U.S. patent application number 13/967086 was filed with the patent office on 2014-06-19 for novel hydrazone-based and oxime-based fluorescent and chromophoric/pro-fluorescent and pro-chromophoric reagents and linkers.
This patent application is currently assigned to Solulink Biosciences, Inc.. The applicant listed for this patent is Solulink Biosciences, Inc.. Invention is credited to Leopold Mendoza, David A. Schwartz.
Application Number | 20140171635 13/967086 |
Document ID | / |
Family ID | 39742311 |
Filed Date | 2014-06-19 |
United States Patent
Application |
20140171635 |
Kind Code |
A1 |
Schwartz; David A. ; et
al. |
June 19, 2014 |
NOVEL HYDRAZONE-BASED AND OXIME-BASED FLUORESCENT AND
CHROMOPHORIC/PRO-FLUORESCENT AND PRO-CHROMOPHORIC REAGENTS AND
LINKERS
Abstract
Novel Hydrazone-based Fluorescent and Pro-Fluorescent Reagents
and Linkers, including conjugationally extended hydrazine
compositions, fluorescent hydrazone compositions, methods of the
formation of hydrazones from the reaction of conjugationally
extended hydrazines with conjugationally extended carbonyls, and
methods of their use in assays systems are described. Use of these
conjugationally compositions for direct colorimetric and
fluorometric assays wherein a chromophore or the fluorophore is
incorporated into the linker that is positioned between a reactive
linking moiety and a biotin molecule.
Inventors: |
Schwartz; David A.;
(Encinitas, CA) ; Mendoza; Leopold; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Solulink Biosciences, Inc. |
San Diego |
CA |
US |
|
|
Assignee: |
Solulink Biosciences, Inc.
San Diego
CA
|
Family ID: |
39742311 |
Appl. No.: |
13/967086 |
Filed: |
August 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11787932 |
Apr 18, 2007 |
8541555 |
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13967086 |
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60792821 |
Apr 18, 2006 |
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60792822 |
Apr 18, 2006 |
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Current U.S.
Class: |
536/26.6 ;
546/273.7 |
Current CPC
Class: |
G01N 33/582 20130101;
C07D 303/02 20130101 |
Class at
Publication: |
536/26.6 ;
546/273.7 |
International
Class: |
G01N 33/58 20060101
G01N033/58 |
Claims
1. A fluorescent hydrazone compound of formula I,
(R.sub.1R.sub.2)NN.dbd.C(R.sub.1R.sub.2) I wherein: R.sub.1 are
independently a substituted or unsubstituted conjugationally
extended moiety wherein the unsubstituted conjugationally extended
moiety is an alkenyl, alkynyl, aromatic, polyaromatic,
heteroaromatic or polyheteroaromatic moiety and wherein the
substituted conjugationally extended moiety may be substituted with
any combination of one or more of the groups hydroxy, alkoxy,
alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and
substituted primary, secondary, tertiary and quaternary amine;
R.sub.2 are independently a hydrogen, a straight chain aliphatic
moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10
carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a
substituted or unsubstituted conjugationally extended moiety
wherein the unsubstituted conjugationally extended moiety is an
alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine and wherein said
composition has an emission frequency equal to or greater than 400
nm.
2. A fluorescent hydrazone compound according to claim 1 wherein
one of R, one of R.sub.1 or one of R.sub.2 further comprises a
linker moiety selected from the group consisting of an amino
reactive moiety, a thiol reactive moiety, an ester moiety and a
modified carbohydrate monomer moiety.
3. A fluorescent hydrazone compound according to claim 2 wherein
said linker further comprises a biomolecule.
4. A fluorescent composition of the formula III, ##STR00006##
wherein; R.sub.1 (which is R.sub.2) is a substituted or
unsubstituted conjugationally extended moiety wherein the
unsubstituted conjugationally extended moiety is an alkenyl,
alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine; R.sub.2 (which
is R.sub.3) is a hydrogen, a straight chain aliphatic moiety of
1-10 carbon atoms, a branched aliphatic moiety of 1-10 carbon
atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a
substituted or unsubstituted conjugationally extended moiety
wherein the unsubstituted conjugationally extended moiety is an
alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine; R.sub.3 (which
is R.sub.4) is H or OH; R.sub.4 (which is R.sub.6) is H or a
nucleic acid moiety; and R.sub.5 (which is R.sub.7) is PO.sub.3 or
a nucleic acid moiety.
5. A profluorescent hydrazine compound of formula IV,
(R.sub.1R.sub.2)N(H).sub.n(NH.sub.2).sub.m IV wherein: R1 is a
substituted or unsubstituted conjugationally extended moiety
wherein the unsubstituted conjugationally extended moiety is an
alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine; R.sub.2 is a
hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a
branched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic
moiety of 1-10 carbon atoms, a substituted or unsubstituted
conjugationally extended moiety wherein the unsubstituted
conjugationally extended moiety is an alkenyl, alkynyl, aromatic,
polyaromatic, heteroaromatic or polyheteroaromatic moiety and
wherein the substituted conjugationally extended moiety may be
substituted with any combination of one or more of the groups
hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo,
unsubstituted amine and substituted primary, secondary, tertiary
and quaternary amine; n is 0 when m is 2 and n is 1 when m is
1.
6. A profluorescent hydrazine compound according to claim 5 wherein
R.sub.1 or R.sub.2 further comprise a linkable moiety selected from
the group consisting of an amino reactive moiety, a thiol reactive
moiety, an ester moiety and a modified carbohydrate monomer
moiety.
7. A profluorescent hydrazine compound according to claim 6 wherein
said linker further comprises a biomolecule.
8. A composition comprising a polymer having one or more
profluorescent hydrazine compounds according to claim 5 bound to
said polymer by one or more linker moieties.
9. A polymer according to claim 8 wherein said polymer is
poly-lysine, poly-ornithine or polyethyleneglycol.
10. A profluorescent oxyamine compound of formula VI,
(R.sub.1R.sub.2)ONH.sub.2 VI wherein: R.sub.1 is a substituted or
unsubstituted conjugationally extended moiety wherein the
unsubstituted conjugationally extended moiety is an alkenyl,
alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine; and R.sub.2 is a
hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a
branched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic
moiety of 1-10 carbon atoms, a substituted or unsubstituted
conjugationally extended moiety wherein the unsubstituted
conjugationally extended moiety is an alkenyl, alkynyl, aromatic,
polyaromatic, heteroaromatic or polyheteroaromatic moiety and
tertiary and wherein the substituted conjugationally extended
moiety may be substituted with any combination of one or more of
the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo,
unsubstituted amine and substituted primary, secondary, quaternary
amine.
11. A profluorescent oxyamine compound according to claim 10
wherein R.sub.1 or R.sub.2 further comprise a linkable moiety
selected from the group consisting of an amino reactive moiety, a
thiol reactive moiety, an ester moiety and a modified carbohydrate
monomer moiety.
12. A profluorescent oxyamine compound according to claim 10
wherein said linker further comprises a biomolecule.
13. A method of preparing a fluorescent hydrazone according to
claim 1 by combining a hydrazine of formula IV,
(R.sub.1R.sub.2)N(H).sub.n(NH.sub.2).sub.m IV with a carbonyl of
formula V: O.dbd.C(R.sub.1R.sub.2) V wherein: R.sub.1 are
independently a substituted or unsubstituted conjugationally
extended moiety wherein the unsubstituted conjugationally extended
moiety is an alkenyl, alkynyl, aromatic, polyaromatic,
heteroaromatic or polyheteroaromatic moiety and wherein the
substituted conjugationally extended moiety may be substituted with
any combination of one or more of the groups hydroxy, alkoxy,
alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and
substituted primary, secondary, tertiary and quaternary amine;
R.sub.2 are independently a hydrogen, a straight chain aliphatic
moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10
carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a
substituted or unsubstituted conjugationally extended moiety
wherein the unsubstituted conjugationally extended moiety is an
alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine; n is 0 when m is
2 and n is 1 when m is 1 for a time and under conditions that allow
hydrazone formation.
14. A method according to claim 13 wherein R.sub.1 or R.sub.2 of
formula IV further comprises a linkable moiety selected from the
group consisting of an amino reactive moiety, a thiol reactive
moiety, an ester moiety and a modified carbohydrate monomer
moiety.
15. A method according to claim 13 wherein R.sub.1 or R.sub.2 of
formula V further comprises a linkable moiety selected from the
group consisting of an amino reactive moiety, a thiol reactive
moiety, an ester moiety and a modified carbohydrate monomer
moiety.
16.-24. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a regular application claiming priority
from provisional patent application Ser. No. 60/792,821 and
60/792,822 both filed 18 Apr. 2006.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] None
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON COMPACT
DISC
[0003] None
BACKGROUND OF THE INVENTION
[0004] (1) Field of the Invention
[0005] The present invention relates to compounds used to label
biomolecules for diagnostic and therapeutic purposes. In
particular, it relates to fluorescent, chromophoric,
pro-fluorescent and pro-chromophoric compounds that may be
conjugated to biomolecules such as proteins and nucleic acids. Such
compounds may be incorporated into linkers that may be used to link
a ligand to a biomolecular probe allowing quantitation of the
ligand bound to that molecular probe.
[0006] (2) Description of Related Art
[0007] Methods to detect interactions between biomolecules
continues to be an area of active research as new and more
sensitive methods are required to increase sensitivity, reduce
costs and enable new detection methods. One of the most widely used
methods is to directly label a biomolecule with a fluorescent
molecule that fluoresces at a desired frequency. For example, a
fluorescent molecule is modified with a thiol- or amino-reactive
moiety such as succinimidyl esters or maleimides that form a
covalent bound in the presence of a sulfhydryl or amine group of a
desired protein. The modified fluorescent molecule is isolated and
reacted with the desired protein. The fluorescently labeled protein
is then used to detect a desired target by monitoring the unique
fluorescent frequency of the fluorophore. A variety of fluorophores
have been modified with these moieties including fluorescein,
rhodamine, Texas Red and cyanine dyes, Cy3 and Cy5. Unfortunately,
the conjugation methods often cause quenching and photobleaching of
the fluorophore and there can be interference with the observed
signal if the unbound labeled biomolecule is not removed from the
reaction mixture.
[0008] Other biomolecules such as nucleic acids such as DNA, RNA,
polynucleotide and oligonucleotides have been labeled with
fluorophores is commonly accomplished by incorporating a
fluorophore on the base moiety of a nucleoside triphosphate. These
fluorescently labeled triphosphates are added to the polymerase
chain reaction (PCR) or reverse transcription reaction wherein the
labeled nucleoside is incorporated in the amplicon yielding a
fluorescently labeled polynucleotide. These fluorescently labeled
polynucleotides are probed using oligonucleotide microarrays
identifying sequences present in the target. Unfortunately, the
fluorophores used for labeling these biomolecules are not often
stable to these synthesis conditions. In addition, the long-term
stability of conjugates are low due to photobleaching,
consequently, retention of the fluorescent signal is difficult when
archiving microarrays.
[0009] A variety of references cite the use of fluorescent
hydrazides, thiosemicarbazides and hydrazides to react with
aldehydes on biological molecules for the detection of the
aldehydes. For example Ahn et al. (B. Ahn, S. G. Rhee and E. R.
Stadtman, Anal. Biochem. 161:245 (1987) describe the use of
fluorescein hydrazide and fluorescein thiosemicarbazide for the
fluorometric determination of protein carbonyl groups and for the
detection of oxidized proteins on polyacrylamide gels. Proudnikov
and Mirzabekov (Nucl. Acids Res. 24:4535 (1996)) describe labeling
of DNA and RNA to identify acid-induced depurination that results
in production of aldehyde moieties detected by reaction of
fluorescent labels containing hydrazide groups in the presence of
sodium cyanoborohydride. Others have labeled the reducing end of
polysaccharides with fluorescent hydrazides. These methods are used
to detect aliphatic aldehyde groups on biomolecules. In each of the
references the fluorescent moiety is incorporated on the hydrazine
or hydrazide that forms a hydrazone on reaction with the aldehyde
present on the biomolecule.
[0010] It has been documented that hydrazones formed between
certain aromatic aldehydes and aromatic hydrazines and not aromatic
hydrazides or aromatic thiosemicarbazides form fluorescent
molecules (J. Wong and F. Bruscato, Tet. Lett. 4593, 1968). It has
also been reported that hydrazones formed specifically from
2-substituted aldehyde heterocycles and 2-substituted hydrazine
heterocycles become fluorescent on chelation to zinc (D. E. Ryan,
F. Snape and M. Winpe, Anal. Chim. Acta 58:101, 1972).
[0011] Schwartz et al. (U.S. Pat. No. 5,420,285; U.S. Pat. No.
5,753,520; U.S. Pat. No. 5,420,285; J. Nucl. Med. 31(12):2022, 1990
and Bioconjug. Chem. 2(5):333, 1991) describe the preparation of
succinimidyl 6-hydraziniumnicotinate hydrochloride for the one-step
modification of amino groups on proteins and other molecules to
incorporate pyridylhydrazine moieties on proteins for the specific
purpose of binding technetium-99m for in vivo diagnostic purpose.
Subsequently Schwartz (U.S. patent application, titled: Functional
Oligonucleotide Modification Reagents and Uses Thereof, filed Aug.
1, 2000) describe novel oligonucleotide aldehyde and hydrazine
phosphoramidite reagents for incorporation of aldehydes and
hydrazines on synthetic oligonucleotides including aromatic and
heteroaromatic aldehydes and hydrazines. Triphosphates
incorporating both aromatic hydrazine and aromatic aldehydes have
been described by Schwartz and Hogrefe (U.S. Pat. No.
6,686,461).
[0012] Cytidine and deoxycytidine moieties in polynucleotides can
be transformed into 4-N-aminocytidine (4-hyd-C), an aromatic
hydrazine, by treatment with hydrazine/bisulfite at neutral pH.
Nitta et al. Eur. J. Biochem. 157(2):427, 1986 has described
crosslinking between 16S ribosomal RNA and protein S4 in E. coli
ribosomal 30S subunits effected by treatment with
bisulfite/hydrazine and bromopyruvate. Also Musso et al., (U.S.
Pat. No. 5,130,466) describe labeling of 4-N-aminocytidine moieties
on hydrazine/bisulfite treated DNA to yield a fluorescently labeled
polynucleotide. Bittner et al. (U.S. Pat. No. 5,491,224) also
describe the labeling of transaminated DNA with fluorescent
moieties possessing moieties that react with the transaminated
cytosine such as fluorophores possessing succinimidyl esters.
[0013] In all of the aforementioned references the biomolecule is
fluorescently labeled with a fluorescent molecule. Unfortunately as
previously stated the processes or methods used to prepare the
conjugate can often times cause quenching or photobleaching of the
fluorophore. In addition, during use the unbound fluorescently
labeled conjugate must be removed to obtain an accurate fluorescent
signal.
[0014] Therefore, there is a need in the field for a fluorescent
label that is resistant to reaction conditions necessary for
producing a labeled biomolecule and does not require removal of the
unbound fluorescently labeled biomolecule from the detection
reaction mixture to obtain a accurate and/or quantitative signal.
There is also a need for fluorophores that may be formed under
standard assay conditions from pro-fluorophores which, are stable
under various laboratory conditions and by a reaction that is
highly specific and efficient.
[0015] To date the most commonly used method to link, immobilize
and detect biomolecules is the biotin/streptavidin ligand/receptor
couple. Biotin (FIG. 1) is a small molecule, MW 250, that binds to
streptavidin with an association constant of 10.sup.15. The
extremely high binding constant and fast kinetics of binding and
the stability of avidin under a variety of conditions make this an
ideal ligand/receptor pair for these purposes. Biotin has been
modified to include amino, thiol and carbohydrate reactive
moieties, i.e. succinimidyl ester, maleimido and hydrazide
respectively, to allow easy incorporation into a large variety of
biomolecules. To accomplish detection of an analyte, biotin is
conjugated to a probing biomolecule such as an antibody or an
oligonucleotide. Following binding of the biotinylated biomolecule
to its receptor or complement, an avidin/reporter conjugate such as
an avidin/fluorophore conjugate or a avidin/reporter enzyme
conjugate is added and allowed to bind to biotinylated probe and
visualized by fluorescence detection or addition of a substrate
that emits light or precipitates a colored insoluble product on
enzymatic processing (Heitzmann H., Richards F. M., Proc. Natl.
Acad. Sci. USA 71:3537-3541, 1974; Diamandis E. P., Christopoulos
T. K., Clin. Chem. 37:625-636, 1991; Wilchek M. Methods Enzymol
Vol. 184, 1990; Savage, M. D. et al., 1992 Avidin-Biotin Chemistry:
A Handbook. Rockford, Ill.: Pierce Chemical Co.).
[0016] Following conjugation it is important to determine that the
probe molecule has been biotinylated and to quantify the number of
biotins now conjugated to the probe molecule. To this end two
multi-step indirect assays have been developed. The first assay is
the HABA ([2-(4'-hydroxyazobenzene)]benzoic acid) assay developed
by Green (Green, N. M. Biochem. J., 94, 23c-24, 1965). To quantify
biotin label incorporation, a solution containing the biotinylated
protein is added to a mixture of HABA and avidin. Because of its
higher affinity for avidin, biotin displaces the HABA from its
interaction with avidin and the absorption at 500 nm decreases
proportionately. By this method, an unknown amount of biotin
present in a solution can be evaluated in a single cuvette by
measuring the absorbance of the HABA-avidin solution before and
after addition of the biotin-containing sample. The change in
absorbance relates to the amount of biotin in the sample.
[0017] The second more sensitive fluorescence-based multi-step
assay developed by Molecular Probes (recently acquired by
Invitrogen Corporation in Carlsbad, Calif.) is the `Fluoreporter
Biotin Quantitation Assay` that is based on fluorescence resonance
energy transfer (FRET) quenching wherein an avidin molecule is
labeled with a fluorophore and its binding sites are occupied with
a fluorescent molecule that quenches the covalently linked
fluorophore until the quencher in the binding site is displaced by
a higher binding biotin molecule resulting in fluorescence of the
covalently attached fluorophore. While this assay is sensitive to
50-100 pmol range it requires many processing steps and a
fluorimeter or multi-well fluorimeter. It is also recommended to
digest the biotinylated protein prior to the assay to expose any
sterically encumbered biotins.
[0018] Consequently there is a need in the field for a assay
wherein the number of biotins covalently linked to a biomolecule
could be determined by direct methods such as spectroscopic
means.
BRIEF SUMMARY OF THE INVENTION
[0019] The present invention provides
profluorescent/prochromophoric hydrazine and aldehyde reagent
compounds for preparing novel hydrazone-based fluorescent
molecules. More specifically conjugationally extended
profluorescent/prochromophoric hydrazine compounds of the formula
(RR.sub.2)N(H).sub.n(NH.sub.2).sub.n, wherein R is independently a
substituted or unsubstituted conjugationally extended moiety
wherein the unsubstituted conjugationally extended moiety is an
alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine; R.sub.2 is
independently a hydrogen, a straight chain aliphatic moiety of 1-10
carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a
cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or
unsubstituted conjugationally extended moiety wherein the
unsubstituted conjugationally extended moiety is an alkenyl,
alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine; n is 0 when m is
and n is 1 when m is 1 may be combined with conjugationally
extended profluorescent/prochromophoric carbonyl compounds of the
formula O.dbd.C(R.sub.1R.sub.2) wherein: R.sub.1 is independently a
substituted or unsubstituted conjugationally extended moiety
wherein the unsubstituted conjugationally extended moiety is an
alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine; R.sub.2 is
independently a hydrogen, a straight chain aliphatic moiety of 1-10
carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a
cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or
unsubstituted conjugationally extended moiety wherein the
unsubstituted conjugationally extended moiety is an alkenyl,
alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine; n is 0 when m is
2 and n is 1 when m is 1 to form fluorescent hydrazone compounds of
the formula (RR.sub.2)NN.dbd.C(R.sub.1R.sub.2).
[0020] In one embodiment the hydrazone compound has the
formula:
##STR00001##
wherein R.sub.1 (which is R.sub.2) is independently a substituted
or unsubstituted conjugationally extended moiety wherein the
unsubstituted conjugationally extended moiety is an alkenyl,
alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine; R.sub.2 (which
is R.sub.3) is independently a hydrogen, a straight chain aliphatic
moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10
carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a
substituted or unsubstituted conjugationally extended moiety
wherein the unsubstituted conjugationally extended moiety is an
alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine; R.sub.3 (which
is R.sub.4) is H or OH; R.sub.4 (which is R.sub.6) is H or a
nucleic acid moiety; and R.sub.5 (which is R.sub.7) is PO.sub.3 or
a nucleic acid moiety.
[0021] In another embodiment these novel profluorophore hydrazine
and carbonyl compounds may further comprise a linkable moiety at
one of the R or R.sub.2 positions wherein the linkable moiety is
selected from the group consisting of an amino reactive moiety, a
thiol reactive moiety, an ester moiety and a modified carbohydrate
monomer moiety.
[0022] In yet another embodiment a biomolecule such as for example
a nucleic acid, a nucleotide, a protein, an amino acid, a
carbohydrate monomer or a polysaccharide is linked to the
profluorescent/prochromophoric hydrazine and/or
profluorescent/prochromophoric carbonyl by a linkable moiety. If
the biomolecule is a nucleic acid it may be DNA, cDNA, RNA, or PNA
and can comprise natural or unnatural bases or internucleotide
linkages selected from the group consisting of phosphodiesters,
phosphorothioates, phosphoramidites and peptide nucleic acids.
[0023] In still another embodiment one or more of the
profluorescent/prochromophoric hydrazine or carbonyl compounds may
be bound to a polymer such as poly-lysine, poly-ornithine or
polyethyleneglycol by one or more linkable moieties.
[0024] In another aspect of the present invention methods of
forming a hydrazone compound are provided by combining the
conjugationally extended profluorescent/prochromophoric hydrazine
of formula (RR.sub.2)N(H).sub.n(NH.sub.2) with conjugationally
extended profluorescent/prochromophoric carbonyl of the formula
O.dbd.C(R.sub.1R.sub.2) for a time and under conditions that allow
hydrazone formation.
[0025] In one embodiment of this aspect of the invention the
conjugationally extended profluorescent/prochromophoric hydrazine
and/or the conjugationally extended profluorescent/prochromophoric
carbonyl may further comprise a linkable moiety at either the
R.sub.1 or R.sub.2 position.
[0026] In yet another aspect of the invention a method for labeling
a biomolecule with a fluorescent hydrazone compound is
provided.
[0027] In still another aspect the present invention provides
oxyamine and aldehyde reagent compounds for preparing novel
oxime-based fluorescent molecules. More specifically
conjugationally extended profluorescent/prochromophoric oxyamine
compound of formula: (R.sub.1R.sub.2)ONH.sub.2 are provided
wherein: R.sub.1 is a substituted or unsubstituted conjugationally
extended moiety wherein the unsubstituted conjugationally extended
moiety is an alkenyl, alkynyl, aromatic, polyaromatic,
heteroaromatic or polyheteroaromatic moiety and wherein the
substituted conjugationally extended moiety may be substituted with
any combination of one or more of the groups hydroxy, alkoxy,
alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and
substituted primary, secondary, tertiary and quaternary amine; and
R.sub.2 is a hydrogen, a straight chain aliphatic moiety of 1-10
carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a
cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or
unsubstituted conjugationally extended moiety wherein the
unsubstituted conjugationally extended moiety is an alkenyl,
alkynyl, aromatic, polyaromatic, heteroaromatic or
polyheteroaromatic moiety and wherein the substituted
conjugationally extended moiety may be substituted with any
combination of one or more of the groups hydroxy, alkoxy, alkene,
alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted
primary, secondary, tertiary and quaternary amine.
[0028] In one embodiment a profluorescent/prochromophoric oxyamine
compound is provided wherein R.sub.1 or R.sub.2 further comprise a
linkable moiety selected from the group consisting of an amino
reactive moiety, a thiol reactive moiety, an ester moiety and a
modified carbohydrate monomer moiety.
[0029] In another embodiment a profluorescent/prochromophoric
oxyamine compound is provided wherein the linker further comprises
a biomolecule selected from the group consisting of a nucleic acid,
a nucleotide, a protein an amino acid, a carbohydrate monomer and a
polysaccharide. The nucleic acid may be selected from the group
consisting of DNA, cDNA, RNA and PNA and may comprise natural or
unnatural bases or internucleotide linkages selected from the group
consisting of phosphodiesters, phosphorothioates, phosphoramidites
and peptide nucleic acids.
[0030] In another aspect of the invention a spectrophotometrically
quantifiable linker is provided comprising of formula: A-B-C-D
wherein A is an amino, thiol or carbohydrate reactive moiety; B is
a chromophoric or fluorescent moiety; C is a flexible linker; and D
is biotin or a receptor ligand. When A is an amino reactive moiety
it may be selected from the group consisting of
N-hydroxysuccinimidyl, p-nitrophenyl, pentafluorophenyl and
N-hydroxybenzotriazolyl. When A is a thiol reactive moiety it may
be selected from the group consisting of maleimido,
.alpha.-haloacetamido and pyridylsulfides. When A is a carbohydrate
reactive moiety it may be aminooxy. B may be a compound that
fluoresces, emits light or precipitates a colored insoluble product
on enzymatic processing. C is a flexible linker and may be a PEG
flexible linker having no less than 8 carbon atoms and no more than
34 carbon atoms. D is a receptor ligand selected from the group
consisting of receptor ligand pairs biotin/avidin, peptide
S/ribonuclease, complimentary oligonucleotide pairs or
antibody/ligand pairs, and digoxigenin/anti-digoxigenin
antibody.
[0031] In one embodiment of the present invention wherein the
spectrophotometrically quantifiable linker is bound to a
biomolecule via a amino, thiol or carbohydrate reactive moiety and
wherein the biomolecule is selected from the group consisting of a
protein, a peptide, an oligonucleotide and a polynucleotide.
Alternatively the spectrophotometrically quantifiable linker may be
bound to a biomolecule via receptor ligand pairs such as
biotin/avidin, peptide S/ribonuclease, digoxigenin/anti-digoxigenin
antibody complimentary oligonucleotide pairs or antibody/ligand
pairs. Correspondingly, a first biomolecule may be bound via an
amino, thiol or carbohydrate reactive moiety and a second
biomolecule may be bound via a receptor ligand pair to the
spectrophotometrically quantifiable linker.
[0032] In another aspect of the present invention a method of
preparing a spectrophotometrically quantifiable linker is provided
by the steps of preparing a first conjugate of a first biomolecule
bound to one profluorescent/prochromophoric compound of a
fluorescent pair via an amino, thiol or carbohydrate reactive
moiety and preparing a second conjugate of a second biomolecule
bound to a flexible linker via a biotin or a receptor ligand and
the other profluorescent/prochromophoric compound of a fluorescent
pair and combining the first conjugate with the second conjugate
for a time thereby forming a hydrazone bond between the
profluorescent/prochromophoric compound pair forming a fluorescent
moiety.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0033] FIG. 1: A diagrammatic representation of the chemistry for
the formation of fluorescent hydrazones from conjugationally
extended aldehydes and hydrazines;
[0034] FIG. 2: A diagrammatic representation of the tautomerization
of bis-(2-heteroaromatic)hydrazone chelates;
[0035] FIG. 3: Hydrazine and aldehyde succinimidyl ester reagents,
SANH and SFB respectively, developed for modification of amino
moieties on biomolecules and a diagrammatic representation of the
conjugation of a hydrazine-modified biomolecules with a
benzaldehyde-modified biomolecule;
[0036] FIG. 4: (A) PAGE gel of the results of the conjugation of a
5'-benzaldehyde-modified oligonucleotide to a hydrazine-modified
antibody visualized by coomassie blue (CB) staining; (B) the same
gel visualized by UV backshadowing to visualize the oligonucleotide
conjugated to the protein; (C) nitrocellulose membrane of the
blotted conjugate following hybridization of the
fluorescein-labeled complementary oligonucleotide demonstrating
retention of hybridization functionality of conjugated
oligonucleotide;
[0037] FIG. 5: A diagrammatic representation of fluorescent
hydrazone (3) formed from 6-hydrazinonicotinic acid (1; R.dbd.OH)
and 4-dimethylaminocinnamaldehyde (2);
[0038] FIG. 6: (A) Chemical structure of benzaldehyde
phosphoramidite used to incorporate benzaldehyde moieties on the
5'-terminus of oligonucleotides during their solid phase synthesis;
(B) PAGE gel of purified oligonucleotide (Lane 1) and the product
of the reaction of the oligonucleotide with
trans-4-hydrazinostilbazole (1; Fluka Chemical Co.);
[0039] FIG. 7: Absorbance and emission spectra of a 22mer
oligonucleotide modified on the 5'-end with the hydrazone formed
from the reaction of benzaldehyde and,
trans-4'-Hydrazino-2-stilbazole;
[0040] FIG. 8: Chemical structure of bifunctional hydrazido amine
modification reagent SHTH;
[0041] FIG. 9: A diagrammatic representation showing hydrazones
prepared from conjugationally extended hydrazines and aldehydes
form fluorescent species while hydrazones prepared from
conjugationally extended hydrazides and aldehydes do not form
substantially fluorescent species.
5'-(6-Hydrazinylpyridine)-modified oligonucleotide is reacted with
4-dimethylaminocinnamaldehyde (Reaction A) and
naphthalene-1,2-dicarboxaldehyde (Reaction B) form fluorescent
species. The hydrazone formed from the reaction of
5'-(6-hydrazidoterephalate)-modified oligonucleotide with
4-dimethylaminocinnamaldehyde is not fluorescent and the product
with NDA forms a weakly fluorescent species based on the
pyrollo-fused naphthalene product without conjugation through
hydrazide moiety;
[0042] FIG. 10: A diagrammatic representation of the conversion of
cytidine to 4-N-aminocytidine with hydrazine/bisulfite;
[0043] FIG. 11: A diagrammatic representation of the incorporation
of fluorescence into DNA wherein salmon sperm DNA was treated with
hydrazine/bisulfite to convert cytidine moieties to
4-aminocytidine, an aromatic hydrazine. The modified DNA was
treated with dimethylaminocinnamaldehyde (DAC; top reaction; Lane
2) or naphthalene-1,2-dicarboxladehyde (NDA; bottom reaction; Lane
4) and visualized following electrophoresis on an agarose gel (at
left). Control reactions wherein untreated DNA was reacted with DAC
and NDA were not fluorescent (Lanes 1 and 3 respectively);
[0044] FIG. 12: Chemical structure of commercially available
aromatic hydrazines;
[0045] FIG. 13: Chemical structure of commercially available
aldehydes;
[0046] FIG. 14: Chemical structure of cyanine dyes Cy3 and Cy5;
[0047] FIG. 15: Chemical structure of cyanine profluors and their
parent fluorophores targeted for synthesis;
[0048] FIG. 16: A diagrammatic representation of the synthetic
methods for the preparation of hydrazinoheterocyles; and
[0049] FIG. 17: Chemical structure of benzimidazole profluors and
synthesis schemes of their parent fluorophores targeted for
synthesis.
[0050] FIG. 18: Chemical structure of biotin;
[0051] FIG. 19: A diagrammatic representation showing hydrazones
prepared from conjugationally extended hydrazines and aldehydes
that form fluorescent species while hydrazones prepared from
conjugationally extended hydrazides and aldehydes do not form
substantially fluorescent species.
5'-(6-Hydrazinylpyridine)-modified oligonucleotide is reacted with
4-dimethyl-aminocinnamaldehyde (Reaction A) and
naphthalene-1,2-dicarboxaldehyde (Reaction B) form fluorescent
species. The hydrazone formed from the reaction of
5'-(6-hydrazidoterephalate)-modified oligonucleotide with
4-dimethylaminocinnamaldehyde is not fluorescent and the product
with NDA forms a weakly fluorescent species based on the
pyrollo-fused naphthalene product without conjugation through
hydrazide moiety;
[0052] FIG. 20: A schematic representation of the synthesis of
amino-reactive biotin/hydrazone chromophore 6;
[0053] FIG. 21: A graph showing amino-reactive biotin/hydrazone
chromophore 6 and overlaid spectra of equivalent amounts (20 .mu.g)
native bIgG and bIgG modified with 5.times., 10.times. and
15.times. amino-reactive biotin/hydrazone chromophore demonstrating
the incorporation of chromophore/PEG4/biotin moiety by their
absorbency at A354.
[0054] FIG. 22: Structure of a thiol-reactive chromophore linker of
the present invention (7), aldehyde-reactive chromophore linker of
the present invention (8) and an oxidized carbohydrate-reactive
chromophore linker of the present invention (9);
[0055] FIG. 23: A schematic representation of the incorporation of
a conjugationally extended aldehyde cytosine triphosphate 10 in a
DNA amplicon (R.dbd.H) or RNA amplicon (R.dbd.OH) and labeling the
modified amplicon with a linker of the present invention 11 and
[0056] FIG. 24: Schematic representation of the synthesis of a
linker of the present invention (11).
DETAILED DESCRIPTION OF THE INVENTION
[0057] Unless defined otherwise, all terms used herein have the
same meaning as are commonly understood by one of skill in the art
to which this invention belongs. All patents, patent applications
and publications referred to throughout the disclosure herein are
incorporated by reference in their entirety. In the event that
there is a plurality of definitions for a term herein, those in
this section prevail.
[0058] The term "biomolecule" as used herein refers to a compound
of biological origin, or of biological activity, that may have, or
may be modified to have, an amine group or carbonyl group that may
be harnessed in the formation of a hydrazone bond with a novel
carbonyl profluorophore or novel hydrazine profluorophore of the
present invention. Biomolecules include for example a nucleic acid,
a nucleotide, a protein, an amino acid, a carbohydrate monomer and
a polysaccharide. If the biomolecule is a nucleic acid it may be
DNA, cDNA, RNA, or PNA and may comprise natural or unnatural bases
or internucleotide linkages such as for example phosphodiesters,
phosphorothioates, phosphoramidites or peptide nucleic acids.
[0059] The term "profluorophore" as used herein refers to a
compound that may, or may not fluoresce, but when joined with its
corresponding profluorophore pair compound produces a fluorescent
hydrazone compound that has a peak emission wavelength
substantially separate from the peak emission wavelength of either
of the profluorophores that they may that make up the fluorescent
hydrazone compound. A profluorophore pair comprises a
hydrazine-based profluorophore and a carbonyl-based profluorophore
that when combined form a fluorescent hydrazone compound.
[0060] The term "pro-chromophore" as used herein refers to a
compound that may, or may not produce a visible color, but when
joined with its corresponding pro-chromophoric pair compound
produces a chromophoric compound that has a peak observable
wavelength substantially separate from the peak observable
wavelength of either of the prochromophores that make up the
chromophoric hydrazone compound. A pro-chromophoric pair comprises
a hydrazine-based pro-chromophore and a carbonyl-based
pro-chromophore that when combined form a chromophoric hydrazone
compound.
[0061] The term "reactive linking moiety" as used herein refers to
molecules used commercially for binding one molecule to another
based on the presence of a particular chemical group on the
molecule of interest. Some commercially sold molecules referred to
herein as linking moieties include those that react with free
amines on the target molecule, such as N-hydroxysuccinimidyl,
p-nitrophenyl, pentafluorophenyl and N-hydroxybenzotriazolyl ester
and those that react with free sulfhydryls present on the target
molecule such as maleimido, .alpha.-haloacetamido and
pyridyldisulfides.
[0062] The term "ligand/receptor couple" as used herein refers to a
pair of molecules having a substantially high affinity of binding
specifically to one another. One example of such a binding pair
would be a receptor on a cell and the ligand that binds that
receptor. Another example would be biotin and avidin, which are two
molecules that have a strong affinity for binding each other having
an association constant of around 10.sup.15. Other pairs include
Peptide S and ribonuclease A, digoxigenin and it receptor and
complementary oligonucleotide pairs.
[0063] To achieve the optimal signal from a fluorescent label it is
important that the structural integrity of the fluorophore is
retained throughout processing of the labeled reporter molecule. A
disadvantage with commercially available fluorophores is their
propensity to be hydrolytically unstable or photobleach. The
ability to efficiently form fluorescent species in situ in
biological media in contrast to present methods wherein a labile
fluorescent species is present throughout all protocols would be
extremely advantageous in yielding products with fully retained
fluorescence for improved limits of detection. In one current
example in DNA microarrays, fluorescently labeled triphosphates,
e.g. Cy3 and Cy5 triphosphosphates (Amersham Biosciences,
Piscataway, N.J.), are incorporated during PCR or reverse
transcriptase amplification however quenching of the fluorophores
through photobleaching or hydrolysis occurs during the many
manipulations required to isolate the desired fluorescently labeled
polynucleotide. To overcome this problem a less than ideal two-step
method has been developed wherein a 3-aminoallylcytidine
triphosphate is incorporated during polynucleotide amplification
with subsequent purification, labeling with fluorescent
succinimidyl esters and final purification to remove excess
unincorporated fluorescent molecules. This chemistry is based on a
amino/succinimidyl ester reaction that requires large excess of
succinimidyl ester due to its instability in water and steps to
remove the excess hydrolyzed reagent. This reaction proceeds over a
small pH range, i.e. 7.2-8.0 and is concentration dependent.
[0064] It would be advantageous to have a method wherein a stable
non-fluorescent species is used to label a biomolecule that
following all required processing in techniques such as PCR,
2-dimensional electrophoresis or immunohistochemistry can be
reacted efficiently with a second non-fluorescent molecule to form
a fluorescent species. The present invention describes a chemistry
wherein a conjugationally extended hydrazine reacts with a
conjugationally extended carbonyl in situ in aqueous media to form
a fluorescent molecule (FIG. 1). Both aldehydes and hydrazines are
stable in aqueous media and react efficiently to form stable
hydrazones. The hydrazone formation is acid catalyzed and has an
optimum pH of 4.7 but proceeds up to pH 8.0. This methodology could
be extended to use with biosensors for biowarfare and pathogen
detection, brand security and Near-IR products. These fluorophores
may also be engineered for use in laser and photonics
applications.
[0065] D. E. Ryan, F. Snape and M. Winpe (Ligand Structure and
Fluorescence of Metal chelates; N-Heterocyclic Hydrazones with
Zinc, Anal, Chim. Acta 58:101, 1972) described a series of
hydrazone chelates (Table 1) and that upon addition of Zn.sup.2+
the chelates complex the metal yielding a fluorescent metal chelate
(FIG. 2). It was postulated how the non-complexed chelate can exist
in two different tautomers that have different fluorescent
properties due to disrupted aromatic bonding. The addition of the
zinc ion `locks in` the tautomer with better conjugation and higher
fluorescence. These authors further described the use of these
chelates as analytical tools for determination of trace amounts,
i.e. parts per million and parts per billion, of zinc.
TABLE-US-00001 Abbreviated Relative Full Name Form
.lamda..sub.excitation .lamda..sub.emission Fluorescence*
Pyridine-2-aldehyde-2-pyridyl hydrazone PAPH 455 515 1
Quinoline-2-aldehyde-2-pyridylhydrazone QAPH 490 540 2
Phenanthridine-2-aldehyde-2-pyridylhydrazone PDAPH 490 545 7
Pyridine-2-aldehyde-2-quinolylhydrazone PAQH 470 535 660
Quinoline-2-aldehyde-2-quinolylhydrazone QAQH 495 595 30
Phenanthridine-2-aldehyde-2- PDAQH 525 610 16 quinolylhydrazone
Pyridine-2-aldehyde-2- PAPDH 450 540 100 phenanthrdinylhydrazone
Quinoline-2-aldehyde-2- QAPDH 510 600 110 phenanthrdinylhydrazone
Phenanthridine-2-aldehyde-2- PDAPDH 580 620 230
phenanthrdinylhydrazone Benzimidazole-2-aldehyde-2-pyridylhydrazone
BAPH 440 510 140 470B550enzimldazole-2-aldehyde-2- BAQH 470 520
2000 quinolylhydrazone Benzimidazole-2-aldehyde-2- BAPDH 480 530
440 phenanthrdinylhydrazone
Phenyl-2-pyridylketone-2-pyridylhydrazone PPKPH 420 470 8
Phenyl-2-pyridylketone-2-quinolylhydrazone PPKQH 470 550 450
Phenyl-2-pyridylketone-2- PPKPDH 490 575 1520
phenanthrdinylhydrazone
Table 1 lists the bis-(2-heteroaromatic)hydrazones prepared by Ryan
et al, supra. and including their excitation and emission
wavelengths and relative fluorescence properties.
[0066] Bifunctional hydrazine and carbonyl reagents to modify
biomolecules have been prepared. FIG. 3 outlines this chemistry.
The hydrazine/carbonyl bioconjugation couple has significant
advantages over currently used maleimido/thiol couple in that both
the aldehyde and hydrazine moieties are stable following
incorporation on biomolecules, simple addition of an
aldehyde-modified biomolecule to a hydrazine-modified biomolecule
yields a stable hydrazone without the requirement of a reduction
reaction to stabilize the bond, the stability of the functional
groups allows conjugations to be performed at low concentrations,
i.e. <100 microgram/mL and the chemistry has been engineered to
prepare conjugates from all biomolecules.
[0067] FIG. 4 shows the conjugation of an 5'-[4
formalbenzamide]-modified oligonucleotide to a hydrazine-modified
antibody. The results demonstrate complete conversion of modified
protein to conjugate by the simple addition of the stable 5'-[4
formalbenzamide]-modified oligonucleotide to the modified-hydrazine
modified protein forming a stable hydrazone mediated conjugate.
[0068] The linkers have been prepared as reagents for the solid
phase syntheses of peptides (hydrazino carboxylic acids) and
oligonucleotides (aldehyde phosphoramidites). Aldehyde-modified
deoxy and ribo-triphosphates have also been prepared and
demonstrated to be incorporated into polynucleotide amplicons.
[0069] In the initial demonstration of the fluorescence of
conjugationally extended hydrazones, 6-hydrazinonicotinic acid (1)
(Solulink Biosciences, San Diego, Calif.) was reacted with
4-dimethylcinnamaldehyde (2) (Aldrich Chemical Co., Milwaukee,
Wis.) to yield fluorescent hydrazone (3) (FIG. 5). Hydrazone (3)
absorbed at 397 nm and emitted at 508 nm a Stokes shift of 109 nm.
Other hydrazones prepared from commercially available
conjugationally extended hydrazines and aldehydes were prepared and
their respective excitation and emission wavelengths are presented
in Table 2 below. It should be noted that the Stokes shifts for
hydrazones 2, 3 and 4 all are 100 nm or greater.
TABLE-US-00002 absorbance emission nm nm ##STR00002## 385 407
##STR00003## 355 472 ##STR00004## 397 508 ##STR00005## 450 550
Table 2 shows the fluorescent hydrazones and their absorbance and
emission maxima.
[0070] In another demonstration benzaldehyde phosphoramidite has
been prepared that is used to incorporate benzaldehyde moieties
directly on the 5'-end of oligonucleotides during solid phase
oligonucleotide synthesis. The incorporation of this moiety is
accomplished with similar identical procedures and yields as
incorporation of DMT-amino modified phosphoramidites. Reaction of
an oligonucleotide with trans-4'-hydrazino-2-stilbazole
dihydrochloride quantitatively yields a fluorescent oligonucleotide
(FIG. 6). The emission and absorbance spectra of hydrazone (4) (see
Table 2 above) linked to a 22mer oligonucleotide are presented in
FIG. 7.
[0071] Methods have been developed to prepare both hydrazino- and
hydrazido-modified oligonucleotides. Hydrazinopyridine-modified
oligonucleotides can be prepared by the reaction of amino-modified
oligonucleotides with SANH and hydrazido-modified oligonucleotides
can be prepared using SHTH (FIG. 8). To demonstrate that hydrazones
prepared from conjugationally extended hydrazines but not
conjugationally extended hydrazides both oligonucleotides were
reacted with 4-dimethylaminocinnamaldehyde (FIG. 9, reactions A and
C) but only the hydrazine derived hydrazone was fluorescent. In
another demonstration both hydrazino- and hydrazido-modified
oligonucleotides were reacted with 1,2-naphthalene-dicarboxaldehyde
(NDA; reactions B and D). It is known that amines react with NDA
yield a fluorescent species. The products from the reaction of
these oligonucleotides were both fluorescent however the hydrazine
derived product absorbed and emitted qualitatively more intensely
and at longer wavelengths than the hydrazido-modified
oligonucleotide.
[0072] In another demonstration salmon sperm DNA was treated with
hydrazine/bisulfite to convert cytidine moieties to
4-N-aminocytidine, an aromatic hydrazine (FIG. 10; Negishi, K.,
Harada, C., Ohara, Y., Oohara, K., Nitta, N. and Hayatsu, H.,
4-N-aminocytidine, a nucleoside analog that has an exceptionally
high mutagenic activity, Nucleic Acids Res. 1983, 11, 5223-33)).
The reaction of the modified DNA with both
4-dimethylaminocinnamaldehyde and naphthalene-1,2-dicarboxaldehyde
(NDA) yielded fluorescent DNA. (FIG. 11).
[0073] It should be noted that the hydrazine-modified cytidine is a
component of the fluorophore and not solely a linkage point. It is
anticipated that conjugationally extended aldehydes that yield
hydrazones with more intensely fluorescent properties can be
developed to convert reverse transcribed DNA to fluorescent species
thereby using all natural triphosphates in the reverse
transcription reaction and not substituted triphosphates whose
incorporation is random and not quantitatively reproducible batch
to batch.
[0074] A library of hydrazone fluorophores may be prepared from
commercially available aromatic hydrazines and aldehydes using the
methods described. FIG. 12 below presents structures of
commercially available hydrazines that will be purchased to be
reacted to form hydrazone fluorophores.
[0075] FIG. 13 presents structures of commercially available
aldehydes that will be purchased to be reacted to form hydrazone
fluorophores.
[0076] The initial pro-fluorophore structures targeted for
syntheses in this program are based on cyanine dyes. These dyes are
extremely sensitive and have been developed for a variety of
commercial uses including life sciences applications as well as
photographic uses (A. Mishra, R. K. Behera, P. K. Behera, .K.
Mishra and G. B. Behera, Cyanines during the 1990's: A Review,
Chem. Rev., 100:1973, 2000). FIG. 14 below presents the structures
of the most used cyanine dyes, Cy3 and Cy5, for life science
applications. These dyes are routinely used as reporter molecules
in both gene and protein microarrays.
[0077] FIG. 15 presents aldehyde and hydrazine cyanine-based
profluorophores and their parent fluorophores targeted for
synthesis.
[0078] Two methods have been developed for the preparation of
hydrazino-substituted aromatic compounds (FIG. 16). The classical
method for the synthesis of 2-hydrazinoheteroaromatic compounds is
direct nucleophilic aromatic substitution of 2-chloro-heterocycles
with hydrazine. Arterburn et al. (J. B. Arterburn, K. V. Rao, R.
Ramdaa and B. R. Dible, Org. Lett, 2001, 3, 1351 and J. B.
Arterburn, B. D. Bryant and D. Chen, Chem. Comm. 2003, 1890) have
developed palladium-catalyzed protocols to convert 2-substituted
bromo, chloro and trifluoro substituted pyridines to
2-hydrazinylpyridines.
[0079] Aromatic aldehydes can be prepared by a variety of methods
including direct oxidation of methyl-substituted aromatic moieties
and reduction of aromatic nitriles. Aromatic aldehydes can be
conjugationally extended using the Mannich reaction.
[0080] Due to the fluorescence of benzimidazole-quinoline hydrazone
(5) a variety of pro-fluorophores based on this parent core
structure have been investigated. FIG. 17 presents target
pro-fluorophores and their respective parent fluorophores.
[0081] Diverse libraries with varying fluorescent properties can be
readily prepared as any carbonyl and any hydrazine prepared or
commercially available can be combined to yield a fluorescent
hydrazone. The excitation and emission characteristics desired can
be tailored by incorporation of substituents such as dimethylamino,
alkoxy and nitro groups.
[0082] The photophysical characteristics of the fluorophores may be
observed using a QM-2 Spectrofluorimeter (Photon Technologies
International, Inc.), with a nitrogen-dye laser/second harmonic
generator excitation source. A Xe arc lamp may be utilized having
excitation that allows for the collection of steady state
excitation and emission spectra, the characterization of quantum
yield, photo-bleaching, and an degradation of fluorescence from
these species. The response of this instrument may be characterized
by fluorescence quantum yield standards (i.e. quinine sulfate) to
determine the quantum yield of the various fluorophores. The laser
system with the laser-strobe detection attachment allows for the
collection of sub nanosecond time-decays. The time decay curves may
be analyzed to determine the excited-state lifetimes of these
fluorophores.
[0083] In addition a Nd:YAG laser pumped OPO system, will allow for
tunable excitation between 400 nm and 3000 nm. The detection system
includes a Jobin-Yvon 0.5 m monochromator with both PMT and CCD
detection. The CCD camera is sensitive in the visible and Near
Infrared regions of the electromagnetic spectrum. This system may
be used for the characterization of fluorophores in the far-red
region of the visible spectrum and in the NIR region. The tunable
excitation will provide a means to excite fluorophores, regardless
of their absorption spectra in the visible/NIR regions
[0084] The stability of the commercially available fluorophores has
limited the full range of development of a variety of applications.
The advantageous characteristics of this technology includes:
elimination of the need to remove the excess second moiety from the
in situ formed fluorescent species as it is either not fluorescent
or has completely different fluorescent properties that do not
interfere with detection of the new fluorescent species; increased
efficiency of the formation of the fluorescent species >90%, in
buffered aqueous media, pH 5.0-8.0.; the ability to prepare a wide
variety of fluorophores of different absorbance and emission
wavelengths by varying the structures of the two moieties of the
final fluorescent molecule; utilizing a linker moiety that may be
incorporated on either of the pro-fluorescent species for covalent
linking to a biomolecule or a surface; significant reduction in
photobleaching or increased hydrolytic stability of the initial
pro-fluorophore as has it will be in a lower energy state than
fully conjugated fluorophores currently employed; and the
development of fluorescent species having well separate spectral
absorbance and emission properties, i.e. a Stoke's shift >100
nm.
[0085] U.S. patent application Ser. No. 60/546,104 to Schwartz
incorporated herein in its entirety has described the in situ
preparation of hydrazone fluorophores by the reaction of a
conjugationally extended aldehyde with a conjugationally extended
hydrazine one of which is linked to biomolecular probe such as an
antibody or an oligonucleotide. FIG. 19 presents the reaction
scheme for the reaction of a conjugationally extended hydrazine
with a conjugationally extended aldehyde linked to an
oligonucleotide forming an oligonucleotide linked fluorescent
hydrazone. The scheme also presents results that demonstrated that
the reaction is specific for a conjugationally extended hydrazine
and not a hydrazide. In contrast to forming
chromophore/fluorophores in situ the present invention incorporates
a pre-formed chromophoric/fluorescent hydrazone into the linker
comprising the ligand for direct spectrophotometric quantitation of
the level of incorporation of the ligand when bound to a
biomolecule such as a protein or nucleic acid.
[0086] FIG. 20 presents the construction of an amino-reactive
biotin moiety that has incorporated in its chain a chromophoric
hydrazone for spectrophotometric quantitiation and a short PEG
linker that is required to retain the binding affinity of biotin to
streptavidin. This tri-functional molecule can be readily
quantified spectrophotometrically following conjugation to a
biomolecule because of its unique molar extinction coefficient
(generally >20000) and its unique absorbance or fluorescence
(generally at wavelengths greater than 300 nm and at frequencies
having no, or only minimal, observable signals prior to
conjugation). It is anticipated that more highly conjugated systems
than presented in FIG. 20 will absorb at longer wavelengths with
greater extinction coefficients or fluorescence allowing even
greater sensitivity. FIG. 21 presents constructions of thiol and
oxidized carbohydrate-reactive linkers of the present
invention.
[0087] The incorporation of labels into nucleic acids such as cDNA
or cRNA using polymerases and reverse transcriptases respectively
for gene expression analysis by microarrays is a multi-step
procedure that requires high levels of reproducibility so results
can be reliably compared between experiments. One current method
for labeling cDNA or cRNA is the use of a nucleoside modified to
incorporate a biotin molecule on the minor groove side. One of the
most commonly used methods to label and detect labeled cDNA and
cRNA is using a biotinylated nucleoside triphosphate (NTP). As
there are only labor-intensive methods to quantitate the level of
biotin incorporation in the amplicon, the biotin-modified amplicon
is used directly without quantitation. It would be extremely
advantageous to be able to directly quantitate the level of biotin
incorporated into cDNA or cRNA. FIG. 22 is a schematic diagram of
the synthesis of a nucleoside triphosphate modified with a
conjugationally extended aldehyde such as a benzaldehyde moiety and
to label the amplicon after elongation by reaction with a
biotinylated conjugationally extended hydrazine. U.S. Pat. No.
6,686,461 to D. Schwartz and R. Hogrefe which is incorporated
herein by reference in its entirety more fully discloses this
synthesis. The chemistry described herein is advantageous in that
the formation of the hydrazone is high yielding at near
stoichiometric amounts, a chromophore is formed that will allow
batch-to-batch quantitiation of levels of incorporation of biotin
and a short polyethylene linker is incorporated is necessary to
retain the affinity of the biotin to its cognate receptor
avidin.
[0088] In another protocol the amplicon may be hybridized prior to
reaction with the biotin hydrazide and subsequently detected with a
fluorescently-labeled avidin or anti-biotin antibody. The
benzaldehyde-labeled amplicon can be quantitated by removing an
aliquot and treating it with a hydrazide pro-fluorophore to form a
fluorescent hydrazone and spectrophotometrically quantitating the
level of aldehyde incorporation. This may be advantageous as the
hybridization reaction will have minimal modification resulting in
less sterically encumbered hybridization.
[0089] In use the linker moiety reacts with a biomolecule such as
an antibody under appropriate reaction conditions. The conjugate is
then purified and the protein concentration determined. The number
of biotin molecules/protein molecule is determined by observing the
absorbance of a known concentration of the conjugate in solution at
a wavelength>300 nm. The concentration of the chromophore and
therefore the biotin is determined by dividing the absorbance
reading by the extinction coefficient of the chromophore
incorporated in the chain. This concentration is divided by the mM
concentration of the protein and the number of biotin molecules per
conjugated is determined.
EXAMPLES
Example 1
Synthesis of Biotin/PEG/hydrazone succinimidyl ester 6 (FIG.
20)
[0090] PMR spectra were obtained on a Bruker 500 MHz NMR at NuMega
Laboratories (San Diego, Calif.) and electrospray mass spectral
data was obtained at HT Laboratories (San Diego, Calif.),
1. Synthesis of Mono-Boc-1,13-diamino-4,7,10-trioxatetradecane (1;
(3-{2-[2-(3-Amino-propoxy)-ethoxy]-ethoxy}-propyl)-carbamic acid
tert-butyl ester), Amine 1
[0091] To a solution of 4,7,10-trioxa-1,13-tridecanediamine (FIG.
20) (30 g; mmol) in dichloromethane (1000 mL) was added a solution
of di-t-butyl dicarbonate (10 g; mmol; Aldrich Chemical Co.,
Milwaukee, Wis.) in dichloromethane (200 mL) over 2 h. The reaction
mixture was stirred at room temperature for 4 hours. Thin layer
chromatography (TLC, silica gel) using
dichloromethane/methanol/triethylamine (90/10/1); ninhydrin
development) indicated the presence of two new spots, a minor spot
at Rf 0.8 ascribed to the bis-BOC product and a major spot at Rf
(0.2) for the desired product. The reaction mixture was washed with
water (4.times.500 mL) to remove the excess diamine and the organic
phase was dried over magnesium sulfate, filtered and concentrated
to give a viscous oil that was purified by flash chromatography
over silica gel using DCM/MeOH/TEA (95/5/1) to give 10.5 g of
desire Amine 1 as an oil.
2. Synthesis of
((3-{2-[2-(3-{[6-(N'-Isopropylidene-hydrazino)-pyridine-3-carbonyl]-amino-
}-propoxy)-ethoxy]-ethoxy}propyl)-carbamic acid tert-butyl ester),
Hydrazone 2
[0092] To a solution of Amine 1 (1.05 g; 3.28 mmol) in DCM (20 mL)
was added a solution of succinimidyl 6-hydrazinonicotiniate acetone
hydrazone (0.951 g; 3.28 mmol; Solulink Biosciences, Inc., San
Diego, Calif.) in DCM (10 mL). The reaction mixture was stirred at
room temperature for 6 hours. Subsequently the reaction mixture was
washed with water and brine. The organic phase was dried (magnesium
sulfate), filtered and concentrated to give 1.2 g of Hydrazone 2 as
a colorless thick oil.
3. Synthesis of
(4-{[5-(3-{2-[2-(3-tert-Butoxy-carbonylamino-propoxy)-ethoxy]-ethoxy}-pro-
pylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzoic acid),
Hydrazone 3
[0093] To Hydrazone 2 (0.405 g: 0.81 mmol) in MeOH (5 mL) and 100
mM MES, 150 mM NaCl (5 mL) was added a solution of
4-carboxybenzaldehyde (0.121; 0.81 mmol) in MeOH (3 mL). The
reaction mixture is allowed to stir at room temperature overnight.
Copious precipitate formed. The reaction mixture was centrifuged
and the solids were washed with a 1/1 solution of MeOH/MES. The
solids were dried under vacuum to yield 0.42 g of Hydrazone 3 as a
pale yellow solid and used directly in the next step.
4. Synthesis of
(4-{[5-(3-{2-[2-(3-Amino-propoxy)-ethoxy]-ethoxy}-propylcarbamoyl)-pyridi-
n-2-yl]-hydrazonomethyl}-benzoic acid hydrochloride salt),
chromophore Hydrazone 4
[0094] A solution of Hydrazone 3 (0.388 g; 0.66 mmol) in dioxane
(15 mL) was prepared with heating. The solution was cooled to room
temperature and 4 N HCl in dioxane (4 mL; Aldrich Chemical Co.,
Milwaukee, Wis.) was added succinimidyl and the reaction was
stirred at room temperature for 16 h. A precipitate formed on
stirring. The reaction mixture was centrifuged and the solids were
washed with dioxane (3.times.10 mL). The solids were resuspended in
dioxane and concentrated under vacuum to yield 240 mg of
amino/PEG4/Hydrazone 4 as a pale yellow solid. Electrospray mass
spec: expected m/e 487. found positive mode 488 (M+H), negative
mode 486 (M-H) and 522 (M+Cl.sup.-).
5. Synthesis of Biotin/PEG4/chromophore succinimidyl ester 6
(5-(N'-{4-[2-(2,5-Dioxo-pyrrolidin-1-yl)-2-oxo-acetyl]-benzylidene}-hydra-
zino)-pyridine-2-carboxylic acid
{3-[2-(2-{3-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamin-
o]-propoxy}-ethoxy)-ethoxy]-propyl}-amide)
[0095] To a solution of amino/PEG4/hydrazone 4 (0.780 g; 1.60 mmol)
in DMF (25 mL) was added biotin succinimidyl ester (0.546 g; 1.60
mmol) followed by the addition of triethylamine (0.726 mL; 4.80
mmol). The solution was stirred at room temperature until complete
as determined by silica gel TLC using DCM/MeOH/TEA (90/10/1) as
eluant (developed by UV to visualize the pyridine chromophore and
dimethylaminocinnamaldehyde/sulfuric acid/ethanol spray followed by
heating to visualize the biotin moiety). To the reaction mixture
N-hydroxysuccinimide (0.184 g; 1.60 mmol) and DCC (0.330 g; 1.60
mmol) were added and stirred at room temperature for 16 hours. The
reaction mixture was concentrated to dryness and partitioned
between DCM and water. The organic phase was further washed with
brine, dried (magnesium sulfate), filtered and concentrated to give
a yellow sticky solid. The solids were triturated with ethyl
acetate. The solids were isolated by filtration to give 830 mg of a
yellow solid. TLC (DCM/MeOH/TEA (90/10/1) indicated one major spot
(visualized by UV and dimethylaminocinnamaldehyde/sulfuric
acid/ethanol solution) and HPLC analysis (YMC C-18, 150.times.4.6
cm; 5 .quadrature.m; 120 A; gradient mobile phase A:
water/acetonitrile/trifluoroacetic acid (20/80/0.1), mobile phase
B: 0.1% TFA in water; gradient 10% A/90% B to 100% A over 20 min;
retention time 8.8 min, detection @A254 and A350. PMR
(DMSO-d.sub.6) .delta.: 11.64, s (1H), 8.65, d, (1H), 8.37 t, (1H)
NH, 8.12 dd (1H), 7.95 and 8.11 ab system (4H), 7.73 t (1H) NH,
7.36 d (1H), 6.41 s (1H), 6.35 s (1H), 5.57 d (1H), 4.29 br. t
(1H), 4.11 br. t (1H), 3.3-3.55 m (12H), 3.08 m (4H), 2.90 s (4H),
2.88 dd (1H), 2.57 d (1H), 2.03 t (2H), 1.75 m (2H), 1.59 m (2H),
1.2-1.5 m (8H). The extinction coefficient of
Biotin/PEG4/chromophore succinimidyl ester 6 was determined by
dissolving Biotin/PEG4/chromophore succinimidyl ester 6 (1.0 mg) in
DMF (1 mL) and diluting into PBS. The absorbance maximum was A354
and the molar extinction coefficient was determined to be
23,250.
Example 2
Protein labeling with Biotin/PEG4/chromophore/succinimidyl ester
6
[0096] Bovine immunoglobulin (bIgG; Sigma Chemical Co., St. Louis,
Mo.) was dissolved in modification buffer (100 mM phosphate, 150 mM
NaCl, pH 7.2) to prepare a 5 mg/mL solution. A solution of
Biotin/PEG4/chromophore/succinimidyl ester 6 (1 mg) dissolved in
DMF (100 mL) was prepared. Three separate reactions were performed
wherein 5 mole equiv., 10 mol equiv. and 15 mol equiv. of
Biotin/PEG4/chromophore/succinimidyl ester 6 (1.3, 2.6 and 3.9
.mu.L,) respectively were added to 0.5 mg bIgG solution. The
reaction was allowed to incubate at room temperature for 2 hours.
The reaction mixtures were desalted into PBS using Biomax
diafiltration apparatuses (Millipore, Inc., Bedford, Mass.).
Protein concentrations of all the modified proteins were determined
using the BCA assay (Pierce Chemical Co., Rockford, Ill.). Spectral
analyses of each product were performed by diluting 20 mg of
modified protein to 100 mL in PBS. The number of moles of
chromophore incorporated was calculated by determining the
absorbance of the protein at A354 dividing by the molar extinction
coefficient, i.e. 29000, of the chromophore. The overlaid spectra
of the products as well as unmodified IgG are present in FIG. 21A.
The number of incorporated biotins in the modified proteins was
further analyzed by the HABA assay (Pierce Chemical Co., Rockford,
Ill.). The results, both tabular and graphically, from both the UV
spectral assay and the HABA assay are presented below.
TABLE-US-00003 IgG/HABA IgG/A354 5X 1.03 2.45 10X 1.60 4.71 15X
2.22 6.25
A further experiment to demonstrate retention of binding activity
of the chromophore/biotinylated bIgG the modified proteins were
incubated with streptavidin and the reaction products were analyzed
by PAGE gel electrophoresis. FIG. 21B presents the results.
Example 3
Synthesis of Biotin/PEG/hydrazone 10 (FIG. 24)
1. Synthesis of
({3-[2-(2-{3-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylami-
no]-propoxy}-ethoxy)-ethoxy]-propyl}-carbamic acid tert-butyl
ester),1-biotinamido/PEG/BOC-amino 14
[0097] To a solution of Amine 1 (0.544 g; 1.70 mmol) in DMF (15 mL)
was added a solution of biotin succinimidyl ester (0.580 g; 1.70
mmol) in DMF followed by the addition of TEA (0.75 mL; 5.09 mmol).
The reaction mixture was stirred at room temperature for 16 h. The
solvent was removed on the rotavap and the residue was partitioned
between DCM and water. The organic phase was further washed with
brine, dried (magnesium sulfate), filtered and concentrated to give
415 mg of 1-biotinamido/PEG/BOC-amino 14 as an amorphous solid. The
product was a single spot by TLC (DCM/MeOH/TEA (90/10/1); developed
by dimethylcinnamaldehyde/ethanol/sulfuric acid/heat to visualize
the biotin moiety). The product was used directly in the next
step.
2. Synthesis of
(5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic acid
(3-{2-[2-(3-amino-propoxy)-ethoxy]-ethoxy}-propyl)-amide),
1-biotinamido/PEG/amino 15
[0098] To a solution of 1-biotinamido/PEG/BOC-amino 14 (400 mg;
0.73 mmol) was dissolved in dioxane (20 mL) with mild heating. The
solution was cooled to room temperature and a solution of 4 N HCl
in dioxane (10 mL; Aldrich Chemical Co., Milwaukee, Wis.) was
added. The reaction was stirred for 14 h. The solvent was removed
on the rotavap and the residue was co-evaporated twice from dry
dioxane. The product, 1-biotinamido/PEG/amino 15, was used directly
without purification.
3. Synthesis of (5-(N'-Methylene-hydrazino)-pyridine-2-carboxylic
acid
{3-[2-(2-{3-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamin-
o]-propoxy}-ethoxy)-ethoxy]-propyl}-amide),
1-biotinamido/PEG/amido-6-hydrazino-4-nicotinamide 11
[0099] To a solution of 1-biotinamido/PEG/amino 15 (0.375 g; 0.78
mmol) in DMF (25 mL) was added a solution of SANH (0.225 g; 0.78
mmol) and triethylamine ((0.645 mL; 4.66 mmol)). The reaction
mixture was stirred at room temperature for 16 h. The solvent was
removed on the rotavap and the residue was partitioned between DCM
and water. The organic phase was further washed with brine, dried
(magnesium sulfate), filtered and concentrated to give 290 mg of
1-biotinamido/PEG/amido-6-hydrazino-4-nicotinamide 11 as an
amorphous solid. The product was a single spot, Rf 0.33, by TLC
(DCM/MeOH/TEA (90/10/1) developed by
dimethylcinnamaldehyde/ethanol/sulfuric acid/heat to visualize the
biotin moiety). Mass spectral data: exptd m/e 621; pos mod exptd
m/e 622 (M+H). found 622 and exptd 644 (M+Na). found 644; neg mode
exptd m/e (M-H) 620. found 620 and (M+Cl.sup.-) 656. found 656
* * * * *