution further comprises a copper
chelator. In some of these, the copper chelator is a copper I
chelator. In some of these, the copper chelator I is a compound of
formula:
##STR00014##
wherein X, Y, and Z each independently have the formula:
##STR00015##
wherein:
[0105] R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are
independently selected from hydrogen, halogen, --SO.sub.3X, a
carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl,
amino, alkyl, heteroalkyl, alkoxy, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted alkoxy,
alkylthio, alkanoylamino, alkylaminocarbonyl, substituted
cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, arylcarboxamido, alkyl and aryl portions are optionally
substituted one or more times by halogen, --SO.sub.3X, a carboxylic
acid, a salt of carboxylic acid, CN, nitro, hydroxyl, amino, alkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,
substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido; and
[0106] B, C, D, and E are C or N; and
[0107] L.sub.1, L.sub.2, and L.sub.3 are covalent linkage having
1-5 non-hydrogen atoms selected from the group consisting of C, N,
O, P and S and composed of any combinations of single, double,
triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds,
nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds,
and phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
and substituted heterocycloalkyl.
[0108] In some of these, R.sub.1, R.sub.2, R.sub.3, R.sub.4 and
R.sub.5 for at least one substituent selected from X, Y, and Z are
each H. In some of these, R.sub.1, R.sub.2, R.sub.3, R.sub.4 and
R.sub.5 for each of X, Y, and Z are each H.
[0109] In some of these, L.sub.1, L.sub.2 and L.sub.3 are each
alkyl groups having a chain length of 1-5 atoms.
[0110] In some of these, L.sub.1, L.sub.2 and L.sub.3 are each
--CH.sub.2CH.sub.2--.
[0111] In others, the copper chelator is
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA,
neocuproine, N-(2-acetamido)iminodiacetic acid (ADA),
pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine
(SCMC), 1,10 phenanthroline, or a derivative thereof, trientine,
glutathione, histidine, polyhistidine,
tris-(hydroxypropyltriazolylmethyl)amine (THPTA), or
tetra-ethylenepolyamine (TEPA).
[0112] In others, the copper chelator is 1,10 phenanthroline,
bathophenanthroline disulfonic acid
(4,7-diphenyl-1,10-phenanthroline disulfonic acid), or
bathocuproine disulfonic acid
(2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate).
[0113] In another aspect, the provides a kit comprising a compound
of the formula (I), (II), (III), (IV), (XIII), (V), (VI), (VII),
(VIII), (XIV), (IX), (X), (XI) or (XII).
[0114] In some embodiments, the kit further comprises a copper ion
source.
[0115] In some embodiments, the kit further comprises at least one
reducing agent. In some of these, the at least one reducing agent
is ascorbate, Tris(2-Carboxyethyl)Phosphine (TCEP), TCP
(2,4,6-trichlorophenol), NADH, NADPH, thiosulfate,
2-mercaptoethanol, dithiothreitol, glutathione, cysteine, metallic
copper, quinone, hydroquinone, vitamin K1, Fe2+, Co2+, or an
applied electric potential. In others, the at least one reducing
agent is ascorbate.
[0116] In some embodiments, the kit further comprises a copper
chelator. In some of these, the copper chelator is a copper I
chelator. In some of these, the copper chelator I is a compound of
formula:
##STR00016##
wherein X, Y, and Z each independently have the formula:
##STR00017##
wherein:
[0117] R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are
independently selected from hydrogen, halogen, --SO.sub.3X, a
carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxyl,
amino, alkylthio, alkanoylamino, alkylaminocarbonyl,
arylcarboxamido, alkyl aryl portions are optionally substituted one
or more times by halogen, --SO.sub.3X, a carboxylic acid, a salt of
carboxylic acid, CN, nitro, hydroxyl, amino, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, alkylthio, alkanoylamino,
alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, arylcarboxamido;
and
[0118] B, C, D, and E are C or N; and
[0119] L.sub.1, L.sub.2, and L.sub.3 are covalent linkage having
1-5 non-hydrogen atoms selected from the group consisting of C, N,
O, P and S and composed of any combinations of single, double,
triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds,
nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds,
and phosphorus-nitrogen bonds in the form of alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
and substituted heterocycloalkyl.
[0120] In some of these, R.sub.1, R.sub.2, R.sub.3, R.sub.4 and
R.sub.5 for at least one substituent selected from X, Y, and Z are
each H. In some of these, R.sub.1, R.sub.2, R.sub.3, R.sub.4 and
R.sub.5 for each of X, Y, and Z are each H.
[0121] In some of these, L.sub.1, L.sub.2 and L.sub.3 are each
alkyl groups having a chain length of 1-5 atoms. In some of these,
L.sub.1, L.sub.2 and L.sub.3 are each --CH.sub.2CH.sub.2--.
[0122] In others, the copper chelator is
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA,
neocuproine, N-(2-acetamido)iminodiacetic acid (ADA),
pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine
(SCMC), 1,10 phenanthroline, or a derivative thereof, trientine,
glutathione, histidine, polyhistidine,
tris-(hydroxypropyltriazolylmethyl)amine (THPTA), or
tetra-ethylenepolyamine (TEPA).
[0123] In others, the copper chelator is 1,10 phenanthroline,
bathophenanthroline disulfonic acid
(4,7-diphenyl-1,10-phenanthroline disulfonic acid), or
bathocuproine disulfonic acid
(2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate).
[0124] In another aspect, the invention provides a compound
selected from the group consisting of:
##STR00018##
[0125] In another aspect, the invention provides a composition
comprising a compound of the formula (I), (II), (III), (IV),
(XIII), (V), (VI), (VII), (VIII), (XIV), (IX), (X), (XI) or (XII);
and a copper chelator I of the formula (XV).
BRIEF DESCRIPTION OF THE DRAWINGS
[0126] FIG. 1 shows a reaction scheme for (A) the synthesis of
tert-butyl(2-(6-(azidomethyl)nicotinamido)ethyl)carbamate (6), and
(B) fluorescent-labeling of compound (6), as described in Example
1.
[0127] FIG. 2 shows a reaction scheme for the synthesis of
tert-butyl(2-(6-((prop-2-yn-1-yloxy)methyl)nicotinamido)ethyl)carbamate
(12), and fluorescent labeling of compound (12), as described in
Example 1.
[0128] FIG. 3 shows rates of click reactions between reagents (A)
QSY pAzide and Oregon Green.RTM. alkyne, and (B) QSY Azide and
Oregon Green.RTM. alkyne, at various concentrations of Cu, as
described in Example 2.
[0129] FIG. 4 shows the effect of increasing concentrations of
Cu.sup.2+, and increasing concentrations of Cu.sup.2+ in the
presence of copper chelator THPTA on the click reaction between
Oregon Green.RTM. alkyne and QSY Azide or QSY pAzide, as described
in Example 2.
[0130] FIG. 5 shows the results of click reactions between a
coumarin-alkyne (16) and various azides in the presence of (A) 125
.mu.M Cu, or (B) 31 .mu.M Cu, with or without THPTA at a ratio of
4:1 (THPTA:Cu), as described in Example 4.
[0131] FIG. 6 shows the stability of GFP in the presence of (A)
various concentrations of Cu(II), and (B) various concentrations of
Cu(II) with sodium ascorbate, as described in Example 5.
[0132] FIG. 7 shows (A) the stability of GFP in the presence of
Cu(II), with or without ascorbate, and with or without THPTA, and
(B) the rate of a click reaction between Oregon Green.RTM. alkyne
and QSY azide under the same concentrations shown in (A), as
described in Example 5.
[0133] FIG. 8 shows Click Labeling of RNA with EU in presence of
GPF with AF647-picolyl azide or AF647 azide with or without THPTA
as ligand as described in Example 6.
[0134] FIG. 9 shows Click Labeling of RNA with EU in presence of
GPF with AF647-picolyl azide or AF647 azide with THPTA as ligand
with various molar ratio of Cu:THPTA. as described in Example
6.
[0135] FIG. 10 shows Graphical representation of GFP stability and
progression of click reaction at 2 mM copper as described in
Example 6.
[0136] FIG. 11 shows Graphical representation of click reaction
rate with or without THPTA at different molar ratios of Cu:THPTA as
described in Example 6
[0137] FIG. 12 shows Graphical representation of GFP stability and
progression of click reaction at 200 .mu.M copper
[0138] FIG. 13 shows Click Labeling of HPG with AF647-picolyl azide
or AF647 azide in presence of GPF with THPTA as described in
Example 7.
[0139] FIG. 14 shows click Labeling of HPG with AF647-picolyl azide
or AF647 azide in presence of GPF with THPTA as ligand with various
molar ratio of Cu:THPTA as described in Example 7.
[0140] FIG. 15 shows graphical representation of GFP stability and
progression of click reaction with HPG at 2 mM copper as described
in Example 7.
[0141] FIG. 16 shows Click Labeling of HPG with AF647-picolyl azide
followed by phalloidin staining in presence of GPF with THPTA as
described in Example 8.
[0142] FIG. 17 shows click Labeling of HPG with AF647-picolyl azide
or AF647 azide in presence of GPF with THPTA as ligand with various
molar ratio of Cu:THPTA as described in Example 8.
[0143] FIG. 18 shows a SDS-PAGE gel GalNAz-labeled monoclonal
anti-TSH click reacted with either DIBO or click reacted
THPTA/Picolyl, where in the latter the THPTA is varied with respect
to the Cu concentration.
[0144] FIG. 19 shows the ligation of a picolyl azide derivative
bearing a carboxylate terminal onto recombinant proteins expressed
on the surface of living mammalian cells, as described in Example
10.
[0145] FIG. 20 shows the HPLC analysis of .sup.W37VLp1A-catalyzed
ligation of picolyl azide (5-(6-(azidomethyl)nicotinamido)pentanoic
acid) onto LAP peptide, as described in Example 10.
[0146] FIG. 21 shows the chelation-assisted copper(I) catalyzed
azide-alkyne cycloaddition for tagging of Alexa Fluor.RTM. 647 onto
neurexin in living HEK cells. Negative controls are shown with ATP
omitted (second column) or wild-type Lp1A in place of .sup.W37VLp1A
(third column). H2B-YFP is the nuclear-localized YFP transfection
marker, as described in Example 10.
[0147] FIG. 22 shows the chelation-assisted copper(I) catalyzed
azide-alkyne cycloaddition for tagging of Alexa Fluor.RTM. 647 onto
neuroligin-1 in living hippocampal neurons, as described in Example
10.
DETAILED DESCRIPTION
[0148] The present invention has utility in the study of
biomolecules, both in vivo and in vitro.
[0149] The present invention provides compositions, methods, and
kits for the labeling, detecting, isolating and/or analysis of
biomolecules modified by attachment of chemical handles.
DEFINITIONS AND ABBREVIATIONS
[0150] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
compositions or process steps, as such may vary. It must be noted
that, as used in this specification and the appended claims, the
singular form "a", "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a ligand" includes a plurality of ligands and
reference to "an antibody" includes a plurality of antibodies and
the like.
[0151] Certain compounds of the present invention can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are encompassed within the scope of the present
invention. Certain compounds of the present invention may exist in
multiple crystalline or amorphous forms. In general, all physical
forms are equivalent for the uses contemplated by the present
invention and are intended to be within the scope of the present
invention.
[0152] Certain compounds of the present invention possess
asymmetric carbon atoms (optical centers) or double bonds; the
racemates, diastereomers, geometric isomers and individual isomers
are encompassed within the scope of the present invention.
[0153] The compounds described herein may be prepared as a single
isomer (e.g., enantiomer, cis-trans, positional, diastereomer) or
as a mixture of isomers. In a preferred embodiment, the compounds
are prepared as substantially a single isomer. Methods of preparing
substantially isomerically pure compounds are known in the art. For
example, enantiomerically enriched mixtures and pure enantiomeric
compounds can be prepared by using synthetic intermediates that are
enantiomerically pure in combination with reactions that either
leave the stereochemistry at a chiral center unchanged or result in
its complete inversion. Alternatively, the final product or
intermediates along the synthetic route can be resolved into a
single stereoisomer. Techniques for inverting or leaving unchanged
a particular stereocenter, and those for resolving mixtures of
stereoisomers are well known in the art and it is well within the
ability of one of skill in the art to choose and appropriate method
for a particular situation. See, generally, Furniss et al. (eds.),
VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5.sup.TH ED.,
Longman Scientific and Technical Ltd., Essex, 1991, pp. 809-816;
and Heller, Acc. Chem. Res. 23: 128 (1990).
[0154] The compounds disclosed herein may also contain unnatural
proportions of atomic isotopes at one or more of the atoms that
constitute such compounds. For example, the compounds may be
radiolabeled with radioactive isotopes, such as for example tritium
(.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C). All
isotopic variations of the compounds of the present invention,
whether radioactive or not, are intended to be encompassed within
the scope of the present invention.
[0155] Where a disclosed compound includes a conjugated ring
system, resonance stabilization may permit a formal electronic
charge to be distributed over the entire molecule. While a
particular charge may be depicted as localized on a particular ring
system, or a particular heteroatom, it is commonly understood that
a comparable resonance structure can be drawn in which the charge
may be formally localized on an alternative portion of the
compound.
[0156] Selected compounds having a formal electronic charge may be
shown without an appropriate biologically compatible counterion.
Such a counterion serves to balance the positive or negative charge
present on the compound. As used herein, a substance that is
biologically compatible is not toxic as used, and does not have a
substantially deleterious effect on biomolecules. Examples of
negatively charged counterions include, among others, chloride,
bromide, iodide, sulfate, alkanesulfonate, arylsulfonate,
phosphate, perchlorate, tetrafluoroborate, tetraarylboride, nitrate
and anions of aromatic or aliphatic carboxylic acids. Preferred
counterions may include chloride, iodide, perchlorate and various
sulfonates. Examples of positively charged counterions include,
among others, alkali metal, or alkaline earth metal ions, ammonium,
or alkylammonium ions.
[0157] The term "alkyl," by itself or as part of another
substituent means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
may be fully saturated, mono- or polyunsaturated and can include
divalent ("alkenyl") and multivalent radicals, having the number of
carbon atoms designated (i.e. C.sub.1-C.sub.10 means one to ten
carbons). Examples of saturated hydrocarbon radicals include, but
are not limited to, groups such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,
(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for
example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An
unsaturated alkyl group is one having one or more double bonds or
triple bonds. Examples of unsaturated alkyl groups include, but are
not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The
term "alkyl," unless otherwise noted, is also meant to include
derivatives of alkyl, such as those defined below, including
heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,
substituted heteroalkyl, substituted cycloalkyl, and substituted
heterocycloalkyl. Alkyl groups that are limited to hydrocarbon
groups are termed "homoalkyl".
[0158] In some embodiments, an alkyl group contains between 1 and
25 carbons, between 1 and 20 carbons (i.e., C.sub.1 to C.sub.20
alkyl), between 1 and 15 carbons (i.e., C.sub.1 to C.sub.15 alkyl),
between 1 and 10 carbons (i.e., C.sub.1 to C.sub.10 alkyl), or
between 1 and 8 carbons (i.e., C.sub.1 to C.sub.8 alkyl). Straight,
branched or cyclic hydrocarbon chains having eight or fewer carbon
atoms may also be referred to herein as "lower alkyl". In addition,
the term "alkyl" as used herein may further include one or more
substitutions at one or more carbon atoms of the hydrocarbon chain
fragment.
[0159] The term "carboxyalkyl" as used herein refers to a straight
or branched-chain alkyl including cycloalkyl comprising at least
one --COOH substituent. The terms "alkoxy," "alkylamino" and
"alkylthio" (or thioalkoxy) are used in their conventional sense,
and refer to heteroalkyl groups attached to the remainder of the
molecule via an oxygen atom, an amino group, or a sulfur atom,
respectively.
[0160] The term "acyl" or "alkanoyl" by itself or in combination
with another term, means, unless otherwise stated, a stable
straight or branched chain, or cyclic alkyl, or combinations
thereof, with an acyl radical on at least one terminus of the
alkyl. An "acyl radical" is a group derived from a carboxylic acid
by removing the --OH moiety therefrom.
[0161] The term "amino" or "amine group" refers to the group
--NR'R'' where R' and R'' are independently selected from hydrogen,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl. In a primary
amine group, both R' and R'' are hydrogen, whereas in a secondary
amine group, either, but not both, R' or R'' is hydrogen. In a
tertiary amine group, neither R' nor R'' is a hydrogen. A
substituted amine is an amine group wherein R' and/or R'' is other
than hydrogen. In addition, the terms "amine" and "amino" can
include protonated and quaternized versions of nitrogen, comprising
the group --NRR'R'' and its biologically compatible anionic
counterions.
[0162] As used herein, the term "heteroatom" includes oxygen (O),
nitrogen (N), sulfur (S), phosphorus (P), silicon (Si), and
selenium (Se).
[0163] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a straight or
branched chain, or cyclic carbon-containing radical, or
combinations thereof, consisting of the stated number of carbon
atoms and at least one heteroatom selected from the group
consisting of O, N, Si, P, S, and Se and wherein the nitrogen,
phosphorous, sulfur, and selenium atoms are optionally oxidized,
and the nitrogen heteroatom is optionally be quaternized. The
heteroatom(s) O, N, P, S, Si, and Se may be placed at any interior
position of the heteroalkyl group or at the position at which the
alkyl group is attached to the remainder of the molecule. Examples
include, but are not limited to, --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3. Similarly, the term
"heteroalkylene" by itself or as part of another substituent means
a divalent radical derived from heteroalkyl, as exemplified, but
not limited by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula
--C(O).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--.
[0164] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include, but are not limited
to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include,
but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),
1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,
3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,
2-piperazinyl, and the like.
[0165] The term "aryl" means, unless otherwise stated, a
polyunsaturated, aromatic moiety that can be a single ring or
multiple rings (preferably from 1 to 3 rings), which are fused
together or linked covalently. In some embodiments, an aryl group
contains twenty or fewer carbon atoms, e.g., phenyl, naphthyl,
biphenyl, and anthracenyl. One or more carbon atoms of the aryl
group may also be substituted with, e.g., alkyl; aryl; heteroaryl;
a halogen; nitro; cyano; hydroxyl, alkoxyl or aryloxyl; thio or
mercapto, alkyl-, or arylthio; amino, alkylamino, arylamino,
dialkyl-, diaryl-, or arylalkylamino; aminocarbonyl,
alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl,
diarylaminocarbonyl, or arylalkylaminocarbonyl; carboxyl, or alkyl-
or aryloxycarbonyl; aldehyde; aryl- or alkylcarbonyl; iminyl, or
aryl- or alkyliminyl; sulfo; alkyl- or alkylcarbonyl; sulfo; alkyl-
or arylsufonyl; hydroximinyl, or aryl- or alkoximinyl. In addition,
two or more alkyl or heteroalkyl substituents of an aryl group may
be combined to form fused aryl-alkyl or aryl-heteroalkyl ring
systems (e.g., tetrahydronaphthyl). Substituents including
heterocyclic groups (e.g., heteroaryloxy, and heteroaralkylthio)
are defined by analogy to the above-described terms.
[0166] The term "heteroaryl" refers to aryl groups (or rings) that
contain from one to four heteroatoms selected from N, O, S, and Se,
wherein the nitrogen, sulfur, and selenium atoms are optionally
oxidized, and the nitrogen atom(s) are optionally quaternized. A
heteroaryl group can be attached to the remainder of the molecule
through a heteroatom. Non-limiting examples of aryl and heteroaryl
groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl,
1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,
4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,
2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,
5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl,
3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, tetrazolyl,
benzo[b]furanyl, benzo[b]thienyl, 2,3-dihydrobenzo[1,4]dioxin-6-yl,
benzo[1,3]dioxol-5-yl and 6-quinolyl. Substituents for aryl and
heteroaryl ring systems are selected from the group of acceptable
substituents described below.
[0167] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxyl)propyl, and the like).
[0168] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl," "heteroaryl," etc.) includes both substituted and
unsubstituted forms of the indicated radical. Nonlimiting exemplary
substituents for each type of radical are provided below.
[0169] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are
generically referred to as "alkyl group substituents," and they can
be one or more of a variety of groups selected from, but not
limited to: --OR, .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR',
-halogen, --SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and --NO.sub.2 in a number
ranging from zero to (2m'+1), where m' is the total number of
carbon atoms in such radical. R, R'', R''' and R'''' each
preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g.,
aryl substituted with 1-3 halogens, substituted or unsubstituted
alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a
compound includes more than one R group, for example, each of the R
groups is independently selected as are each R', R'', R''' and
R'''' groups when more than one of these groups is present. When R'
and R'' are attached to the same nitrogen atom, they can be
combined with the nitrogen atom to form a 5-, 6-, or 7-membered
ring. For example, --NR'R'' is meant to include, but not be limited
to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of
substituents, one of skill in the art will understand that the term
"alkyl" is meant to include groups including carbon atoms bound to
groups other than hydrogen groups, such as haloalkyl (e.g.,
--CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g., --C(O)CH.sub.3,
--C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the like).
[0170] Similar to the substituents described for the alkyl radical,
substituents for the aryl and heteroaryl groups are generically
referred to as "aryl group substituents." The substituents are
selected from, for example: halogen, --OR', .dbd.O, .dbd.NR',
.dbd.N--OR', --NR'R'', --SR', -halogen, --SiR'R''R''', --OC(O)R',
--C(O)R', --CO.sub.2R', --CONR'R'', --OC(O)NR'R'', --NR''C(O)R',
--NR'--C(O)NR''R''', --NR''C(O).sub.2R',
--NR--C(NR'R''R''').dbd.NR'''', --NR--C(NR'R'').dbd.NR''',
--S(O)R', --S(O).sub.2R', --S(O).sub.2NR'R'', --NRSO.sub.2R', --CN
and --NO.sub.2, --R', --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxy, and fluoro(C.sub.1-C.sub.4)alkyl, in
a number ranging from zero to the total number of open valences on
the aromatic ring system; and where R', R'', R''' and R'''' are
preferably independently selected from hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl and substituted or unsubstituted
heteroaryl. When a compound includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R'', R''' and R'''' groups when more than one of these groups
is present. In the schemes that follow, the symbol X represents "R"
as described above.
[0171] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CRR').sub.q--U--, wherein T and U are
independently --NR--, --O--, --CRR'-- or a single bond, and q is an
integer of from 0 to 3. Alternatively, two of the substituents on
adjacent atoms of the aryl or heteroaryl ring may optionally be
replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CRR'--, --O--, --NR--, --S--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 4. One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula
--(CRR').sub.s--X--(CR''R''').sub.d--, where s and d are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituents R, R', R'' and R''' are preferably independently
selected from hydrogen or substituted or unsubstituted
(C.sub.1-C.sub.6)alkyl.
[0172] The term "chain length," as used herein, refers to the
smallest number of carbon and/or heteroatoms between two
substituents. As a nonlimiting example, the chain length between X
and Y in the molecule
X--(CH.sub.2).sub.3--CH(CH.sub.2CH.sub.3)--NH--Y is 5.
[0173] The term "activated alkyne," as used herein, refers to a
cyclooctyne that selectively reacts with an azide on another
molecule to form a covalent chemical bond between the activated
alkyne group and the alkyne reactive group. Activated alkynes
include, but are not limited to, cyclooctynes and
difluorocyclooctynes, described, e.g., in Agard et al., J. Am.
Chem. Soc., 2004, 126 (46):15046-15047; dibenzocyclooctynes,
described, e.g., in Boon et al., WO2009/067663 A1 (2009); and
aza-dibenzocyclooctynes, described, e.g., in Debets et al., Chem.
Comm, 2010, 46:97-99. These dibenzocyclooctynes (including the
aza-dibenzocyclooctynes) described above are collectively referred
to herein as cyclooctyne groups.
[0174] The term "affinity," as used herein, refers to the strength
of the binding interaction of two molecules, such as an antibody
and an antigen, or a positively charged moiety and a negatively
charged moiety. For bivalent molecules such as antibodies, affinity
is typically defined as the binding strength of one binding domain
for the antigen, e.g. one Fab fragment for the antigen. The binding
strength of both binding domains together for the antigen is
referred to as "avidity". As used herein "high affinity" refers to
a ligand that binds to an antibody having an affinity constant
(K.sub.a) greater than 10.sup.4 M.sup.-1, typically
10.sup.5-10.sup.11 M.sup.-1; as determined by inhibition ELISA or
an equivalent affinity determined by comparable techniques such as,
for example, Scatchard plots or using K.sub.d/dissociation
constant, which is the reciprocal of the K.sub.a.
[0175] The term "alkyne reactive," as used herein, refers to a
chemical moiety that selectively reacts with an alkyne, such as a
terminal alkyne or an activated alkyne, on another molecule to form
a covalent chemical bond between the alkyne modified group and the
alkyne reactive group. Examples of alkyne-reactive groups include,
but are not limited to, azide and nitrones. "Alkyne-reactive" can
also refer to a molecule that contains a chemical moiety that
selectively reacts with an alkyne group.
[0176] The term "antibody," as used herein, refers to a protein of
the immunoglobulin (Ig) superfamily that binds noncovalently to
certain substances (e.g. antigens and immunogens) to form an
antibody-antigen complex. Antibodies can be polyclonal or
monoclonal. Antibodies can also be chimeric, humanized, or human
antibodies. It is understood that the term "antibody" as used
herein includes within its scope any of the various classes or
sub-classes of immunoglobulin derived from any of the animals
conventionally used, or from human.
[0177] The term "antibody fragments," as used herein, refers to
fragments of antibodies that retain the principal selective binding
characteristics of the whole antibody. Nonlimiting exemplary
antibody fragment include Fab, Fab', F(ab').sub.2, Fv, and
single-chain Fv (scFv). Further nonlimiting exemplary antibody
fragments include (i) the Fd fragment, consisting of the VH and CH1
domains; (ii) the dAb fragment (Ward, et al., Nature 341, 544
(1989)), which consists of a VH domain; and (iii) isolated CDR
regions. In addition, arbitrary fragments can be made using
recombinant technology that retains antigen-recognition
characteristics.
[0178] The term "antigen," as used herein, refers to a molecule or
molecules to which an antibody binds selectively. An antigen may
comprise any type of molecule, such as, for example, protein,
oligonucleotide, polysaccharide, or small molecule. In some
embodiments, an antigen comprises more than one molecule, such as
for example, a heterodimeric receptor, a receptor bound to its
ligand, or a complex comprising a protein and a small molecule or
oligonucleotide. In some embodiments, a target is an antigen.
[0179] The term "aqueous solution," as used herein, refers to a
solution that is at least 50% water. In some embodiments, an
aqueous solution retains the solution characteristics of water.
[0180] The term "azide reactive," as used herein, refers to a
chemical moiety that selectively reacts with an azide on another
molecule to form a covalent chemical bond between the azido
modified group and the azide reactive group. Examples of
azide-reactive groups include, but are not limited to, alkyne,
including, but not limited to, terminal alkynes and activated
alkynes; and phosphines, including, but not limited to,
triarylphosphines. "Azide-reactive" can also refer to a molecule
that contains a chemical moiety that selectively reacts with an
azido group.
[0181] The term "biomolecule," as used herein, refers to proteins,
peptides, amino acids, glycoproteins, nucleic acids, nucleotides,
nucleosides, oligonucleotides, sugars, oligosaccharides, lipids,
hormones, proteoglycans, carbohydrates, polypeptides,
polynucleotides, polysaccharides, drugs, prodrugs, etc., which may
be found in a living organism (including an isolated cell). A
biomolecule need not be a naturally-occurring molecule, but may be
a molecule that has been introduced into the living organism or an
ancestor of the living organism, e.g., directly, through transgenic
methods, or otherwise.
[0182] The term "carrier molecule," as used herein, refers to a
biological or a non-biological moiety that is covalently bonded to
a compound of the present invention, and which confers a desirable
property on the compound and/or on a biomolecule conjugated
thereto. Nonlimiting exemplary such desirable properties include
binding properties, such as, for example, the ability to
specifically bind to another moiety (e.g., a member of a binding
pair); increasing half-life; increasing solubility; and directing
the compound to a particular location in a cell or organism. Such
moieties include, but are not limited to, amino acids, peptides,
proteins, polysaccharides, nucleosides, nucleotides,
oligonucleotides, nucleic acids, haptens, psoralens, drugs,
hormones, lipids, lipid assemblies, synthetic polymers, polymeric
microparticles, biological cells, viruses, and combinations
thereof.
[0183] The term, "chemical handle," as used herein, refers to a
functional group that is capable of undergoing a click reaction, a
1,3-dipolar cycloaddition, and/or a Staudinger ligation.
Nonlimiting exemplary chemical handles include alkyne-reactive
moieties, such as azide; and azide-reactive moieties, such as
alkynes, including, but not limited to, terminal alkynes and
activated alkynes; and phosphines, including, but not limited to, a
triarylphosphine; and the like.
[0184] The term "complementary chemical handle," as used herein,
refers to a functional group that is capable of undergoing a click
reaction, a 1,3-dipolar cycloaddition, and/or a Staudinger ligation
with a specified chemical handle. For example, for an azide
chemical handle, complementary chemical handles include, but are
not limited to, alkynes, such as terminal alkynes and activated
alkynes, and phosphines, such as triarylphosphines.
[0185] The terms "click chemistry" and "click reaction," as used
herein, refer to copper ion-catalyzed 1,3-dipolar cycloadditions
between an azide and a terminal alkyne to form a
1,2,3-triazole.
[0186] The term "1,3-dipolar cycloaddition," as used herein, refers
to reactions between an azide and an alkyne to form a
1,2,3-triazole.
[0187] The term "copper ion source," as used herein, refers to any
source of Cu(I) ions, whether or not formation of Cu(I) ions
involves other agents, such as reducing agents. Nonlimiting
exemplary copper ion sources include copper salts, such as
Cu(NO.sub.3).sub.2Cu(OAc).sub.2 or CuSO.sub.4; copper halides, such
as CuBr and CuI; and copper-containing metals, such as copper
wire.
[0188] The terms "copper ion chelator" and "copper chelator," as
used herein, refer to a moiety that binds to, and stabilizes, Cu(I)
ions. Nonlimiting exemplary copper chelators are discussed
herein.
[0189] The term "halogen," as used herein, refers to an atom
selected from F, Cl, Br, and I.
[0190] The term "linker" or "L", as used herein, refers to a single
covalent bond or a series of stable covalent bonds incorporating
1-30 nonhydrogen atoms selected from the group consisting of C, N,
O, S, P, Si, and Se. Exemplary linking members include moieties
that includes --C(O)NH--, --C(O)O--, --NH--, --S--, --O--, and the
like. In some embodiments, a linker has a chain length of 1-30
atoms, or 1-25 atoms, or 1-20 atoms, or 1-15 atoms, or 1-10 atoms,
or 1-5 atoms. A "cleavable linker" is a linker that has one or more
covalent bonds that can be broken under particular reaction
conditions or in the presence of a particular molecule or enzyme,
such that the moiety on one side of the cleavable linker is no
longer covalently bound to the moiety on the other side of the
cleavable linker. The term "cleavable group" refers to a moiety
that allows for release of a portion, e.g., a reporter molecule,
carrier molecule or solid support, of a conjugate from the
remainder of the conjugate by cleaving a bond linking the released
moiety to the remainder of the conjugate. Such cleavage (for both
cleavable linkers and cleavable groups) is either chemical in
nature, or enzymatically mediated. Exemplary enzymatically
cleavable linkers and groups include natural amino acids or peptide
sequences that end with a natural amino acid. In addition to
enzymatically cleavable linkers and groups, it is within the scope
of the present invention to include one or more sites that are
cleaved by the action of an agent other than an enzyme. Exemplary
non-enzymatic cleavage agents include, but are not limited to,
acids, bases, light (e.g., nitrobenzyl derivatives, phenacyl
groups, benzoin esters), and heat. Many cleavable groups are known
in the art. See, for example, Jung et al., Biochem. Biophys. Acta,
761: 152-162 (1983); Joshi et al., J. Biol. Chem., 265: 14518-14525
(1990); Zarling et al., J. Immunol., 124: 913-920 (1980); Bouizar
et al., Eur. J. Biochem., 155: 141-147 (1986); Park et al., J.
Biol. Chem., 261: 205-210 (1986); Browning et al., J. Immunol.,
143: 1859-1867 (1989). Moreover a broad range of cleavable,
bifunctional (both homo- and hetero-bifunctional) spacer arms are
commercially available. An exemplary cleavable linker or group, an
ester, may be cleaved by a reagent, e.g. sodium hydroxide,
resulting in a carboxylate-containing product and a
hydroxyl-containing product.
[0191] The term "low copper," as used herein, refers to a copper
concentration of less than 1 millimolar.
[0192] The term "modified biomolecule" as used herein refers to a
biomolecule which has been modified by covalent attachment of at
least one chemical handle. A biomolecule may be modified in vitro
or in vivo.
[0193] The term "phosphine reactive" as used herein refers to a
chemical moiety that selectively reacts via Staudinger ligation
with a phosphine group, including but not limited to a
triarylphosphine group, on another molecule to form a covalent
chemical bond. Examples of phosphine reactive groups include, but
are not limited to, azide.
[0194] The terms "protein" and "polypeptide" are used herein in a
generic sense to refer to polymers of amino acid residues of any
length. The term "peptide" is used herein to refer to polypeptides
having fewer than 100 amino acid residues, typically fewer than 10
amino acid residues. The amino acid residues in a polypeptide,
protein, or peptide may be naturally-occurring amino acid residues
or non-naturally occurring amino acid residues.
[0195] The term "reducing agent," as used herein, refers to an
agent that is capable of reducing Cu(II) to Cu(I). Nonlimiting
exemplary reducing agents include ascorbate,
tris(2-carboxyethyl)phosphine (TCEP), NADH, NADPH, thiosulfate,
metallic copper, hydroquinone, vitamin K.sub.1, glutathione,
cysteine, 2-mercaptoethanol, dithiothreitol, and an applied
electric potential. Nonlimiting exemplary metals that may act as
reducing agents include Al, Be, Co, Cr, Fe, Mg, Mn, Ni, Zn, Au, Ag,
Hg, Cd, Zr, Ru, Fe, Co, Pt, Pd, Ni, Rh, and W.
[0196] The term "reporter molecule" refers to a moiety that is
directly or indirectly detectable. In some embodiments, and as a
non-limiting example, a reporter molecule may be directly
detectable, e.g., due to its spectral properties. In some
embodiments, and as a non-limiting example, a reporter molecule may
be indirectly detectable, e.g., due to its enzymatic activity,
wherein the enzymatic activity produces a directly detectable
signal. Such reporter molecules include, but are not limited to,
radiolabels; pigments, dyes, and other chromogens; spin labels;
fluorescent labels (i.e., fluorophores such as coumarins, cyanines,
benzofurans, quinolines, quinazolinones, indoles, benzazoles,
borapolyazaindacenes, and xanthenes, including fluoresceins,
rhodamines, and rhodols); chemiluminescent substances, wherein the
detectable signal is generated by chemical modification of
substance; metal-containing substances; enzymes, wherein the enzyme
activity generates a signal (such as, for example, by forming a
detectable product from a substrate; haptens that can bind
selectively to another molecule (such as, for example, an antigen
that binds to an antibody; or biotin, which binds to avidin and
streptavidin). Many reporter molecules are known in the art, some
of which are described, e.g., in Richard P. Haugland, Molecular
Probes Handbook of Fluorescent Probes and Research Products
(9.sup.th edition, CD-ROM, September 2002), supra.
[0197] The term "solid support," as used herein, refers to a
material that is substantially insoluble in a selected solvent
system, or which can be readily separated (e.g., by precipitation)
from a selected solvent system in which it is soluble. Solid
supports useful in practicing the present invention may include
groups that are activated or capable of activation such that one or
more compounds described herein will bind to the solid support.
[0198] The terms "structural integrity of the [biomolecule] is not
reduced" or "preservation of the structural integrity of the
[biomolecule]", as used herein, mean that either: 1) when analyzed
by gel electrophoresis and detection (such as staining), a band or
spot arising from the labeled biomolecule is not reduced in
intensity by more than 20%, and preferably not reduced by more than
10%, with respect to the corresponding band or spot arising from
the same amount of the electrophoresed unlabeled biomolecule,
arising from the labeled biomolecule analyzed; or 2) when analyzed
by gel electrophoresis, a band or spot arising from the labeled
biomolecule is not observed to be significantly less sharp than the
corresponding band or spot arising from the same amount of the
electrophoresed unlabeled biomolecule, where "significantly less
sharp" (synonymous with "significantly more diffuse") means the
detectable band or spot takes up at least 5% more, preferably 10%
more, more preferably 20% more area on the gel than the
corresponding unlabeled biomolecule. Other reproducible tests for
structural integrity of labeled biomolecules include, without
limitation detection of released amino acids or peptides, or mass
spectrometry.
[0199] The term "therapeutic molecule" refers to a molecule that
can be used to treat and/or alleviate a condition and/or symptom in
a subject, and/or can be used to affect biological processes in
cells in vitro. Therapeutic molecules include, but are not limited
to, antimetabolites, alkylating agents, anthracyclines,
antibiotics, and anti-mitotic agents. Nonlimiting exemplary
therapeutic molecules include taxol, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine,
lidocaine, propranolol, puromycin, abrin, ricin A, pseudomonas
exotoxin, diphtheria toxin, tumor necrosis factor,
.gamma.-interferon, .alpha.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator,
interleukin-1, interleukin-2, interleukin-6, granulocyte macrophage
colony stimulating factor, or granulocyte colony stimulating
factor, and analogs or homologs thereof.
[0200] The present invention provides low-copper click reactions,
1,3-dipolar cycloadditions, and Staudinger ligations involving a
modified biomolecule and a compound of any one of Formulas (I) to
(XIII). In some embodiments, the modified biomolecule comprises an
azide moiety and the compound of any one of Formulas (I) to (XIII)
comprises a terminal alkyne. In some embodiments, the modified
biomolecule comprises an alkyne, such as a terminal alkyne or an
activated alkyne, or a phosphine, such as a triarylphosphine, and
the compound of any one of Formulas (I) to (XIII) comprises an
azide moiety.
[0201] Accordingly, provided herein are compounds, compositions,
methods, and kits for the labeling, detecting, isolating and/or
analysis of biomolecules. In some embodiments, presented are novel
compounds comprising an azide moiety or an alkyne moiety. In some
embodiments, methods are provided for covalently attaching the
novel compounds to modified biomolecules using a click reaction, a
1,3-dipolar cycloaddition, or a Staudinger ligation. In some such
embodiments, the method comprises labeling, detecting, isolating
and/or analyzing the biomolecule.
Click Chemistry
[0202] Azides and terminal alkynes can undergo Cu(I)-catalyzed
Azide-Alkyne Cycloaddition (CuAAC) at room temperature. Such
Cu(I)-catalyzed azide-alkyne cycloadditions, sometimes referred to
as click chemistry, typically results in formation of a
1,2,3-triazole. Various exemplary click reactions are known in the
art, and are described, e.g., in U.S. Publication No.
2005/0222427.
[0203] Click reactions can be performed in a variety of aqueous
solutions, including, but not limited to, water, and mixtures of
water and various miscible or partially miscible organic solvents.
Nonlimiting such organic solvents include alcohols, dimethyl
sulfoxide (DMSO), dimethyl formamide (DMF), tert-butanol (tBuOH)
and acetone.
[0204] In some embodiments, the copper used as a catalyst in a
click reaction is Cu(I) ions. Exemplary sources of Cu(I) ions
include, but are not limited to, cuprous halides such as cuprous
bromide or cuprous iodide. In some embodiments, a click reaction is
carried out in the presence of Cu(II) ions and a reducing agent,
which reduces the Cu(II) to Cu(I) in situ. Exemplary sources of
Cu(II) ions include, but are not limited to, Cu(NO.sub.3).sub.2,
Cu(OAc).sub.2, and CuSO.sub.4. Nonlimiting exemplary reducing
agents include ascorbate, tris(2-carboxyethyl)phosphine (TCEP),
NADH, NADPH, thiosulfate, metallic copper, hydroquinone, vitamin
K.sub.1, glutathione, cysteine, 2-mercaptoethanol, dithiothreitol,
Fe.sup.2+, Co.sup.2+, and an applied electric potential. In some
embodiments, a reducing agent is a metal selected from Al, Be, Co,
Cr, Fe, Mg, Mn, Ni, Zn, Au, Ag, Hg, Cd, Zr, Ru, Fe, Co, Pt, Pd, Ni,
Rh, and W.
[0205] In some embodiments, the reducing agent is included in a
click reaction in a micromolar to millimolar range. In some
embodiments, the concentration of the reducing agent is between 100
.mu.M and 100 mM, between 10 .mu.M and 10 mM, or between 1 .mu.M
and 1 mM.
[0206] In some embodiments, a click reaction includes a chelator
that stabilizes Cu(I) ions. Nonlimiting exemplary such chelators
are described herein.
[0207] In some embodiments, at least one copper chelator is
included in a click reaction. In some such embodiments, the copper
chelator is added after a Cu(II) source has been contacted with a
reducing agent. In some embodiments, the copper chelator is added
at the same time the Cu(II) source is contacted with a reducing
agent. In some embodiments, a copper chelator is added to a
solution containing one or both of the click reactants (i.e., a
solution containing one or both of the azide-containing reactant
and the alkyne-containing reactant), and a solution containing the
Cu(II) source and the reducing agent is subsequently added to
initiate the click reaction.
[0208] In some embodiments, a click reaction comprises a compound
of any one of Formulas (I) to (XIII) and a modified biomolecule. In
some such embodiments, the compound of any one of Formulas (I) to
(XIII) comprises a terminal alkyne and the modified biomolecule
comprises an azide. In some embodiments, the compound of any one of
formulas (I) to (XIII) comprises an azide and the modified
biomolecule comprises a terminal alkyne. In some embodiments, a
click reaction further comprises Cu(I) ions. In some embodiments, a
click reaction further comprises Cu(II) ions and at least one
reducing agent. In some embodiments, a click reaction further
comprises a copper chelator.
Activated Alkyne Chemistry (1,3-Dipolar Cycloadditions)
[0209] In some instances, azides and alkynes can undergo
catalyst-free 1,3-dipolar cycloaddition when an activated alkyne is
used. In some embodiments, alkynes can be activated by ring strain
such as, by way of example only, eight membered ring structures,
including seven to ten-membered ring structures with
electron-withdrawing groups appended thereon. In some embodiments,
alkynes can be activated by the addition of a Lewis acid such as,
by way of example only, Au(I) or Au(III). Nonlimiting exemplary
activated alkynes include cyclooctynes and difluorocyclooctynes,
which are described, e.g., in Agard et al., J. Am. Chem. Soc.,
2004, 126 (46):15046-15047; dibenzocyclooctynes, which are
described, e.g., in Boon et al., WO2009/067663 A1; and
aza-dibenzocyclooctynes, which are described, e.g., in Debets et
al., Chem. Comm., 2010, 46:97-99.
[0210] Typically, an activated alkyne conjugated with fluorophores
or antibody undergoes cycloaddition to azide in one to twelve hour
at room temperature. The reaction can be carried out in organic or
aqueous solvents, buffers like PBS, TRIS or mixtures of buffers and
organic solvents.
[0211] In some embodiments of the methods described herein, a
modified biomolecule comprises an activated alkyne and a compound
of any one of Formulas (I) to (XIII) comprises an azide.
Staudinger Ligation
[0212] In a Staudinger ligation, an azide is reacted with a
triarylphosphine comprising an electrophilic trap (typically, a
methyl ester). Following formation of an aza-ylide intermediate,
the intermediate rearranges to produce a ligated product having an
amide linkage, and a phosphine oxide. Such ligations are described,
e.g., in U.S. Publication No. 2006/0276658. In some embodiments,
the phosphine comprises an acyl group such as an ester, thioester
or N-acyl imidazole (i.e. a phosphinoester, phosphinothioester,
phosphinoimidazole) to trap the aza-ylide intermediate and form an
amide bond upon hydrolysis. In some embodiments, the phosphine can
be a di- or triarylphosphine to stabilize the phosphine. The
phosphines used in Staudinger ligation methods described herein
include, but are not limited to, cyclic or acyclic, halogenated,
bisphosphorus, or polymeric phosphines.
[0213] A typical procedure for a Staudinger ligation is as follows
(J. Am. Chem. Soc. 2002, 124, 14893-14902): The cells were pelleted
(3500 rpm, 3 min) and washed twice with 200 .mu.L of labeling
buffer (1% FBS in PBS, pH) 7.4). After the second wash, cells were
typically resuspended in a volume of 50 .mu.L of labeling buffer
and 50 .mu.L of 2 in solution (0.5 mM in PBS, pH) 7.4). After
incubation at room temperature for 1 h, the cells were pelleted
(3500 rpm, 3 min) and washed three times with ice-cold labeling
buffer, the cells were pelleted, washed with 200 .mu.L of ice-cold
labeling buffer, and then diluted to a volume of 400 .mu.L for flow
cytometry analysis.
[0214] In some embodiments of the methods described herein, a
modified biomolecule comprises a phosphine and a compound of any
one of Formulas (I) to (XIII) comprises an azide.
Compounds for Conjugating Biomolecules
[0215] In some embodiments, the present invention provides
compounds having the formula:
##STR00019##
wherein:
[0216] A is a carbon, or A, R.sub.5, and R.sub.6 are absent;
[0217] R.sub.1, R.sub.2, R.sub.3, and R.sub.4, are independently
selected from hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido, alkyl and aryl portions are optionally substituted
one or more times by halogen, --SO.sub.3X, a carboxylic acid, a
salt of carboxylic acid, CN, nitro, hydroxyl, amino, hydrazine,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, alkylthio,
alkanoylamino, alkylaminocarbonyl, aryl, heteroaryl, substituted
aryl, arylalkyl, substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido; or two substituents selected from R.sub.1,
R.sub.2, R.sub.3, and R.sub.4, wherein each of the at least two
substituents are on different carbon atoms together form a fused
moiety selected from cycloalkyl, heterocycloalkyl, substituted
cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, and all of the remaining substituents are independently
selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl; or at least two
of the remaining substituents together form a fused moiety selected
from cycloalkyl, heterocycloalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl, and
any remaining substituents are independently selected from
hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl;
[0218] R.sub.5, and R.sub.6, are independently selected from
hydrogen, halogen, --SO.sub.3X, a carboxylic acid, a salt of
carboxylic acid, CN, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, alkylthio, alkanoylamino, alkylaminocarbonyl,
aryl, heteroaryl, substituted aryl, arylalkyl, substituted
arylalkyl, and substituted heteroaryl, arylcarboxamido, alkyl and
aryl portions are optionally substituted one or more times by
halogen, --SO.sub.3X, a carboxylic acid, a salt of carboxylic acid,
CN, nitro, hydroxyl, amino, hydrazine, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, alkylthio, alkanoylamino,
alkylaminocarbonyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl,
arylcarboxamido;
[0219] at least one substituent selected from R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5 and R.sub.6 comprises X-L-, wherein:
[0220] X is selected from a reporter molecule, a carrier molecule,
a solid phase, a therapeutic molecule such as peptide, a protein,
an antibody, a polysaccharide, a nucleic acid polymer, an ion
complexing moiety, a lipid or a non-biological organic polymer or
polymeric micro or nano particle, that are optionally bound to one
or more additional fluorophores; or [0221] X is a reactive group
such as carboxylic acid, an activated ester of carboxylic acid, an
amine, a hydrazine, a haloacetamide, an alkyl halide, an
isothiocynate or a maleimide group; and [0222] L is an
independently a single covalent bond or L is covalent linkage
having 1-24 non-hydrogen atoms selected from the group consisting
of C, N, O, P and S and composed of any combinations of single,
double, triple or aromatic carbon-carbon bonds, carbon-nitrogen
bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur
bonds, phosphorus-oxygen bonds and phosphorus-nitrogen bonds in the
form of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy,
substituted alkoxy, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl;
[0223] Z is an independently a single covalent bond or Z is
covalent linkage having 1-10 non-hydrogen atoms selected from the
group consisting of C, N, O, P and S and composed of any
combinations of single, double, triple or aromatic carbon-carbon
bonds, carbon-nitrogen bonds, carbon-oxygen bonds, and
carbon-sulfur bonds in the form of a straight- or branched-chain
alkyl or heteroalkyl chain;
[0224] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0225] In some embodiments, the compound is of the formula:
##STR00020##
[0226] In some embodiments, the compound is of the formula:
##STR00021##
wherein
[0227] R.sub.2, R.sub.3, R.sub.4, and R.sub.7 to R.sub.12 are
independently selected from hydrogen, halogen, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl; or
two substituents selected from R.sub.2, R.sub.3, R.sub.4, and
R.sub.7 to R.sub.12, wherein the two substituents are on different
carbon atoms, together form a fused moiety selected from
cycloalkyl, heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, and all of the
remaining substituents are independently selected from hydrogen,
halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl; or two of the remaining substituents also
together form a fused moiety selected from cycloalkyl,
heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, and the
remaining substituents are independently selected from hydrogen,
halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl.
[0228] In some embodiments, the present invention provides
compounds having the formula:
##STR00022##
wherein:
[0229] R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently
selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl; or two
substituents selected from R.sub.1, R.sub.2, R.sub.3, and R.sub.4
together form a fused moiety selected from cycloalkyl,
heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, and the
remaining two substituents also together form a fused moiety
selected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl, or
the remaining two substituents are independently selected from
hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl;
at least one substituent selected from R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, and R.sub.6 comprises X-L-, wherein: [0230] X is
selected from a reporter molecule, a carrier molecule, a solid
phase, or a therapeutic molecule; and [0231] L is a group selected
from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy,
substituted alkoxy, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl, having a chain length of 0-20 atoms; [0232]
A is a carbon, and R.sub.5 and R.sub.6 are independently selected
from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl; or A, R5, and R6
are absent;
[0233] B is selected from O, S, and NR.sub.7, wherein R.sub.7 is
selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl
[0234] Z is a straight- or branched-chain alkyl or heteroalkyl
having a chain length of 1-10 atoms, or is absent; and
[0235] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0236] In some embodiments, the present invention provides
compounds having the formula:
##STR00023##
wherein:
[0237] A is a carbon, or A, R.sub.5, and R.sub.6 are absent;
[0238] R.sub.1, R.sub.2, R.sub.5, and R.sub.6 are independently
selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl; or two
substituents selected from R.sub.1, R.sub.2, R.sub.5, and R.sub.6,
wherein the two substituents are on different carbon atoms,
together form a fused moiety selected from cycloalkyl,
heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, and the
remaining substituents are independently selected from hydrogen,
halogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, alkoxy, substituted alkoxy, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl; at least one substituent selected from
R.sub.1, R.sub.2, R.sub.5, and R.sub.6 comprises X-L-, wherein:
[0239] X is selected from a reporter molecule, a carrier molecule,
a solid phase, or a therapeutic molecule; and [0240] L is a group
selected from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
alkoxy, substituted alkoxy, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
aryl, heteroaryl, substituted aryl, arylalkyl, substituted
arylalkyl, and substituted heteroaryl, having a chain length of
0-20 atoms;
[0241] B is selected from O, S, and NR.sub.3, wherein R.sub.3 is
selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl;
Z is a straight- or branched-chain alkyl or heteroalkyl having a
chain length of 1-10 atoms, or is absent; and
[0242] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0243] In some embodiments, when a compound is of Formula (VI),
R.sub.1, R.sub.2, R.sub.5, and R.sub.6 are independently selected
from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl; or R.sub.1 and
R.sub.2 together form a fused moiety selected from cycloalkyl,
heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, and R.sub.5 and
R.sub.6 are independently selected from hydrogen, halogen, alkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,
substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl.
[0244] In some embodiments, the present invention provides
compounds having the formula:
##STR00024##
wherein:
[0245] A is a carbon, or A, R.sub.5, and R.sub.6 are absent;
[0246] R.sub.1, R.sub.2, R.sub.3, R.sub.5, and R.sub.6 are
independently selected from hydrogen, halogen, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl; or
two substituents selected from R.sub.1, R.sub.2, R.sub.3, R.sub.5,
and R.sub.6, wherein the two substituents are on different carbon
atoms, together form a fused moiety selected from cycloalkyl,
heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, and the
remaining substituents are selected from hydrogen, halogen, alkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, substituted alkyl,
substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl;
at least one substituent selected from R.sub.1, R.sub.2, R.sub.3,
R.sub.5, and R.sub.6 comprises X-L-, wherein: [0247] X is selected
from a reporter molecule, a carrier molecule, a solid phase, or a
therapeutic molecule; and [0248] L is a group selected from alkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, substituted
alkoxy, substituted alkyl, substituted heteroalkyl, substituted
cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl, having a chain length of 0-20 atoms;
[0249] Z is a straight- or branched-chain alkyl or heteroalkyl
having a chain length of 1-10 atoms, or is absent; and
[0250] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0251] In some embodiments, the present invention provides
compounds having the formula:
##STR00025##
wherein:
[0252] A is a carbon, or A, R.sub.5, and R.sub.6 are absent;
[0253] R.sub.1, R.sub.2, R.sub.5, and R.sub.6 are independently
selected from hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl; at least one
substituent selected from R.sub.1, R.sub.2, R.sub.5, and R.sub.6
comprises X-L-, wherein: [0254] X is selected from a reporter
molecule, a carrier molecule, a solid phase, or a therapeutic
molecule; and [0255] L is a group selected from alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, alkoxy, substituted alkoxy,
substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl,
having a chain length of 0-20 atoms; Z is a straight- or
branched-chain alkyl or heteroalkyl having a chain length of 1-10
atoms, or is absent; and
[0256] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0257] In some embodiments, the present invention provides
compounds having the formula:
##STR00026##
wherein:
[0258] A is a carbon, or A, R.sub.5, and R.sub.6 are absent;
[0259] m and n is an integer between 4 and 8;
[0260] R.sub.7 is selected from hydrogen, alkyl, heteroalkyl,
substituted alkyl, and substituted heteroalkyl;
[0261] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, each
R', and each R'' are independently selected from hydrogen, halogen,
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, alkoxy, substituted alkoxy, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl; or two substituents selected from R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, an R', and an R'', wherein the
two substituents are on different carbon atoms, together form a
fused moiety selected from cycloalkyl, heterocycloalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, aryl,
heteroaryl, substituted aryl, arylalkyl, substituted arylalkyl, and
substituted heteroaryl, and all of the remaining substituents are
independently selected from hydrogen, halogen, alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted alkyl, substituted
heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,
alkoxy, substituted alkoxy, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl; or
two of the remaining substituents also together form a fused moiety
selected from cycloalkyl, heterocycloalkyl, substituted cycloalkyl,
substituted heterocycloalkyl, aryl, heteroaryl, substituted aryl,
arylalkyl, substituted arylalkyl, and substituted heteroaryl, and
the remaining substituents are independently selected from
hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, substituted alkyl, substituted heteroalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, alkoxy,
substituted alkoxy, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl;
[0262] at least one substituent selected from R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, an R', and an R'' comprises
X-L-, wherein: [0263] X is selected from a reporter molecule, a
carrier molecule, a solid phase, or a therapeutic molecule; and
[0264] L is a group selected from alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, alkoxy, substituted alkoxy, substituted alkyl,
substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, heteroaryl, substituted aryl, arylalkyl,
substituted arylalkyl, and substituted heteroaryl, having a chain
length of 0-20 atoms;
[0265] Z is a straight- or branched-chain alkyl or heteroalkyl
having a chain length of 1-10 atoms, or is absent; and
[0266] G is a chemical handle selected from an azide-reactive
group, an alkyne-reactive group, and a phosphine-reactive
group.
[0267] In some embodiments of compounds of Formulas (I) to (XIII),
one of the R substituents comprises X-L-, and the remaining R
substituents are each H. In some embodiments compounds of Formulas
(I) to (XIII), L is an alkyl group having a chain length of 0 to 15
atoms, 0 to 10 atoms, or 0 to 5 atoms. In some embodiments
compounds of Formulas (I) to (XIII), L is
--NH--(CH.sub.2).sub.n--NH--C(O)--, wherein n is 1 to 12. In some
embodiments, n is 1 to 10, 1 to 8, or 1 to 5.
[0268] In some embodiments compounds of Formulas (I) to (XIII), G
is an azide or a terminal alkyne. In some embodiments, when a
compound of any one of Formulas (I) to (XIII) is to be used in a
click reaction, G is an azide or terminal alkyne. In some
embodiments, when a compound of any one of Formulas (I) to (XIII)
is to be used in a 1,3-dipolar cycloaddition with an activated
alkyne, G is an azide. In some embodiments, when a compound of any
one of Formulas (I) to (XIII) is to be used in a Staudinger
ligation, G is an azide.
[0269] In some embodiments, the reporter molecule comprises a
chromophore, fluorophore, fluorescent protein, phosphorescent dye,
tandem dye, particle, hapten, enzyme, or radioisotope. In some
embodiments, the fluorophore is a xanthene, coumarin, cyanine,
pyrene, oxazine, borapolyazaindacene, or carbopyranine. In some
embodiments, the enzyme is horseradish peroxidase, alkaline
phosphatase, beta-galactosidase, or beta-lactamase. In some
embodiments, the particle is a semiconductor nanocrystal.
[0270] In some embodiments, the carrier molecule is an amino acid,
peptide, protein, polysaccharide, nucleoside, nucleotide,
oligonucleotide, nucleic acid, hapten, psoralen, drug, hormone,
lipid, lipid assembly, tyramine, synthetic polymer, polymeric
microparticle, biological cell, cellular component, ion chelating
moiety, enzymatic substrate, or virus. In some embodiments, the
carrier molecule is an antibody, antibody fragment, antigen,
avidin, streptavidin, biotin, dextran, IgG binding protein,
fluorescent protein, agarose, or non-biological microparticle.
[0271] In some embodiments, the solid support is an aerogel,
hydrogel, resin, bead, biochip, microfluidic chip, silicon chip,
multi-well plate, membrane, conducting metal, nonconducting metal,
glass, or magnetic support. In some embodiments, the solid support
is a silica gel, polymeric membrane, particle, derivatized plastic
film, glass bead, cotton, plastic bead, alumina gel,
polysaccharide, poly(acrylate), polystyrene, poly(acrylamide),
polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin,
glycogen, amylopectin, mannan, inulin, nitrocellulose,
diazocellulose, polyvinylchloride, polypropylene, polyethylene,
nylon, latex bead, magnetic bead, paramagnetic bead,
superparamagnetic bead, or starch.
[0272] In some embodiments, the therapeutic molecule is taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid,
procaine, tetracaine, lidocaine, propranolol, puromycin, or analogs
or homologs thereof. In some embodiments, the therapeutic molecule
is an antimetabolite, alkylating agent, anthracycline, antibiotic,
or anti-mitotic agent. In some embodiments, the therapeutic
molecule is abrin, ricin A, pseudomonas exotoxin, diphtheria toxin,
tumor necrosis factor, .gamma.-interferon, .alpha.-interferon,
nerve growth factor, platelet derived growth factor, tissue
plasminogen activator, interleukin-1, interleukin-2, interleukin-6,
granulocyte macrophage colony stimulating factor, or granulocyte
colony stimulating factor.
[0273] The compounds of the present invention may be made, for
example, using the exemplary reaction schemes shown in FIG. 1 and
FIG. 2, and described in Example 1.
Reporter Molecules
[0274] In some embodiments, a compound of any one of Formulas (I)
to (XIII) comprises a reporter molecule. The reporter molecules
used in the methods and compositions provided herein include any
directly or indirectly detectable reporter molecule that can be
covalently attached as a substituent of a compound of any one of
Formulas (I) to (XIII).
[0275] Reporter molecules used in the methods and compositions
described herein include, but are not limited to, chromophores,
fluorophores, fluorescent proteins, phosphorescent dyes, tandem
dyes, particles, haptens, enzymes, and radioisotopes. In some
embodiments, a reporter molecule is a fluorophore, a fluorescent
protein, a hapten, or an enzyme.
[0276] A fluorophore is any chemical moiety that exhibits an
absorption maximum at wavelengths greater than 280 nm, and retains
its spectral properties when covalently attached to a biomolecule
following reaction of a compound of any one of Formulas (I) to
(XIII) comprising the fluorophore with the modified biomolecule.
Fluorophores include, without limitation, pyrenes; anthracenes;
naphthalenes; acridines; stilbenes; indoles and benzindoles;
oxazoles and benzoxazoles; thiazoles and benzothiazoles;
4-amino-7-nitrobenz-2-oxa-1,3-diazoles (NBD); cyanines;
carbocyanines; carbostyryls; porphyrina; salicylates;
anthranilates; azulenes; perylenes; pyridines; quinolines;
borapolyazaindacenes; xanthenes (including, but not limited to,
fluoresceins (such as benzo- or dibenzofluoresceins,
seminaphthofluoresceins, or naphthofluoresceins), rhodols (such as
eminaphthorhodafluors), and rhodamine); oxazines and benzoxazines
(including, but not limited to, resorufins, aminooxazinones,
diaminooxazines, and their benzo-substituted analogs); carbazines;
phenalenones; coumarins; benzofurans; benzphenalenones;
carbopyranines, semiconductor nanocrystals; and derivatives of any
of the above.
[0277] In some embodiments, a reporter molecule is selected from a
xanthene (including, but not limited to, sulfonated xanthenes,
fluorinated xanthenes, rhodol, rhodamine, fluorescein and
derivatives thereof), coumarin (including, but not limited to,
sulfonated coumarin and fluorinated coumarin), cyanine (including,
but not limited to, sulfonated cyanine), pyrene, oxazine,
borapolyazaindacene, carbopyranine, and semiconductor
nanocrystal.
[0278] In some embodiments, a reporter molecule is a xanthene that
is bound as a substituent of a compound of any one of Formulas (I)
to (XIII) via a single covalent bond at the 9-position of the
xanthene. In some embodiments, the xanthene is selected from
3H-xanthen-6-ol-3-one attached through the 9-position,
6-amino-3H-xanthen-3-one attached through the 9-position, and
6-amino-3H-xanthen-3-imine attached through the 9-position.
[0279] One skilled in the art can select a fluorophore to be
included as a substituent of a compound of any one of Formulas (I)
to (XIII) according to the particular application. Physical
properties of a fluorophore that can be used for detection of
modified biomolecules include, but are not limited to, spectral
characteristics (absorption, emission, and stokes shift),
fluorescence intensity, lifetime, polarization and photo-bleaching
rate, and combinations thereof. In various embodiments, one or more
of the physical properties can be used to distinguish one
fluorophore from another, and thereby allow for multiplexed
analysis. In some embodiments, the fluorophore has an absorption
maximum at wavelengths greater than 480 nm, at wavelengths between
488 nm to 514 nm (particularly suitable for excitation by the
output of the argon-ion laser excitation source), or at wavelengths
near 546 nm (particularly suitable for excitation by a mercury arc
lamp).
[0280] Many of fluorophores can also function as chromophores and
thus the described fluorophores may also be used as chromophore
reporter molecules in the methods and compositions described
herein.
[0281] In some embodiments, a reporter molecule is an enzyme. In
some embodiments, an enzyme is a desirable label because it can
amplify the detectable signal, thus increasing assay sensitivity.
In some embodiments, the enzyme itself is not directly detectable,
but its activity can be used to create a detectable signal when the
enzyme is contacted with an appropriate substrate, such that the
converted substrate produces, for example, a fluorescent,
colorimetric, or luminescent signal. Various substrates are known
in the art, some of which are described in the Molecular Probes
Handbook, supra.
[0282] In some embodiments, when an enzyme reporter molecule is an
oxidoreductase such as, by way of example only, horseradish
peroxidase, suitable substrates include, but are not limited to,
3,3'-diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC),
2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),
o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB),
o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol,
4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and
reduced benzothiazines, including Amplex.RTM. Red reagent and its
variants (U.S. Pat. No. 4,384,042), Amplex UltraRed and its
variants (WO05/042504), reduced dihydroxanthenes, including
dihydrofluoresceins and dihydrorhodamines, including
dihydrorhodamine 123. Peroxidase substrates that may be used with
the enzymatic reporter molecules described herein also include, but
are not limited to, tyramides (U.S. Pat. Nos. 5,196,306; 5,583,001
and 5,731,158), which can be intrinsically detectable before action
of the enzyme but are "fixed in place" by the action of a
peroxidase in the process described as tyramide signal
amplification (TSA). In various embodiments, such substrates may be
used, for example, to label targets in samples that are cells,
tissues or arrays for their subsequent detection by microscopy,
flow cytometry, optical scanning and fluorometry.
[0283] In some embodiments, when an enzyme reporter molecule is a
phosphatase enzyme such as, by way of example only, an acid
phosphatases or an alkaline phosphatase, suitable substrates
include, but are not limited to, 5-bromo-6-chloro-3-indolyl
phosphate (BCIP), 6-chloro-3-indolyl phosphate,
5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenyl phosphate, and
o-nitrophenyl phosphate. Nonlimiting fluorogenic substrates
include, but are not limited to, 4-methylumbelliferyl phosphate,
6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S.
Pat. No. 5,830,912), fluorescein diphosphate, 3-O-methylfluorescein
phosphate, resorufin phosphate,
9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate (DDAO
phosphate), ELF 97, ELF 39, and related phosphates (U.S. Pat. Nos.
5,316,906 and 5,443,986).
[0284] In some embodiments, when an enzyme reporter molecule is a
glycosidase such as, by way of example only, a beta-galactosidase,
beta-glucuronidase, or beta-glucosidase, suitable substrates
include, but are not limited to,
5-bromo-4-chloro-3-indolylbeta-D-galactopyranoside (X-gal) and
similar indolyl galactosides, glucosides, and glucuronides,
o-nitrophenyl beta-D-galactopyranoside (ONPG), p-nitrophenyl
beta-D-galactopyranoside, resorufin beta-D-galactopyranoside,
fluorescein digalactoside (FDG), fluorescein diglucuronide and
their structural variants, 4-methylumbelliferyl
beta-D-galactopyranoside, carboxyumbelliferyl
beta-D-galactopyranoside, and fluorinated coumarin
beta-D-galactopyranosides.
[0285] Enzyme reporter molecules also include, but are not limited
to, hydrolases such as cholinesterases and peptidases, oxidases
such as glucose oxidase and cytochrome oxidases, and reductases,
for which suitable substrates are known. Additional nonlimiting
exemplary enzyme reporter molecules include luciferases and
aequorins. In addition, the chemiluminescence-producing substrates
for phosphatases, glycosidases and oxidases such as those
containing stable dioxetanes, luminol, isoluminol and acridinium
esters can also be used with the enzyme reporter molecules
described herein.
[0286] In some embodiments, a reporter molecule is a hapten.
Nonlimiting exemplary haptens include hormones, naturally occurring
and synthetic drugs, pollutants, allergens, affector molecules,
growth factors, chemokines, cytokines, lymphokines, amino acids,
peptides, chemical intermediates, nucleotides, biotin and the like.
In some embodiments, a hapten is not directly detectable, but it
can bind to another molecule that is detectable. As a nonlimiting
example, a hapten may be an antigen that can be bound by an
antibody specific to that antigen, wherein the antibody comprises a
detectable label, or wherein the antibody can be bound by a
secondary antibody comprising a detectable label.
[0287] In some embodiments, a reporter molecule is a fluorescent
protein. Nonlimiting exemplary fluorescent proteins include green
fluorescent protein (GFP) and the phycobiliproteins and derivatives
thereof. In some embodiments, a fluorescent protein is used in
conjunction with a fluorophore in order to obtain a larger stokes
shift from the fluorescent protein's absorption spectra. In some
embodiments, the fluorescent protein and fluorophore function as an
energy transfer pair, wherein the fluorescent protein emits at the
wavelength at which the fluorophore absorbs and the fluorophore
then emits at a wavelength farther from the fluorescent protein's
emission wavelength than could have been obtained with only the
fluorescent protein. In some such embodiments, a compound of any
one of Formulas (I) to (XIII) comprises a fluorescent protein as
one substituent and a fluorophore as another substituent. In some
embodiments, a compound of any one of Formulas (I) to (XIII)
comprises both the fluorescent protein and the fluorophore as a
single substituent, wherein the fluorescent protein and the
fluorophore are connected to one another by a linker. Nonlimiting
exemplary fluorescent protein/fluorophore pairs include
phycobiliproteins and sulforhodamine fluorophores, sulfonated
cyanine fluorophores, or sulfonated xanthene fluorophores. In some
embodiments, the fluorophore functions as the energy donor and the
fluorescent protein as the energy acceptor. Nonlimiting exemplary
radioisotopes that may be used as reporter molecules include For
example, the compounds may be radiolabeled with radioactive
isotopes, such as for example tritium (.sup.3H), iodine-125
(.sup.125I) or carbon-14 (.sup.14C), sulfur-35 (.sup.35S), etc. All
isotopic variations of the compounds of the present invention,
whether radioactive or not, are intended to be encompassed within
the scope of the present invention.
[0288] Methods of attaching reporter molecules as substituents of
compounds of Formulas (I) to (XIII) are known in the art.
Nonlimiting exemplary methods include the methods shown in FIGS. 1
and 2, in which a reporter molecule comprising an
N-hydroxysuccinimidyl (NHS) ester is reacted with a precursor of a
compound of any one of Formulas (I) to (XIII) bearing a primary
amine on at least one substituent. SDP esters, TFP, PFP,
carbamates, thiocarbamates and maleimides may also be used in place
of NHS esters.
Carrier Molecules
[0289] In some embodiments, a compound of any one of Formulas (I)
to (XIII) comprises a carrier molecule as a substituent.
[0290] Carrier molecules include, but are not limited to, antigens,
steroids, vitamins, drugs, haptens, metabolites, toxins,
environmental pollutants, amino acids, peptides, proteins, nucleic
acids, nucleic acid polymers, carbohydrates, lipids, and polymers.
In some embodiments, a carrier molecule comprises an amino acid, a
peptide, a protein, an antibody or fragment thereof, an antigen,
avidin, streptavidin, biotin, a dextran, an IgG binding protein
(such as protein A or protein G), agarose, a polysaccharide, a
nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a
hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a
synthetic polymer, a non-biological microparticle (such as a
polymeric microparticle), an ion chelating moiety, an enzymatic
substrate, a biological cell, a cellular component, a virus, or
combinations thereof.
[0291] In some embodiments, when the carrier molecule is an
enzymatic substrate, the enzymatic substrate is selected from an
amino acid, a peptide, a sugar, an alcohol, alkanoic acid,
4-guanidinobenzoic acid, a nucleic acid, a lipid, sulfate,
phosphate, --CH.sub.2OCO-alkyl, and combinations thereof. In
certain embodiments, such enzyme substrates can be cleaved by
enzymes selected from peptidases, phosphatases, glycosidases,
dealkylases, esterases, guanidinobenzotases, sulfatases, lipases,
peroxidases, histone deacetylases, exonucleases, reductases,
endoglycoceramidases and endonucleases.
[0292] In some embodiments, when the carrier molecule comprises an
amino acid, a peptide, or protein, the carrier molecule is selected
from a neuropeptide, a cytokine, a toxin, a protease substrate, and
a protein kinase substrate. In some embodiments, a carrier is a
peptide that functions as an organelle localization peptide, that
is, a peptide that serves to target the conjugated compound for
localization within a particular cellular substructure by cellular
transport mechanisms, including, but not limited to, a nuclear
localization signal sequence.
[0293] In some embodiments, a carrier molecule is a protein
selected from an enzyme, an antibody, a lectin, a glycoprotein, a
histone, an albumin, a lipoprotein, protein A, protein G, a
phycobiliprotein or other fluorescent protein, a hormone, a toxin,
and a growth factor. In some embodiments, a carrier molecule is a
protein selected from an antibody, an antibody fragment, avidin,
streptavidin, a toxin, a lectin, or a growth factor. In some
embodiments, a carrier molecule comprises a hapten such as, for
example, biotin, digoxigenin, or a fluorophore.
[0294] In some embodiments, a carrier molecule comprises a nucleic
acid base, nucleoside, nucleotide or a nucleic acid polymer, a
peptide nucleic acid (PNA), or a locked nucleic acid (LNA), single-
or multi-stranded, natural or synthetic DNA or RNA oligonucleotide,
or DNA/RNA hybrid, optionally containing an additional linker or
spacer for attachment of a fluorophore or other ligand. In some
embodiments, a nucleic acid carrier molecule (including, but not
limited to, LNA, PNA, DNA, and RNA) comprises fewer than 50
nucleotides, or fewer than 25 nucleotides.
[0295] In some embodiments, a carrier molecule comprises a
carbohydrate or polyol, including a polysaccharide, such as
dextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin,
starch, agarose and cellulose, or a polymer such as a poly(ethylene
glycol). In some embodiments, a carrier molecule comprises dextran,
agarose, or FICOLL.
[0296] In some embodiments, a carrier molecule comprises a lipid
including, but not limited to, glycolipids, phospholipids, and
sphingolipids. In some embodiments, such lipids contain 6-25
carbons. In some embodiments, a carrier molecule includes a lipid
vesicle, such as a liposome, or is a lipoprotein. Some lipophilic
substituents are useful, in some embodiments, for facilitating
transport of a conjugated molecule into cells or cellular
organelles.
[0297] In some embodiments, a carrier molecule is a cell, cellular
fragment, or subcellular particle, including virus particles,
bacterial particles, virus components, biological cells (such as
animal cells, plant cells, bacteria, or yeast), or cellular
components. Non-limiting examples of such cellular components
include lysosomes, endosomes, cytoplasm, nuclei, histones,
mitochondria, Golgi apparatus, endoplasmic reticulum and
vacuoles.
[0298] In some embodiments, a carrier molecule comprises a specific
binding pair member. In some such embodiments, the presence of the
carrier molecule, and therefore the biomolecule to which it is
conjugated through a compound of any one of Formulas (I) to (XIII),
can be detected using a complementary specific binding pair member
comprising a detectable label. Nonlimiting exemplary binding pairs
are set forth in Table 2.
TABLE-US-00001 TABLE 2 Exemplary Specific Binding Pairs Antigen
Antibody Biotin avidin (or streptavidin or anti-biotin) IgG*
protein A or protein G Drug drug receptor Folate folate binding
protein Toxin toxin receptor Carbohydrate lectin or carbohydrate
receptor Peptide peptide receptor Protein protein receptor enzyme
substrate Enzyme DNA (RNA) cDNA (cRNA).dagger. Hormone hormone
receptor Ion Chelator *IgG is an immunoglobulin .sup..dagger.cDNA
and cRNA are the complementary strands used for hybridization
[0299] In some embodiments, a carrier molecule is an
antibody-binding moiety, such as, but not limited to, anti-Fc, an
anti-Fc isotype, anti-J chain, anti-kappa light chain, anti-lambda
light chain, or a single-chain fragment variable protein, an
anti-Fc Fab fragment; or a non-antibody peptide or protein, such
as, for example but not limited to, soluble Fc receptor, protein G,
protein A, protein L, lectins, or a fragment thereof.
[0300] Methods of attaching carrier molecules as substituents of
compounds of Formulas (I) to (XIII) are known in the art.
Nonlimiting exemplary methods include as examples amides,
thioamides, ethers, thioethers, carbamates, thiocarbamates,
sulfhydryl groups, amino groups, etc.
Solid Supports
[0301] In some embodiments, a compound of any one of Formulas (I)
to (XIII) comprises a solid support as a substituent.
[0302] A large number of solid supports are known in the art and
can be used, in some embodiments, as a substituent of a compound of
any one of Formulas (I) to (XIII). Nonlimiting exemplary solid
supports include solid and semi-solid matrixes, such as aerogels
and hydrogels, resins, beads, biochips (including thin film coated
biochips), microfluidic chip, a silicon chip, multi-well plates
(also referred to as microtiter plates or microplates), membranes,
conducting and nonconducting metals, glass (including microscope
slides) and magnetic supports. Other nonlimiting examples of solid
supports include silica gels, polymeric membranes, particles,
derivatized plastic films, derivatized glass, derivatized silica,
glass beads, cotton, plastic beads, alumina gels, polysaccharides
such as Sepharose, poly(acrylate), polystyrene, poly(acrylamide),
polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin,
glycogen, amylopectin, mannan, inulin, nitrocellulose,
diazocellulose, polyvinylchloride, polypropylene, polyethylene
(including poly(ethylene glycol)), nylon, latex bead, magnetic
bead, paramagnetic bead, superparamagnetic bead, starch and the
like. In some embodiments, the solid supports used in the methods
and compositions described herein are substantially insoluble in
liquid phases.
[0303] In some embodiments, a solid support may comprise a reactive
functional group, including, but not limited to, hydroxyl,
carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido,
urea, carbonate, carbamate, isocyanate, sulfone, sulfonate,
sulfonamide, sulfoxide, azide, alkyne, or phosphine, wherein such
functional groups are used to covalently attach the solid support
to a precursor of a compound of any one of Formulas (I) to
(XIII).
[0304] A suitable solid phase support used in the methods and
compositions described herein, can be selected on the basis of
desired use. By way of example only, where amide bond formation is
desirable to attach the precursor of a compound of any one of
Formulas (I) to (XIII) to the solid support, resins generally
useful in peptide synthesis may be employed, such as polystyrene,
POLYHIPE.TM. resin, polyamide resin, polystyrene resin grafted with
polyethylene glycol, polydimethyl-acrylamide resin, or PEGA beads.
In some embodiments, precursors to compounds of Formulas (I) to
(XIII) are deposited onto a solid support in an array format. In
some such deposition is accomplished by direct surface contact
between the support surface and a delivery mechanism, such as a pin
or a capillary, or by ink jet technologies which utilize
piezoelectric and other forms of propulsion to transfer liquids
from miniature nozzles to solid surfaces.
Modified Biomolecules
[0305] The modification of biomolecules to incorporate chemical
handles allows chemical attachment of another moiety (such as a
reporter molecule or solid support) through a subsequent click
reaction. In some embodiments, the chemical handle of the modified
biomolecule is selected from azide, alkyne (such as a terminal
alkyne or an activated alkyne), and phosphine. In some embodiments,
a biomolecule is modified in vivo, for example, using cellular
biosynthetic pathways, such as, for example, glycosylation of
proteins, DNA replication, or transcription of RNA. In some
embodiments, a biomolecule is modified in vivo by contacting a cell
with a reagent that modifies a particular biomolecule or class of
biomolecules. In some embodiments, a biomolecule is modified in
vitro using a reagent that modifies a biomolecule.
[0306] Various methods and reagents for modifying biomolecules in
vivo are known in the art. For example, in some embodiments,
glycoproteins may be modified in vivo by contacting a cell with
non-native glycans that comprise chemical handles. The non-native
glycans are used by the cell to glycosylate glycoproteins,
resulting in covalent attachment of chemical handles to such
glycoproteins. Nonlimiting exemplary non-native glycans that may be
used to modify glycoproteins with chemical handles include
tetraacetylated N-azidoacetylglucosamine, tetraacetylated
N-azidoacetylgalactosamine, tetraacetylated
N-azidoacetylmannosamine, and tetraacetylfucose alkyne.
[0307] In some embodiments, a protein may be modified by
incorporating non-native amino acids comprising chemical handles.
Such modification may occur in vivo, during protein synthesis, or
in an in vitro protein translation system. Nonlimiting exemplary
non-native amino acids that may be used to modify proteins with
chemical handles include, but are not limited to,
4-azido-L-phenylalanine, L-azidohomoalanine, and
L-homopropargylglycine.
[0308] In some embodiments, a prenylated protein may be modified,
for example, by contacting a cell with a farnesyl alcohol azide or
a geranylgeranyl alcohol azide.
[0309] In some embodiments, a protein may be modified during fatty
acid acylation of the protein, for example, by contacting a cell
with a non-native fatty acid comprising a chemical handle.
Nonlimiting exemplary non-native fatty acids that may be used to
modify proteins with chemical handles include, but are not limited
to, palmitic acid azide, myristic acid azide, and the fatty acid
analogs described, e.g., in International Application No.
PCT/US10/61768.
[0310] In some embodiments, DNA may be modified in vivo or in vitro
using various non-native nucleoside triphosphates that comprise
chemical handles. In some embodiments, the DNA is modified during
replication through incorporation of a non-native nucleoside by DNA
polymerase. In some embodiments, the DNA is modified during
apoptosis through incorporation of a non-native nucleoside by
terminal nucleotidyl transferase (TdT). Nonlimiting exemplary such
non-native nucleoside triphosphates include C-8-alkyne-dUTP and/or
C8-alkyne-dCTP. Following incorporation, the DNA comprises one or
more covalently attached alkyne groups. In some embodiments, DNA
may be modified during chemical DNA synthesis using, for example,
phophoramidites comprising chemical handles.
[0311] In some embodiments, RNA may be modified in vivo or in vitro
using various non-native nucleoside triphosphates that comprise
chemical handles. In some embodiments, the RNA is modified during
replication through incorporation of a non-native nucleoside by RNA
polymerase. Nonlimiting exemplary such non-native nucleoside
triphosphates include C-8-alkyne-UTP and/or C8-alkyne-CTP.
Following incorporation, the RNA comprises one or more covalently
attached alkyne groups. In some embodiments, RNA may be modified
during chemical RNA synthesis using, for example, phophoramidites
comprising chemical handles.
[0312] In some embodiments, a biomolecule may be modified in vitro
using a reagent that covalently attaches a chemical handle through
a particular group on the biomolecule. For example, in some
embodiments, a biomolecule that comprises a primary amine
(--NH.sub.2) may be modified using a reagent such as NHS-azide,
NHS-phosphine, and sulfo-NHS-phosphine, SDP-azide, TFP-azide,
PFP-azide, carbamate-azide, thiocarbamate-azide and maleimide-azide
may also be used in place of NHS-azides.
Copper Ion Sources
[0313] In some embodiments, a click reaction comprises a copper ion
source that provides Cu(I) ions. In some embodiments, a copper ion
source provides Cu(I) ions in the presence of a reducing agent. In
some such embodiments, a copper ion source provides Cu(II) ions,
which are reduced to Cu(I) ions in the presence of a reducing
agent. Nonlimiting exemplary copper ion sources that produce Cu(I)
ions include CuBr, CuI, tetrakis(acetonitrile)Cu(I)
hexafluorophosphate, tetrakis(acetonitrile)Cu(I) tetrafluoroborate,
tetrakis(acetonitrile)Cu(I) triflate, CuCN, Cu(I) butanethiolate,
Cu(I) thiophenolate, Cu(I) triflate. In some embodiments, a copper
ion source that produces Cu(I) ions is included in a click reaction
at a concentration between 0.01 mM and 10 mM, between 0.01 mM and 5
mM, between 0.05 mM and 5 mM, between 0.1 mM and 5 mM, between 0.5
mM and 5 mM, between 0.5 mM and 4 mM, or between 0.5 mM and 3 mM.
Nonlimiting exemplary copper ion sources that produce Cu(II) ions
include Cu(NO.sub.3).sub.2 Cu(OAc).sub.2 or CuSO.sub.4, metallic Cu
and metallic Cu with sonication. In some embodiments, a copper ion
source that produces Cu(II) ions is included in a click reaction at
a concentration between 0.01 mM and 10 mM, between 0.01 mM and 5
mM, between 0.05 mM and 5 mM, between 0.1 mM and 5 mM, between 0.5
mM and 5 mM, between 0.5 mM and 4 mM, or between 0.5 mM and 3
mM.
[0314] In some embodiments, a copper ion source is
copper-containing metal, such as copper wire.
[0315] Nonlimiting exemplary reducing agents that may be used to
reduce Cu(II) ions to Cu(I) ions include ascorbate,
tris(2-carboxyethyl)phosphine (TCEP), NADH, NADPH, thiosulfate,
metallic copper, hydroquinone, vitamin K.sub.1, glutathione,
cysteine, 2-mercaptoethanol, and dithiothreitol. Nonlimiting
exemplary metals that may act as reducing agents include Al, Be,
Co, Cr, Fe (including Fe.sup.2+), Mg, Mn, Ni, Zn, Au, Ag, Hg, Cd,
Zr, Ru, Fe, Co (including Co.sup.2+), Pt, Pd, Ni, Rh, and W. In
some embodiments, a reducing agent is included in a click reaction
at a concentration 1 micromolar to 5 molar.
[0316] In some embodiments, a reducing agent is an applied electric
potential. In this case, a ligand such as TBTA, THPTA,
benxzimidazole, BCS, etc is used employed and an electric potential
of -30 to -300 mV is applied in a two compartment cell using a
combination of working and reference electrodes. Standard buffers
can be used (HEPES, Tris, etc) and the electric potential may be
applied during the course of the reaction. See Chem Bio Chem 2008,
9, 1481-1486. for further details and experimental information.
Copper Ion Chelators
[0317] Without limitation to any specific mechanism, it is known
that copper can promote the cleavage of biomolecules, such as
proteins and nucleic acids. The addition of a copper chelator in a
click reaction may reduce the detrimental effects of copper,
thereby preserves the structural integrity of biomolecules.
[0318] In some embodiments, a click reaction comprises a copper
chelator. In some embodiments, a copper chelator stabilizes Cu(I)
ions against oxidation, precipitation, and/or disproportionation.
By including a copper chelator, in some embodiments, a lower
concentration of copper ions can be used in a click reaction to
achieve the same efficiency as would be obtained in the presence of
higher concentrations of copper ions in the absence of a
chelator.
[0319] Nonlimiting exemplary copper ion chelators include compounds
of formula (V):
##STR00027##
wherein X, Y, and Z each independently have the formula:
##STR00028##
wherein: R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are
independently selected from hydrogen, halogen, alkyl, heteroalkyl,
alkoxy, cycloalkyl, heterocycloalkyl, substituted alkyl,
substituted heteroalkyl, substituted alkoxy, substituted
cycloalkyl, substituted heterocycloalkyl, aryl, heteroaryl,
substituted aryl, arylalkyl, substituted arylalkyl, and substituted
heteroaryl; and L.sub.1, L.sub.2, and L.sub.3 are independently
selected from alkyl, heteroalkyl, substituted alkyl, and
substituted heteroalkyl, having a chain length of 1-5 atoms.
[0320] In some embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4 and
R.sub.5 for at least one substituent selected from X, Y, and Z are
each H. In some embodiments, L.sub.1, L.sub.2 and L.sub.3 are each
alkyl groups having a chain length of 1-5 atoms. In some
embodiments, L.sub.1, L.sub.2 and L.sub.3 are each
--CH.sub.2CH.sub.2--.
[0321] Nonlimiting exemplary copper ion chelators also include 1,10
phenanthroline-containing copper (I) chelators, such as, for
example, bathophenanthroline disulfonic acid
(4,7-diphenyl-1,10-phenanthroline disulfonic acid) and
bathocuproine disulfonic acid (BCS;
2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate).
Nonlimiting exemplary chelators also include
tris-(hydroxypropyltriazolylmethyl)amine (THPTA; see, e.g.,
Jentzsch et al., Inorganic Chemistry, 48(2): 9593-9595 (2009)) and
the Cu(I) chelators described in U.S. Publication No.
US2010/0197871, the disclosure of which is incorporated herein by
reference. Nonlimiting exemplary chelators also include
N-(2-acetamido)iminodiacetic acid (ADA), pyridine-2,6-dicarboxylic
acid (PDA), S-carboxymethyl-L-cysteine (SCMC), trientine,
tetra-ethylenepolyamine (TEPA),
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA,
neocuproine, N-(2-acetamido)iminodiacetic acid (ADA),
pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine
(SCMC), tris-(benzyl-triazolylmethyl)amine (TBTA), and derivatives
thereof. In some embodiments, histidine is used as a chelator. In
some embodiments, glutathione is used as a chelator and a reducing
agent.
[0322] In some embodiments, a copper chelator, such as a compound
of formula (V), is included in a click reaction at molar ratio of
1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or greater than
10:1, relative to the concentration of copper in the click
reaction. That is, in some embodiments, if copper is included in a
click reaction at a concentration of 2 mM, a copper chelator, such
as a compound of formula (V), may be included in the click reaction
at a concentration of 2 mM (1:1), 4 mM (2:1), 6 mM (3:1), etc. In
some embodiments, the concentration of a copper chelator, such as a
compound of formula (V), in a click reaction is between 1 .mu.M and
100 mM, between 10 .mu.M and 10 mM, between 50 .mu.M and 10 mM, or
between 1 mM and 10 mM.
Compositions
[0323] In some embodiments, compositions are provided. In some
embodiments, a composition comprises a compound of any one of
Formulas (I) to (XIII). In some embodiments, a composition
comprises a compound of any one of Formulas (I) to (XIII) and a
modified biomolecule. In some such embodiments, the compound of any
one of Formulas (I) to (XIII) comprises an azide and the
biomolecule comprises an alkyne, such as a terminal alkyne or an
activated alkyne, or a phosphine, such as a triarylphosphine. In
some embodiments, the compound of any one of Formulas (I) to (XIII)
comprises an alkyne and the biomolecule comprises an azide.
[0324] In some embodiments, a composition comprises a first
compound of any one of Formulas (I) to (XIII) and a second compound
of any one of Formulas (I) to (XIII), wherein the first and second
compounds of Formulas (I) to (XIII) are distinguishable from one
another. For example, in some embodiments, the first compound of
any one of Formulas (I) to (XIII) comprises a first reporter
molecule and the second compound of any one of Formulas (I) to
(XIII) comprises a second reporter molecule, wherein the first and
second reporter molecules are detectably different. In some
embodiments, the first compound of any one of Formulas (I) to
(XIII) comprises an alkyne and the second compound of any one of
Formulas (I) to (XIII) comprises an azide. In some such
embodiments, the composition comprises a first biomolecule
comprising an alkyne reactive group and a second biomolecule
comprising an azide reactive group. In some embodiments, a
composition comprise three, four, five, or more compounds of
Formulas (I) to (XIII). In some such embodiments, the compounds of
Formulas (I) to (XIII) in a composition can each be independently
detected. That is, in some embodiments, two or more of the
compounds comprise detectably different reporter molecules and/or
can be separated from one another prior to detection, etc.
[0325] In some embodiments, a composition further comprises a
copper ion source and/or a reducing agent and/or a copper ion
chelator. In some such embodiments, the copper ion chelator is a
compound of formula (V).
[0326] Various buffering agents can be included in the compositions
described herein, including inorganic and organic buffering agents.
In some embodiments buffering agent is a zwitterionic buffering
agent. Exemplary buffering agents include phosphate (such as, for
example, in phosphate buffered saline (PBS)), succinate, citrate,
borate, maleate, cacodylate, N-(2-Acetamido)iminodiacetic acid
(ADA), 2-(N-morpholino)-ethanesulfonic acid (MES),
N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),
piperazine-N,N'-2-ethanesulfonic acid (PIPES),
2-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES),
3-(N-morpholino)-propanesulfonic acid (MOPS),
N-tris-(hydroxymethyl)-2-ethanesulfonic acid (TES),
N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES),
3-(N-tris-(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid
(TAPSO), 3-(N,N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic
acid (DIPSO),
N-(2-Hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid)
(HEPPSO), 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid
(EPPS), N-[Tris(hydroxymethyl)methyl]glycine (Tricine),
N,N-Bis(2-hydroxyethyl)glycine (Bicine),
(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic
acid (TAPS),
N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), tris(hydroxy methyl)amino-methane (Tris),
TRIS-Acetate-EDTA (TAE), glycine,
bis[2-hydroxyethyl]iminotris[hydroxymethyl]methane (BisTris), and
combinations thereof. In some embodiments, a composition further
comprises ethylene diamine tetraacetic acid (EDTA).
[0327] The concentration of such buffering agents in a composition,
in some embodiments, is between 0.1 mM and 1 M, between 10 mM and 1
M, between 20 mM and 500 mM, between 50 mM and 300 mM, between 0.1
mM and 50 mM, and between 0.5 mM and 20 mM.
[0328] One skilled in the art can select a suitable composition pH
according to the intended application. In order to retain the
structural integrity of biomolecules, in some embodiments, the pH
is maintained in a physiological range, such as, for example,
between about 6.5 and 8. In some embodiments, a composition has a
pH of between 5 and 9 at 25.degree. C., between 6 and 8.5 at
25.degree. C., between 6 and 8 at 25.degree. C., between 6.5 and 8
at 25.degree. C., or between 6.5 and 7.5 at 25.degree. C.
[0329] In some embodiments, a composition comprises one or more
non-ionic detergents. Non-limiting examples of such non-ionic
detergents include polyoxyalkylene diols, ethers of fatty alcohols
(such as alcohol ethoxylates), alkyl phenol ethoxylates, ethylene
oxide/propylene oxide block copolymers, polyoxyethylene ester of a
fatty acids, alkyl phenol surfactants, polyoxyethylene mercaptan
analogs of alcohol ethoxylates, polyoxyethylene adducts of alkyl
amines, polyoxyethylene alkyl amides, sorbitan esters, and alcohol
phenol ethoxylate. Non-limiting examples of sorbitan esters include
polyoxyethylene(20) sorbitan monolaurate (TWEEN20),
polyoxyethylene(20) sorbitan monopalmitate (TWEEN40),
polyoxyethylene(20) sorbitan monostearate (TWEEN60) and
polyoxyethylene(20) sorbitan monooleate (TWEEN 80). In some
embodiments, the concentration of such non-ionic detergents in a
composition is between 0.005 and 0.5%, between 0.01 and 0.4%,
between 0.01 and 0.3%, between 0.01 and 0.2%, or between 0.01 and
0.2%.
Conjugation of Modified Biomolecules
[0330] In various embodiments, the modified biomolecules described
herein may be linked to at least one moiety selected from a
reporter molecule, a carrier molecule, a solid phase, and a
therapeutic molecule, by conjugating the modified biomolecule to a
compound of any one of Formulas (I) to (XIII) using a click
reaction, a 1,3-dipolar cycloaddition reaction, or Staudinger
ligation reaction. In some embodiments, the reaction is carried out
at room temperature in aqueous solution.
[0331] In some embodiments, a click reaction is carried out in the
presence of copper, such as Cu(I) ions. In some embodiments, a
click reaction is carried out in the presence of a reducing agent.
In some embodiments, the click reaction is carried out in the
presence of a copper chelator. In some embodiments, the resulting
conjugated product is stable in an aqueous environment for
sufficient time to allow manipulation, quantification, and/or
detection of the biomolecule.
[0332] In some embodiments, the modified biomolecule comprises an
azide moiety. In some such embodiments, the compound of any one of
Formulas (I) to (XIII) comprises a terminal alkyne at substituent
G. In some embodiments, the modified biomolecule comprises an
alkyne moiety, such as a terminal alkyne or an activated alkyne. In
some such embodiments, the compound of any one of Formulas (I) to
(XIII) comprises an azide at substituent G. In some embodiments,
the modified biomolecule comprises a phosphine moiety, such as a
triarylphosphine. In some such embodiments, the compound of any one
of Formulas (I) to (XIII) comprises an azide at substituent G.
[0333] In some embodiments, the click reaction, 1,3-dipolar
cycloaddition reaction, or Staudinger ligation reaction is carried
out in a cell, in a cell lysate, in a solution comprising an
isolated modified biomolecule, or with a modified biomolecule
immobilized on a solid support.
[0334] In some embodiments, a modified biomolecule comprises more
than one type of chemical handle. As a nonlimiting example, in some
embodiments, a modified biomolecule comprises an azide and an
alkyne, such as a terminal alkyne or an activated alkyne. In some
such embodiments, the modified biomolecule may be conjugated to a
compound of any one of Formulas (I) to (XIII) comprising a terminal
alkyne and/or a compound of any one of Formulas (I) to (XIII)
comprising an azide, using click chemistry. In some embodiments,
the modified biomolecule may be conjugated to a compound of any one
of Formulas (I) to (XIII) comprising an azide using click
chemistry, and may be conjugated to another compound that comprises
a phosphine using a Staudinger ligation or comprises an alkyne,
such as a terminal alkyne or activated alkyne, using a 1,3-bipolar
cycloaddition. Alternatively, in some embodiments, the modified
biomolecule may be conjugated to a compound of any one of Formulas
(I) to (XIII) comprising a terminal alkyne, and may be conjugated
to another compound that comprises an azide, both using click
chemistry. Numerous combinations of chemical handles and
conjugating reagents are possible, and can be selected according to
the intended application by one skilled in the art.
[0335] In some embodiments, a method comprises two or more
conjugation reactions. In some such embodiments, two or more
conjugation reactions occur using the same reaction chemistry
(i.e., two or more occur using click chemistry, 1,3-dipolar
cycloaddition, or Staudinger ligation). As a nonlimiting example, a
first modified biomolecule comprises an azide and a second modified
biomolecule comprises an alkyne. Both modified biomolecules may be
conjugated using click chemistry, either simultaneously or
sequentially. In some such embodiments, the modified biomolecules
are contacted with a first compound of any one of Formulas (I) to
(XIII) comprising an alkyne and a second compound of any one of
Formulas (I) to (XIII) comprising an azide. The first biomolecule
comprising the azide will be conjugated to the first compound of
any one of Formulas (I) to (XIII) comprising an alkyne, and the
second biomolecule comprising the alkyne will be conjugated to the
second compound of any one of Formulas (I) to (XIII) comprising an
azide. In some embodiments, in order to reduce the occurrence of
the first biomolecule conjugating to the second biomolecule, the
concentration of the compounds of Formulas (I) to (XIII) can be
controlled appropriately (e.g., such that they are in excess with
respect to the concentration of the biomolecules) and/or the
biomolecules can be spatially distinct (e.g., in different cellular
compartments).
[0336] In some embodiments, two or more conjugation reactions occur
using different reaction chemistry. As a nonlimiting example, a
first modified biomolecule comprises an azide and a second modified
biomolecule comprises a phosphine. The first biomolecule may be
conjugated to a first compound of any one of Formulas (I) to (XIII)
comprising an alkyne using click chemistry, and the second
biomolecule may be conjugated to a second compound of any one of
Formulas (I) to (XIII) comprising an azide using Staudinger
ligation. The click reaction and the Staudinger ligation may be
carried out either simultaneously or sequentially. In some
embodiments, in order to reduce the occurrence of the first
biomolecule conjugating to the second biomolecule, the
concentration of the compounds of Formulas (I) to (XIII) can be
controlled appropriately (e.g., such that they are in excess with
respect to the concentration of the biomolecules) and/or the
biomolecules can be spatially distinct (e.g., in different cellular
compartments).
Conjugation in a Cell
[0337] In some embodiments, methods of conjugating a modified
biomolecule to a compound of any one of Formulas (I) to (XIII) in a
cell are provided. In some such embodiments, the conjugated
biomolecule is separated from the cell following conjugation. In
some embodiments, the conjugated biomolecule is identified,
detected, and/or quantified in the cellular environment following
conjugation (such as, for example, in the live cell, or in a cell
that has been fixed and/or permeabilized prior to identification,
detection and/or quantification of the biomolecule).
[0338] In some embodiments, a method of conjugating a modified
biomolecule to a compound of any one of Formulas (I) to (XIII) in a
cell comprises contacting a cell comprising a modified biomolecule
with a compound of any one of Formulas (I) to (XIII) under
conditions allowing the compound of any one of Formulas (I) to
(XIII) to come into contact with the modified biomolecule. In some
embodiments, if the modified biomolecule is located on the surface
of the cell, contacting the cell with a composition comprising the
compound of any one of Formulas (I) to (XIII) allows conjugation of
the modified biomolecule. In some embodiments, when the modified
biomolecule is located inside the cell, the cell may be contacted
with a composition comprising the compound of any one of Formulas
(I) to (XIII) with or without prior fixing and/or permeabilization
of the cell. In some embodiments, for example when the conjugation
occurs via click reaction, the cell may also be contacted with a
copper ion source, a reducing agent, and/or a copper ion chelator.
Additional components, such as buffers, detergents, salts, and the
like, can also be included in the conjugation reaction. One skilled
in the art can select suitable additional components depending on
the application.
[0339] The conjugation can be performed under aerobic or anaerobic
conditions, such as under nitrogen or argon gas, and can be
performed for any suitable length of time, such as, for example,
from five minutes to six hours, from 10 minutes to 3 hours, from 20
minutes to 3 hours, or from 30 minutes to 2 hours. The reaction can
be performed at a wide range of temperatures, for example, between
4.degree. C. and 50.degree. C., between 10.degree. C. and
40.degree. C., or between 15.degree. C. and 30.degree. C.
[0340] Cells may be fixed using any method, including, but not
limited to treatment with 4% formaldehyde or methanol.
[0341] Cells may be permeabilized by any method, including but not
limited to treatment with NP-40 buffer or 0.1% Triton buffer.
[0342] In some embodiments, a cell comprising more than one
modified biomolecule is contacted with more than one compound of
any one of Formulas (I) to (XIII), wherein the compounds of
Formulas (I) to (XIII) are detectably different. In some such
embodiments, the cell is contacted with two or more compounds of
Formulas (I) to (XIII) simultaneously or sequentially. Nonlimiting
exemplary chemical handles that may be used in such multiplex
reactions are described above.
[0343] Following conjugation, the conjugated biomolecules may be
separated and/or detected according to methods known in the art.
Exemplary such methods are discussed herein.
[0344] In some embodiments, a method of comprises:
(a) contacting a cell comprising a modified biomolecule with a
compound of any one of Formulas (I) to (XIII) under conditions
allowing conjugation of the modified biomolecule to the compound of
formula compound of any one of Formulas (I) to (XIII) to form a
conjugated biomolecule; and (b) detecting the conjugated
biomolecule. In some embodiments, the modified biomolecule
comprises an azide and the compound of any one of Formulas (I) to
(XIII) comprises a terminal alkyne. In some embodiments, the
modified biomolecule comprises a terminal alkyne, an activated
alkyne, or a phosphine, and the compound of any one of Formulas (I)
to (XIII) comprises an azide. In some embodiments, the method
comprises separating the conjugated biomolecule, before or after
(b). In some embodiments, the method comprises fixing the cell
before (a). In some embodiments, the compound of any one of
Formulas (I) to (XIII) comprises a reporter molecule. In some
embodiments, the compound of any one of Formulas (I) to (XIII)
comprises a fluorophore. In some embodiments, detecting comprises
illuminating the conjugated biomolecule with an appropriate
wavelength of light, such that the reporter molecule emits light,
and observing the emitted light.
Conjugation in Solution
[0345] In some embodiments, methods of conjugating a modified
biomolecule to a compound of any one of Formulas (I) to (XIII) in
solution are provided. Such solutions include, but are not limited
to, cell lysates, solutions of isolated biomolecules (in which the
biomolecules are separated from at least some of the components of
cells in which the biomolecules are ordinarily found), cell
supernatants, liquid biological samples (described below), and the
like.
[0346] In some embodiments, a method of conjugating a modified
biomolecule to a compound of any one of Formulas (I) to (XIII) in
solution comprises contacting the modified biomolecule with a
compound of any one of Formulas (I) to (XIII) under conditions
allowing the compound of any one of Formulas (I) to (XIII) to react
with the modified biomolecule via a click reaction, a 1,3-dipolar
cycloaddition, or a Staudinger ligation. In some embodiments, for
example when the conjugation occurs via click reaction, a copper
ion source, a reducing agent, and/or a copper ion chelator may also
be included in the solution. Additional components, such as
buffers, detergents, salts, and the like, can also be included in
the conjugation reaction. One skilled in the art can select
suitable additional components depending on the application.
[0347] In some embodiments, more than one modified biomolecule is
present in solution. In some such embodiments, more than one
compound of any one of Formulas (I) to (XIII) is also added to the
solution and conjugated to the more that one modified biomolecules.
In some embodiments, two or more compounds of Formulas (I) to
(XIII) are added to the solution sequentially or simultaneously. In
some embodiments, the compounds of Formulas (I) to (XIII) are
detectably different. Nonlimiting exemplary chemical handles that
may be used in such multiplex reactions are described above.
[0348] The conjugation can be performed under aerobic or anaerobic
conditions, such as under nitrogen or argon gas, and can be
performed for any suitable length of time, such as, for example,
from five minutes to six hours, from 10 minutes to 3 hours, from 20
minutes to 3 hours, or from 30 minutes to 2 hours. The reaction can
be performed at a wide range of temperatures, for example, between
4.degree. C. and 50.degree. C., between 10.degree. C. and
40.degree. C., or between 15.degree. C. and 30.degree. C.
[0349] Following conjugation, the conjugated biomolecules may be
separated and/or detected according to methods known in the art.
Exemplary such methods are discussed herein.
[0350] In some embodiments, a method of comprises:
(c) contacting a modified biomolecule with a compound of any one of
Formulas (I) to (XIII) under conditions allowing conjugation of the
modified biomolecule to the compound of formula compound of any one
of Formulas (I) to (XIII) to form a conjugated biomolecule; and (d)
detecting the conjugated biomolecule. In some embodiments, the
modified biomolecule comprises an azide and the compound of any one
of Formulas (I) to (XIII) comprises a terminal alkyne. In some
embodiments, the modified biomolecule comprises a terminal alkyne,
an activated alkyne, or a phosphine, and the compound of any one of
Formulas (I) to (XIII) comprises an azide. In some embodiments, the
method comprises separating the conjugated biomolecule. In some
embodiments, the compound of any one of Formulas (I) to (XIII)
comprises a reporter molecule. In some embodiments, the compound of
any one of Formulas (I) to (XIII) comprises a fluorophore. In some
embodiments, detecting comprises illuminating the conjugated
biomolecule with an appropriate wavelength of light, such that the
reporter molecule emits light, and observing the emitted light.
Conjugation on a Solid Support
[0351] In some embodiments, methods of conjugating a modified
biomolecule to a compound of any one of Formulas (I) to (XIII) on a
solid support are provided. Nonlimiting exemplary such solid
supports include the various solid supports discussed herein,
including, but not limited to, solid and semi-solid matrixes, such
as glass, slides, arrays, silica particles, polymeric particles,
microtiter plates and polymeric gels. In some embodiments, the
compound of any one of Formulas (I) to (XIII) comprises a solid
support as a substituent. In some embodiments, the modified
biomolecule is bound to a solid support.
[0352] The modified biomolecule may be bound to a solid support
through any means. For example, in some embodiments, the modified
biomolecule may have been adsorbed onto a solid support through
non-covalent interactions. In some embodiments, the modified
biomolecule comprises a member of a binding pair, and is bound to a
solid support that comprises the other member of the binding pair.
In some embodiments, the modified biomolecule has been conjugated
to a solid support through a prior reaction, which may be a click
reaction, 1,3-dipolar cycloaddition, a Staudinger ligation, or
other type of reaction. Thus, in some embodiments, the modified
biomolecule is attached to a solid support using a functional group
other than the chemical handle used for a click reaction,
1,3-dipolar cycloaddition, or Staudinger ligation, whereupon the
attached modified biomolecule is then conjugated to a compound of
any one of Formulas (I) to (XIII) through the chemical handle in a
click reaction, 1,3-dipolar cycloaddition, or Staudinger ligation.
By way of example only, the modified biomolecule can be immobilized
to a solid support using hydroxyl, carboxyl, amino, thiol,
aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate,
isocyanate, sulfone, sulfonate, sulfonamide or sulfoxide functional
groups.
[0353] When conjugation of the biomolecule to a compound of any one
of Formulas (I) to (XIII) occurs on a solid support, in some
embodiments, the reaction is carried out in a similar composition
as is used for solution-phase conjugation.
[0354] In some embodiments, a method of comprises:
(e) contacting a modified biomolecule with a compound of any one of
Formulas (I) to (XIII) under conditions allowing conjugation of the
modified biomolecule to the compound of formula compound of any one
of Formulas (I) to (XIII) to form a conjugated biomolecule, wherein
the modified biomolecule or the compound of any one of Formulas (I)
to (XIII) is immobilized on a solid support; and (f) detecting the
conjugated biomolecule. In some embodiments, the modified
biomolecule comprises an azide and the compound of any one of
Formulas (I) to (XIII) comprises a terminal alkyne. In some
embodiments, the modified biomolecule comprises a terminal alkyne,
an activated alkyne, or a phosphine, and the compound of any one of
Formulas (I) to (XIII) comprises an azide. In some embodiments, the
method comprises separating the conjugated biomolecule. In some
embodiments, the compound of any one of Formulas (I) to (XIII)
comprises a reporter molecule. In some embodiments, the compound of
any one of Formulas (I) to (XIII) comprises a fluorophore. In some
embodiments, detecting comprises illuminating the conjugated
biomolecule with an appropriate wavelength of light, such that the
reporter molecule emits light, and observing the emitted light.
Separation of Conjugated Biomolecules
[0355] In some embodiments, a conjugated biomolecule is separated
following conjugation via a click reaction, a 1,3-dipolar
cycloaddition, or a Staudinger ligation. Nonlimiting exemplary
methods of separating conjugated biomolecules include
sedimentation, centrifugation, magnetic attraction, chromatographic
methods, and electrophoretic methods.
[0356] In some embodiments, separation of the conjugated
biomolecule is facilitated by a substituent on a compound of any
one of Formulas (I) to (XIII) that has been conjugated to the
biomolecule. As a nonlimiting example, the compound of any one of
Formulas (I) to (XIII) may comprise a member of a binding pair,
which is then bound to the complementary member of the binding pair
to separate the conjugated biomolecule. For example, in some
embodiments, the compound of any one of Formulas (I) to (XIII)
comprises biotin such that the conjugated biomolecule may be
separated by binding to a streptavidin-containing solid support,
such as streptavidin-coated multiwell plates or streptavidin-coated
microparticles. As a further non-limiting example, the compound of
any one of Formulas (I) to (XIII) may comprise a microparticle
(including, for example, a magnetic microparticle) as a
substituent, such that the conjugated biomolecule may be separated
by centrifugation (or contact with a magnet if the microparticle is
magnetic).
[0357] In some embodiments, conjugated biomolecules are separated
by thin layer or column chromatography. Nonlimiting exemplary such
chromatography includes size exclusion, ion exchange, and affinity
chromatography. In some embodiments, conjugated biomolecules are
separated using isoelectric focusing. In some embodiments,
conjugated biomolecules are separated using electrophoresis.
Nonlimiting exemplary electrophoresis includes gel electrophoresis
(such as, for example, agarose gel electrophoresis and acrylamide
gel electrophoresis), capillary electrophoresis, capillary gel
electrophoresis, and slab gel electrophoresis. Gel electrophoresis
can be denaturing or nondenaturing, and can include denaturing gel
electrophoresis followed by nondenaturing gel electrophoresis
(e.g., "2D" gels). The conjugated biomolecules may be detected at
any time before, during, or after separation. In some embodiments,
such as when the conjugated biomolecules are separated by gel
electrophoresis, the conjugated biomolecules may be detected in the
separation medium (e.g., the gel), either during or after
separation.
[0358] One skilled in the art can select a suitable separation
method according to the moieties conjugated to the conjugated
biomolecule, the identity or type of biomolecule, and the
particular application.
Detection of Conjugated Biomolecules
[0359] In some embodiments, the conjugated biomolecules are
detected following conjugation. In some embodiments, a reporter
molecule that is a substituent of a compound of any one of Formulas
(I) to (XIII) that has been conjugated to a biomolecule is used for
detection. In some embodiments, a carrier molecule that is a
substituent of a compound of any one of Formulas (I) to (XIII) that
has been conjugated to a biomolecule is used for detection. In some
embodiments, a solid support that is a substituent of a compound of
any one of Formulas (I) to (XIII) that has been conjugated to a
biomolecule is used for detection. The phrase "used for detection"
encompasses direct or indirect detection of the reporter molecule,
carrier molecule, or solid support. The conjugated biomolecules may
be detected by any method. Many methods of detection are known in
the art, and some non-limiting exemplary methods will be discussed
below by way of illustration only. One skilled in the art can
select a suitable detection method depending on the identity and/or
properties of the reporter molecule, carrier molecule, solid
support, biomolecule, and any other moieties associated
therewith.
[0360] Detection of conjugated biomolecules may occur at any time
following conjugation, and at any time before, during, or after
separation, if such separation is carried out.
[0361] In some embodiments, the moieties used for detection are any
fluorophores described herein that can be used as substituents on
compounds of Formulas (I) to (XIII). Nonlimiting exemplary such
fluorophores include fluoresceins, rhodamines, TAMRA, Alexa dyes,
SYPRO dyes, and BODIPY dyes.
[0362] In some embodiments, a method comprises multiplexed
detection of modified biomolecules, for example, by conjugating the
modified biomolecules to compounds of Formulas (I) to (XIII)
comprising different reporter molecules. In some embodiments, the
conjugation reaction can be carried out such that modified
biomolecule comprising particular chemical handles are conjugated
to compounds of Formulas (I) to (XIII) comprising particular
reporter molecules.
[0363] By way of illustration only, as a nonlimiting example, a
composition comprising a first modified biomolecule, a second
modified biomolecule, and a third modified biomolecule is provided,
wherein the first modified biomolecule comprises a phosphine, the
second modified biomolecule comprises an azide, and the third
modified biomolecule comprises a terminal alkyne. The composition
is contacted with a first compound of any one of Formulas (I) to
(XIII) comprising a first reporter molecule and an azide moiety in
the absence of Cu(I) ions. The first compound conjugates to the
first biomolecule through a Staudinger ligation. In some
embodiments, unconjugated first compound is rendered inactive
and/or removed from the composition. Thereafter, a second compound
of any one of Formulas (I) to (XIII) comprising a second reporter
molecule and a terminal alkyne, and a third compound of any one of
Formulas (I) to (XIII) comprising a third reporter molecule and an
azide are added to the composition in the presence of Cu(I) ions.
The second compound conjugates to the second modified biomolecule
and the third compound conjugates to the third modified biomolecule
through a click reaction. Following conjugation, each of the three
conjugated biomolecules comprises a different reporter molecule
and, in some embodiments, can be detected in a multiplex detection
method.
[0364] In some embodiments, in-gel fluorescence detection allows
for quantitative differential analysis of biomolecules and is
amenable to multiplexing with other protein gel stains. In some
embodiments, utilizing fluorescent- and/or UV-excitable reporter
molecules as substituents of compounds of Formulas (I) to (XIII)
allows for the multiplexed detection of biomolecules (such as, for
example, glycoproteins, phosphoproteins, and total proteins) in the
same 1-D or 2-D gels.
[0365] In some embodiments, detection of modified biomolecules
(such as, for example, proteins) can be by Western blot, in which
the modified biomolecules are separated by gel electrophoresis and
transferred to a blotting membrane. The modified biomolecules may
be conjugated on the blotting membrane to a compound of any one of
Formulas (I) to (XIII), and then detected. Alternatively, in some
embodiments, modified biomolecules that have been previously
conjugated to a compound of any one of Formulas (I) to (XIII) can
be separated by gel electrophoresis and transferred to a blotting
membrane, and then detected.
[0366] Another potential aspect of "in gel" detection is the total
detection of proteins in electrophoresis gels or Western blot
membranes using a "universal" click chemistry, in which
phenylboronic acid-containing molecules are tethered via a linker
to an azide moiety or an alkyne moiety. The phenylboronic acid
stably associates with the cis-diol moieties on glycoproteins under
certain conditions. Such phenylboronic acid-containing molecules
can be used, in some embodiments, to modify glycoproteins with
either azide or alkyne moieties after electrophoretic separation.
The azide or alkyne moieties can then be used to conjugate the
glycoproteins to a compound of any one of Formulas (I) to (XIII)
comprising, for example, a reporter molecule, via click chemistry,
activated alkyne chemistry, or Staudinger ligation. In some
embodiments, the conjugated glycoproteins may then be detected,
either directly or indirectly, using, for example, the reporter
molecule. In some embodiments, glycoproteins of interest can then
be isolated by excising portions of the gel comprising the modified
glycoproteins, and the phenylboronic acid dissociated from the
glycoproteins under acidic conditions, thereby releasing the
conjugated compound of any one of Formulas (I) to (XIII) from the
glycoprotein. In some embodiments, the glycoprotein may then be
identified using, for example, mass spectrometry.
[0367] In some embodiments, when detection comprises detecting an
optical response, the conjugated biomolecules may be detected at
any time by illumination with a wavelength of light that results in
a detectable optical response, and observation with a means for
detecting the optical response. In some embodiments, such
illumination is by a violet or visible wavelength emission lamp, an
arc lamp, a laser, or even sunlight or ordinary room light, wherein
the wavelength of such sources overlap the absorption spectrum of
the moiety being detected, such as a fluorophore or chromophore. In
some embodiments, such illumination is by a violet or visible
wavelength emission lamp, an arc lamp, a laser, or even sunlight or
ordinary room light, wherein a fluorescent compound displays
intense visible absorption as well as fluorescence emission.
[0368] In some embodiments, the illumination sources include, but
are not limited to, hand-held ultraviolet lamps, mercury arc lamps,
xenon lamps, argon lasers, laser diodes, blue laser diodes, and YAG
lasers. These illumination sources are optionally integrated into
laser scanners, flow cytometer, fluorescence microplate readers,
standard or mini fluorometers, or chromatographic detectors. The
fluorescence emission following illumination is optionally detected
by visual inspection, or by use of any of the following devices:
CCD cameras, video cameras, photographic film, laser scanning
devices, fluorometers, photodiodes, photodiode arrays, quantum
counters, epifluorescence microscopes, scanning microscopes, flow
cytometers, fluorescence microplate readers, or by means for
amplifying the signal such as photomultiplier tubes.
[0369] In some embodiments, for example, when a sample is examined
using a flow cytometer, a fluorescence microscope, or a
fluorometer, the instrument is optionally used to distinguish
and/or discriminate between multiple fluorophores having detectably
different optical properties. In some embodiments, when a sample is
examined using a flow cytometer, examination of the sample
optionally includes isolation of particles within the sample based
on the fluorescence response by using a sorting device.
[0370] In certain embodiments, fluorescence is optionally quenched
using either physical or chemical quenching agents.
Samples
[0371] The end user will determine the choice of the sample and the
way in which the sample is prepared. Samples that can be used with
the methods and compositions described herein include, but are not
limited to, any biological derived material or aqueous solution
that contains a modified biomolecule. In certain embodiments, a
samples also includes material in which a modified biomolecule has
been added. The sample that can be used with the methods and
compositions described herein can be a biological fluid including,
but not limited to, whole blood, plasma, serum, nasal secretions,
sputum, saliva, urine, sweat, transdermal exudates, cerebrospinal
fluid, or the like. In other embodiments, the sample are biological
fluids that include tissue and cell culture medium wherein modified
biomolecule of interest has been secreted into the medium. Cells
used in such cultures include, but are not limited to, prokaryotic
cells and eukaryotic cells that include primary cultures and
immortalized cell lines. Such eukaryotic cells include, without
limitation, ovary cells, epithelial cells, circulating immune
cells, .beta. cells, hepatocytes, and neurons. In certain
embodiments, the sample may be whole organs, tissue or cells from
an animal, including but not limited to, muscle, eye, skin, gonads,
lymph nodes, heart, brain, lung, liver, kidney, spleen, thymus,
pancreas, solid tumors, macrophages, mammary glands, mesothelium,
and the like.
Kits
[0372] In some embodiments, kits are provided, wherein the kits
comprise a compound of any one of Formulas (I) to (XIII). In some
embodiments, a kit further comprises a copper ion source. In some
embodiments, a kit further comprises a reducing agent. In some
embodiments, a kit further comprises a copper ion chelator. In some
embodiments, a kit further comprises a reagent for modifying a
biomolecule. Nonlimiting exemplary such copper ion sources,
reducing agents, copper ion chelators, and reagents for modifying
biomolecules are described herein.
[0373] In some embodiments, a kit further comprises a copper ion
chelator of formula (V).
[0374] A detailed description of the invention having been provided
above, the following examples are given for the purpose of
illustrating the invention and shall not be construed as being a
limitation on the scope of the invention or claims.
[0375] The following examples are intended to illustrate but not
limit the invention.
Example 1
[0376] FIG. 1A shows an exemplary method of preparing a reagent for
making a compound of any one of Formulas (I) to (XIII). The
exemplary method is as follows.
[0377] General Synthetic Methods
[0378] Chemicals were purchased from Sigma-Aldrich, Alfa Aesar, TCI
America, Fisher Scientific, Adesis Inc, or EMD unless specified
otherwise. Analytical thin-layer chromatography was performed using
0.25 mm silica gel 60.sub.F254 plates and visualized with 254 nm UV
light or with bromocresol green. .sup.1H NMR spectra were recorded
on a Bruker Avance 400 MHz or a Varian Inova 500 MHz spectrometer.
All samples were dissolved in CDCl.sub.3, CD.sub.3OD, D.sub.2O, or
d.sub.6-DMSO and chemical shifts (.delta.) are expressed in parts
per million relative to residual solvent peak as an internal
standard. Abbreviations are: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad. Coupling constants (J) are
reported in hertz (Hz). Mass spectra were recorded using
electrospray ionization (ESI) on an Applied Biosystems 200 QTRAP
mass spectrometer or an Agilent 1100 MSD ion trap mass
spectrometer. Absorbance and fluorescence properties for selected
compounds were determined on a Perkin Elmer LS50B Luminescence
Spectrometer in HPLC-grade methanol.
[0379] Analytic LC-MS data were acquired using Waters 2695 Affiance
HPLC coupled to a single-quadrupole Waters Micromass ZQ mass
spectrometer, and an Xterra MS C18 column (2.5 .mu.m particle size,
4.6.times.50 mm dimension). The elution gradient is 5-95%
acetonitrile/10 mM NH.sub.4OAc, pH=7 over 20 minutes. MS data were
recorded simultaneously in negative and positive ionization modes.
Preparative HPLC purification was performed using Waters 600 HPLC
equipped with Waters 996 diode array detector, Waters 717 plus
autosampler, and a Luna C18 column (Phenomenex; 5 .mu.m particle
size, 4.6 mm.times.250 mm dimension).
Preparation of picolyl 6-(methoxycarbonyl)nicotinic acid (2)
[0380] 12.6 g of pyridine-2,5-dicarboxylic acid was suspended in
150 mL of methanol and 4.5 g of 95% sulfuric acid was slowly added.
The reaction was heated to reflux for 2.5 h, cooled to 25.degree.
C. and the whole was poured into 750 mL of deionized (DI) water at
room temperature. A white precipitate formed and the suspension was
stirred for 20 minutes, filtered through a Buchner funnel with
filter paper. The precipitate was rinsed with deionized and
collected. This material was dissolved in 150 mL of dichloromethane
heated to 35.degree. C., washed once with 150 mL of saturated
sodium bicarbonate solution. The two layers were separated, and 2 N
HCl was added to the organic layer to until the pH was 1-2. A white
precipitate formed and was filtered with a Buchner funnel fitted
with filter paper. The product was rinsed with deionized water,
collected, and dried under vacuum to provide 4.75 g of compound 2.
TLC (80:20 ACN:H.sub.2O, uv): R.sub.f=0.53 (bis-methyl ester
R.sub.f=0.83). .sup.1H NMR (400 MHz, d.sub.6-DMSO): 9.12 (br s,
1H), 8.42 (t, 1H), 8.15 (t, 1H), 3.89 (s, 3H).
Preparation of 5-(2,5-dioxopyrrolidin-1-yl) 2-methyl
pyridine-2,5-dicarboxylate (3)
[0381] 100 mg of 2 was dissolved in 10 mL of dichloromethane at
ambient temperature. N-hydroxysuccinimide (95 mg) was added,
followed by 133 mg of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide) hydrochloride and the mixture stirred at room
temperature for 2 hours. The mixture was then diluted with 15 mL of
chloroform, followed by the addition of 15 mL of 2% HCl solution.
The organic layer was successively washed once with 15 mL of DI
water. The organic layer was then dried with MgSO.sub.4, filtered
and 3 used directly in the next step. TLC (ethyl acetate, uv):
R.sub.f=0.53.
Preparation of methyl
5-((2-((tert-butoxycarbonyl)amino)ethyl)carbamoyl)picolinate
(4)
[0382] Compound 3 is used directly by dissolving in 15 mL of
dichloromethane. 0.30 mL of N,N-diisopropylethylamine was added at
room temperature followed by 119 mg of
tert-butyl(2-aminoethyl)carbamate hydrochloride was then added and
the reaction was stirred for 50 minutes, at which time 30 mL of
dichloromethane was added followed by 20 mL of 2 M
Na.sub.2CO.sub.3. The layers were separated and the organic layer
was dried with MgSO.sub.4, filtered and concentrated to a residue
which was taken up in 3 mL of warm methanol and loaded onto a
silica gel chromatography column and eluted with ethyl acetate and
methanol to provide 104 mg of 4 as a white solid. TLC (ethyl
acetate, uv): R.sub.f=0.32.). .sup.1H NMR (400 MHz, CDCl.sub.3):
9.15 (d, 1H), 8.30 (d, 1H), 8.14 (complex, 2H), 5.32 (br s, 1H),
4.00 (s, 3H), 3.56 (m, 2H), 3.41 (m, 2H), 1.39 (s, 9H).
Preparation of
tert-butyl(2-(6-(hydroxymethyl)nicotinamido)ethyl)carbamate (5)
[0383] Compound 4 (2.4 g) was dissolved in 50 mL of methanol and
567 mg of NaBH.sub.4 was carefully added to control gas evolution.
THF (5 mL) was added the reaction was placed in a preheated bath at
65.degree. C. The reaction was stirred at this temperature for 35
minutes, at which time 6 mL of 2 M Na.sub.2CO.sub.3 was added
dropwise followed by 6 mL of water. The mixture was then
concentrated to 1/5 original volume and 100 mL of ethyl acetate was
added. The layers were separated and the organic layer was dried
with MgSO.sub.4, filtered and concentrated to an oil, which was
taken up in dichloromethane and flashed with dichloromethane with
0.5% triethylamine and methanol to provide 0.91 g of 5 as a white
solid. TLC (ethyl acetate, uv): R.sub.f=0.13.). .sup.1H NMR (400
MHz, d.sub.6-DMSO): 8.89 (s, 1H), 8.61 (s, 1H), 8.17 (d, 1H), 7.54
(d, 1H), 6.93 (m, 1H), 5.53 (s, 1H), 4.61 (s, 2H), 3.29 (m, 2H),
3.12 (m, 2H), 2.51 (s, 2H), 1.7 (s, 9H).
Preparation of
tert-butyl(2-(6-(azidomethyl)nicotinamido)ethyl)carbamate (6)
[0384] Compound 5 (132 mg) was dissolved in 5 mL of DMF. Sodium
azide (218 mg) was added, followed by triphenylphosphine (129 mg)
and carbon tetrabromide (189 mg) and the reaction was stirred for 1
hour at which time 10 mL of 1 M Na.sub.2CO.sub.3 was added followed
by 30 mL of ethyl acetate. The organic layer was washed twice with
15 mL of satt'd NaHCO.sub.3 solution (each), the organic layer was
dried with MgSO.sub.4, filtered, and concentrated to a clear oil.
This material was then purified by flash chromatography suing ethyl
acetate as eluent. TLC (ethyl acetate, uv): R.sub.f=0.30. .sup.1H
NMR (400 MHz, CDCl.sub.3): 9.04 (d, 1H), 8.19 (dd, 1H), 7.72 (br s,
1H), 7.43 (d, 1H), 5.17 (t, 1H), 4.55 (s, 2H), 3.58 (q, 2H), 3.43
(q, 2H), 1.43 (s, 9H).
[0385] FIG. 1B shows an exemplary method of preparing a compound of
any one of Formulas (I) to (XIII) from
tert-butyl(2-(6-(azidomethyl)nicotinamido)ethyl)carbamate (6). The
exemplary method is as follows.
Preparation of a representative azido-dye conjugated compound,
6-(azidomethyl)-N-(2-(6,8-difluoro-7-hydroxy-2-oxo-2H-chromene-3-carboxam-
ido)ethyl)nicotinamide (8)
[0386] Compound 6 (5.2 mg) was dissolved in 1 mL of DCM, cooled to
5.degree. C., then 1 mL of TFA was added and the mixture was
stirred for 15 minutes, then allowed to warm gradually to ambient
temperature. The mixture was then concentrated to an orange oil and
dried under vacuum for 2 hours at which time the mixture was taken
up in 0.8 mL of DMF. 0.1 mL of N,N-diisopropylethylamine was added,
followed by 4.9 mg of Pacific Blue SE (7) at ambient temperature.
The mixture was stirred for 1 hour, then the reaction mixture was
loaded directly onto a silica gel column and chromatographed with
dichloromethane and methanol to provide a pale yellow oil. This
material was then purified by preparative HPLC using a gradient of
10 mM ammonium acetate and methanol to obtain the product of
>99% purity by HPLC analysis. .sup.1H NMR (400 MHz,
d.sub.3-MeOD): 9.21 (t, 1H), 8.98 (d, 1H), 8.68 (d, 1H), 8.26 (d,
1H), 7.57 (d, 1H), 7.26 (dd, 1H), 4.58 (s, 2H), 3.67-3.64 (m,
4H).
Preparation of QSYp Azide
[0387] QSY picolyl azide ("QSYp Azide") having the structure:
##STR00029##
was prepared substantially as described above for compound (8),
except QSY-SE (Life Technologies catalog # Q-10193):
##STR00030##
was used in place of Pacific Blue SE (7). Other labeled azide
compounds, such as AF488-pAzide, AF647-pAzide, and
biotin-PEG-pAzide, where PEG is one or more repeating units of
polyethylene glycol. These compounds can be made using a
substantially similar method as with compound 8. Selected examples
of compounds synthesized in this manner include the following
structures:
##STR00031##
N-Ethyl biotin picolyl azide: MS (ESI): MH+=475.3, 473.3 (negative
mode).
##STR00032##
AF 488-picolyl azide: LCMS (ESI): Xterra C8, 50 mm.times.2.1 mm at
0.2 mL/min 40-100% ACN/10 mM NH.sub.4OAc, pH=7 over 20 minutes.
T.sub.r=2.1 mM, MH+=737.05.
TAMRA Picolyl Azide:
##STR00033##
[0388] AF 647-picolyl azide: LCMS (ESI): Xterra C8, 50 mm.times.2.1
mm at 0.2 mL/min 40-100% ACN/10 mM NH.sub.4OAc, pH=7 over 20
minutes. T.sub.r=2.1 min, MH+=737.05.
##STR00034##
AF 594-picolyl azide: LCMS (ESI): Xterra C8, 50 mm.times.2.1 mm at
0.2 mL/min 0-60% ACN/10 mM NH.sub.4OAc, pH=7 over 20 minutes.
T.sub.r=2.1 min, MH+=925.3.
##STR00035##
AF 555-picolyl alkyne (this compound was prepared in analogy to
compound 15. AF 488-picolyl azide: LCMS (ESI): Xterra C8, 50
mm.times.2.1 mm at 0.2 mL/min 40-100% ACN/10 mM NH.sub.4OAc, pH=7
over 20 minutes. T.sub.r=6.17 min, MH+=1048.39.
[0389] FIG. 2 shows an exemplary method of preparing a compound of
any one of Formulas (I) to (XIII). The exemplary method is as
follows.
Preparation of 6-((prop-2-yn-1-yloxy)methyl)nicotinic acid (10)
[0390] Methyl 6-(hydroxymethyl)nicotinate (9, 303 mg) was dissolved
in THF. 0.28 mL of a 9 M solution of propargyl bromide in toluene
was added to the reaction vessel at ambient temperature. After 12
hours, the reaction mixture was diluted with 50 mL of Et.sub.2O and
washed once with 30 mL of a saturated solution of sodium
bicarbonate. The organic layer was dried with MgSO.sub.4, filtered,
and concentrated to a residue, which was chromatographed on a
silica gel column with ethyl acetate and hexanes to obtain an oil.
TLC (ethyl acetate, uv): R.sub.f=0.64. .sup.1H NMR (400 MHz,
CDCl.sub.3): 9.16 (d, 1H), 8.32 (dd, 1H), 7.57 (d, 1H), 4.80 (s,
2H), 4.33 (dd, 2H), 3.96 (s, 3H), 2.50 (t, 1H).
Preparation of 6-((prop-2-yn-1-yloxy)methyl)nicotinic acid (11)
[0391] Compound 10 (18 mg) was dissolved in 0.5 mL of methanol.
LiOH (0.131 mL of a 2 M aqueous solution) was added to the mixture
at ambient temperature and the reaction was stirred for 30 minutes
at which time the reaction mixture was directly applied to a silica
gel column and chromatographed with dichloromethane (DCM) and
methanol to provide 11 as a light yellow oil. TLC (ethyl acetate,
uv): R.sub.f=0.10. .sup.1H NMR (400 MHz, CDCl.sub.3): 9.21 (s, 1H),
8.36 (d, 2H), 7.48 (s, 1H), 4.80 (s, 2H), 4.31 (s, 2H), 2.49 (s,
1H).
Preparation of
tert-butyl(2-(6-((prop-2-yn-1-yloxy)methyl)nicotinamido)ethyl)carbamate
(12)
[0392] Compound 11 (11 mg) was dissolved in 2 mL of DCM. 0.12 mL of
triethylamine was added at ambient temperature followed by 27 .mu.L
ethylchloroformate and the mixture was stirred for 2 hours, at
which time the mixture was cooled to -15.degree. C. and 57 mg of
tert-butyl(2-aminoethyl)carbamate hydrochloride was added and the
mixture was stirred for 12 hours. The mixture was then applied
directly to silica gel column and chromatographed with hexanes and
ethyl acetate to furnish 12 as a white powder. TLC (ethyl acetate,
uv): R.sub.f=0.43. .sup.1H NMR (400 MHz, CDCl.sub.3): 9.02 (s, 1H),
8.17 (dd, 1H), 7.60 (br s, 1H), 7.54 (d, 1H), 5.07 (br s, 1H), 4.79
(s, 2H), 4.32 (d, 2H), 3.58 (q, 2H), 3.44 (m, 2H), 2.50 (t, 1H),
2.06 (s, 1H), 1.44 (s, 9H).
Preparation of Compound 15
[0393] Compound 12 (3.5 mg) was dissolved in 0.3 mL of DCM and 0.5
mL of trifluoroacetic acid was added at 0-5.degree. C. After 1.4
hours, the solvent was removed and the sample placed under vacuum
for 3 hours. Compound 14 (4.9 mg) was then added, followed by 0.60
ml of DCM, and finally 0.10 mL of N,N-diisopropylethylamine. After
2 hours, the reaction mixture was concentrated directly under
vacuum and 0.30 mL of methanol and 0.20 mL of a solution of 8%
triethylamine in water was added. The reaction was stirred for 1
hour. 0.20 mL of a solution of 8% triethylamine in water was then
added and the reaction was stirred for 30 minutes, then
concentrated to a residue. This material was purified by HPLC using
a gradient of 10 mM NH.sub.4OAc/MeOH. The fractions containing
product were collected and concentrated, taken up in water and
lyophilized to provide 15 as a red powder. Analytical HPLC: Luna
C18 250 mm.times.4.6 mm, 5 .mu.m column at 1.0 mL/min with 10 mM
NH.sub.4OAc/Methanol gradient of 5-95% over 30 minutes.
T.sub.R=20.8 minutes, >99% pure at 254 nm and 490 nm .sup.1H NMR
(400 MHz, D.sub.2O): 8.46 (s, 1H), 7.83 (dd, 2H), 7.74 (dd, 1H),
7.26 (s, 1H), 7.08 (d, 1H), 6.60 (dd, 4H), 4.42 (s, 2H), 4.12 (d,
2H), 3.54 (m, 4H), 2.80 (m, 1H), [amide NH's exchanged with
D.sub.2O].
Example 2
[0394] In order to compare the click reaction rates using a picolyl
azide reactant, to a similar reactant that lacks a pyridyl group,
Cu(I)-catalyzed azide-alkyne cycloaddition reactions were carried
out between Oregon Green.RTM. 488 ("OG") alkyne (Life Technologies,
Carlsbad, Calif.) and either QSY.RTM. Azide having the
structure:
##STR00036##
or QSY.RTM. picolyl azide ("QSYp Azide;" see Example 1) having the
structure:
##STR00037##
[0395] Each reaction contained 10 .mu.M OG alkyne, 40 .mu.M of QSY
Azide or QSYp Azide, 5 mM sodium ascorbate, and various
concentrations of CuSO.sub.4 were added in a buffer containing 100
mM Tris, pH 8, and 25% 1,2 propane diol. QSY acts as a quencher of
OG. Thus, the click reaction brings the dyes in close proximity,
resulting in quenching of the OG fluorescent signal.
[0396] As shown in FIG. 3, click reaction rates using picolyl
reagent QSY pAzide (A) were faster than click reaction rates using
reagent QSY Azide (B) at all Cu concentrations tested. Thus, the
presence of the picolyl moiety increased the rate of the click
reaction in that experiment.
[0397] Next, the effect of a Cu(I)-stabilizing copper chelators on
the click reactions between OG alkyne and QSY pAzide or QSY Azide
was determined. In this experiment, reactions were carried out for
30 minutes in the presence of sodium ascorbate and 2 mM, 1 mM, 0.5
mM, or 0.25 mM Cu.sup.2+; or in the presence of sodium ascorbate
and 0.25 mM, 0.125 mM, 0.0625 mM, or 0.03125 mM Cu.sup.2+ and THPTA
having the structure:
##STR00038##
at a molar ratio of THPTA:Cu of 0:1 to 8:1.
[0398] The results are shown in FIG. 4. As shown in that figure, at
higher concentrations of Cu.sup.2+ and in the absence of THPTA,
very little click reaction takes place between OG alkyne and QSY
Azide in 30 minutes. In contrast, greater than 50% of the OG alkyne
is quenched in 30 minutes when QSY pAzide is used as a reactant, in
the presence of at least 0.5 mM Cu.sup.2+. The presence of THPTA
markedly increases the extent of the click reaction in 30 minutes
for both QSY pAzide and QSY Azide (compare, for example, 0.25 mM
Cu.sup.2+ in the presence and absence of THPTA). Thus, THPTA not
only reduces the requirement for Cu.sup.2+ ions, which can be
detrimental to biological systems, it greatly increases the
reaction rate for both picolyl and non-picolyl click reactants.
Example 3
[0399] In order to determine whether picolyl azides showed similar
enhancement in click reaction rates, various alkynes were used in
the following click reaction:
##STR00039##
[0400] The time to completion of the reaction was determined by
taking aliquots of the reaction mixture at time points and applying
sample to thin-layer chromatography (TLC) plates. The TLC plates
were then developed using mixtures of organic solvents. Reaction
progress was estimated by the intensity of spots corresponding to
starting material and product. Reaction times are estimated.
[0401] The results of that experiment are shown below
TABLE-US-00002 R ##STR00040## ##STR00041## ##STR00042##
##STR00043## Time to >2 h 10-16 min 10-16 min No reaction
completion
[0402] In that experiment, use of the picolyl alkyne reactants
resulted in much shorter reaction times than the non-picolyl
alkynes under the same reaction conditions.
Example 4
[0403] A fluorogenic click assay for determining the relative rate
of click reactions was used to test various azide compounds in a
procedure adapted from Zhou and Fahmi, J. Am. Chem. Soc., 2004,
126, 8862-8863:
##STR00044##
[0404] General reactions conditions: 20 .mu.M azide, 40 .mu.M
7-ethynyl coumarin 16, and 4 mM sodium ascorbate in 100 .mu.M Tris
buffer at pH 7.4 with 25% v/v 1,2-propanediol at 25.+-.1.degree. C.
Reactions were initiated by the addition of CuSO4: 125 .mu.M.
Coumarin fluorescence was recorded on a Molecular Devices
SpectraMax M5 microplate reader with excitation at 320 nm and
emission detection at 430 nm with a cutoff at 420 nm. The turn-on
fluorescence of coumarin triazole was correlated to its
concentration using a calibration curve made from known
concentrations (1.25-40 .mu.M) of the triazole adduct between
2-picolyl azide and 7-ethynylcoumarin 16. Fluorescence measurements
were taken for at least 30 min at 30 sec intervals. Measurements
for each azide were performed in triplicates or more. Error was
determined from the standard deviation from data sets at each time
point. Background fluorescence was subtracted from all data for
normalization.
[0405] Coumarin-alkyne 16 was prepared and characterized as
previously described in Thou and Fahrni, J. Am. Chem. Soc., 2004,
126, 8862-8863.
[0406] The various organic azide compounds used were synthesized as
described below.
[0407] Benzyl azide (as shown below) is commercially available:
##STR00045##
Synthesis of 2-azidomethylpyrdine (2-picolyl azide)
##STR00046##
[0409] This compound was prepared and characterized according to a
published literature procedure of Brotherton, W. S.; Michaels, H.
A.; Simmons, J. T.; Clark, R. J.; Dalai, N. S.; Zhu, L. Organic
Letters 2009, 11, 4954 4957.
Synthesis of 4-azidomethylbenzoic acid
##STR00047##
[0411] This compound was prepared and characterized as described in
Zhou and Fahrni, J. Am. Chem. Soc., 2004, 126, 8862-8863.
Synthesis of 4-azidomethylnicotinic acid
##STR00048##
[0413] Methyl 5-(azidomethyl)nicotinate (114 mg, 0.59 mmol) was
dissolved in methanol (2.5 mL). A 1.0 M solution of LiOH in water
(1.78 mL, 1.78 mmol) was then added and the mixture was stirred for
25 minutes, at which time acetic acid (60 .mu.L) was added and the
mixture was loaded directly onto a silica gel column equilibrated
with ethyl acetate+1% acetic acid and chromatographed with ethyl
acetate+1% acetic acid to 4% acetonitrile/ethyl acetate+1% acetic
acid to provide 101 mg (96%) of this compound as a yellow solid.
Rf=0.35 (ethyl acetate+1% acetic acid, 254 nm UV). .sup.1H NMR (400
MHz, 1:1 CDCl.sub.3:CD.sub.3OD): 9.75 (dd, J=2.0, 0.4 Hz, 1H), 8.98
(dd, J=8.0. 2.0 Hz, 1H), 8.12 (d, J=8.0 Hz, 1H), 5.35 (s, 1H), 5.18
(s, 2H). MS (ESI-): 177 (M.sup.+, 100%; 133.0 (60%).
Synthesis of methyl-5-(azidomethyl)nicotinate
##STR00049##
[0415] This compound was prepared and characterized as described in
Khilevich, A., Liu, B., Mayhugh, D. R., Schekeryantz, J. M., and
Zhang, D. Imidazolecarboxamide derivatives as mGluR2 receptor
potentiators and their preparation, pharmaceutical compositions and
use in the treatment of depression. WO2010/009062. Jan. 21,
2010.
Synthesis of 2-azidomethyl-4-methoxypyridine
##STR00050##
[0417] 2-Hydroxymethyl-4-methoxypyridine (278 mg, 2.0 mmol) was
dissolved in tetrahydrofuran (15 mL) in a 50 mL round-bottomed
flask under argon. The flask was cooled to 0-5.degree. C. with an
ice/water bath for 10 minutes at which time, powdered KOH (157 mg,
2.8 mmol) was added followed by para-toluenesulfonyl chloride
(pTsCl). The reaction was stirred for 12 hours, at which time
diethyl ether (30 mL) was added. The mixture was transferred to a
separatory funnel, and a saturated solution of NaHCO.sub.3 (40 mL)
was added. The organic layer was dried with MgSO.sub.4, filtered,
and concentrated to a residue, which was chromatographed on a
silica gel column with a 10% to 50% gradient of ethyl
acetate/hexanes. Rf=0.69 (ethyl acetate, 254 nm UV). This material
was then dissolved in N,N-dimethylformamide (5 mL), and sodium
azide (266 mg, 4.09 mmol) was added and the reaction was stirred at
ambient temperature for 16 hours, at which time the reaction
mixture was diluted with diethyl ether (30 mL) and washed with a
saturated solution of NaHCO.sub.3 (3.times.30 mL), then with brine
(25 mL), dried with MgSO.sub.4, filtered and concentrated in vacuo.
The resulting residue was chromatographed over silica gel with a
15% to 50% gradient of ethyl acetate/hexanes to furnish 100 mg (30%
yield) of this compound as a light yellow oil. Rf=0.68 (ethyl
acetate, 254 nm UV). .sup.1H NMR (400 MHz, CDCl.sub.3): 8.34 (d,
J=5.6 Hz, 1H), 6.81 (d, J=2.4 Hz 1H), 4.39 (s, 2H), 3.81 (s,
3H).
Synthesis of 2-azidomethyl-4-chloropyridine
##STR00051##
[0419] 2-azidomethyl-4-chloropyridine was prepared and
characterized as described in Jung, F. H., Cephalosporin
derivatives. EP Patent No. 0127992. Dec. 12, 1984.
Synthesis of tert-Butyl(6-azidomethylpyridin-3-yl)carbamate
##STR00052##
[0421] tert-Butyl(6-(hydroxymethyl)pyridin-3-yl)carbamate (24 mg,
0.107 mmol) was dissolved in DMF (2 mL) and sodium azide (35 mg,
0.54 mmol) was added followed by the simultaneous addition of
carbon tetrabromide (179 mg, 0.54 mmol) and triphenylphosphine (142
mg, 0.54 mol). The mixture was stirred for 2 hours at ambient
temperature, then diluted in ethyl acetate (20 mL) and saturated
NaHCO.sub.3 solution (20 mL). The layers were separated, and the
organic layer was dried with MgSO.sub.4, filtered, and concentrated
to a yellow oil, which was chromatographed (15-90% ethyl
acetate/hexanes on silica gel) to afford this compound as a film
16.8 mg (63%). Rf=0.92 (ethyl acetate, 254 nm UV). MS (ESI+): 250.0
(M+H+, 100%; 194.0, 25%).
Synthesis of 5-carbomethoxy nicotinic acid
##STR00053##
[0423] This compound was prepared and characterized as described in
Luk, K-C., So, S-S., Zhang, J., and Zhang, Z. Preparation of
oxindoles as inhibitors of MDM2-p53 interaction for the treatment
of cancer. WO2006136606. Dec. 28, 2006.
Synthesis of 5-(2,5-dioxopyrrolidin-1-yl) 2-methyl
pyridine-2,5-dicarboxylate
##STR00054##
[0425] 5-carbomethoxy nicotinic acid (100 mg, 0.55 mmol) was
dissolved in dichloromethane (10 mL) at ambient temperature.
N-Hydroxysuccinimide (NHS; 95 mg, 0.83 mmol) was added, followed by
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) hydrochloride (133
mg, 0.69 mol) and the mixture stirred at ambient temperature for 2
hours. The mixture was then diluted with chloroform (15 mL),
followed by the addition of 2% HCl solution (15 mL). The organic
layer was washed with water (15 mL), dried with MgSO.sub.4, and
filtered. This compound was used directly in the next step without
further purification. Rf=0.53 (ethyl acetate, 254 nm UV).
Synthesis of methyl 5-(2-(tert-butoxycarbonylamino)ethyl)carbamoyl
picolinate
##STR00055##
[0427] 5-(2,5-dioxopyrrolidin-1-yl) 2-methyl
pyridine-2,5-dicarboxylate (155 mg, 0.55 mmol) was dissolved in
dichloromethane (15 mL). N,N-diisopropylethylamine (0.30 mL, 1.65
mmol) was added at ambient temperature followed by tert-butyl (2
aminoethyl)carbamate hydrochloride (119 mg, 0.61 mmol). The
reaction was stirred for 50 minutes, then diluted with
dichloromethane (30 mL) and 2 M Na.sub.2CO.sub.3 solution (20 mL).
The layers were separated and the organic layer was dried with
MgSO.sub.4, filtered and concentrated in vacuo. The residue was
dissolved in warm methanol (3 mL), loaded onto a silica gel
chromatography column and eluted with ethyl acetate then 95:5 ethyl
acetate:methanol to provide 104 mg (58%) of this compound as a
white solid. Rf=0.32 (ethyl acetate, 254 nm UV). .sup.1H NMR (400
MHz, CDCl.sub.3): 9.15 (d, J=2 Hz, 1H), 8.30 (d, J=1.6 Hz, 1H),
8.14 (complex, 2H), 5.32 (br s, 1H), 4.00 (s, 3H), 3.56 (m, 2H),
3.41 (m, 2H), 1.39 (s, 9H).
Synthesis
tert-butyl(2-(6-(hydroxymethyl)nicotinamido)ethyl)carbamate
##STR00056##
[0429] NaBH.sub.4 (55 mg, 1.46 mmol) was added slowly to methyl
5-(2-(tert-butoxycarbonylamino)ethyl) carbamoyl picolinate (157 mg,
0.49 mmol) dissolved in methanol (3 mL). THF (10 mL) was added and
the reaction flask was placed in an oil bath preheated to
65.degree. C. The reaction was stirred at 65.degree. C. for 15
minutes, at which time 2 M Na.sub.2CO.sub.3 solution (6 mL) was
added over 10 minutes followed by water (6 mL). The mixture was
then concentrated to 1/5 of its original volume and chromatographed
directly with 0.5% triethylamine and ethyl acetate/methanol to
provide this compound (132 mg, 91%) as a white solid. Rf=0.10
(ethyl acetate, 254 nm UV). .sup.1H NMR (400 MHz, d6-DMSO): 8.89
(s, 1H), 8.61 (s, 1H), 8.18 (d, J=8.0 Hz, 1H), 7.55 (d, J=8.0 Hz,
1H), 6.93 (m, 1H), 5.53 (br s, 1H), 4.61 (s, 2H), 3.29 (m, 2H),
3.12 (d, J=6.0 Hz, 2H), 2.51 (s, 2H), 1.7 (s, 9H).
Synthesis of
tert-butyl(2-(6-(azidomethyl)nicotinamido)ethyl)carbamate
##STR00057##
[0431] tert-butyl(2-(6-(hydroxymethyl)nicotinamido)ethyl)carbamate
(132 mg, 0.45 mmol) was dissolved in DMF (5 mL). Sodium azide (218
mg, 3.36 mmol) was added, followed by triphenylphosphine (129 mg,
0.492 mmol) and carbon tetrabromide (189 mg, 185 mg, 0.56 mmol).
The reaction was stirred for 1 hour at ambient temperature, at
which time 2 M Na.sub.2CO.sub.3 solution (10 mL) was added followed
by ethyl acetate (30 mL). The organic layer was washed with
saturated NaHCO.sub.3 solution (2.times.15 mL), dried with
MgSO.sub.4, filtered, and concentrated in vacuo. The resulting
clear oil was purified by silica gel chromatography using ethyl
acetate as eluent to furnish this compound as a white solid.
Rf=0.30 (ethyl acetate, 254 nm UV). .sup.1H NMR (400 MHz,
CDCl.sub.3): 9.04 (d, J=1.6 Hz, 1H), 8.19 (dd, J=8.1, 2.3 Hz, 1H),
7.72 (br s, 1H), 7.43 (d, J=8.1 Hz, 1H), 5.17 (t, J=8.1 Hz, 5.8 Hz,
1H), 4.55 (s, 2H), 3.58 (q, J=4.9 Hz, 2H), 3.43 (q, J=5.9 Hz, 2H),
1.43 (s, 9H). LCMS (ESI+): 296.50 (MH+, 100%), 240.38 (86%); TR=8.8
min.
Synthesis of tert-butyl 2-(2-(hydroxymethyl)pyridin-4
ylamino)ethylcarbamate
##STR00058##
[0433] To a 40 mL pressure tube equipped with a stir bar was added
4-chloro-2-pyridylmethanol (0.25 g, 1.74 mmol),
N,N-diisopropylethylamine (1.52 mL, 8.71 mmol), and toluene (0.925
mL; 8.71 mmol) was added tert-butyl(2-aminoethyl)carbamate (0.55 g,
3.50 mmol). The vessel was purged with argon and then placed in an
oil bath pre-heated to 125.degree. C. The temperature of the oil
bath was increased to 140.degree. C. over 30 minutes. After 2
hours, TLC (conditions below) indicated near complete consumption
of the starting material. The reaction was cooled to ambient
temperature and the toluene layer was decanted. Methanol (15 mL)
was added and the mixture was heated to ca. 40.degree. C. for 5
minutes to break up the solid residual. After concentrating the
mixture down to 1/5 of its original volume, CHCl.sub.3 (5 mL) was
added and the residue was loaded onto a 5 cm.times.15 cm silica gel
column equilibrated with dichloromethane (DCM)+0.5% triethylamine.
Flash chromatography with DCM to DCM/10% Methanol with 0.5%
triethylamine provided an inseparable mixture of the compound and
starting tert-butyl(2-aminoethyl)carbamate (1:1.4 molar ratio).
Rf=0.38 (15% MeOH/DCM+0.1% TEA, 254 nm UV). This mixture was used
in the next step without further purification. .sup.1H NMR (400
MHz, CD.sub.3OD): (9) 7.93 (d, J=5.6 Hz, 1H), 6.75 (d, J=2.4 z,
1H), 6.48 (dd, J=6.0, 2.4 Hz, 1H), 4.54 (s, 2H), 3.66 (s, 1H), 3.26
(m, 4H), 1.46 (s, 9H); (tert-butyl 2-aminoethylcarbamate) 3.33 (m,
2H), 3.12 (t, J=6.4 Hz, 2H), 2.71 (t, J=6.4 Hz, 2H), 1.44 (s,
9H).
Synthesis of tert-butyl
2-(2-(azidomethyl)pyridin-4-ylamino)ethylcarbamate
##STR00059##
[0435] The mixture of tert-butyl
2-(2-(hydroxymethyl)pyridin-4-ylamino)ethylcarbamate and
tert-butyl(2-aminoethyl)carbamate was dissolved in DMF (3.5 mL) in
a 20 mL screw top vial. Sodium azide (91 mg, 1.40 mmol) was added
followed by the simultaneous addition of triphenylphosphine (370
mg, 1.40 mmol) and carbon tetrabromide (470 mg, 1.40 mmol) at
ambient temperature. After 1 hour, additional sodium azide 91 mg
(1.40 mmol) was added followed by the simultaneous addition of
triphenylphosphine (370 mg, 1.40 mmol) and carbon tetrabromide (470
mg, 1.40 mmol). The reaction was stirred for another hour and
concentrated under a stream of nitrogen to remove the solvent. The
resulting residue was chromatographed with 1% to 10%
MeOH/CHCl.sub.3 to provide this compound as a white solid (75 mg,
27% over 2 steps). Rf=0.22 (5% MeOH/DCM+0.1% TEA, 254 nm UV).
.sup.1H NMR (400 MHz, CD.sub.3OD): 8.00 (d, J=6.4 Hz, 1H), 6.71 (d,
J=1.6 Hz, 1H), 6.63 (dd, J=6.0, 2.0 Hz, 1H), 4.38 (s, 2H), 3.33 (m,
2H; overlaps with residual MeOH), 3.25 (m, 2H), 1.96 (s, 2H), 1.44
(s, 9H). MS (ESI+): 293.3 (M+H+).
Synthesis of 2-azidoethylpyridine
##STR00060##
[0437] 2-azidoethylpyridine was prepared and characterized as
described by Brotherton, W. S.; Michaels, H. A.; Simmons, J. T.;
Clark, R. J.; Dalal, N. S.; Zhu, L. Organic Letters 2009, 11, 4954
4957.
Synthesis of 3-azido-1-(pyridin-2-yl)propan-1-one
##STR00061##
[0439] 2-picolinic acid (74 mg, 0.60 mmol) was dissolved in
anhydrous dichloromethane (4 mL) under Argon. Triethylamine (0.84
mL, 6.0 mmol) was then added followed by ethyl chloroformate (86
.mu.L, 0.90 mmol). The reaction mixture was stirred for 30 minutes
at ambient temperature, at which time 2-azidoethylamine
hydrochloride (11 mg, 0.90 mmol) was added. After further stirring
at ambient temperature for 12 hours, the solvent was removed in
vacuo and the resulting residue was taken up in ethyl acetate,
loaded onto a preparatory TLC plate, and developed using 4:1
hexanes:ethyl acetate solvent system. The product-containing band
was collected and rinsed with 10:1 chloroform:methanol, filtered,
and concentrated to a yellow oil (25 mg, 22%). Rf=0.64 (ethyl
acetate, u.v.). .sup.1H NMR (400 MHz, CDCl.sub.3): 8.76 (d, J=4 Hz,
1H), 8.14 (d, J=8 Hz, 1H), 7.83 (dd, J=7.6, 6.4 Hz, 1H), 7.47 (m,
1H), 4.49 (dd, J=7.2, 6.8 Hz, 2H), 3.95 (s, 1H), 1.45 (t, J=7.2 Hz,
3H).
Synthesis of 2-Azidomethylquinoline
##STR00062##
[0441] 2-Azidomethylquinoline was prepared and characterized as
described by Brotherton, W. S.; Michaels, H. A.; Simmons, J. T.;
Clark, R. J.; Dalal, N. S.; Zhu, L. Organic Letters 2009, 11, 4954
4957.
Synthesis of 6-bromomethyl-2,2'-bipyridine
##STR00063##
[0443] 6-bromomethyl-2,2'-bipyridine was prepared and characterized
as described by Murashima, T.; Tsukiyama, S.; Fujii, S.; Hayata,
K.; Sakai, H.; Miyazawa, T.; Yamada, T. Organic & Biomolecular
Chemistry 2005, 3, 4060-4064.
Synthesis of 6-Azidomethyl-2,2'-bipyridine
##STR00064##
[0445] To the solution of 6-bromomethyl-2,2'-bipyridine (75 mg,
0.30 mmol) in DMF (2 mL) was added sodium azide (59 mg, 0.90 mmol).
The reaction mixture was stirred for 2 hours, then diluted with
diethyl ether (20 mL) and saturated NaHCO.sub.3 solution (15 mL).
The layers were separated and the organic layer was further washed
with saturated NaHCO.sub.3 solution (2.times.15 mL), dried with
MgSO.sub.4, filtered, and concentrated to an oil. LC-MS (ESI+):
221.39 (MH+, 44%); TR=17.4 min
Synthesis of 2-azidomethylbenzimidazole
##STR00065##
[0447] 2-azidomethylbenzimidazole was prepared and characterized as
described by Hideg, K.; Hankovszky, H. O. Synthesis-Stuttgart 1978,
313-315.
Synthesis of 5-(6-(Azidomethyl)nicotinamido)pentanoic acid
##STR00066##
[0449] To a solution of 6-azidomethylnicotinic acid (30 mg, 0.168
mmol) in anhydrous DMF (500 .mu.L) was added disuccinimidyl
carbonate (DSC; 65 mg, 0.253 mmol) and triethylamine (TEA; 120
.mu.L, 0.840 mmol). The reaction was allowed to proceed for 3 hours
at ambient temperature. The reaction mixture was diluted with
chloroform and water. Layers were separated, and the aqueous layer
was extracted with chloroform three times. The combined organic
layer was washed with brine, dried over MgSO.sub.4, and
concentrated in vacuo. The residual mixture was purified by silica
chromatography (1:1 hexanes:ethyl acetate) to afford the
succinimidyl ester of 6-azidomethylnicotinic acid. Rf=0.67 in 9:1
chloroform:methanol.
[0450] To a solution of 5-azidomethylnicotinic acid succinimidyl
ester (15 mg, 0.055 mmol) in anhydrous DMF (500 .mu.L) was added
5-aminovaleric acid (32 mg, 0.273 mmol) and TEA (38 .mu.L, 0.273
mmol). The reaction proceeded for 12 hours at ambient temperature.
TEA and DMF were then removed in vacuo, and the resulting residue
was dry-loaded in 9:1 chloroform:methanol onto a silica column, and
purified using 9:1 chloroform:methanol as eluent. Rf=0.19 in 9:1
chloroform:methanol. .sup.1H NMR (D.sub.2O, 500 MHz): 8.83 (s, 1H),
8.18 (d, 1H, J=8.5 Hz), 7.59 (d, 1H, J=8 Hz), 4.62 (s, 2H), 3.42
(m, 2H), 2.32 (m, 2H), 1.65 (m, 4H).
Synthesis of ALEXA FLUOR-647 (AF-647)-picolyl azide conjugate
##STR00067##
[0452] To a solution of
8-(2-aminoethyl)-6-(azidomethyl)nicotinamide (5.5 mg, 0.019 mmol)
in DMF (0.95 mL) was added DIPEA (100 .mu.L) and ALEXA FLUOR 647
succinimidyl ester (ALEXA FLUOR 647-SE; 20 mg, 0.016 mmol). After
stirring at ambient temperature for 10 hours, the reaction mixture
was concentrated and directly purified by preparative HPLC using a
30 minute gradient of 5-95% 10 mM NH4OAc/MeOH at a 1 mL/min flow
rate. Fractions containing the product were combined and
concentrated in vacuo. The residual was then dissolved in water (10
mL), flash-frozen, then lyophilized to yield 13.6 mg of ALEXA
FLUOR-647-picolyl azide as a bright blue powder (83%). Tr=20.8 min
at 647 nm MS (ESI+): 1061.3 (M+H.sup.+; 2%), 531.2, 6%); (ESI-):
1060.3 (Zwitterion, 17%), 540.3 (52%), 529.3 (M.sup.2-. 100%).
HPLC: >99% purity at 254 nm and 644 nm.
Synthetic Preparation of Fluorogenic Assay Reagents
[0453] To prepare the triazole adduct between 16 and, for example,
2-azidomethylpyridine, 16 (20 mg, 0.073 mmol) was dissolved in DMSO
(4 mL). 2-azidomethylpyridine (20 mg, 0.15 mmol) was added followed
by a 0.50 M solution of sodium ascorbate in water (59 .mu.L, 0.029
mmol), and a 0.25 M copper(II) sulfate solution in water (30 .mu.L,
0.007 mmol). The reaction was stirred for 1 hour and the solvent
removed in vacuo. The crude reaction mixture was taken up in
methanol and loaded directly onto a preparative TLC plate (0.25 mm
thickness) and the plate was developed with 95:5 acetonitrile:H2O.
The product-containing silica was collected and sonicated in
chloroform (30 mL) for 3 minutes and filtered. The filtrate was
concentrated to deliver 8.9 mg of II (30% yield) as a tan solid.
Rf=0.80 (97:3 acetonitrile:H2O). .sup.1H NMR (400 MHz, CDCl3):
12.27 (br s, 1H), 8.58 (dd, J=4.8, 4.0 Hz, 1H), 7.92-7.82 (m, 3H),
7.44-7.35 (m, 3H), 5.81 (s, 1H), 5.44 (s, 1H), 4.51 (s, 2H), 3.37
(s, 4H), 2.71 (J=7.6, 6.0 Hz, 1H), 2.57 (t, J=7.6, 6.0 Hz, 1H).
Excitation maxima=325 nm, emission maxima=415 nm (100 mM Tris
buffer with 25% 1,2-propanediol). LC-MS (ESI+): 435.48 (MH+, 100%),
407.49 (65%); TR=8.4 min. FIG. 5B shows the results of certain
click reactions in the presence of 31 .mu.M Cu and THPTA at a ratio
of 4:1 (THPTA:Cu).
[0454] Various azides (R--N.sub.3), including those shown
below:
##STR00068##
were reacted with 16.
[0455] Stock solutions of 16 and the azide compounds were prepared
in 100 mM Tris buffer containing 25% 1,2-propanediol, unless
otherwise noted. Data were recorded on a SpectraMax M5 instrument
with excitation performed at 320 nm and emission detection at 430
nm, with a cutoff at 420 nm. The instrument was set to kinetic
mode. 96-well plates were used in the top-read mode for
fluorescence measurement. The total volume in each well was 200
.mu.L. CuSO.sub.4/water solutions were always added last to the
plate to initiate reaction and measurements were taken at least
every minute for 60 minutes. When THPTA was included in the
reaction, the THPTA:Cu ratio was 4:1. A fixed ratio of 16:1 sodium
ascorbate:Cu was also employed. Reagent concentrations in the stock
and final solutions were as follows:
TABLE-US-00003 Component Initial concentration Final concentration
16 100 .mu.M 40 .mu.M azides 100 .mu.M 20 .mu.M
[0456] The results of that experiment are shown in FIG. 5.
[0457] FIG. 5A shows the results of certain click reactions in the
presence of 125 .mu.M Cu. From FIG. 5A, it can be seen that
6-(azidomethyl)nicotinic acid undergoes click reactions much faster
than 4-(azidomethyl)benzoic acid. This is believed to be due to the
heteroatom of 6-(azidomethyl)nicotinic, which coordinates to Cu
ions and in turn accelerates the rate of reaction.
[0458] FIG. 5B shows the additive effect that ligands such as THPTA
have when added to the reaction of picolyl azides. Click reactions
are faster when a combination of especially groups and ligands are
used. Even when 4-azidomethylbenzoic acid is reacted with an alkyn
in the presence of THPTA, the Click reaction is much slower than
when a picolyl azide is used with our without a ligand such as
THPTA.
[0459] A summary of azide heterocyclic structures and their
copper(I) catalyzed azide-alkyne cycloaddition reaction conversions
after 5 min and 30 min reaction times is shown below.
Concentrations used were 20 .mu.M azide, 40 .mu.M 7-ethynyl
coumarin, 125 .mu.M CuSO4, and 4 mM sodium ascorbate. Reactions
were performed in 100 .mu.M Tris pH 7.4 with 25% v/v 1,2
propanediol at 25.degree. C.
TABLE-US-00004 Conversion (%) Conversion (%) Azide Structure after
5 min after 30 min ##STR00069## <1 <1 ##STR00070## 35 80
##STR00071## <1 1.4 ##STR00072## 19 49 ##STR00073## 9.4 22
##STR00074## 41 94 ##STR00075## 14 40 ##STR00076## 2.6 5.5
##STR00077## 14 37 ##STR00078## 1.8 2.7 ##STR00079## 12 24
##STR00080## <1 <1 ##STR00081## 7.8 20 ##STR00082## <1
<1 ##STR00083## <1 <1
[0460] Several trends are apparent.
[0461] First, 2-picolyl azide and 6-(azidomethyl)nicotinic acid are
much faster reactants than their carbocyclic analogues, benzyl
azide and 4-(azidomethyl)benzoic acid, giving >35-fold and
>19-fold improvements in initial CuAAC rates under these
conditions.
[0462] Second, among the picolyl azide structures, an azide with an
electron donating group gives a faster rate than one with electron
withdrawing groups, presumably because the former enhances the
chelating strength of pyridyl nitrogen. An exception is the picolyl
azide with an alkylamino substituent, which gives little conversion
after 30 min.
[0463] A third observation is that azido compounds with other
copper-coordinating motifs generally did not have as strong an
accelerative effect as 2-picolyl azide. 2-azidoethylpyridine, which
contains an additional methylene spacer between the pyridyl ring
and the azido group (to give a six-membered chelate ring), gave at
least 3-fold less product after 5 min than picolyl azide.
6-azidomethyl-2,2'-bipyridine and 2-(azidomethyl)benzimidazole each
did not give any product after 30 min, despite being strong copper
chelators.
Example 5
[0464] The stability of GFP in the presence of various
concentrations of Cu(II), Cu(II) plus sodium ascorbate (to produce
Cu(I)), and Cu(II) plus sodium ascorbate and THPTA, was determined
as follows.
[0465] GFP (5 nM) was incubated in 100 mM Tris buffer with 25%
1,2-propanediol at 25.degree. C. in the presence of 2 mM, 1 mM, 0.5
mM, 0.25 mM, 0.125 mM, or 0.0625 mM CuSO.sub.4. No ascorbate was
included in the incubation mixtures. GFP fluorescence was then
measured at various time intervals. As shown in FIG. 6A, at the
highest concentration of CuSO.sub.4, 2 mM, the GFP signal was 60%
of maximum after 30 minutes in that experiment. GFP was found to be
essentially stable at <0.5 mM Cu(II).
[0466] In the next experiment, 5 nM GFP was incubated in 100 mM
Tris buffer with 25% 1,2-propanediol at 25.degree. C. in the
presence of 2 mM, 1 mM, 0.5 mM, 0.25 mM, 0.125 mM, or 0.0625 mM
CuSO.sub.4. In each mixture, sodium ascorbate was included at a
concentration 10-fold greater than the Cu(II) concentration (i.e.,
for 1 mM Cu(II), 10 mM sodium ascorbate was included). GFP
fluorescence was then measured at various time intervals. As shown
in FIG. 6B, at the highest concentration of Cu(II) and sodium
ascorbate (2 mM Cu(II) plus 20 mM sodium ascorbate), the GFP signal
was 53% of maximum after 38 minutes in that experiment. GFP was
found to be essentially stable at <0.25 mM Cu(II) plus 2.5 mM
sodium ascorbate.
[0467] Finally, GFP stability was determined in the presence of
Cu(II), with and without sodium ascorbate, and with or without
THPTA. In this experiment, 5 nM GFP was incubated in 100 mM Tris
buffer with 25% 1,2-propanediol at 25.degree. C. in the presence of
(i) 2 mM Cu(II); (ii) 1 mM Cu(II), 10 mM sodium ascorbate; (iii) 1
mM Cu(II), 4 mM THPTA, 10 mM sodium ascorbate; or (iv) 0.125 mM
Cu(II), 0.5 mM THPTA, and 10 mM sodium ascorbate. In this
experiment, the mixtures were incubated for 30 minutes typically.
As shown in FIG. 7A, the GFP signal gradually declined under all of
the tested conditions in that experiment. The mixtures comprising
THPTA and lower concentrations of Cu(II) showed less degradation of
the GFP signal, compared to the mixtures with Cu(II) alone or
Cu(II) plus ascorbate.
[0468] To determine whether the GFP degradation would be
significant during the time frame for completing a click reaction,
click reactions between Oregon Green.RTM. ("OG") alkyne and
QSY-azide were carried under the same four conditions (plus a
copper-less control) described above. As shown in FIG. 7B, the
click reactions in the presence of THPTA and low copper, which
resulted in the least degradation of GFP signal in FIG. 7A, were
the fastest. At the lowest concentration of Cu(II) with THPTA
(0.125 mM Cu(II) plus 0.5 mM THPTA and 10 mM sodium ascorbate), the
click reaction was complete in about 20 minutes, at which time the
GFP signal was still about 95% of maximum (see FIG. 7A). These
results show that copper-catalyzed click reactions can be carried
out in biological systems without significant protein
degradation.
Example 6
General Procedure for EU Labeling of RNA in Cells
A375 Cells Stably Transfected with GFP Expressing Erk2 are Plated
Overnight at 5000 Cells/Well Cell Density
[0469] Human malignant melanoma (A375) cells expressing Erk2-GFP
(Life Technologies) were cultured in L-glutamine-containing
Dulbecco's modified Eagle Medium (Life Technologies) supplemented
with 10% v/v fetal bovine serum (Life Technologies), non-essential
amino acids (Life Technologies), and 5 ng/mL blasticidin. All cells
were maintained at 37.degree. C. under 5% CO.sub.2.
[0470] These cells are pulse with 200 nM 5-ethynyl uridine (EU) for
1 hour followed by fixation with 3.7% formalin in PBS for 15
minutes. The cells are then washed twice with 3% BSA in PBS
followed by permeabilization with 0.5% Triton in PBS for 20
minutes. The permeabilized cells were washed twice with 3% BSA in
PBS. Click reaction was carried out for 1 hr at RT followed by
washing the cells twice with 3% BSA in PBS and rinsed twice with
PBS. The click conditions were combinations of with or without
chelate and AF647-picolyl azide or AF647-azide. The clicked cells
were then stained with Hoechst stain (1 ug/mL in PBS) for 30 min at
RT followed by three times washing with PBS. The cells were imaged
on ArrayScan.
Example 7
General Procedure for HPG Labeling of Proteins in Cells
[0471] A375 cells stably transfected with GFP expressing Erk2 are
plated overnight at 5000 cells/well cell density.
[0472] These cells were pulse with 50 .mu.M L-homopropargylglycine
(HPG) for 1 h in presence or absence of 40 .mu.M cycloheximide
followed by fixation with 3.7% formalin in PBS for 15 minutes. The
cells are then washed twice with 3% BSA in PBS followed by
permeabilization with 0.5% Triton in PBS for 20 minutes. The
permeabilized cells were washed twice with 3% BSA in PBS. Click
reaction was carried out for 1 hr at RT using 2 mM CuSO.sub.4 and
10 mM Sodium Ascorbate, followed by washing the cells twice with 3%
BSA in PBS and rinsed twice with PBS. The click reactions were
carried out following combinations of chelate, Cu and AF647-picolyl
azide or AF647-azide.
a. 4:1 chelate/copper+picolyl azide b. 2:1 chelate/copper+picolyl
azide c. 1:1 chelate/copper+picolyl azide d. No chelate NO
picolyl=Classic click
[0473] The clicked cells were then stained with Hoechst stain (1
ug/mL in PBS) for 30 min at RT followed by three times washing with
PBS. The cells were imaged on ArrayScan.
Example 8
General Procedure for Labeling with Phalloidin AF546 after Click
HPG Labeling of Proteins in Cells
[0474] A375 cells stably transfected with GFP expressing Erk2 are
plated overnight at 5000 cells/well cell density. Prior to
incubation with HPG containing medium, cells were washed once with
DPBS with calcium and magnesium, then grown in methionine-free DMEM
(Life Technologies) for 30 min. These cells were pulsed with 50
.mu.M HPG for 1 hr in presence or absence of 40 .mu.M cycloheximide
followed by fixation with 3.7% formalin in PBS for 15 minutes. The
cells are then washed twice with 3% BSA in PBS followed by
permeabilization with 0.5% Triton in PBS for 20 minutes. The
permeabilized cells were washed twice with 3% BSA in PBS. Click
reaction was carried out for 1 hr at RT using 2 mM CuSO.sub.4 and
10 mM Sodium Ascorbate, followed by washing the cells twice with 3%
BSA in PBS and rinsed twice with PBS. The click reactions were
carried out using 4:1 chelate/copper and AF647-picolyl azide The
clicked cells were then stained with Phalloidin-AF546 (30 min at
rt) conjugate followed by Hoechst stain (1 ug/mL in PBS) for 30 min
at RT followed by three times washing with PBS. The cells were
imaged on ArrayScan.
[0475] FIGS. 13 through 17 further illustrate the generality of the
use of chelation-assisted copper (I)-catalyzed azide-alkyne
cycloaddition using picolyl azides to label metabolically alkynes
in proteins and found much higher detection sensitivity compared
without chelation assistance.
Example 9
[0476] Monoclonal antibody anti-TSH was modified at its GlcNAc
sites with UDPGalNAz using galactosyltransferase. The GalNAz groups
were then click reacted either with the standard click reaction
using BCS, with DIBO click reaction or with THPTA/Picolyl click
reaction. Cu 2 mM is the original Cu Click reaction with 2 mM Cu+10
mM Ascorbate for 4 min, followed by addition of 10 mM BCS with
incubation for 30 min. THPTA/Picolyl click reactions were carried
out with 0.25 mM Cu, supplemented with THPTA at a molar ratio of
0:1, 1:1, 2:1, 4:1, and 8:1 for 40 minutes in the presence of 10 mM
ascorbate. For all Cu containing reactions 10 .mu.M picolyl Oregon
green alkyne was used. DIBO click reactions were carried out with
DIBO-Oregon Green 488 at 20 .mu.M for 3 h. In FIG. 18, samples of
control antibodies are indicated with galT- (no
galactosyltransferase was added in the GalNAz modification step).
SBP (SeeBlue.RTM. Plus 2 Pre-Stained Standard; LC5925) and M12
(Mark 12.TM. Unstained Standard; LC5677) were used as molecular
weight standards. Gels were imaged with a FUJIFILM FLA-9000 for
fluorescence detection for 488 nm dyes followed by SYPRO.RTM. Ruby
General Protein Stain.
Example 10
Site Specific Labeling of Cell Surface Proteins with an Engineered
Picolyl Azide Ligase and Chelation-Assisted Copper(I)-Catalyzed
Azide-Alkyne Cycloaddition
[0477] The picolyl azide structure was converted into a substrate
(for example, 5-(6-(azidomethyl)nicotinamido)pentanoic acid as
shown below) for lipoic acid ligase, an Escherichia coli enzyme
useful for site-specific protein labeling in cells. The data showed
that the W37V mutant of E. coli lipoic acid ligase (Lp1A) was found
to efficiently catalyze the ligation of a picolyl azide derivative
bearing a carboxylate terminal onto recombinant proteins expressed
on the surface of living mammalian cells as shown in FIG. 19. The
ligated picolyl azide was then chemoselectively derivatized with
various alkyne-fluorophores, under extremely mild conditions with
50 .mu.M copper, in high yield and with minimal observable
cytotoxicity, even on living neurons. A side-by-side comparison of
our chelation-assisted copper(I)-catalyzed azide-alkyne
cycloaddition protocol for specific protein labeling on the cell
surface, against an analogous labeling protocol with alkyl azide
followed by chelation-assisted copper(I)-catalyzed azide-alkyne
cycloaddition showed that the former gives a 9-fold higher signal,
with minimal background, after only 5 min of labeling (for the
second step).
Synthesis of 5-(6-(azidomethyl)nicotinamido)pentanoic acid
##STR00084##
[0479] To a solution of 6-azidomethylnicotinic acid (30 mg, 0.168
mmol; from Example 4) in anhydrous DMF (500 .mu.L) was added
disuccinimidyl carbonate (DSC; 65 mg, 0.253 mmol) and triethylamine
(TEA; 120 .mu.L, 0.840 mmol). The reaction was allowed to proceed
for 3 hours at ambient temperature. The reaction mixture was
diluted with chloroform and water. Layers were separated, and the
aqueous layer was extracted with chloroform three times. The
combined organic layer was washed with brine, dried over
MgSO.sub.4, and concentrated in vacuo. The residual mixture was
purified by silica chromatography (1:1 hexanes:ethyl acetate) to
afford the succinimidyl ester of 6-azidomethylnicotinic acid.
Rf=0.67 in 9:1 chloroform:methanol.
[0480] To a solution of 5-azidomethylnicotinic acid succinimidyl
ester (15 mg, 0.055 mmol) in anhydrous DMF (500 .mu.L) was added
5-aminovaleric acid (32 mg, 0.273 mmol) and TEA (38 .mu.L, 0.273
mmol). The reaction proceeded for 12 hours at ambient temperature.
TEA and DMF were then removed in vacuo, and the resulting residue
was dry-loaded in 9:1 chloroform:methanol onto a silica column, and
purified using 9:1 chloroform:methanol as eluent. Rf=0.19 in 9:1
chloroform:methanol. .sup.1H NMR (D.sub.2O, 500 MHz): 8.83 (s, 1H),
8.18 (d, 1H, J=8.5 Hz), 7.59 (d, 1H, J=8 Hz), 4.62 (s, 2H), 3.42
(m, 2H), 2.32 (m, 2H), 1.65 (m, 4H).
[0481] In Vitro Lp1A-Catalyzed Picolyl Azide Ligation for
Preparation of a LAP-Picolyl Azide Adduct.
[0482] The enzymatic reaction was assembled as follows: 150 .mu.M
LAP (amino acid sequence: GFEIDKVWYDLDA), 5 .mu.M W37VLp1A, 500
.mu.M 5-(6-(azidomethyl)nicotinamido)pentanoic acid, 1 mM ATP, and
5 mM Mg(OAc)2 in 20% v/v glycerol in Dulbecco's phosphate-buffered
saline (DPBS) at 30.degree. C. for 30 min. The reaction was
quenched with EDTA (final concentration 50 mM) to give the
LAP-picolyl azide adduct and analyzed on a Varian Prostar HPLC
using a reverse phase C18 Microsorb-MV100 column (250.times.4.6
mm). Chromatograms were recorded at 210 nm. A 10-min gradient of
30-60% acetonitrile in water with 0.1% trifluoroacetic acid at a
flow rate of 1 mL/min was used. FIG. 20 shows these chromatograms.
LAP had a retention time of 7.5 min; after ligation to the picolyl
azide, the retention time increased to 11 min. The data showed that
the W37Vlp1A catalyzes the attachment of the picolyl azide to
LAP.
[0483] Cell Culturing.
[0484] Human embryonic kidney (HEK) cells were cultured in minimal
essential medium (MEM, Mediatech) supplemented with 10% v/v fetal
bovine serum (PAA Laboratories).
[0485] For hippocompal neuron cultures, Spague Dawley rat pups were
sacrificed at embryonic day 18. Hippocampal tissue was digested
with papain (Worthington) and DNaseI (Roche) and plated on glass
coverslips pretreated with poly-D-lysine (Sigma) and mouse laminin
(Life Technologies) in L-glutamine-containing MEM (Sigma)
supplemented with 10% v/v fetal bovine serum (PAA Laboratories) and
B27 (Life Technologies). At 3 days in vitro, half of the growth
medium was replaced with Neurobasal medium (Life Technologies)
supplemented with B27 and GlutaMAX (Life Technologies).
[0486] Genetic Constructs.
[0487] Complete nucleotide sequences of the following constructs
are known in the art: Lp1A variants in pYFJ16 for expression in E.
coli; LAP-CFP in pDisplay; LAP-neurexin-1.beta. in pECFP-N1; and
LAP-neuroligin-1 in pNICE.
[0488] Imaging
[0489] Fluorescence imaging. For FIGS. 21 and 22, cells were imaged
in Tyrode's buffer or DPBS in epifluorescence or confocal modes.
For epifluorescence imaging, we used a Zeiss AxioObserver inverted
microscope with a 40.times. oil-immersion objective. CFP (420Y20
excitation, 425 dichroic, 475Y40 emission), Alexa Fluor.RTM. 647
(630Y20 excitation, 660 dichroic, 680Y30 emission) and differential
interference contrast (DIC) images were collected and analyzed
using Slidebook software (Intelligent Imaging Innovations). For
confocal imaging, we used a Zeiss Axiovert 200M inverted microscope
with a 40.times. oil-immersion objective. The microscope was
equipped with a Yokogawa spinning disk confocal head, a Quad-band
notch dichroic mirror (405Y488Y568Y647), and 491 (DPSS), 561 nm
(DPSS), 640 nm (DPSS) lasers (all 50 mW). YFP/Alexa Fluor.RTM. 488
(491 laser excitation, 528Y38 emission), Alexa Fluor.RTM. 568 (561
laser excitation, 617Y73 emission), Alexa Fluor.RTM. 647 (640 laser
excitation, 680/30 emission), and DIC images were collected using
Slidebook software. Fluorescence images in each experiment were
normalized to the same intensity ranges. Acquisition times ranged
from 10-1000 milliseconds.
[0490] General Protocol for Cell-Surface Protein Labeling with
PRIME Method Followed by Chelation-Assisted Copper(I)-Catalyzed
Azide-Alkyne Cycloaddition.
[0491] Human embryonic kidney (HEK) cells were transfected at
.about.80% confluency with expression plasmids for LAP-tagged
neurexin-1.beta. (400 ng for a 0.95 cm2 dish) and yellow
fluorescent protein-tagged histone 2B protein (H2B-YFP; 100 ng)
using lipofectamine 2000 (Invitrogen). 24 hr after transfection,
cells were treated with 10 .mu.M purified W37VLp1A, 200 .mu.M
5-(6-(azidomethyl)nicotinamido)pentanoic acid made above, 1 mM ATP,
and 5 mM Mg(OAc)2 in cell growth medium for 20 min at room
temperature. After excess Lp1A labeling reagents had been removed
by quickly replacing the medium 2-3 times, cells were further
labeled with 20 .mu.M Alexa Fluor.RTM. 647-alkyne, 50 .mu.M CuSO4,
250 .mu.M THPTA, and 2.5 mM sodium ascorbate in DPBS for 5 min at
room temperature. Cells were immediately imaged after excess CuAAC
labeling reagents were removed by 2-3 quick washes with fresh
growth medium.
[0492] FIG. 21 shows the results of chelation-assisted copper(I)
catalyzed azide-alkyne cycloaddition for tagging of Alexa
Fluor.RTM. 647 onto neurexin in living HEK cells. Negative controls
are shown with ATP omitted (second column) or wild-type Lp1A in
place of .sup.W37VLp1A (third column). H2B-YFP is the
nuclear-localized YFP transfection marker. Negative controls are
shown with ATP omitted (second column) or wild-type Lp1A in place
of .sup.W37VLp1A (third column). H2B-YFP is the nuclear-localized
YFP transfection marker. Confocal images are shown. Scale bars for
all images, 10 .mu.m.
[0493] Labeling of LAP-Neuroligin-1 in Live Dissociated Neurons
with PRIME Followed by Chelation-Assisted CuAAC.
[0494] Neurons were transfected at 5 days in vitro with expression
plasmids for LAP-tagged neuroligin-1 (500 ng for a 1.9 cm2 dish)
and green fluorescent protein-tagged Homer1b (Homer-GFP; 100 ng for
a 1.9 cm2 dish) using Lipofectamine 2000, using half the amount of
the manufacturer's recommended reagent quantity. Neurons were
labeled at 11 days in vitro with 10 .mu.M purified W37VLp1A, 200
.mu.M of 5-(6-(azidomethyl)nicotinamido)pentanoic acid made above,
1 mM ATP, and 5 mM Mg(OAc)2 in preconditioned supplemented
Neurobasal medium for 20 min at 37.degree. C. After brief rinsing
in supplemented preconditioned medium, neurons were further labeled
with 20 .mu.M Alexa Fluor.RTM. 647-alkyne, 50 .mu.M Tempol, 50
.mu.M CuSO4, 250 .mu.M THPTA, and 2.5 mM sodium ascorbate in
Tyrode's buffer for 5 min at room temperature. The labeling
solution was then replaced with supplemented Neurobasal medium
containing 500 .mu.M bathocuproin sulfonate, which was incubated
with neurons for 30 sec. Neurons were imaged live in Tyrode's
buffer after 2 further washes with supplemented Neurobasal
medium.
[0495] FIG. 22 shows chelation-assisted copper(I) catalyzed
azide-alkyne cycloaddition for tagging of Alexa Fluor.RTM. 647 onto
neuroligin-1 in living hippocampal neurons. DIV 11 (11 days in
vitro) rat hippocampal neurons expressing LAP-neuroligin-1 and
GFP-tagged Homer1b were labeled with
5-(6-(azidomethyl)nicotinamido)pentanoic acid via .sup.W37VLp1A,
then with Alexa Fluor.RTM. 647-alkyne via chelation-assisted
copper(I) catalyzed azide-alkyne cycloaddition, and imaged live
after brief rinsing. Labeling conditions were the same as in the
above general protocol for cell-surface protein labeling with PRIME
method, except: 1) higher [CuSO4] of 300 .mu.M was used for the
bottom row; 2) a radical scavenger Tempol (50 .mu.M) was added to
the CuAAC labeling solution; and 3) a biocompatible copper chelator
bathocuproine sulfonate (500 .mu.M) was used during the first rinse
to immediately quench the click reaction. Alexa Fluor.RTM. 647
images in the second column correspond to the boxed regions 1 and
2, shown at higher zoom. White arrows denote regions of focal
swelling when 300 .mu.M CuSO4 is used. Confocal images are shown.
Scale bars for all images, 10 .mu.m.
* * * * *