U.S. patent application number 16/381831 was filed with the patent office on 2019-12-05 for preparation of oligo conjugates.
The applicant listed for this patent is Emerald Therapeutics, Inc.. Invention is credited to Brian M. Frezza, Daniel J. Kleinbaum, Courtney E. Webster.
Application Number | 20190367554 16/381831 |
Document ID | / |
Family ID | 51488584 |
Filed Date | 2019-12-05 |
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United States Patent
Application |
20190367554 |
Kind Code |
A1 |
Frezza; Brian M. ; et
al. |
December 5, 2019 |
PREPARATION OF OLIGO CONJUGATES
Abstract
Conjugated molecules are prepared that comprise a predetermined
number of oligo conjugation components. The conjugated molecules
also may comprise one or more detectable labels. Preparation of
these molecules can be implemented according to an asymmetric or a
symmetric conjugation strategy.
Inventors: |
Frezza; Brian M.; (Redwood
City, CA) ; Webster; Courtney E.; (South San
Frrancisco, CA) ; Kleinbaum; Daniel J.; (Redwood
City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Emerald Therapeutics, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
51488584 |
Appl. No.: |
16/381831 |
Filed: |
April 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15042064 |
Feb 11, 2016 |
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16381831 |
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13790922 |
Mar 8, 2013 |
9289502 |
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15042064 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y10S 977/932 20130101;
C07H 21/00 20130101; A61K 47/549 20170801; C07K 14/003 20130101;
C07H 21/04 20130101 |
International
Class: |
C07H 21/04 20060101
C07H021/04; C07H 21/00 20060101 C07H021/00; C07K 14/00 20060101
C07K014/00 |
Claims
1. A method of preparing a compound of the formula ##STR00226##
comprising (i) attaching a conjugation component of the formula
##STR00227## wherein R.sup.2 is R.sup.2a or R.sup.2ap, to a solid
support ##STR00228## to form a compound of the formula ##STR00229##
(ii) when R.sup.2 is R.sup.2ap, converting ##STR00230## to
##STR00231## and (iii) contacting ##STR00232## with a conjugation
component of the formula ##STR00233## wherein R.sup.3 is R.sup.3a,
R.sup.3ap, hydrogen, a detectable label or an oligo; to form
##STR00234## wherein: ##STR00235## is a solid support material;
##STR00236## and are independently an oligo; R.sup.1a and R.sup.1b
are complementary conjugation functionalities and L.sup.1 is
conjugate linker, and R.sup.1a, R.sup.1b, and L.sup.1 are (a)
R.sup.1a is azido, R.sup.1b is --C.ident.C--CR.sup.23, and L.sup.1
is ##STR00237## or (b) R.sup.1a is --NHR.sup.23, R.sup.1b is
carboxy, and L.sup.1 is --NR.sup.23C(.dbd.O)--, or (c) R.sup.1a is
carboxy, R.sup.1b is --NHR.sup.23, and L.sup.1 is
--C(.dbd.O)NR.sup.23--, or (d) R.sup.1a is --NHR.sup.23, R.sup.1b
is halo, and L.sup.1 is --NR.sup.23--, or (e) R.sup.1a is
--O--P(.dbd.O)(OH)(X), R.sup.1b is hydroxy, and L.sup.1 is
--O--P(.dbd.O)(OH)--O--, or (f) R.sup.1a is --O--P(.dbd.O)(OH)(X),
R.sup.1b is --NHR.sup.23, and L.sup.1 is --O--P(.dbd.O)(OH)--
NR.sup.23--, or (g) R.sup.1a is --O--P(.dbd.O)(OH)(X), R.sup.1b is
thio, and L.sup.1 is --O--P(.dbd.O)(OH)--S--, or (h) R.sup.1a is
halo, R.sup.1b is thio, and L.sup.1 is --S--, or (i) R.sup.1a is
##STR00238## R.sup.1b is thio, and L.sup.1 is ##STR00239## or (j)
R.sup.1a is --C.ident.C--R.sup.23, R.sup.1b is azido, and L.sup.1
is ##STR00240## or (k) R.sup.1a is halo, R.sup.1b is --NHR.sup.23,
and L.sup.1 is --NR.sup.23--, or (l) R.sup.1a is hydroxy, R.sup.1b
is --O--P(.dbd.O)(OH)(X), and L.sup.1 is --O--P(.dbd.O)(OH)--O--,
or (m) R.sup.1a is --NHR.sup.23, R.sup.1b is --O--P(.dbd.O)(OH)(X),
and L.sup.1 is --NR.sup.23--P(.dbd.O)(OH)--O--, or (n) R.sup.1a is
thio, R.sup.1b is --O--P(.dbd.O)(OH)(X), and L.sup.1 is
--S--P(.dbd.O)(OH)--O--, or (o) R.sup.1a is --O--P(.dbd.O)(OH)SH,
R.sup.1b is --X, and L.sup.1 is --O--P(.dbd.O)(OH)--S--, or (p)
R.sup.1a is --X, R.sup.1b is --O--P(.dbd.O)(OH)SH, and L.sup.1 is
--S--P(.dbd.O)(OH)--O--, or (q) R.sup.1a is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, R.sup.1b is
--NHR.sup.23, L.sup.1 is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)NR.sup.23--, or (r) R.sup.1a is
--NHR.sup.23, R.sup.1b is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, L.sup.1 is
--NR.sup.23C(.dbd.O)--(CR.sup.25R.sup.25).sub.s--, or (s) R.sup.1a
is thio, R.sup.1b is halo, and L.sup.1 is --S--, or (t) R.sup.1a is
thio, R.sup.1b is ##STR00241## and L.sup.1 is ##STR00242## (u)
R.sup.1a is --SH, R.sup.1b is
--O--P(.dbd.O)(OH)(O--(CH.sub.2).sub.n--SH) and L.sup.1 is
--S--S--(CH.sub.2).sub.n--O--P(OH)(.dbd.O)--O--; R.sup.2a and
R.sup.2b are complementary conjugation functionalities and L.sup.2
is conjugate linker, and R.sup.2ap, R.sup.2a, R.sup.2b, and L.sup.2
are (a') R.sup.2ap is halo, R.sup.2a is azido, R.sup.2b is
--C.ident.C--R.sup.23, and L.sup.2 is ##STR00243## or (b')
R.sup.2ap is --NR.sup.23Pr, R.sup.2a is --NHR.sup.23, R.sup.2b is
carboxy, and L.sup.2 is --NR.sup.23C(.dbd.O)--, or (c') R.sup.2ap
is carboxy ester, R.sup.2a is carboxy, R.sup.2b is --NHR.sup.23,
and L.sup.2 is --C(.dbd.O)NR.sup.23--, or (d') R.sup.2ap is
--NR.sup.23Pr, R.sup.2a is --NHR.sup.23, R.sup.2b is halo, and
L.sup.2 is --NR.sup.23--, or (e') R.sup.2ap is --OH, phosphate or
phosphate ester, R.sup.2a is --O--P(.dbd.O)(OH)(X), R.sup.2b is
hydroxy, and L.sup.2 is --O--P(.dbd.O)(OH)--O--, or (f') R.sup.2ap
is --OH, phosphate or phosphate ester, R.sup.2a is
--O--P(.dbd.O)(OH)(X), R.sup.2b is --NHR.sup.23, and L.sup.2 is
--O--P(.dbd.O)(OH)--NR.sup.23--, or (g') R.sup.2ap is --OH,
phosphate or phosphate ester, R.sup.2a is --O--P(.dbd.O)(OH)(X),
R.sup.2b is thio, and L.sup.2 is --O--P(.dbd.O)(OH)--S--, or (h')
R.sup.2a is --X, R.sup.2b is thio, and L.sup.2 is --S--, or (i')
R.sup.2a is ##STR00244## R.sup.2b is thio, and L.sup.2 is
##STR00245## or (j') R.sup.2a is --C.ident.C--R.sup.23, R.sup.2b is
azido, and L.sup.2 is ##STR00246## or (k') R.sup.2a is halo,
R.sup.2b is --NHR.sup.23, and L.sup.2 is --NR.sup.23--, or (l')
R.sup.2a is hydroxy, R.sup.2b is --O--P(.dbd.O)(OH)(X), and L.sup.2
is --O--P(.dbd.O)(OH)--O--, or (m') R.sup.2a is --NHR.sup.23,
R.sup.2b is --O--P(.dbd.O)(OH)(X), and L.sup.2 is
--NR.sup.23--P(.dbd.O)(OH)--O--, or (n') R.sup.2a is thio, R.sup.2b
is --O--P(.dbd.O)(OH)(X), and L.sup.2 is --S--P(.dbd.O)(OH)--O--,
or (o') R.sup.2ap is --O--P(.dbd.O)(OH)SR.sup.24, R.sup.2a is
--O--P(.dbd.O)(OH)SH, R.sup.2b is --X, and L.sup.2 is
--O--P(.dbd.O)(OH)--S--, or (p') R.sup.2a is --X, R.sup.2b is
--O--P(.dbd.O)(OH)SH, and L.sup.2 is --S--P(.dbd.O)(OH)--O--, or
(q') R.sup.2a is --(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23,
R.sup.2b is --NHR.sup.23, L.sup.2 is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)NR.sup.23--, or (r') R.sup.2a
is --NHR.sup.23, R.sup.2b is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, L.sup.2 is
--NR.sup.23C(.dbd.O)--(CR.sup.25R.sup.25).sub.s--, or (s') R.sup.2a
is thio, R.sup.2b is halo, and L.sup.2 is --S--, or (t') R.sup.2a
is thio, R.sup.2b is ##STR00247## and L.sup.2 is ##STR00248## or
(u') R.sup.2a is --SH, R.sup.2b is
--O--P(.dbd.O)(OH)(O--(CH.sub.2).sub.n--SH) and L.sup.2 is
--S--S--(CH.sub.2).sub.n--O--P(OH)(.dbd.O)--O--; X is selected from
chlorine, bromine, fluorine, tosylate, mesylate, triflate, or
dimethoxy triflate, n is 1, 2, 3, 4, 5, or 6; R.sup.23 is selected
from the group consisting of hydrogen, alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, cycloalkynyl, substituted cycloalkynyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic; Pr is an amino protecting group; R.sup.24 is trityl
or benzyl; R.sup.25 is hydrogen or C.sub.1-6 alkyl; n is 1, 2, 3,
4, 5, or 6; s is an integer of greater than 1; R.sup.3ap is
selected from the group consisting of halo, --OH, --NR.sup.23Pr,
--O--P(.dbd.O)(OH)SR.sup.24, carboxy ester, phosphate and phosphate
ester; and R.sup.3a is selected from the group consisting of azido,
--C.ident.C--R.sup.23, --NHR.sup.23, carboxy, halo, hydroxy,
--C(.dbd.O)OR.sup.23, --O--P(.dbd.O)(OH)SH and
--O--P(.dbd.O)(OH)Br; provided that R.sup.2 does not react with
R.sup.1a or R.sup.1b, and R.sup.3 does not react with R.sup.2a or
R.sup.2b; --- represents the point of connection to the part of the
solid support-bound conjugated molecule that is closer to
##STR00249## and represents the point of connection to the part of
the solid support-bound conjugated molecule that is further away
from ##STR00250##
2. The method of claim 1 for preparing a compound of the formula
##STR00251## comprising (i) attaching a compound of the formula
##STR00252## to a solid support ##STR00253## to form a compound of
the formula ##STR00254## and (iii) reacting ##STR00255## with a
compound of the formula ##STR00256## to form ##STR00257## wherein:
##STR00258## is a solid support material; ##STR00259## and are
independently an oligo; R.sup.1a and R.sup.1b are complementary
conjugation functionalities and L.sup.1 is conjugate linker,
R.sup.2a and R.sup.2b are complementary conjugation functionalities
and L.sup.2 is conjugate linker, and R.sup.1a, R.sup.1bL.sup.1,
R.sup.2a, R.sup.2b, L.sup.2, and R.sup.3a are selected from
TABLE-US-00005 R.sup.1a, R.sup.2a, or R.sup.3a R.sup.1b or R.sup.2b
L.sup.1 or L.sup.2 --C.ident.C--R.sup.23 azido ##STR00260## azido
--C.ident.C--R.sup.23 ##STR00261## carboxy --NHR.sup.23
--C(.dbd.O)NR.sup.23-- --NHR.sup.23 carboxy --NR.sup.23C(.dbd.O)--
halo --NHR.sup.23 --NR.sup.23-- --NR.sup.23 halo --NR.sup.23--
hydroxy --O--P(.dbd.O)(OH)(X) --O--P(.dbd.O)(OH)--O--
--O--P(.dbd.O)(OH)(X) hydroxy --O--P(.dbd.O)(OH)--O-- --NHR.sup.23
--O--P(.dbd.O(OH)(X) --NR.sup.23--P(.dbd.O)(OH)--O--
--O--P(.dbd.O)(OH)(X) --NHR.sup.23 --O--P(.dbd.O)(OH)--NR.sup.23--
thio --O--P(.dbd.O)(OH)(X) --S--P(.dbd.O)(OH)--O--
--O--P(.dbd.O)(OH)(X) thio --O--P(.dbd.O)(OH)--S-- thio --X --S--
--X thio --S-- --O--P(.dbd.O)(OH)SH --X --O--P(.dbd.O)(OH)--S-- --X
--O--P(.dbd.O)(OH)SH --S--P(.dbd.O)(OH)--O--
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23 --NHR.sup.23
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)NR.sup.23-- --NHR.sup.23
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23
--NR.sup.23C(.dbd.O)--(CR.sup.25R.sup.25).sub.s-- thio ##STR00262##
##STR00263## ##STR00264## thio ##STR00265## --SH
--O--P(.dbd.O)(OH)(O--(CH.sub.2).sub.n--SH)
--S--S--(CH.sub.2).sub.n--O--P(OH)(.dbd.O)--O--
wherein the selection of R.sup.1a, R.sup.2a, or R.sup.3a is
independent of one another provided that L.sup.1 and L.sup.2 are
different, R.sup.2a does not react with R.sup.1a or R.sup.1b, and
R.sup.3a does not react with R.sup.2a or R.sup.2b; X is selected
from chlorine, bromine, fluorine, tosylate, mesylate, triflate, or
dimethoxy triflate; R.sup.23 is selected from the group consisting
of hydrogen, alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, cycloalkynyl, substituted cycloalkynyl, heteroaryl,
substituted heteroaryl, heterocyclic, and substituted heterocyclic;
and R.sup.25 is hydrogen or C.sub.1-6 alkyl; s is an integer of
greater than 1; --- represents the point of connection to the part
of the solid support-bound conjugated molecule that is closer to
##STR00266## and represents the point of connection to the part of
the solid support-bound conjugated molecule that is further away
from ##STR00267##
3. A method of preparing a compound of the formula ##STR00268##
comprising (i) attaching a compound of the formula ##STR00269## to
a solid support ##STR00270## to form a compound of the formula
##STR00271## (ii) converting ##STR00272## to ##STR00273## and (iii)
contacting ##STR00274## with a compound of the formula ##STR00275##
to form ##STR00276## wherein: ##STR00277## is a solid support
material; ##STR00278## and are independently an oligo; R.sup.ap,
R.sup.a, R.sup.b, and L are selected from (a) R.sup.ap is halo,
R.sup.a is azido, R.sup.b is --C.ident.C--R.sup.23, and L is
##STR00279## or (b) R.sup.ap is --NR.sup.23Pr, R.sup.a is
--NHR.sup.23, R.sup.b is carboxy, and L is --NR.sup.23C(.dbd.O)--,
or (c) R.sup.ap is carboxy ester, R.sup.a is carboxy, R.sup.b is
--NHR.sup.23, and L is --C(.dbd.O)NR.sup.23--, or (d) R.sup.ap is
--NR.sup.23Pr, R.sup.a is --NHR.sup.23, R.sup.b is halo, and L is
--NR.sup.23--, or (e) R.sup.ap is --OH, phosphate or phosphate
ester; R.sup.a is --O--P(.dbd.O)(OH)(X), R.sup.b is hydroxy, and L
is --O--P(.dbd.O)(OH)--O--, or (f) R.sup.ap is --OH, phosphate or
phosphate ester; R.sup.a is --O--P(.dbd.O)(OH)(X), R.sup.b is
--NHR.sup.23, and L is --O--P(.dbd.O)(OH)--NR.sup.23--, or (g)
R.sup.ap is --O--P(.dbd.O)(OH)SR.sup.24, R.sup.a is
--O--P(.dbd.O)(OH)SH, R.sup.b is --X, and L is
--O--P(.dbd.O)(OH)--S--, or (h) R.sup.ap is --OH, phosphate or
phosphate ester; R.sup.a is --O--P(.dbd.O)(OH)Br, R.sup.b is thio,
and L is --O--P(.dbd.O)(OH)--S--, or (i) (i) R.sup.ap is
--SR.sup.24, R.sup.a is --SH, R.sup.b is --O--P(.dbd.O)(OH)SH and L
is --O--P(.dbd.O)(OH)S--S--; wherein R.sup.23 is selected from the
group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, cycloalkynyl, substituted cycloalkynyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic; X is selected from chlorine, bromine, fluorine,
tosylate, mesylate, triflate, or dimethoxy triflate; R.sup.24 is
trityl or benzyl; --- represents the point of connection to the
part of the solid support-bound conjugated molecule that is closer
to ##STR00280## and represents the point of connection to the part
of the solid support-bound conjugated molecule that is further away
from ##STR00281##
4. A method of preparing a compound of the formula ##STR00282##
comprising cleaving the bond between ##STR00283## and L.sup.1 of
the compound of the formula ##STR00284## thereby obtaining
##STR00285## wherein: the compound of ##STR00286## is prepared
according to the method of claim 1; and ##STR00287## L.sup.1,
L.sup.2, R.sup.3, ##STR00288## are as defined in claim 1; and Z is
selected from --OH, OH--(C.sub.1-C.sub.10)alkylene-, --COOH,
NH.sub.2C(O)--, NH.sub.2NH--C(O)--,
COOH--(C.sub.1-C.sub.10)alkylene-,
NH.sub.2C(O)--(C.sub.1-C.sub.10)alkylene-,
NH.sub.2NH--C(O)--(C.sub.1-C.sub.10)alkylene-,
CH.sub.2.dbd.CH--(C.sub.1-C.sub.10)alkylene-,
C.ident.C--(C.sub.1-C.sub.10)alkylene- or
HS--(C.sub.1-C.sub.10)alkylene- and any alkylene is optionally
substituted by one or more groups selected from --OH, halogen,
--NHR'', --NHC(O)--(C.sub.1-C.sub.10)alkylene-C.ident.CH, or
--NHC(O)--(C.sub.1-C.sub.10)alkylene-CH.dbd.CH.sub.2; R'' is
selected from (C.sub.1-C.sub.10)alkyl,
(C.sub.3-C.sub.10)cycloalkyl, or (C.sub.3-C.sub.10)aryl.
5.-8. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. Ser. No.
15/042,064, filed Feb. 11, 2016, which is continuation of U.S.
application Ser. No. 13/790,922, filed Mar. 8, 2013, now U.S. Pat.
No. 9,289,502, issued Mar. 22, 2016, which is hereby incorporated
by reference in its entirety.
FIELD
[0002] The present technology relates generally to the preparation
of oligo conjugates, which can be constituents of nano-scale
information processing systems. In this context, the category of
oligos encompasses polymers of 2'-deoxyribosenucleotide residues
(DNA), ribonucleotide residues (RNA), or both DNA residues and RNA
residues. The oligo category also includes polymers of peptide
nucleic acids (PNAs) as well as heteropolymers that comprise both
PNA monomers and RNA and/or DNA monomers.
BACKGROUND
[0003] Nucleic acids have been used to implement nano-scale
information processing systems suitable for solving computational
problems in a test tube or in a cell, as illustrated in U.S. patent
application publications No. 20050112614, No. 20100069621 and No.
20110294687 and in U.S. Pat. No. 7,745,594. Such nano-scale systems
typically must be compatible with a biological environment,
particularly, if their potential for use in diagnostic assays or
for treatment of diseases is to be realized.
[0004] A nano-scale information processing system suitable for such
uses requires several nucleic acid segments that can serve as
computation units that are capable of performing logical
operations. Thus, there exists a need to develop synthetic
strategies that will permit the synthesis of a diverse array of
oligos, at high purity, as well as strategies for conjugating or
annealing such oligos in an efficient, well-controlled manner.
SUMMARY
[0005] The methodology of the invention accommodates various
conjugation components, discussed in detail below, to yield
conjugated molecules that comprise a predetermined number of the
components, as desired. The inventive methodology thus can be used
to develop nano-scale information processing systems, as described
above. In particular, the invention is also suitable for producing
biological transistors, which can be part of integrated circuits
capable of executing compound logic functions, e.g., in diagnostic
or therapeutic contexts that entail targeting of neoplastic or
virus-infected cells.
[0006] Accordingly, the invention provides a method for preparing a
compound of the formula
##STR00001##
[0007] The inventive method comprises
(i) attaching a conjugation component of formula
##STR00002##
wherein R.sup.2 is R.sup.2a or R.sup.2ap, to a solid support
##STR00003##
to form a compound of formula
##STR00004##
(ii) when R.sup.2 is R.sup.2ap, converting
##STR00005##
to
##STR00006##
and (iii) reacting
##STR00007##
with a conjugation component of formula
##STR00008##
where R.sup.3 can be a protected conjugation functionality
R.sup.3ap or an unprotected conjugation functionality R.sup.3a.
When R.sup.3 is R.sup.3a then (iv) the latter further reacts with
an oligo. As noted above, the oligo can comprise (a) a sequence of
2'-deoxyribosenucleotide residues (DNA), (b) a sequence of
ribonucleotide residues (RNA), or (c) a sequence containing both
2'-deoxyribosenucleotide residues and ribosenucleotide residues.
Alternatively, the oligo can be comprised of peptide nucleic acid
monomers (PNAs), linked by amide bonds, or it can be a
heteropolymer that has both PNA monomers and RNA and/or DNA
monomeric units. An oligo may optionally comprise a label.
[0008] The reaction (iv) forms
##STR00009##
where
##STR00010##
is a solid support material and
##STR00011##
are independently selected from the category of oligos, defined
above. In addition, ----- represents the point of connection to the
part of the solid support-bound conjugated molecule that is closer
to
##STR00012##
and represents the point of connection to the part of the solid
support-bound conjugated molecule that is further away from
##STR00013##
[0009] The inventive methodology thus described is qualified in
that R.sup.2 does not react with R.sup.1a or R.sup.1b and R.sup.3
does not react with R.sup.2a or R.sup.2b as further described
below. Furthermore, substituents R.sup.1a and R.sup.1b are
complementary conjugation functionalities and L.sup.1 is conjugate
linker formed by reaction of R.sup.1a and R.sup.1b. In keeping with
this characterization of their respective chemical roles,
selections of R.sup.1a, R.sup.1b and L.sup.1 can be grouped as
follows: [0010] (a) R.sup.1a is azido, R.sup.1b is
--C.ident.C--R.sup.23, and L.sup.1 is
##STR00014##
[0010] or [0011] (b) R.sup.1a is --NHR.sup.23, R.sup.1b is carboxy,
and L.sup.1 is --NR.sup.23C(.dbd.O)--, or [0012] (c) R.sup.1a is
carboxy, R.sup.1b is --NHR.sup.23, and L.sup.1 is
--C(.dbd.O)NR.sup.23--, or [0013] (d) R.sup.1a is --NHR.sup.23,
R.sup.1b is halo, and L.sup.1 is --NR.sup.23--, or [0014] (e)
R.sup.1a is --O--P(.dbd.O)(OH)(X), R.sup.1b is hydroxy, and L.sup.1
is --O--P(.dbd.O)(OH)--O--, or [0015] (f) R.sup.1a is
--O--P(.dbd.O)(OH)(X), R.sup.1b is --NHR.sup.23, and L.sup.1 is
--O--P(.dbd.O)(OH)--NR.sup.23--, or [0016] (g) R.sup.1a is
--O--P(.dbd.O)(OH)(X), R.sup.1b is thio, and L.sup.1 is
--O--P(.dbd.O)(OH)--S--, or [0017] (h) R.sup.1a is --X, R.sup.1b is
thio, and L.sup.1 is --S--, or (i) R.sup.1a is
##STR00015##
[0017] R.sup.1b is thio, and L.sup.1 is
##STR00016##
or [0018] (j) R.sup.1a is --C.ident.C--R.sup.23, R.sup.1b is azido,
and L.sup.1 is
##STR00017##
[0018] or [0019] (k) R.sup.1a is --X, R.sup.1b is --NHR.sup.23, and
L.sup.1 is --NR.sup.23--, or [0020] (l) R.sup.1a is hydroxy,
R.sup.1b is --O--P(.dbd.O)(OH)(X), and L.sup.1 is
--O--P(.dbd.O)(OH)--O--, or [0021] (m) R.sup.1a is --NHR.sup.23,
R.sup.1b is --O--P(.dbd.O)(OH)(X), and L.sup.1 is
--NR.sup.23--P(.dbd.O)(OH)--O--, or [0022] (n) R.sup.1a is thio,
R.sup.1b is --O--P(.dbd.O)(OH)(X), and L.sup.1 is
--S--P(.dbd.O)(OH)--O--, or [0023] (o) R.sup.1a is
--O--P(.dbd.O)(OH)SH, R.sup.1b is --X, and L.sup.1 is
--O--P(.dbd.O)(OH)--S--, or [0024] (p) R.sup.1a is --X, R.sup.1b is
--O--P(.dbd.O)(OH)SH, and L.sup.1 is --S--P(.dbd.O)(OH)--O--, or
[0025] (q) R.sup.1a is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, R.sup.1b is
--NHR.sup.23, L.sup.1 is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)NR.sup.23--, or [0026] (r)
R.sup.1a is --NHR.sup.23, R.sup.1b is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, L.sup.1 is
--NR.sup.23C(.dbd.O)--(CR.sup.25R.sup.25).sub.s--, or [0027] (s)
R.sup.1a is thio, R.sup.1b is --X, and L.sup.1 is --S--, or [0028]
(t) R.sup.1a is thio, R.sup.1b is
##STR00018##
[0028] and L.sup.1 is
##STR00019##
[0029] or [0030] (u) R.sup.1a is --SH, R.sup.1b is
--O--P(.dbd.O)(OH)(O--(CH.sub.2).sub.n--SH) and L.sup.1 is
--S--S--(CH.sub.2).sub.n--O--P(OH)(.dbd.O)--O--.
[0031] In the above characterization of R.sup.1a and R.sup.1b group
X is selected from chlorine, bromine, fluorine, tosylate, mesylate,
triflate, or dimethoxy triflate and n is 1, 2, 3, 4, 5, or 6.
[0032] Similarly, R.sup.2a and R.sup.2b are complementary
conjugation functionalities and L.sup.2 is conjugate linker formed
by reacton of R.sup.2a and R.sup.2b, and they likewise can be
groups as above. Thus, [0033] (a') R.sup.2ap is halo, R.sup.2a is
azido, R.sup.2b is --C.ident.C--R.sup.23, and L.sup.2 is
##STR00020##
[0033] or [0034] (b') R.sup.2ap is --NR.sup.23Pr, R.sup.2a is
--NHR.sup.23, R.sup.2b is carboxy, and L.sup.2 is
--NR.sup.23C(.dbd.O)--, or [0035] (c') R.sup.2ap is carboxy ester,
R.sup.2a is carboxy, R.sup.2b is --NHR.sup.23, and L.sup.2 is
--C(.dbd.O)NR.sup.23--, or [0036] (d') R.sup.2ap is --NR.sup.23Pr,
R.sup.2a is --NHR.sup.23, R.sup.2b is halo, and L.sup.2 is
--NR.sup.23--, or [0037] (e') R.sup.2ap is --OH, phosphate or
phosphate ester, R.sup.2a is --O--P(.dbd.O)(OH)(X), R.sup.2b is
hydroxy, and L.sup.2 is --O--P(.dbd.O)(OH)--O--, or [0038] (f')
R.sup.2ap is --OH, phosphate or phosphate ester, R.sup.2a is
--O--P(.dbd.O)(OH)(X), R.sup.2b is --NHR.sup.23, and L.sup.2 is
--O--P(.dbd.O)(OH)--NR.sup.23--, or [0039] (g') R.sup.2ap is --OH,
phosphate or phosphate ester, R.sup.2a is --O--P(.dbd.O)(OH)(X),
R.sup.2b is thio, and L.sup.2 is --O--P(.dbd.O)(OH)--S--, or [0040]
(h') R.sup.2a is halo, R.sup.2b is thio, and L.sup.2 is --S--, or
[0041] (i') R.sup.2a is
##STR00021##
[0041] R.sup.2b is thio, and L.sup.2 is
##STR00022##
or [0042] (j') R.sup.2a is --C.ident.C--R.sup.23, R.sup.2b is
azido, and L.sup.2 is
##STR00023##
[0042] or [0043] (k') R.sup.2a is --X, R.sup.2b is --NHR.sup.23,
and L.sup.2 is --NR.sup.23--, or [0044] (l') R.sup.2a is hydroxy,
R.sup.2b is --O--P(.dbd.O)(OH)(X), and L.sup.2 is
--O--P(.dbd.O)(OH)--O--, or [0045] (m') R.sup.2a is --NHR.sup.23,
R.sup.2b is --O--P(.dbd.O)(OH)(X), and L.sup.2 is
--NR.sup.23--P(.dbd.O)(OH)--O--, or [0046] (n') R.sup.2a is thio,
R.sup.2b is --O--P(.dbd.O)(OH)(X), and L.sup.2 is
--S--P(.dbd.O)(OH)--O--, or [0047] (o') R.sup.2ap is
--O--P(.dbd.O)(OH)SR.sup.24, R.sup.2a is --O--P(.dbd.O)(OH)SH,
R.sup.2b is --X, and L.sup.2 is --O--P(.dbd.O)(OH)--S--, or [0048]
(p') R.sup.2a is --X, R.sup.2b is --O--P(.dbd.O)(OH)SH, and L.sup.2
is --S--P(.dbd.O)(OH)--O--, or [0049] (q') R.sup.2a is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, R.sup.2b is
--NHR.sup.23, L.sup.2 is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)NR.sup.23--, or [0050] (r')
R.sup.2a is --NHR.sup.23, R.sup.2b is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, L.sup.2 is
--NR.sup.23C(.dbd.O)--(CR.sup.25R.sup.25).sub.s--, or [0051] (s')
R.sup.2a is thio, R.sup.2b is --X, and L.sup.2 is --S--, or [0052]
(t') R.sup.2a is thio, R.sup.2b is
##STR00024##
[0052] and L.sup.2 is
##STR00025##
[0053] or [0054] (u') R.sup.2a is --SH, R.sup.2b is
--O--P(.dbd.O)(OH)(O--(CH.sub.2).sub.n--SH) and L.sup.2 is
--S--S--(CH.sub.2).sub.n--O--P(OH)(.dbd.O)--O--.
[0055] In the above characterization of R.sup.2a and R.sup.2b group
X is selected from chlorine, bromine, fluorine, tosylate, mesylate,
triflate, or dimethoxy triflate.
[0056] In the foregoing description, R.sup.23 is selected from the
group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, cycloalkynyl, substituted cycloalkynyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic. It also is the case that:
[0057] Pr is an amino protecting group;
[0058] R.sup.24 is trityl or benzyl;
[0059] R.sup.25 is hydrogen or C.sub.1-6 alkyl;
[0060] s is an integer greater than 1;
[0061] R.sup.3ap is selected from the group consisting of halo,
--NR.sup.23Pr, --O--P(.dbd.O)(OH)SR.sup.24, carboxy ester,
phosphate and phosphate ester; and
[0062] R.sup.3a is selected from the group consisting of --X,
azido, --C.ident.C--R.sup.23, --NHR.sup.23, carboxy, hydroxy,
--C(.dbd.O)OR.sup.23, --O--P(.dbd.O)(OH)SH and
--O--P(.dbd.O)(OH)(X), and group X is selected from chlorine,
bromine, fluorine, tosylate, mesylate, triflate, or dimethoxy
triflate.
[0063] In one of its aspects, the inventive methodology embodies an
asymmetric conjugation strategy, illustrated in FIG. 1. As shown,
conjugation functionalities .alpha., .beta. are present at each end
of a first oligo and conjugation functionalities .alpha.', .beta.'
at each end of a second oligo. FIG. 1 shows that (i) .alpha. reacts
to form a new bond with .alpha.' and (ii) .beta. reacts to form a
new bond with .beta.', but (iii) each of conjugation
functionalities .alpha. or .alpha.' cannot react with .beta. or
.beta.', respectively. Thus, two oligos, each having a different
conjugation functionalities at their respective 5'-end and 3'-end,
are linked to obtain a di-oligo product. In this way the asymmetric
conjugation strategy of the invention permits the conjugation of
components in a desired sequence and orientation, as exemplified by
the product depicted in FIG. 1.
[0064] The inventive methodology also can proceed in accordance
with a symmetric conjugation strategy, depicted in FIG. 2. Pursuant
to this approach, every oligo has the same set of 5'- and
3'conjugation functionalities. Thus, oligo A and oligo B in FIG. 2
are shown to have the same set of .alpha. and .alpha.'
functionalities at their respective termini.
[0065] Pursuant to the symmetric strategy of the invention, a
polymer product containing oligo A and oligo B is obtained the by
conjugating the unprotected a conjugation functionality of oligo A
to solid support, the surface of which is functionalized with an
.alpha.' group. The .alpha.' group at the other end of oligo A is
protected, preventing unwanted conjugation between .alpha. and
.alpha.' groups of separate oligo A molecules present in the
reaction mixture. After oligo A is tethered to the solid support
and the terminal .alpha.' group is deprotected, the tethered
product is then allowed to come into contact with a molecule of
oligo B, which has an unprotected a conjugation functionality at
one end and a protected .alpha.' group at the other end.
[0066] By either the asymmetric approach or the symmetric approach,
the present invention permits the "programmed" construction of a
conjugated molecule of prescribed length and sequence. That is,
production of a conjugated molecule pursuant to the invention can
be designed beforehand and controlled in practice to determine, via
the particular manner chosen by which the oligos are conjugated,
the numbers and types of the oligos in the resultant conjugated
molecule.
[0067] In a variation of inventive method, a compound of the
formula
##STR00026##
is produced by steps that comprise: (i) attaching a compound of
formula
##STR00027##
to a solid support
##STR00028##
to form a compound of the formula
##STR00029##
and (iii) reacting
##STR00030##
with a compound of the formula
##STR00031##
to form
##STR00032##
where:
##STR00033##
is a solid support material;
##STR00034##
and are independently selected from the group oligo;
[0068] R.sup.1a and R.sup.1b are complementary conjugation
functionalities and L.sup.1 is conjugate linker formed by reacton
of R.sup.1a and R.sup.1b, R.sup.2a and R.sup.2b are complementary
conjugation functionalities and L.sup.2 is conjugate linker formed
by reacton of R.sup.2a and R.sup.2b. R.sup.1a, R.sup.1b, L.sup.1,
R.sup.2a, R.sup.2b, L.sup.2, and R.sup.3a are selected from
TABLE-US-00001 R.sup.1a, R.sup.2a, or R.sup.3a R.sup.1b or R.sup.2b
L.sup.1 or L.sup.2 --C.ident.C--R.sup.23 azido ##STR00035## azido
--C.ident.C--R.sup.23 ##STR00036## carboxy --NHR.sup.23
--C(.dbd.O)NR.sup.23-- --NHR.sup.23 carboxy --NR.sup.23C(.dbd.O)--
halo --NHR.sup.23 --NR.sup.23-- --NR.sup.23Pr halo --NR.sup.23--
hydroxy --O--P(.dbd.O)(OH)(X) --O--P(.dbd.O)(OH)--O--
--O--P(.dbd.O)(OH)(X) hydroxy --O--P(.dbd.O)(OH)--O-- --NHR.sup.23
--O--P(.dbd.O)(OH)(X) --NR.sup.23--P(.dbd.O)(OH)--O--
--O--P(.dbd.O)(OH)(X) --NHR.sup.23 --O--P(.dbd.O)(OH)--NR.sup.23--
thio --O--P(.dbd.O)(OH)(X) --S--P(.dbd.O)(OH)--O--
--O--P(.dbd.O)(OH)(X) thio --O--P(.dbd.O)(OH)--S-- thio --X --S--
--X thio --S-- --O--P(.dbd.O)(OH)SH --X --O--P(.dbd.O)(OH)--S-- --X
--O--P(.dbd.O)(OH)SH --S--P(.dbd.O)(OH)--O--
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23 --NHR.sup.23
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)NR.sup.23-- --NHR.sup.23
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23
--NR.sup.23C(.dbd.O)--(CR.sup.25R.sup.25).sub.s-- thio ##STR00037##
##STR00038## ##STR00039## thio ##STR00040## --SH
--O--P(.dbd.O)(OH)(O--(CH.sub.2).sub.n--SH)
--S--S--(CH.sub.2).sub.n--O--P(OH)(.dbd.O)--O--
where the selection of R.sup.1a, R.sup.2a, or R.sup.3a is
independent of each other provided that L.sup.1 and L.sup.2 are
different, R.sup.2a does not react with R.sup.1a or R.sup.1b, and
R.sup.3a does not react with R.sup.2a or R.sup.2b;
[0069] X is selected from chlorine, bromine, fluorine, tosylate,
mesylate, triflate, or dimethoxy triflate;
[0070] R.sup.23 is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
cycloalkynyl, substituted cycloalkynyl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic;
[0071] R.sup.25 is hydrogen or C.sub.1-6 alkyl;
[0072] s is an integer of greater than 1;
[0073] ----- represents the point of connection to the part of the
solid support-bound conjugated molecule that is closer to
##STR00041##
and represents the point of connection to the part of the solid
support-bound conjugated molecule that is further away from
##STR00042##
[0074] Pursuant to another variation of the inventive methodology,
a compound is produced of the formula
##STR00043##
by steps comprising (i) attaching a compound of the formula
##STR00044##
to a solid support
##STR00045##
to form a compound of the formula
##STR00046##
(ii) converting
##STR00047##
to
##STR00048##
and (iii) reacting
##STR00049##
with a compound of the formula
##STR00050##
to form
##STR00051##
where:
##STR00052##
is a solid support material;
##STR00053##
and are each independently selected from the group oligo;
[0075] R.sup.ap is protected conjugation functionality which when
deprotected is converted to R.sup.a, R.sup.a and R.sup.b are
complementary conjugation functionalities and L is conjugate linker
formed by reacton of R.sup.a and R.sup.b. R.sup.ap, R.sup.a,
R.sup.b, and L are selected from [0076] (a) R.sup.ap is halo,
R.sup.a is azido, R.sup.b is --C.ident.C--R.sup.23, and L is
##STR00054##
[0076] or [0077] (b) R.sup.ap is --NR.sup.23Pr, R.sup.a is
--NHR.sup.23, R.sup.b is carboxy, and L is --NR.sup.23C(.dbd.O)--,
or [0078] (c) R.sup.ap is carboxy ester, R.sup.a is carboxy,
R.sup.b is --NHR.sup.23, and L is --C(.dbd.O)NR.sup.23--, or [0079]
(d) R.sup.ap is --NR.sup.23Pr, R.sup.a is --NHR.sup.23, R.sup.b is
halo, and L is --NR.sup.23--, or [0080] (e) R.sup.ap is --OH,
phosphate or phosphate ester, R.sup.a is --O--P(.dbd.O)(OH)(X),
R.sup.b is hydroxy, and L is --O--P(.dbd.O)(OH)--O--, or [0081] (f)
R.sup.ap is --OH, phosphate or phosphate ester, R.sup.a is
--O--P(.dbd.O)(OH)(X), R.sup.b is --NHR.sup.23, and L is
--O--P(.dbd.O)(OH)--NR.sup.23--, or [0082] (g) R.sup.ap is
--O--P(.dbd.O)(OH)SR.sup.24, R.sup.a is --O--P(.dbd.O)(OH)SH,
R.sup.b is halo, and L is --O--P(.dbd.O)(OH)--S--, or [0083] (h)
R.sup.ap is --OH, phosphate or phosphate ester, R.sup.a is
--O--P(.dbd.O)(OH)(X), R.sup.b is thio, and L is
--O--P(.dbd.O)(OH)--S-- or [0084] (i) R.sup.ap is --SR.sup.24,
R.sup.a is --SH, R.sup.b is --O--P(.dbd.O)(OH)SH and L is
--O--P(.dbd.O)(OH)S--S--; where R.sup.23 is selected from the group
consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, cycloalkynyl, substituted cycloalkynyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic;
[0085] X is selected from chlorine, bromine, fluorine, tosylate,
mesylate, triflate, or dimethoxy triflate;
[0086] Pr is an amino protecting group;
[0087] R.sup.24 is trityl or benzyl; and
[0088] ----- represents the point of connection to the part of the
solid support-bound conjugated molecule that is closer to
##STR00055##
while represents the point of connection to the part of the solid
support-bound conjugated molecule that is further away from
##STR00056##
[0089] In yet another variation, the method of the invention
entails preparing a compound of the formula
##STR00057##
To this end the method comprises
[0090] cleaving the bond between
##STR00058##
and L.sup.1 of the compound of the formula
##STR00059##
thereby obtaining
##STR00060##
[0091] Z in the polymer product is selected from --OH,
OH--(C.sub.1-C.sub.10)alkylene-, --COOH, NH.sub.2C(O)--,
NH.sub.2NH--C(O)--, COOH--(C.sub.1-C.sub.10)alkylene-,
NH.sub.2C(O)--(C.sub.1-C.sub.10)alkylene-,
NH.sub.2NH--C(O)--(C.sub.1-C.sub.10)alkylene-,
CH.sub.2.dbd.CH--(C.sub.1-C.sub.10)alkylene-,
C.ident.C--(C.sub.1-C.sub.10)alkylene- or
HS--(C.sub.1-C.sub.10)alkylene- and the alkylene can be optionally
substituted by one or more groups selected from --OH, halogen,
--NHR'', --NHC(O)--(C.sub.1-C.sub.10)alkylene-C.ident.CH, or
--NHC(O)--(C.sub.1-C.sub.10)alkylene-CH.dbd.CH.sub.2. When the
alkylene is substituted with an --NHR'' group, variable R'' is
selected from (C.sub.1-C.sub.10)alkyl,
(C.sub.3-C.sub.10)cycloalkyl, or (C.sub.3-C.sub.10)aryl
[0092] Pursant to the present invention, conjugation functionality
R.sup.3 can be labeled by converting a compound of the formula
##STR00061##
to a compound of the formula
##STR00062##
In this context R.sup.3 is brought into contact with an optically
detectable group or a radiolabeled group, thereby to obtain a
product that carries a detectable label. The labeled product can be
cleaved from the solid support, in the manner described immediately
above.
[0093] The invention also provides a method for producing a
conjugated molecule that is bound to a solid support, as
represented by the formula
##STR00063##
In the formula
##STR00064##
is a solid support material,
##STR00065##
is selected from the category of oligo defined above; and
[0094] R.sup.2 is either a protected conjugation functionality
R.sup.2ap, or an unprotected conjugation functionality R.sup.2a.
When R.sup.2 is R.sup.2ap then R.sup.2ap is selected from the group
consisting of halo, NR.sup.23Pr, carboxy ester, phosphate,
phosphate ester, and --O--P(.dbd.O)(OH)SR.sup.24. Following removal
of the protection group, R.sup.2ap is converted to R.sup.2a.
R.sup.2a is a group selected from halo, azido, hydroxy, thio,
--NHR.sup.23, carboxy,
##STR00066##
--O--P(.dbd.O)(OH)(X), --C.ident.C--R.sup.23, --O--P(.dbd.O)(OH)SH
and
[0095] --(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, and X is
selected from chlorine, bromine, fluorine, tosylate, mesylate,
triflate, or dimethoxy triflate. Furthermore,
[0096] L.sup.1 is selected from the group consisting of
--C(.dbd.O)NR.sup.23--, --NR.sup.23C(.dbd.O)--,
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)NR.sup.23--,
--NR.sup.23C(.dbd.O)--(CR.sup.25R.sup.25).sub.s--, --NR.sup.23--,
--O--P(.dbd.O)(OH)--O--, --NR.sup.23--P(.dbd.O)(OH)--O--,
--O--P(.dbd.O)(OH)--NR.sup.23--, --S--, --S--P(.dbd.O)(OH)--O--,
and --O--P(.dbd.O)(OH)--S--,
##STR00067##
[0097] R.sup.23 is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
cycloalkynyl, substituted cycloalkynyl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic,
[0098] R.sup.24 is trityl or benzyl,
[0099] R.sup.25 is hydrogen or C.sub.1-6 alkyl,
[0100] s is an integer of greater than 1,
[0101] ----- represents the point of connection to the part of the
solid support-bound conjugated molecule that is closer to
##STR00068##
and represents the point of connection to the part of the solid
support-bound conjugated molecule that is further away from
##STR00069##
[0102] In yet another aspect, the invention provides a compound
according formula:
##STR00070##
where R.sup.2 is a protected conjugation functionality R.sup.2ap,
or an unprotected conjugation functionality R.sup.2a.
##STR00071##
is selected from the oligo category defined above, and substituent
R.sup.1b is selected from the group consisting of
--C.ident.C--R.sup.23, carboxy, --NHR.sup.23, halo, hydroxy, thio,
azido, --O--P(.dbd.O)(OH)(X), --O--P(.dbd.O)(OH)SH,
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, and
##STR00072##
Group X is selected from chlorine, bromine, fluorine, tosylate,
mesylate, triflate, or dimethoxy triflate.
[0103] Furthermore, R.sup.2ap is selected from the group consisting
of halo, NR.sup.23Pr, carboxy ester, phosphate, phosphate ester,
and --O--P(.dbd.O)(OH)SR.sup.24,
[0104] R.sup.2a is selected from the group consisting of halo,
azido, hydroxy, thio, --NHR.sup.23, carboxy, --NHR.sup.23,
--O--P(.dbd.O)(OH)(X),
##STR00073##
--C.ident.C--R.sup.23, --O--P(.dbd.O)(OH)SH and
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23,
[0105] R.sup.23 is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic,
[0106] R.sup.24 is alkyl or benzyl, and
[0107] represents the point of connection to
##STR00074##
Group X is as defined above.
[0108] These and other aspects and embodiments of the invention are
further described in the text that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0109] The invention will be more fully understood by reference to
the following drawings, which are for illustrative purposes
only:
[0110] FIG. 1 illustrates an asymmetric conjugation strategy of the
invention.
[0111] FIG. 2 illustrates a symmetric conjugation strategy of the
invention.
[0112] FIGS. 3A and 3B Reagents for functionalizing the N-terminal
of a PNA.
[0113] FIG. 4 Reagents for functionalizing the C-terminal of a
PNA
DETAILED DESCRIPTION
[0114] In the following detailed description, reference is made to
the accompanying drawings, which form a part hereof. In the
drawings, similar symbols typically identify similar components,
unless context dictates otherwise. The illustrative embodiments
described in the detailed description, drawings, and claims are not
meant to be limiting. Other embodiments may be utilized, and other
changes may be made, without departing from the spirit or scope of
the subject matter presented here.
Definitions
[0115] Certain terms employed in this description have the
following defined meanings. Terms that are not defined have their
art-recognized meanings.
[0116] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise.
[0117] The term "comprising" is intended to mean that the devices
and methods include the recited components or steps, but not
excluding others. "Consisting essentially of" when used to define
devices and methods, shall mean excluding other components or steps
that would materially affect the basic and novel characteristics of
the technology. "Consisting of" shall mean excluding any components
or steps not specified in the claim. Embodiments defined by each of
these transition terms are within the scope of this disclosure.
[0118] "Solid support" refers to a solid material on which a
compound can be attached during solid phase synthesis. This class
of materials is exemplified as polystyrene, such as styrene
cross-linked with 1-2% divinylbenzene, polyacrylamide,
PEG-polystyrene (PEG-PS), PEG-based supports, which are composed of
a PEG-polypropylene glycol network or PEG with polyamide or
polystyrene, controlled pore glass, cellulose fibers, and highly
cross-linked polystyrene, gel-type polymers supported by rigid
matrices.
[0119] The term "oligo" denotes a category, discussed above, that
encompasses a polymer, having from 2 to about 150 covalently linked
monomer units, that is characterized by (i) a sequence of
2'-deoxyribosenucleotide residues (DNA), (ii) a sequence of
ribonucleotide residues (RNA), or (iii) a sequence containing both
2'-deoxyribosenucleotide residues and ribosenucleotide residues,
which may be referred to as an oligonucleotide. The oligo category
also encompasses polymers of peptide nucleic acid monomers (PNAs),
in which the monomeric units are linked to each other by an amide
bond. In the present context an oligo also can be a heteropolymer
that has both PNA monomers and RNA and/or DNA monomeric units.
[0120] The monomeric units of an oligonucleotide can be linked
through a phosphodiester bond, a phosphorothioate bond, a
methylphosphonate bond, or an amide (--C(O)--NH--) bond, as a
function of the chemical nature of monomers used to synthesize the
oligo. In a given embodiment the oligo is functionalized through
the conjugation of another group. The latter group also can be
labeled with any manner of detectable group.
[0121] "Peptide" refers to a polymer amino acid monomers (whether
or not naturally occurring) linked by peptide bonds (also known as
amide bonds) formed when the carboxyl carbon atom of the carboxylic
acid group bonded to the alpha-carbon of one amino acid (or amino
acid residue) becomes covalently bound to the amino nitrogen atom
of the amino group bonded to the alpha-carbon of an adjacent amino
acid (or amino acid residue). Amino acids which have been
incorporated into a peptide are termed amino acid residues. Every
peptide has an N-terminus and C-terminus residue on the ends of the
peptide (except for cyclic peptides). Peptides typically have fewer
than 50 amino acid residues. "Protein" refers polymers of amino
acid monomers linked by peptide bonds that have more amino acid
residues than peptides.
[0122] "Peptide nucleic acid" or PNA refers to synthetic polymers
comprising repeating N-(2-aminoethyl)-glycine units linked by amide
bonds. The purine (adenine (A) guanine (G)) and pyrimidine (thymine
(T) and cytosine (C)) bases are attached to the backbone through
methylene carbonyl linkages. PNAs do not contain any pentose sugar
moieties or phosphate groups. Examples of peptide nucleic acids are
described in U.S. Pat. Nos. 5,539,082 and 6,395,474.
[0123] "Peptide nucleic acid derivative" refers to a peptide
nucleic acid wherein the N-(2-aminoethyl)-glycine backbone or one
or more bases are modified, or which comprises additional moieties,
as a metal complex or a detectable moiety. Illustrative of peptide
nucleic acid derivatives are those described in Hudson et al., Pure
Appl. Chem. 76: 1591-98 (2004), Imoto, Nucleic Acids Symp Ser 52:
391-92 (2008), Ferrer et al., Letters in Peptide Science 7: 195-206
(2000), Kramer et al., Metal Ions Life Sci. 10: 319-40 (2012),
Verheijen et al., Bioconjugate Chem. 11: 741-43 (2000), and Ganesh,
Current Organic Chem. 4: 931-43 (2000).
[0124] "Conjugation functionality" refers to a functional group on
a molecule that can react with a functional group on another
molecule resulting in connection of the two molecules through the
formation of one ore more covalent bonds. Conjugation
functionalities are designated as .alpha., .alpha.', .beta.,
.beta.', R.sup.1a, R.sup.1b, R.sup.2a, R.sup.2b, etc. and described
herein. The two conjugation functionalities that react with each
other are referred to as complementary conjugation functionalities.
For example, in this specification .alpha. and .alpha.', .beta. and
.beta.', R.sup.1a and R.sup.1b, R.sup.2a and R.sup.2b are pairs of
complementary conjugation functionalities.
[0125] "Conjugation component" refers to a molecule, such as an
oligo, having at least one conjugation functionality.
[0126] "Conjugate linker" refers to a linker formed by reaction of
a conjugation functionality of one conjugation component with a
complementary conjugation functionality of another conjugation
component. Conjugate linkers are designated as L.sup.2 and L.sup.3,
etc., and are described herein.
[0127] "Conjugated molecule" refers to a molecule having two or
more conjugation components that are linked via a conjugate linker
described herein. A conjugated molecule may be a conjugation
component if it comprises a conjugation functionality.
[0128] "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl
groups having from 1 to 10 carbon atoms and preferably 1 to 6
carbon atoms. "C.sub.u-v alkyl" refers to alkyl groups having from
u to v carbon atoms, wherein u and v are integers. This term
includes, by way of example, linear and branched hydrocarbyl groups
such as methyl (CH.sub.3--), ethyl (CH.sub.3CH.sub.2--), n-propyl
(CH.sub.3CH.sub.2CH.sub.2--), isopropyl ((CH.sub.3).sub.2CH--),
n-butyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2--), isobutyl
((CH.sub.3).sub.2CHCH.sub.2--), sec-butyl
((CH.sub.3)(CH.sub.3CH.sub.2)CH--), t-butyl ((CH.sub.3).sub.3C--),
n-pentyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and
neopentyl ((CH.sub.3).sub.3CCH.sub.2--).
[0129] "Alkenyl" refers to straight or branched hydrocarbyl groups
having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms
and having at least 1 and preferably from 1 to 2 sites of vinyl
(>C.dbd.C<) unsaturation. Such groups are exemplified, for
example, by vinyl, allyl, and but-3-en-1-yl. Included within this
term are the cis and trans isomers or mixtures of these
isomers.
[0130] "Alkynyl" refers to straight or branched monovalent
hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2
to 3 carbon atoms and having at least 1 and preferably from 1 to 2
sites of acetylenic (--C.ident.C--) unsaturation. Examples of such
alkynyl groups include acetylenyl (--C.ident.CH), and propargyl
(--CH.sub.2C.ident.CH).
[0131] "Substituted alkyl" and "substituted C.sub.u-v alkyl"
encompass an alkyl group having from 1 to 5 and, in some
embodiments, 1 to 3 or 1 to 2 substituents selected from the group
consisting of alkenyl, substituted alkenyl, alkynyl, substituted
alkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,
amino, substituted amino, aminocarbonyl, aminothiocarbonyl,
aminocarbonylamino, aminocarbonyloxy, aminosulfonyl,
aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted
aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio,
azido, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl
ester)oxy, cyano, cycloalkyl, substituted cycloalkyl,
cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio,
substituted cycloalkylthio, guanidino, substituted guanidino, halo,
hydroxy, hydroxyamino, alkoxyamino, hydrazino, substituted
hydrazino, heteroaryl, substituted heteroaryl, heteroaryloxy,
substituted heteroaryloxy, heteroarylthio, substituted
heteroarylthio, heterocyclic, substituted heterocyclic,
heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,
substituted heterocyclylthio, nitro, spirocycloalkyl, SO.sub.3H,
substituted sulfonyl, sulfonyloxy, thioacyl, thiocyanate, thiol,
alkylthio, and substituted alkylthio, where such substituents are
defined in this specification.
[0132] "Substituted alkenyl" refers to alkenyl groups having from 1
to 3 substituents, and preferably 1 to 2 substituents, selected
from the group consisting of alkoxy, substituted alkoxy, acyl,
acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminocarbonyloxy,
aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,
substituted aryl, aryloxy, substituted aryloxy, arylthio,
substituted arylthio, carboxyl, carboxyl ester, (carboxyl
ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted
cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy,
cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,
substituted cycloalkenyl, cycloalkenyloxy, substituted
cycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,
guanidino, substituted guanidino, halo, hydroxy, heteroaryl,
substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy,
heteroarylthio, substituted heteroarylthio, heterocyclic,
substituted heterocyclic, heterocyclyloxy, substituted
heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio,
nitro, SO.sub.3H, substituted sulfonyl, sulfonyloxy, thioacyl,
thiol, alkylthio, and substituted alkylthio, wherein said
substituents are defined herein and with the proviso that any
hydroxy substitution is not attached to a vinyl (unsaturated)
carbon atom.
[0133] "Substituted alkynyl" refers to alkynyl groups having from 1
to 3 substituents, and preferably 1 to 2 substituents, selected
from the group consisting of alkoxy, substituted alkoxy, acyl,
acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminocarbonyloxy,
aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,
substituted aryl, aryloxy, substituted aryloxy, arylthio,
substituted arylthio, carboxyl, carboxyl ester, (carboxyl
ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted
cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy,
cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,
substituted cycloalkenyl, cycloalkenyloxy, substituted
cycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,
guanidino, substituted guanidino, halo, hydroxy, heteroaryl,
substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy,
heteroarylthio, substituted heteroarylthio, heterocyclic,
substituted heterocyclic, heterocyclyloxy, substituted
heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio,
nitro, SO.sub.3H, substituted sulfonyl, sulfonyloxy, thioacyl,
thiol, alkylthio, and substituted alkylthio, wherein said
substituents are defined herein and with the proviso that any
hydroxy substitution is not attached to an acetylenic carbon
atom.
[0134] "Alkoxy" refers to the group --O-alkyl wherein alkyl is
defined herein. Alkoxy includes, by way of example, methoxy,
ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and
n-pentoxy.
[0135] "Substituted alkoxy" refers to the group --O-(substituted
alkyl) wherein substituted alkyl is defined herein.
[0136] "Acyl" refers to the groups H--C(O)--, alkyl-C(O)--,
substituted alkyl-C(O)--, alkenyl-C(O)--, substituted
alkenyl-C(O)--, alkynyl-C(O)--, substituted alkynyl-C(O)--,
cycloalkyl-C(O)--, substituted cycloalkyl-C(O)--,
cycloalkenyl-C(O)--, substituted cycloalkenyl-C(O)--, aryl-C(O)--,
substituted aryl-C(O)--, heteroaryl-C(O)--, substituted
heteroaryl-C(O)--, heterocyclic-C(O)--, and substituted
heterocyclic-C(O)--, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined herein.
Acyl includes the "acetyl" group CH.sub.3C(O)--.
[0137] Acylamino" refers to the groups --NR.sup.15C(O)alkyl,
--NR.sup.15C(O)substituted alkyl, --NR.sup.15C(O)cycloalkyl,
--NR.sup.15C(O)substitutedcy cloalkyl, --NR.sup.15C(O)cycloalkenyl,
--NR.sup.15C(O)substituted cycloalkenyl, --NR.sup.15C(O)alkenyl,
--NR.sup.15C(O)substituted alkenyl, --NR.sup.15C(O)alkynyl,
--NR.sup.15C(O)substituted alkynyl, --NR.sup.15C(O)aryl,
--NR.sup.15C(O)substituted aryl, --NR.sup.15C(O)heteroaryl,
--NR.sup.15C(O)substituted heteroaryl, --NR.sup.15C(O)heterocyclic,
and --NR.sup.15C(O)substituted heterocyclic wherein R.sup.15 is
hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined
herein.
[0138] "Acyloxy" refers to the groups alkyl-C(O)O--, substituted
alkyl-C(O)O--, alkenyl-C(O)O--, substituted alkenyl-C(O)O--,
alkynyl-C(O)O--, substituted alkynyl-C(O)O--, aryl-C(O)O--,
substituted aryl-C(O)O--, cycloalkyl-C(O)O--, substituted
cycloalkyl-C(O)O--, cycloalkenyl-C(O)O--, substituted
cycloalkenyl-C(O)O--, heteroaryl-C(O)O--, substituted
heteroaryl-C(O)O--, heterocyclic-C(O)O--, and substituted
heterocyclic-C(O)O-- wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined
herein.
[0139] "Amino" refers to the group --NH.sub.2.
[0140] "Substituted amino" refers to the group --NR'R'' where R'
and R'' are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, substituted
heterocyclic, --SO.sub.2-alkyl, --SO.sub.2-substituted alkyl,
--SO.sub.2-alkenyl, --SO.sub.2-substituted alkenyl,
--SO.sub.2-cycloalkyl, --SO.sub.2-substituted cycloalkyl,
--SO.sub.2-cycloalkenyl, --SO.sub.2-substituted
cylcoalkenyl,--SO.sub.2-aryl, --SO.sub.2-substituted aryl,
--SO.sub.2-heteroaryl, --SO.sub.2-substituted heteroaryl,
--SO.sub.2-heterocyclic, and --SO.sub.2-substituted heterocyclic
and wherein R' and R'' are optionally joined, together with the
nitrogen bound thereto to form a heterocyclic or substituted
heterocyclic group, provided that R' and R'' are both not hydrogen,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein. When R' is hydrogen and R'' is
alkyl, the substituted amino group is sometimes referred to herein
as alkylamino. When R' and R'' are both alkyl, the substituted
amino group is sometimes referred to herein as dialkylamino. When
referring to a monosubstituted amino, it is meant that either R' or
R'' is hydrogen but not both. When referring to a disubstituted
amino, it is meant that neither R' nor R'' are hydrogen.
[0141] "Aminocarbonyl" refers to the group --C(O)NR.sup.10R.sup.11
where R.sup.10 and R.sup.11 are independently selected from the
group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic and where R.sup.10 and
R.sup.11 are optionally joined together with the nitrogen bound
thereto to form a heterocyclic or substituted heterocyclic group,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
[0142] "Aminocarbonyloxy" refers to the group
--OC(O)NR.sup.10R.sup.11 where R.sup.10 and R.sup.11 are
independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.10 and R.sup.11 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0143] "Amidino" refers to the group
--C(.dbd.NR.sup.12)NR.sup.10R.sup.11 where R.sup.10, R.sup.11, and
R.sup.12 are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R.sup.10 and R.sup.11 are optionally joined
together with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
[0144] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic
group of from 6 to 14 carbon atoms having a single ring (e.g.,
phenyl) or multiple condensed rings (e.g., naphthyl or anthryl)
which condensed rings may or may not be aromatic (e.g.,
2-benzoxazolinone, 2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like)
provided that the point of attachment is at an aromatic carbon
atom. Preferred aryl groups include phenyl and naphthyl.
[0145] "Substituted aryl" refers to aryl groups which are
substituted with 1 to 5, preferably 1 to 3, or more preferably 1 to
2 substituents selected from the group consisting of alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino,
acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminocarbonyloxy,
aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,
substituted aryl, aryloxy, substituted aryloxy, arylthio,
substituted arylthio, carboxyl, carboxyl ester, (carboxyl
ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted
cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy,
cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,
substituted cycloalkenyl, cycloalkenyloxy, substituted
cycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,
guanidino, substituted guanidino, halo, hydroxy, heteroaryl,
substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy,
heteroarylthio, substituted heteroarylthio, heterocyclic,
substituted heterocyclic, heterocyclyloxy, substituted
heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio,
nitro, SO.sub.3H, substituted sulfonyl, sulfonyloxy, thioacyl,
thiol, alkylthio, and substituted alkylthio, wherein said
substituents are defined herein.
[0146] "Aryloxy" refers to the group --O-aryl, where aryl is as
defined herein, that includes, by way of example, phenoxy and
naphthoxy.
[0147] "Substituted aryloxy" refers to the group --O-(substituted
aryl) where substituted aryl is as defined herein.
[0148] "Arylthio" refers to the group --S-aryl, where aryl is as
defined herein.
[0149] "Substituted arylthio" refers to the group --S-(substituted
aryl), where substituted aryl is as defined herein.
[0150] "Carbonyl" refers to the divalent group --C(O)-- which is
equivalent to --C(.dbd.O)--.
[0151] "Carboxy" or "carboxyl" refers to --COOH or a salt
thereof.
[0152] "Carboxy ester" refers to the groups --C(O)O-alkyl,
--C(O)O-substituted alkyl, --C(O)O-alkenyl, --C(O)O-substituted
alkenyl, --C(O)O-alkynyl, --C(O)O-substituted alkynyl,
--C(O)O-aryl, --C(O)O-substituted aryl, --C(O)O-cycloalkyl,
--C(O)O-substituted cycloalkyl, --C(O)O-heteroaryl,
--C(O)O-substituted heteroaryl, --C(O)O-heterocyclic, and
--C(O)O-substituted heterocyclic wherein alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein. "(Carboxyl ester)amino" refers
to the group --NR.sup.16--C(O)O-alkyl,
--NR.sup.16--C(O)O-substituted alkyl, --NR.sup.16--C(O)O-alkenyl,
--NR.sup.16--C(O)O-substituted alkenyl, --NR.sup.16--C(O)O-alkynyl,
--NR.sup.16--C(O)O-- substituted alkynyl, --NR.sup.16--C(O)O-aryl,
--NR.sup.16--C(O)O-substituted aryl, --NR.sup.16--C(O)O-cycloalkyl,
--NR.sup.16--C(O)O-substituted cycloalkyl,
--NR.sup.16--C(O)O-cycloalkenyl, --NR.sup.16--C(O)O-substituted
cycloalkenyl, --NR.sup.16--C(O)O-heteroaryl,
--NR.sup.16--C(O)O-substituted heteroaryl,
--NR.sup.16--C(O)O-heterocyclic, and --NR.sup.16--C(O)O-substituted
heterocyclic wherein R.sup.16 is alkyl or hydrogen, and wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
[0153] "Cyano" refers to the group --CN.
[0154] "Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10
carbon atoms having single or multiple cyclic rings including
fused, bridged, and spiro ring systems. One or more of the rings
can be aryl, heteroaryl, or heterocyclic provided that the point of
attachment is through the non-aromatic carbon. Examples of suitable
cycloalkyl groups include, for instance, adamantyl, cyclopropyl,
cyclobutyl, cyclopentyl, and cyclooctyl. Other examples of
cycloalkyl groups include bicycle[2,2,2,]octanyl, norbornyl, and
spirobicyclo groups such as spiro[4.5]dec-8-yl:
##STR00075##
[0155] "Cycloalkenyl" refers to non-aromatic cyclic alkyl groups of
from 3 to 10 carbon atoms having single or multiple cyclic rings
and having at least one >C.dbd.C<ring unsaturation and
preferably from 1 to 2 sites of >C.dbd.C<ring
unsaturation.
[0156] "Substituted cycloalkyl" and "substituted cycloalkenyl"
refers to a cycloalkyl or cycloalkenyl group having from 1 to 5 or
preferably 1 to 3 substituents selected from the group consisting
of oxo, thione, alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,
acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminocarbonyloxy,
aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,
substituted aryl, aryloxy, substituted aryloxy, arylthio,
substituted arylthio, carboxyl, carboxyl ester, (carboxyl
ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted
cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy,
cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,
substituted cycloalkenyl, cycloalkenyloxy, substituted
cycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,
guanidino, substituted guanidino, halo, hydroxy, heteroaryl,
substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy,
heteroarylthio, substituted heteroarylthio, heterocyclic,
substituted heterocyclic, heterocyclyloxy, substituted
heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio,
nitro, SO.sub.3H, substituted sulfonyl, sulfonyloxy, thioacyl,
thiol, alkylthio, and substituted alkylthio, wherein said
substituents are defined herein.
[0157] "Cycloalkyloxy" refers to --O-cycloalkyl.
[0158] "Substituted cycloalkyloxy refers to --O-(substituted
cycloalkyl).
[0159] "Cycloalkylthio" refers to --S-cycloalkyl.
[0160] "Substituted cycloalkylthio" refers to --S-(substituted
cycloalkyl).
[0161] "Halo" or "halogen" refers to fluoro, chloro, bromo and
iodo.
[0162] "Haloalkyl" refers to an alkyl group in which one or more
hydrogen atoms are replaced by a halogen selected from chlorine,
fluorine, bromine or iodine.
[0163] "Haloalkoxy" refers to --O-alkyl group in which one or more
hydrogen atoms of the alkyl group are replaced by a halogen
selected from chlorine, fluorine, bromine or iodine.
[0164] "Haloalkylthio" refers to --S-alkyl group in which one or
more hydrogen atoms of the alkyl group are replaced by a halogen
selected from chlorine, fluorine, bromine or iodine.
[0165] "Hydroxy" or "hydroxyl" refers to the group --OH.
[0166] "Heteroaryl" refers to a monocyclic aromatic group having 5
to 6 carbon atoms or a bicyclic ring having 8 to 10 carbon atoms
containing 1 to 4 heteroatoms independently selected from the group
consisting of oxygen, nitrogen and sulfur within the ring. Such
heteroaryl groups can have a single ring (e.g., pyridinyl or furyl)
or multiple condensed rings (e.g., indolizinyl or benzothienyl)
wherein the condensed rings may or may not be aromatic and/or
contain a heteroatom provided that the point of attachment is
through an atom of the aromatic heteroaryl group. In one
embodiment, the nitrogen and/or the sulfur ring atom(s) of the
heteroaryl group are optionally oxidized to provide for the N-oxide
(N.fwdarw.O), sulfinyl, or sulfonyl moieties. Preferred heteroaryls
include pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.
[0167] "Substituted heteroaryl" refers to heteroaryl groups that
are substituted with from 1 to 5, preferably 1 to 3, or more
preferably 1 to 2 substituents selected from the group consisting
of the same group of substituents defined for substituted aryl.
[0168] "Heteroaryloxy" refers to --O-heteroaryl.
[0169] "Substituted heteroaryloxy refers to the group
--O-(substituted heteroaryl).
[0170] "Heteroarylthio" refers to the group --S-heteroaryl.
[0171] "Substituted heteroarylthio" refers to the group
--S-(substituted heteroaryl).
[0172] "Heterocycle" or "heterocyclic" or "heterocycloalkyl" or
"heterocyclyl" refers to a saturated or partially saturated, but
not aromatic, group having from 1 to 10 ring carbon atoms and from
1 to 4 ring heteroatoms selected from the group consisting of
nitrogen, sulfur, or oxygen. Heterocycle encompasses single ring or
multiple condensed rings, including fused bridged and spiro ring
systems. In fused ring systems, one or more the rings can be
cycloalkyl, aryl, or heteroaryl provided that the point of
attachment is through the non-aromatic ring. In one embodiment, the
nitrogen and/or sulfur atom(s) of the heterocyclic group are
optionally oxidized to provide for the N-oxide, sulfinyl, or
sulfonyl moieties.
[0173] "Substituted heterocyclic" or "substituted heterocycloalkyl"
or "substituted heterocyclyl" refers to heterocyclyl groups that
are substituted with from 1 to 5 or preferably 1 to 3 of the same
substituents as defined for substituted cycloalkyl.
[0174] "Heterocyclyloxy" refers to the group --O-heterocycyl.
[0175] "Substituted heterocyclyloxy refers to the group
--O-(substituted heterocycyl).
[0176] "Heterocyclylthio" refers to the group --S-heterocycyl.
[0177] "Substituted heterocyclylthio" refers to the group
--S-(substituted heterocycyl).
[0178] Examples of heterocycle and heteroaryls include, but are not
limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine,
pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,
dihydroindole, indazole, purine, quinolizine, isoquinoline,
quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline,
cinnoline, pteridine, carbazole, carboline, phenanthridine,
acridine, phenanthroline, isothiazole, phenazine, isoxazole,
phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine,
piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline,
4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine,
thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also
referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl,
piperidinyl, pyrrolidine, and tetrahydrofuranyl.
[0179] "Nitro" refers to the group --NO.sub.2.
[0180] "Oxo" refers to the atom (.dbd.O) or (--O.sup.-).
[0181] "Spiro ring systems" refers to bicyclic ring systems that
have a single ring carbon atom common to both rings.
[0182] "Thioacyl" refers to the groups H--C(S)--, alkyl-C(S)--,
substituted alkyl-C(S)--, alkenyl-C(S)--, substituted
alkenyl-C(S)--, alkynyl-C(S)--, substituted alkynyl-C(S)--,
cycloalkyl-C(S)--, substituted cycloalkyl-C(S)--,
cycloalkenyl-C(S)--, substituted cycloalkenyl-C(S)--, aryl-C(S)--,
substituted aryl-C(S)--, heteroaryl-C(S)--, substituted
heteroaryl-C(S)--, heterocyclic-C(S)--, and substituted
heterocyclic-C(S)--, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined
herein.
[0183] "Thiol" or "thio" refers to the group --SH.
[0184] "Thioether" refers to the group --S--.
[0185] "Alkylthio" refers to the group --S-alkyl wherein alkyl is
as defined herein.
[0186] "Thiocarbonyl" refers to the divalent group --C(S)-- which
is equivalent to --C(.dbd.S)--.
[0187] "Thione" refers to the atom (.dbd.S).
[0188] "Substituted alkylthio" refers to the group --S-(substituted
alkyl) wherein substituted alkyl is as defined herein.
[0189] "Azido" denotes the group --N.sub.3.
[0190] "Amino protecting" groups are known in the field and
illustrated by N-tert-butoxycarbonyl (t-Boc),
9-fluorenylmethoxycarbonyl (Fmoc), carboxybenzyl (Cbz), acetyl
(Ac), benzoyl (Bz), p-methoxybenzyl carbonyl (Moz or MeOZ), benzyl
(Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM),
p-methoxyphenyl (PMP), etc.
[0191] "Phosphate" means --O--P(.dbd.O)(OH).sub.2.
[0192] "Phosphate ester" refers to --O--P(.dbd.O)(OH)(OR.sup.23),
wherein R.sup.23 is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic.
[0193] "Phosphoramidate" refers to the dianionic form of
phosphoramidic acid [(OH).sub.2P(O)NH.sub.2].
[0194] "Phosphonates" are organic compounds containing
R.sup.23--PO(OH).sub.2 or R.sup.23--PO(OR).sub.2 groups where R is
alkyl or aryl and R.sup.23 is selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic.
[0195] "Phosphorothioate" refers to
##STR00076##
as well as to tautomers of these species, where "tautomer" denotes
alternate forms of a compound that differ in the position of a
proton.
General Conjugation Methodology
[0196] In one aspect, provided herewith is a method of preparing a
solid support-bound conjugated molecule of the formula
##STR00077##
wherein the method comprises (i) attaching a conjugation component
of the formula
##STR00078##
wherein R.sup.2 is R.sup.2a Or R.sup.2ap
[0197] to a solid support
##STR00079##
to form a compound of the formula
##STR00080##
(ii) when R.sup.2 is R.sup.2ap, converting
##STR00081##
to
##STR00082##
and (iii) reacting
##STR00083##
with a conjugation component of the formula
##STR00084##
wherein R.sup.3 is R.sup.3a or R.sup.3ap, or R.sup.3 is selected
from --OH, a detectable label, or another oligo;
[0198] to form
##STR00085##
wherein:
##STR00086##
is a solid support material;
##STR00087##
and are each independently selected from the group oligo;
[0199] R.sup.1a and R.sup.1b are complementary conjugation
functionalities, L.sup.1 is conjugate linker, and R.sup.1a,
R.sup.1b, and L.sup.1 are: [0200] (a) R.sup.1a is azido, R.sup.1b
is --C.ident.C--R.sup.23, and L.sup.1 is
##STR00088##
[0200] or [0201] (b) R.sup.1a is --NHR.sup.23, R.sup.1b is carboxy,
and L.sup.1 is --NR.sup.23C(.dbd.O)--, or [0202] (c) R.sup.1a is
carboxy, R.sup.1b is --NHR.sup.23, and L.sup.1 is
--C(.dbd.O)NR.sup.23--, or [0203] (d) R.sup.1a is --NHR.sup.23,
R.sup.1b is halo, and L.sup.1 is --NR.sup.23--, or [0204] (e)
R.sup.1a is --O--P(.dbd.O)(OH)(X), R.sup.1b is hydroxy, and L.sup.1
is --O--P(.dbd.O)(OH)--O--, or [0205] (f) R.sup.1a is
--O--P(.dbd.O)(OH)(X), R.sup.1b is --NHR.sup.23, and L.sup.1 is
--O--P(.dbd.O)(OH)--NR.sup.23--, or [0206] (g) R.sup.1a is
--O--P(.dbd.O)(OH)(X), R.sup.1b is thio, and L.sup.1 is
--O--P(.dbd.O)(OH)--S--, or [0207] (h) R.sup.1a is --X, R.sup.1b is
thio, and L.sup.1 is --S--, or [0208] (i) R.sup.1a is
##STR00089##
[0208] R.sup.1b is thio, and L.sup.1 is
##STR00090##
or [0209] (j) R.sup.1a is --C.ident.C--R.sup.23, R.sup.1b is azido,
and L.sup.1 is R.sup.23,
##STR00091##
[0209] or [0210] (k) R.sup.1a is halo, R.sup.1b is --NHR.sup.23,
and L.sup.1 is --NR.sup.23--, or [0211] (l) R.sup.1a is hydroxy,
R.sup.1b is --O--P(.dbd.O)(OH)(X), and L.sup.1 is
--O--P(.dbd.O)(OH)--O--, or [0212] (m) R.sup.1a is --NHR.sup.23,
R.sup.1b is --O--P(.dbd.O)(OH)(X), and L.sup.1 is
--NR.sup.23--P(.dbd.O)(OH)--O--, or [0213] (n) R.sup.1a is thio,
R.sup.1b is --O--P(.dbd.O)(OH)(X), and L.sup.1 is
--S--P(.dbd.O)(OH)--O--, or [0214] (o) R.sup.1a is
--O--P(.dbd.O)(OH)SH, R.sup.1b is --X, and L.sup.1 is
--O--P(.dbd.O)(OH)--S--, or [0215] (p) R.sup.1a is --X, R.sup.1b is
--O--P(.dbd.O)(OH)SH, and L.sup.1 is --S--P(.dbd.O)(OH)--O--, or
[0216] (q) R.sup.1a is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, R.sup.1b is
--NHR.sup.23, L.sup.1 is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)NR.sup.23--, or [0217] (r)
R.sup.1a is --NHR.sup.23, R.sup.1b is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, L.sup.1 is
--NR.sup.23C(.dbd.O)--(CR.sup.25R.sup.25).sub.s--, or [0218] (s)
R.sup.1a is thio, R.sup.1b is --X, and L.sup.1 is --S--, or [0219]
(t) R.sup.1a is thio, R.sup.1b is
##STR00092##
[0219] and L.sup.1 is
[0220] ##STR00093## [0221] (u) R.sup.1a is --SH, R.sup.1b is
--O--P(.dbd.O)(OH)(--O--(CH.sub.2).sub.n--SH), and L.sup.1 is
--S--S--(CH.sub.2).sub.n--O--P(OH)(.dbd.O)--O--.
[0222] In the above characterization of R.sup.1a and R.sup.1b group
X is selected from chlorine, bromine, fluorine, tosylate, mesylate,
triflate, or dimethoxy triflate where n is 1, 2, 3, 4, 5, or 6.
[0223] Similarly, R.sup.2a and R.sup.2b are complementary
conjugation functionalities, L.sup.2 is conjugate linker, and
R.sup.2ap, R.sup.2a, R.sup.2b, and L.sup.2 are: [0224] (a')
R.sup.2ap is halo, R.sup.2a is azido, R.sup.2b is
--C.ident.C--R.sup.23, and L.sup.2 is
##STR00094##
[0224] or [0225] (b') R.sup.2ap is --NR.sup.23Pr, R.sup.2a is
--NHR.sup.23, R.sup.2b is carboxy, and L.sup.2 is
--NR.sup.23C(.dbd.O)--, or [0226] (c') R.sup.2ap is carboxy ester,
R.sup.2a is carboxy, R.sup.2b is --NHR.sup.23, and L.sup.2 is
--C(.dbd.O)NR.sup.23--, or [0227] (d') R.sup.2ap is --NR.sup.23Pr,
R.sup.2a is --NHR.sup.23, R.sup.2b is halo, and L.sup.2 is
--NR.sup.23--, or [0228] (e') R.sup.2ap is --OH, phosphate or
phosphate ester, R.sup.2a is --O--P(.dbd.O)(OH)(X), R.sup.2b is
hydroxy, and L.sup.2 is --O--P(.dbd.O)(OH)--O--, or [0229] (f)
R.sup.2ap is --OH, phosphate or phosphate ester, R.sup.2a is
--O--P(.dbd.O)(OH)(X), R.sup.2b is --NHR.sup.23, and L.sup.2 is
--O--P(.dbd.O)(OH)--NR.sup.23--, or [0230] (g') R.sup.2ap is --OH,
phosphate or phosphate ester, R.sup.2a is --O--P(.dbd.O)(OH)(X),
R.sup.2b is thio, and L.sup.2 is --O--P(.dbd.O)(OH)--S--, or [0231]
(h') R.sup.2a is --X, R.sup.2b is thio, and L.sup.2 is --S--, or
[0232] (i') R.sup.2a is
##STR00095##
[0232] R.sup.2b is thio, and L.sup.2 is
##STR00096##
or [0233] (j') R.sup.2a is --C.ident.C--R.sup.23, R.sup.2b is
azido, and L.sup.2 is
##STR00097##
[0233] or [0234] (k') R.sup.2a is --X, R.sup.2b is --NHR.sup.23,
and L.sup.2 is --NR.sup.23--, or [0235] (l') R.sup.2a is hydroxy,
R.sup.2b is --O--P(.dbd.O)(OH)(X), and L.sup.2 is
--O--P(.dbd.O)(OH)--O--, or [0236] (m') R.sup.2a is --NHR.sup.23,
R.sup.2b is --O--P(.dbd.O)(OH)(X), and L.sup.2 is
--NR.sup.23--P(.dbd.O)(OH)--O--, or [0237] (n') R.sup.2a is thio,
R.sup.2b is --O--P(.dbd.O)(OH)(X), and L.sup.2 is
--S--P(.dbd.O)(OH)--O--, or [0238] (o') R.sup.2ap is
--O--P(.dbd.O)(OH)SR.sup.24, R.sup.2a is --O--P(.dbd.O)(OH)SH,
R.sup.2b is halo, and L.sup.2 is --O--P(.dbd.O)(OH)--S--, or [0239]
(p') R.sup.2a is --X, R.sup.2b is --O--P(.dbd.O)(OH)SH, and L.sup.2
is --S--P(.dbd.O)(OH)--O--, or [0240] (q') R.sup.2a is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, R.sup.2b is
--NHR.sup.23, L.sup.2 is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)NR.sup.23--, or [0241] (r')
R.sup.2a is --NHR.sup.23, R.sup.2b is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, L.sup.2 is
--NR.sup.23C(.dbd.O)--(CR.sup.25R.sup.25).sub.s--, or [0242] (s')
R.sup.2a is thio, R.sup.2b is --X, and L.sup.2 is --S--, or [0243]
(t') R.sup.2a is thio, R.sup.2b is
##STR00098##
[0243] and L.sup.2 is
##STR00099##
[0244] or [0245] (u') R.sup.2a is --SH, R.sup.2b is
--O--P(.dbd.O)(OH)(--O--(CH.sub.2).sub.n--SH), and L.sup.2 is
--S--S--(CH.sub.2).sub.n--O--P(OH)(.dbd.O)--O--.
[0246] In the above characterization of R.sup.1a and R.sup.1b group
X is selected from chlorine, bromine, fluorine, tosylate, mesylate,
triflate, or dimethoxy triflate
[0247] Substituent R.sup.23 is selected from the group consisting
of hydrogen, alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, cycloalkynyl, substituted cycloalkynyl, heteroaryl,
substituted heteroaryl, heterocyclic, and substituted heterocyclic,
Pr is an amino protecting group, R.sup.24 can be trityl or benzyl,
substituent R.sup.25 is hydrogen or C.sub.1-6 alkyl and s is an
integer greater than 1, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
[0248] For polymeric compounds synthesized using the inventive
methodology, substituent R.sup.3ap is a protected conjugation
functionality selected from the group consisting of halo,
--NR.sup.23Pr, --O--P(.dbd.O)(OH)SR.sup.24, carboxy ester,
phosphate and phosphate ester; and R.sup.3a is a conjugation
functionality selected from the group consisting of azido,
--C.ident.C--R.sup.23, --NHR.sup.23, carboxy, halo, hydroxy,
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, --O--P(.dbd.O)(OH)SH
and --O--P(.dbd.O)(OH)(X), where X is as defined above. In
accordance with the inventive methodology, R.sup.2 is not permitted
to react with R.sup.1a or R.sup.1b, nor is R.sup.3 permitted to
react with R.sup.2a or R.sup.2b. As discussed above, the symbol
-----represents the point of connection to the part of the solid
support-bound conjugated molecule that is closer to
##STR00100##
and represents the point of connection to the part of the solid
support-bound conjugated molecule that is further away from
##STR00101##
[0249] In accordance with another aspect of the invention, the
method is provided for preparing a solid support-bound conjugated
molecule of the formula:
##STR00102##
where the method comprises the following steps. When R.sup.3 is a
protected conjugation functionality, such as the group R.sup.3ap,
deprotecting R.sup.3ap to convert
##STR00103##
##STR00104##
in a deprotection step. The deprotection step is followed by a
conjugation step in which the solid support-bound conjugated
molecule according to formula
##STR00105##
having a reactive conjugation functionality R.sup.3a is contacted
with a conjugation component according to formula
##STR00106##
to form a solid support-bound conjugated molecule as illustrated
below:
##STR00107##
[0250] Substituent R.sup.4 in the obtained product is a group
selected from halo, --NR.sup.23Pr, --OH,
--O--P(.dbd.O)(OH)SR.sup.24, carboxy ester, phosphate, phosphate
ester, azido, --C.ident.C--R.sup.23, --NHR.sup.23, carboxy,
hydroxy, --(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23,
--O--P(.dbd.O)(OH)SH, or --O--P(.dbd.O)(OH)(X). R.sup.4 can also be
a detectable label or another oligo selected from DNA, RNA, a
polymer containing both DNA and RNA residues, or a polymer composed
entirely of of PNA monomers. In any event, R.sup.4 does not react
with R.sup.3a or R.sup.3b.
[0251] The bond L.sup.1 that binds the desired product to the solid
support can be cleaved via a suitable cleavage method to give a
conjugated molecule of the formula:
##STR00108##
The method used to cleave the oligo product from the resin support
will depend on the type of resin used to synthesize the oligo
product, the chemical structure of L.sup.1, the chemical identity
of the other linkers (e.g., L.sup.2, L.sup.3) in the product, as
well as the stability of monomer groups in Oligos A, B and C to
chemical reagents used to cleave the conjugated molecule from the
solid support. The choice for using a particular resin will depend
on protocols used for coupling the first monomer, the cleavage
conditions to be used to obtain final product in high yield and the
desired functionality, that is, the chemical nature of terminal
group "Z" in the cleaved final product. Illustrative cleavage
protocols are further described below. Several types of resin
supports are commercially available. Exemplary of such supports
without limitation are the controlled pore glass, PAM resin,
benzhydrylamine resin (BHA), Wang resin, oxime resin (Kaiser), HMBA
resin, Rink amide resin and PAL resin. Depending on the resin used,
Z in the cleaved product can be any one of the following groups
--OH, OH--(C.sub.1-C.sub.10)alkylene-, --COOH, NH.sub.2C(O)--,
NH.sub.2NH--C(O)--, COOH--(C.sub.1-C.sub.10)alkylene-,
NH.sub.2C(O)--(C.sub.1-C.sub.10)alkylene-,
NH.sub.2NH--C(O)--(C.sub.1-C.sub.10)alkylene-,
CH.sub.2.dbd.CH--(C.sub.1-C.sub.10)alkylene-,
C.ident.C--(C.sub.1-C.sub.10)alkylene- or
HS--(C.sub.1-C.sub.10)alkylene- and the alkylene can be optionally
substituted by one or more groups selected from --OH, halogen,
--NHR'', --NHC(O)--(C.sub.1-C.sub.10)alkylene-C.ident.CH, or
--NHC(O)--(C.sub.1-C.sub.10)alkylene-CH.dbd.CH.sub.2 with R''
defined as above. Illustrative of reagents used to cleave the oligo
product from the solid support include UV light, acids such as
trifluoromethane sulfonic acid, trifluoroacetic acid, hydrogen
bromide, acetic acid. Nucleophilic reagents such as sodium
hydroxide, hydrazine, alcohols primary amines and hydrides can be
used to cleave the oligo product off HMBA and oxime resins. If the
oligo is conjugated to solid support "S" through a disulfide
linkage, cleavage of the oligo product from the solid support can
be facilitated using a reducing agents. Illustrative of reagents
suitable for cleaving a disulfide bond without limitation are
.beta.-mercaptoethanol dithiothreitol (DTT),
tris-(2-carboxyethyl)phosphine (TCEP), or hydride reagents such as
sodium borohydride and soudin cyanoborohydride. Depending on the
cleaving reagent used oligo products having a terminal amide group,
carboxylic acid group, alcohol group, aldehyde group or hydrazide
group are obtained.
[0252] In some embodiments, the method further comprises repeating
deprotection and conjugation steps described above "n" times to
form a solid support-bound conjugated molecule of the formula:
##STR00109##
wherein n is an integer equal to or greater than 1, for example, an
integer of between 1 and 50, or 1 and 25, or 1 and 20, or 1 and 10,
and each
##STR00110##
is independently selected from the above-discussed oligo
category.
[0253] In the above method, the intermediate product after each
conjugation step has a terminal group R.sup.4. When R.sup.4 is a
conjugation functionality selected from the group consisting of
azido, --C.ident.C--R.sup.23, --NHR.sup.23, carboxy, halo, hydroxy,
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, --O--P(.dbd.O)(OH)SH
and --O--P(.dbd.O)(OH)Br, R.sup.4 is R.sup.3a and the deprotection
step of converting R.sup.4 to R.sup.3a may be omitted. When the
R.sup.4 is a group selected from halo, --NR.sup.23Pr,
--O--P(.dbd.O)(OH)SR.sup.24, carboxy ester, phosphate or phosphate
ester, the terminal protected conjugation functionality R.sup.4 is
first deprotected to obtain an intermediate that has the reactive
conjugation functionality R.sup.3a described above. This
intermediate then can be contacted with the next conjugation
component
##STR00111##
so as to permit a reaction between complementary conjugation
functionalities R.sup.3a and R.sup.3b, so as to covalently bond
another
##STR00112##
to the solid support-bound conjugated molecule.
[0254] After completion of the reaction sequence, the bond L.sup.1
is cleaved under suitable cleavage conditions to give a conjugated
molecule of the formula:
##STR00113##
with Z being a group is as defined above.
[0255] For conjugated molecules that comport with the above
formula, each set of R.sup.3ap, R.sup.3a, R.sup.3b, and L.sup.3 is
independently selected from [0256] (a'') R.sup.3a p is halo,
R.sup.3a is azido, R.sup.3b is --C.ident.C--R.sup.23, and L.sup.3
is
##STR00114##
[0256] or [0257] (b'') R.sup.3ap is --NR.sup.23Pr, R.sup.3a is
--NHR.sup.23, R.sup.3b is carboxy, and L.sup.3 is
--NR.sup.23C(.dbd.O)--, or [0258] (c'') R.sup.3ap is carboxy ester,
R.sup.3a is carboxy, R.sup.3b is --NHR.sup.23, and L.sup.3 is
--C(.dbd.O)NR.sup.23--, or [0259] (d'') R.sup.3ap is --NR.sup.23Pr,
R.sup.3a is --NHR.sup.23, R.sup.3b is halo, and L.sup.3 is
--NR.sup.23--, or [0260] (e'') R.sup.3ap is --OH, phosphate or
phosphate ester, R.sup.3a is --O--P(.dbd.O)(OH)(X), R.sup.3b is
hydroxy, and L.sup.3 is --O--P(.dbd.O)(OH)--O--, or [0261] (f')
R.sup.3ap is --OH, phosphate or phosphate ester, R.sup.3a is
--O--P(.dbd.O)(OH)(X), R.sup.3b is --NHR.sup.23, and L.sup.3 is
--O--P(.dbd.O)(OH)--NR.sup.23--, or [0262] (g'') R.sup.3ap is --OH,
phosphate or phosphate ester, R.sup.3a is --O--P(.dbd.O)(OH)(X),
R.sup.3b is thio, and L.sup.3 is --O--P(.dbd.O)(OH)--S--, or [0263]
(h'') R.sup.3a is --X, R.sup.3b is thio, and L.sup.3 is --S--, or
[0264] (i'') R.sup.3a is
##STR00115##
[0264] R.sup.3b is thio, and L.sup.3 is
##STR00116##
or [0265] (j'') R.sup.3a is --C.ident.C--R.sup.23, R.sup.3b is
azido, and L.sup.3 is
##STR00117##
[0265] or [0266] (k'') R.sup.3a is halo, R.sup.3b is --NHR.sup.23,
and L.sup.3 is --NR.sup.23--, or [0267] (l'') R.sup.3a is hydroxy,
R.sup.2b is --O--P(.dbd.O)(OH)(X), and L.sup.3 is
--O--P(.dbd.O)(OH)--O--, or [0268] (m'') R.sup.3a is --NHR.sup.23,
R.sup.3b is --O--P(.dbd.O)(OH)(X), and L.sup.3 is
--NR.sup.23--P(.dbd.O)(OH)--O--, or [0269] (n'') R.sup.3a is thio,
R.sup.3b is --O--P(.dbd.O)(OH)(X), and L.sup.3 is
--S--P(.dbd.O)(OH)--O--, or [0270] (o'') R.sup.3ap is
--O--P(.dbd.O)(OH)SR.sup.24, R.sup.3a is --O--P(.dbd.O)(OH)SH,
R.sup.3b is --X, and L.sup.3 is --O--P(.dbd.O)(OH)--S--, or [0271]
(p'') R.sup.3a is --X, R.sup.3b is --O--P(.dbd.O)(OH)SH, and
L.sup.3 is --S--P(.dbd.O)(OH)--O--, or [0272] (q'') R.sup.3a is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, R.sup.3b is
--NHR.sup.23, L.sup.3 is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)NR.sup.23--, or [0273] (r'')
R.sup.3a is --NHR.sup.23, R.sup.3b is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, L.sup.3 is
--NR.sup.23C(.dbd.O)--(CR.sup.25R.sup.25).sub.s--, or [0274] (s'')
R.sup.3a is thio, R.sup.3b is --X, and L.sup.3 is --S--, or [0275]
(t'') R.sup.3a is thio, R.sup.3b is
##STR00118##
[0275] and L.sup.3 is
##STR00119##
[0276] or [0277] (u'') R.sup.3a is --SH, R.sup.3b is
--O--P(.dbd.O)(OH)(O--(CH.sub.2).sub.n--SH) and L.sup.3 is
--S--S--(CH.sub.2).sub.n--O--P(OH)(.dbd.O)--O--.
[0278] An embodiment of this general process is illustrated in
Scheme 1.
##STR00120##
[0279] In Scheme 1, m is an integer starting from 1 and increasing
by 1 after each optional deprotection and conjugation steps to a
predetermined value "n". According to the protocol illustrated in
Scheme 1, subscript `n" in the resin bound conjugated molecule is
an integer between 0 and 25, for example, "n" can be 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24. Other variables
are as defined above.
Asymmetric Conjugation Methodology
[0280] In another aspect, is provided a method of preparing a
compound of the formula
##STR00121##
asymmetric conjugation strategy. According to this methodology, a
conjugation component according to formula
##STR00122##
is attached to a solid support
##STR00123##
to form a compound of the formula
##STR00124##
This intermediate then is allowed to react with a conjugation
component of the formula
##STR00125##
thereby to form
##STR00126##
A solid support material
##STR00127##
is used for synthesis, with each of
##STR00128##
being selected independently from the category oligo described
above.
[0281] R.sup.3 is R.sup.3a, a detectable label or an oligo.
R.sup.1a, R.sup.1b and R.sup.2a, R.sup.2b are complementary
conjugation functionalities, and L.sup.1 and L.sup.2 are conjugate
linkers formed by reaction between R.sup.1a and R.sup.1b and
between R.sup.2a and R.sup.2b, respectively. R.sup.1a, R.sup.1b,
L.sup.1, R.sup.2a, R.sup.2b, L.sup.2, and R.sup.3a are selected
from:
TABLE-US-00002 R.sup.1a, R.sup.2a, or R.sup.3a R.sup.1b or R.sup.2b
L.sup.1 or L.sup.2 --C.ident.C--R.sup.23 azido ##STR00129## azido
--C.ident.C--R.sup.23 ##STR00130## carboxy --NHR.sup.23
--C(.dbd.)NR.sup.23-- --NHR.sup.23 carboxy --NR.sup.23C(.dbd.O)--
halo --NHR.sup.23 --NR.sup.23-- --NR.sup.23 halo --NR.sup.23--
hydroxy --O--P(.dbd.O)(OH)(X) --O--P(.dbd.O)(OH)--O--
--O--P(.dbd.O)(OH)(X) hydroxy --O--P(.dbd.O)(OH)--O-- --NHR.sup.23
--O--P(.dbd.O)(OH)(X) --NR.sup.23--P(.dbd.O)(OH)--O--
--O--P(.dbd.O)(OH)(X) --NHR.sup.23 --O--P(.dbd.O)(OH)--NR.sup.23--
thio --O--P(.dbd.O)(OH)(X) --S--P(.dbd.O(OH)--O--
--O--P(.dbd.O)(OH)(X) thio --O--P(.dbd.O(OH)--S-- thiol --X --S--
--X thio --S-- --O--P(.dbd.O)(OH)SH --X --O--P(.dbd.O)(OH)--S-- --X
--O--P(.dbd.O)(OH)SH --S--P(.dbd.O)(OH)--O--
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23 --NHR.sup.23
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)NR.sup.23-- --NHR.sup.23
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23
--NHR.sup.23C(.dbd.O)--(CR.sup.25R.sup.25).sub.2-- thio
##STR00131## ##STR00132## ##STR00133## thio ##STR00134## --SH
--O--P(.dbd.O)(OH)(O--(CH.sub.2).sub.n--SH)
--O--P(.dbd.O)(OH)(O--(CH.sub.2).sub.n--S--S--
X is a group selected from chlorine, bromine, fluorine, tosylate,
mesylate, triflate, or dimethoxy triflate and the selection of
R.sup.1a, R.sup.2a, or R.sup.3a are independent of each other
provided that L.sup.1 and L.sup.2 are different, R.sup.2a does not
react with R.sup.1a or R.sup.1b, and R.sup.3a does not react with
R.sup.2a or R.sup.2b;
[0282] R.sup.23 is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
cycloalkynyl, substituted cycloalkynyl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic; and
[0283] ----- represents the point of connection to the part of the
solid support-bound conjugated molecule that is closer to
##STR00135##
while represents the point of connection to the part of the solid
support-bound conjugated molecule that is further away from
##STR00136##
[0284] In accordance with the invention, a method also is provided
for the manufacture of a solid support-bounded conjugated molecule
according to the following formula:
##STR00137##
According to this method, a compound according to formula
##STR00138##
is reacted with a conjugation component
##STR00139##
to obtain the following polymer
##STR00140##
[0285] R.sup.4 in such as polymer can be a conjugation
functionality selected from the definitions for conjugation
functionalities R.sup.1a, R.sup.2a, or R.sup.3a, provided above, a
detectable label or a polymer encompassed by the category oligo
described above, provided that R.sup.4 does not react with R.sup.3a
or R.sup.3b.
[0286] Upon completion of the synthesis, the target polymer is
cleaved from the solid support using a suitable cleavage method to
give
##STR00141##
where Z is selected from --OH, OH--(C.sub.1-C.sub.10)alkylene-,
--COOH, NH.sub.2C(O)--, NH.sub.2NH--C(O)--,
COOH--(C.sub.1-C.sub.10)alkylene-,
NH.sub.2C(O)--(C.sub.1-C.sub.10)alkylene-,
NH.sub.2NH--C(O)--(C.sub.1-C.sub.10)alkylene-,
CH.sub.2.dbd.CH--(C.sub.1-C.sub.10)alkylene-,
C.ident.C--(C.sub.1-C.sub.10)alkylene- or
HS--(C.sub.1-C.sub.10)alkylene- and the alkylene can be optionally
substituted by one or more groups selected from --OH, halogen,
--NHR'', --NHC(O)--(C.sub.1-C.sub.10)alkylene-C.ident.CH, or
--NHC(O)--(C.sub.1-C.sub.10)alkylene-CH.dbd.CH.sub.2. When the
alkylene is substituted with an --NHR'' group, variable R'' is
selected from (C.sub.1-C.sub.10)alkyl,
(C.sub.3-C.sub.10)cycloalkyl, or (C.sub.3-C.sub.10)aryl. Reagents
that are useful for cleaving the oligo product from the solid
support are similar to the ones described above.
[0287] In some embodiments, the method further comprises repeating
the conjugation_step "n" times to form a solid support-bound
conjugated molecule according to the following formula:
##STR00142##
wherein n is an integer of equal to or greater than 1, and each
##STR00143##
is independently selected from the category oligo described
above.
[0288] In the above method, the intermediate product after each
conjugation step has a terminal group R.sup.4. When the R.sup.4 is
a conjugation functionality R.sup.3a, this group permits the
intermediate to react in subsequent conjugation steps with a
complementary conjugation functionality R.sup.3b that is present on
one end of a conjugation component of the formula:
##STR00144##
to add
##STR00145##
to the solid support-bound conjugated molecule.
[0289] After completion of the reaction sequence, the bond L.sup.1
that binds the desired product to the solid support is cleaved
under suitable cleavage conditions to give a conjugated molecule of
the formula:
##STR00146##
where the terminal group Z is defined above.
[0290] Each set of R.sup.3a, R.sup.3b, and L.sup.3 is independently
selected from
[0291] (a'') R.sup.3a is azido, R.sup.3b is --C.ident.C--R.sup.23,
and L.sup.3 is
##STR00147##
or
[0292] (b'') R.sup.3a is --NHR.sup.23, R.sup.3b is carboxy, and
L.sup.3 is --C(.dbd.O)NR.sup.23--, or
[0293] (c'') R.sup.3a is carboxy, R.sup.3b is --NHR.sup.23, and
L.sup.3 is --C(.dbd.O)NR.sup.23--, or
[0294] (d'') R.sup.3a is --NHR.sup.23, R.sup.3b is halo, and
L.sup.3 is --NR.sup.23--, or
[0295] (e'') R.sup.3a is --O--P(.dbd.O)(OH)(X), R.sup.3b is
hydroxy, and L.sup.3 is --O--P(.dbd.O)(OH)--O--, or
[0296] (f') R.sup.3a is --O--P(.dbd.O)(OH)(X), R.sup.3b is
--NHR.sup.23, and L.sup.2 is --NR.sup.23--P(.dbd.O)(OH)--O--,
or
[0297] (g'') R.sup.3a is --O--P(.dbd.O)(OH)(X), R.sup.3b is thio,
and L.sup.3 is --S--P(.dbd.O)(OH)--O--, or
[0298] (h'') R.sup.3a is --X, R.sup.3b is thio, and L.sup.3 is
--S--, or
[0299] (i'') R.sup.3a is
##STR00148##
R.sup.3b is thio, and L.sup.3 is
##STR00149##
or
##STR00150##
[0300] (j'') R.sup.3a is --C.ident.C--R.sup.23, R.sup.3b is azido,
and L.sup.3 is R.sup.23, or
[0301] (k') R.sup.3a is halo, R.sup.3b is --NHR.sup.23, and L.sup.3
is --NR.sup.23--, or
[0302] (l'') R.sup.3a is hydroxy, R.sup.3b is
--O--P(.dbd.O)(OH)(X), and L.sup.3 is --O--P(.dbd.O)(OH)--O--,
or
[0303] (m'') R.sup.3a is --NHR.sup.23, R.sup.3b is
--O--P(.dbd.O)(OH)(X), and L.sup.3 is
--O--P(.dbd.O)(OH)--NR.sup.23--, or
[0304] (n'') R.sup.3a is thio, R.sup.3b is --O--P(.dbd.O)(OH)(X),
and L.sup.3 is --O--P(.dbd.O)(OH)--S--, or
[0305] (o'') R.sup.3a is --O--P(.dbd.O)(OH)SH, R.sup.3b is --X, and
L.sup.3 is --O--P(.dbd.O)(OH)--S--, or
[0306] (p'') R.sup.3a is --X, R.sup.3b is --O--P(.dbd.O)(OH)SH, and
L.sup.3 is --O--P(.dbd.O)(OH)--S--, or
[0307] (q'') R.sup.3a is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, R.sup.3b is
--NHR.sup.23, L.sup.3 is --C(.dbd.O)NR.sup.23--,
[0308] (r'') R.sup.3a is --NHR.sup.23, R.sup.3b is
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, L.sup.3 is
--NR.sup.23C(.dbd.O)--, or
[0309] (s'') R.sup.3a is thio, R.sup.3b is --X, and L.sup.3 is
--S--, or
[0310] (t'') R.sup.3a is thio, R.sup.3b is
##STR00151##
and L.sup.3 is
##STR00152##
[0311] or
[0312] (u'') R.sup.3a is --SH, R.sup.3b is
--O--P(.dbd.O)(OH)(O--(CH.sub.2).sub.n--SH) and L.sup.3 is
--S--S--(CH.sub.2).sub.n--O--P(OH)(.dbd.O)--O--, where group X in
conjugation functionalities R.sup.3a and R.sup.3b is as defined
above.
[0313] An embodiment of this process is illustrated in Scheme 2. In
Scheme 2, m is an integer starting from 1 and increasing by 1 after
each conjugation step until a conjugated molecule having the
prescribed number ("n") of oligo units is obtained, where "n" is an
integer between 0 and 25, for example, "n" can be 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24. The definitions for
variables R.sup.1a, R.sup.1b, R.sup.2a, R.sup.2b, R.sup.3a,
R.sup.3b and R.sup.4 are as defined above.
##STR00153##
Symmetric Conjugation Methodology
[0314] In still another aspect, provided herewith is a method of
preparing a compound of the formula
##STR00154##
comprising (i) attaching a conjugation component of the formula
##STR00155##
to a solid support
##STR00156##
to form a compound of the formula
##STR00157##
(ii) converting
##STR00158##
to
##STR00159##
and (iii) reacting
##STR00160##
with a conjugation component of the formula
##STR00161##
to form
##STR00162##
where:
##STR00163##
is a solid support material;
##STR00164##
are each independently selected from polymers encompassed by the
category oligo defined above;
[0315] According to this synthetic methodology, R.sup.3 can either
be a protected conjugation functionality R.sup.ap, or R.sup.3 is a
group selected from a detectable label or a polymer of the categoty
oligo. R.sup.a and R.sup.b are complementary conjugation
functionalities and L is conjugate linker formed by reaction of
R.sup.a and R.sup.b. R.sup.ap, R.sup.a, R.sup.b, and L are each
independently selected from [0316] (a) R.sup.ap is halo, R.sup.a is
azido, R.sup.b is --C.ident.C--R.sup.23, and L is
##STR00165##
[0316] or [0317] (b) R.sup.ap is --NR.sup.23Pr, R.sup.a is
--NHR.sup.23, R.sup.b is carboxy, and L is --NR.sup.23C(.dbd.O)--,
or [0318] (c) R.sup.ap is carboxy ester, R.sup.a is carboxy,
R.sup.b is --NHR.sup.23, and L is --C(.dbd.O)NR.sup.23--, or [0319]
(d) R.sup.ap is --NR.sup.23Pr, R.sup.a is --NHR.sup.23, R.sup.b is
halo, and L is --NR.sup.23--, or [0320] (e) R.sup.ap is --OH,
phosphate or phosphate ester, R.sup.a is --O--P(.dbd.O)(OH)(X),
R.sup.b is hydroxy, and L is --O--P(.dbd.O)(OH)--O--, or [0321] (f)
R.sup.ap is --OH, phosphate or phosphate ester, R.sup.a is
--O--P(.dbd.O)(OH)(X), R.sup.b is --NHR.sup.23, and L is
--O--P(.dbd.O)(OH)--NR.sup.23--, or [0322] (g) R.sup.ap is
--O--P(.dbd.O)(OH)SR.sup.24, R.sup.a is --O--P(.dbd.O)(OH)SH,
R.sup.b is --X, and L.sup.2 is --O--P(.dbd.O)(OH)--S--, or [0323]
(h) R.sup.ap is --OH, phosphate or phosphate ester, R.sup.a is
--O--P(.dbd.O)(OH)(X), R.sup.b is thio, and L is
--O--P(.dbd.O)(OH)--S-- [0324] (i) R.sup.ap is --SR.sup.24, R.sup.a
is --SH, R.sup.b is HS--(CH.sub.2).sub.n--O--P(OH)(.dbd.O)--O-- and
L is --S--S--(CH.sub.2).sub.n--O--P(OH)(.dbd.O)--O--;
[0325] R.sup.23 is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
cycloalkynyl, substituted cycloalkynyl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic;
[0326] X is selected from chlorine, bromine, fluorine, tosylate,
mesylate, triflate, or dimethoxy triflate;
[0327] Pr is an amino protecting group;
[0328] R.sup.24 is trityl or benzyl;
[0329] ----- represents the point of connection to the part of the
solid support-bound conjugated molecule that is closer to
##STR00166##
and represents the point of connection to the part of the solid
support-bound conjugated molecule that is further away from
##STR00167##
[0330] In another of its aspects, the present invention provides a
method for preparing a solid support-bound conjugated molecule of
the formula:
##STR00168##
where the compound of formula
##STR00169##
in which R.sup.3 is a protected functional group R.sup.ap is
deprotected to obtain a reactive intermediate
##STR00170##
which subsequently is contacted with a conjugation component
according to formula:
##STR00171##
to form
##STR00172##
[0331] In this embodiment, R.sup.4 is selected from the group
consisting of R.sup.ap, a detectable label or a polymer from
category oligo.
[0332] The bond L that binds the desired product to the solid
support can be cleaved under suitable cleavage conditions to give a
conjugated molecule of the formula:
##STR00173##
[0333] In some embodiments, the method further comprises repeating
the deprotection and conjugation steps "n" times to form a solid
support-bound conjugated molecule of the formula:
##STR00174##
Integer n in the product obtained using the described method can
have a value equal to or greater than 1, and each
##STR00175##
is independently selected from polymers within the category
oligo.
[0334] According to the above-described method, the intermediate
product after each conjugation step has a terminal group R.sup.4.
When the R.sup.4 is R.sup.ap, R.sup.4 is first converted to a
conjugation functionality R.sup.a and then is contacted with a
complementary conjugation functionality R.sup.b that is present on
one end of the conjugation component of the formula:
##STR00176##
to add a
##STR00177##
to the solid support-bound conjugated molecule.
[0335] After completing the synthesis to obtain a solid
support-bound conjugated molecule that has the specified number
of
##STR00178##
groups; the bond L that binds the final product to the solid
support is cleaved under suitable cleavage conditions to give a
resin free conjugated molecule according to formula:
##STR00179##
Depending on the nature of the solid support used for synthesis,
terminal group Z in the resin free product is a group selected from
--OH, OH--(C.sub.1-C.sub.10)alkylene-, --COOH, NH.sub.2C(O)--,
NH.sub.2NH--C(O)--, COOH--(C.sub.1-C.sub.10)alkylene-,
NH.sub.2C(O)--(C.sub.1-C.sub.10)alkylene-,
NH.sub.2NH--C(O)--(C.sub.1-C.sub.10)alkylene-,
CH.sub.2.dbd.CH--(C.sub.1-C.sub.10)alkylene-,
C.ident.C--(C.sub.1-C.sub.10)alkylene- or
HS--(C.sub.1-C.sub.10)alkylene- and the alkylene can be optionally
substituted by one or more groups selected from --OH, halogen,
--NHR'', --NHC(O)--(C.sub.1-C.sub.10)alkylene-C.ident.CH, or
--NHC(O)--(C.sub.1-C.sub.10)alkylene-CH.dbd.CH.sub.2. Reagents
suitable for cleaving the oligo are as defined above.
[0336] In this embodiment, all R.sup.ap are the same, all R.sup.a
are the same, R.sup.b are the same, and each L is the same, and
these variables are as defined above.
[0337] An embodiment of this process is illustrated in Scheme 3. In
Scheme 3, "m" is an integer that starts from 1 and increases by 1,
after each pair of deprotection and conjugation steps, until a
conjugated molecule having the prescribed number ("n") of oligo
units is obtained, where "n" is an integer between 0 and 25, for
example, "n" can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,
18, 20, 22, or 24. Other variables are as defined above.
##STR00180##
[0338] In some embodiments of the inventive methodology, general,
asymmetric or symmetric, R.sup.23 is hydrogen or C.sub.1-6
alkyl.
[0339] In some embodiments of the inventive methodology, general,
asymmetric or symmetric, the conjugation functionalities and the
conjugate linker are:
TABLE-US-00003 R.sup.a, R.sup.1a, R.sup.2a, or R.sup.3a R.sup.b,
R.sup.1b, R.sup.2b or R.sup.3b L, L.sup.1, L.sup.2 or L.sup.3
--C.ident.C--H azido ##STR00181## azido --C.ident.C--H ##STR00182##
carboxy --NH.sub.2 --C(.dbd.O)NH-- --NH.sub.2 carboxy
--NHC(.dbd.O)-- halo --NH.sub.2 --NH-- --NH.sub.2 halo --NH--
hydroxy --O--P(.dbd.O)(OH)(X) --O--P(.dbd.O)(OH)--O--
--O--P(.dbd.O)(OH)(X) hydroxy --O--P(.dbd.O)(OH)--O-- --NH.sub.2
--O--P(.dbd.O)(OH)B(X) --NH--P(.dbd.O)(OH)--O--
--O--P(.dbd.O)(OH)(X) --NH.sub.2 --O--P(.dbd.O)(OH)--NH-- thio
--O--P(.dbd.O)(OH)(X) --S--P(.dbd.O)(OH)--O-- --O--P(.dbd.O)(OH)(X)
thio --O--P(.dbd.O)(OH)--S-- thio --X --S-- --X thio --S--
--O--P(.dbd.O)(OH)SH --X --O--P(.dbd.O)(OH)--S-- --X
--O--P(.dbd.O)(OH)SH --S--P(.dbd.O)(OH)--O-- ##STR00183##
--NH.sub.2 --(CH.sub.2).sub.sC(.dbd.O)NH-- --NH.sub.2 ##STR00184##
--NHC(.dbd.O)--(CH.sub.2).sub.s-- thio ##STR00185## ##STR00186##
##STR00187## thio ##STR00188## --SH
--O--P(.dbd.O)(OH)(O--(CH.sub.2).sub.n--SH)
--O--P(.dbd.O)(OH)(O--(CH.sub.2).sub.n--S--S--
Conjugation Components
[0340] In another aspect, the invention provides a solid support
represented by the formula:
##STR00189##
wherein:
##STR00190##
is a solid support material;
##STR00191##
is selected from the category oligo;
[0341] R.sup.2 is R.sup.2a or R.sup.2ap;
[0342] R.sup.2ap is selected from the group consisting of halo,
NR.sup.23Pr, carboxy ester, --NR.sup.23Pr, --OH, phosphate,
phosphate ester, and --O--P(.dbd.O)(OH)SR.sup.24,
[0343] R.sup.2a is selected from the group consisting of halo,
azido, hydroxy, thio, --NHR.sup.23, carboxy, --NHR.sup.23,
--O--P(.dbd.O)(OH)(SH),
##STR00192##
--C.ident.C--R.sup.23, and
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23;
[0344] R.sup.23 is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
cycloalkynyl, substituted cycloalkynyl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic;
[0345] Pr is an amino protecting group;
[0346] R.sup.24 is trityl or benzyl;
[0347] R.sup.25 is hydrogen or C.sub.1-6 alkyl;
[0348] s is an integer of greater than 1; and
[0349] represents the point of connection to
##STR00193##
[0350] In another aspect, the invention provides a compound
according to formula:
##STR00194##
wherein R.sup.2 is R.sup.2a Or R.sup.2ap,
##STR00195##
is selected from the category oligo; R.sup.1b is selected from the
group consisting of --C.ident.C--R.sup.23, carboxy, --NHR.sup.23,
halo, hydroxy, thio, azido, --O--P(.dbd.O)(OH)(X),
--O--P(.dbd.O)(OH)SH,
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23, and
##STR00196##
[0351] R.sup.2ap is selected from the group consisting of halo,
NR.sup.23Pr, carboxy ester, --OH, phosphate, phosphate ester, and
--O--P(.dbd.O)(OH)SR.sup.24;
[0352] R.sup.2a is selected from the group consisting of halo,
azido, hydroxy, thio, --NHR.sup.23, carboxy,
--O--P(.dbd.O)(OH)Br,
##STR00197##
--C.ident.C--R.sup.23,
--(CR.sup.25R.sup.25).sub.sC(.dbd.O)OR.sup.23 and
--O--P(.dbd.O)(OH)SH;
[0353] R.sup.23 is selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
cycloalkynyl, substituted cycloalkynyl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic;
[0354] R.sup.24 is trityl or benzyl;
[0355] Pr is an amino protecting group;
[0356] and represents the point of connection to
##STR00198##
provided that R.sup.2 does not react with R.sup.1b.
Labels
[0357] Pursuant to the invention, a conjugated molecule or a
components of such molecule can comprise one or more labels that
are attached directly or through a linker. Such labeling can permit
detection of the presence, the whereabouts and/or the quantitiy of
a certain conjugation component or of conjugated molecules. For
instance, the labeling can allow differentiation of different
conjugation components or conjugated molecules in a nano-scale
information processing system as above.
[0358] In this regard, a "label" is a group that can be detected by
any means. Accordingly, the category of suitable labels includes
but is not limited to fluorescent moieties, bioluminescent
moieties, chemiluminescent moieties, colorimetric moieties,
enzymatic moieties that generate a detectable signal when contacted
with a substrate, spectrally resolvable quantum dots, metal
nanoparticles or nanoclusters, paramagnetic, superparamagnetic and
ferromagnetic substances, fluorophores, quenchers, and the like. A
label also can be a group that carries a radioactive atom, such as
Gallium-67 and Indium-111. In addition, a label can be polypeptide
or protein that acts as a receptor or as a ligand for a cognate
molecule the binding of which generates a detectable signal.
[0359] Examples of detectable moieties conjugated to another
molecule are described in U.S. Pat. Nos. 4,855,225 and 5,188,934,
in U.S. patent application publications No. 2005/0112065, No.
2007/0110798 and No. 2011/0077169, in published international
application WO1991/005060, and in Lee et al., Nucleic Acids Res.
20: 2471-483 (1992). Additionally, multiple detectable groups can
be attached to a single conjugation component or to a conjugated
molecule to provide a combined signal that allows the conjugation
component or conjugated cmolecule to be identified and
distinguished from others to which are attached a different
detectable moiety or a different set of detectable groups. That
such combinations of detectable groups are known is evidenced, for
example, those described in U.S. Pat. No. 6,632,609 and in Speicher
et al., Nature Genetics 12: 368-75 (1996).
[0360] Fluorophores, also known as fluorescent dyes, are detectable
groups that absorb light energy at a defined excitation wavelength
and emit light energy at a different wavelength. Different
fluorophores can be selected for use to give a mixture detectable
groups that can be detected based on their spectral
characteristics, particularly fluorescence emission wavelength
and/or intensity, under certain detection conditions.
[0361] Quenchers are detectable groups that are capable of
absorbing the energy of an excited fluorescent detectable group
when located in close proximity with the excited fluorescent
detectable group and of dissipating that energy without the
emission of visible light. Examples of quenchers include, but are
not limited to, DABCYL (4-(4'-dimethylaminophenylazo)benzoic acid)
succinimidyl ester, diarylrhodamine carboxylic acid, succinimidyl
ester (QSY-7), and 4',5'-dinitrofluorescein carboxylic acid,
succinimidyl ester (QSY-33) (all available from Molecular Probes),
quencher1 (Q1; available from Epoch), or "black hole quenchers"
BHQ-1, BHQ-2, and BHQ-3 (available from BioSearch, Inc.).
[0362] In some embodiments, the detectable group is selected
from:
TABLE-US-00004 Name Structure pyrene azide (N-(3-Azidopropyl)-
4-pyren-1-yl-butyramide) ##STR00199## hydroxy-coumarin azide (3-
Azido-7-hydroxycoumarin) ##STR00200## 5-FAM azide
(N-(3-azidopropyl)- 3',6'-dihydroxy-3-oxo-3H-
spiro[isobenzofuran-1,9'- xanthene]-5-carboxamide) ##STR00201## Cy3
azide ##STR00202## Cy5-azide ##STR00203## TAMRA alkyne (5-
Carboxytetramethylrhodamine, Propargylamide) ##STR00204##
5-(Bromomethyl) Fluorescein ##STR00205## Dabcyl Succinimidyl Ester
(4-((4- (dimethylamino)phenyl)azo)benz- oic Acid ##STR00206## Alexa
Fluor 430 Carboxylic acid, Succinimidyl Ester ##STR00207##
Use of the Conjugated Molecules
[0363] The conjugated molecules prepared by the methods described
herein can be used to develop molecular processing networks, of
different sizes and complexities, that are comprised of conditional
nucleic acid-exchange reactions. The conjugated molecules also can
be useful as biological transistors, which can constitute
integrated circuits for executing compound logic functions, e.g.,
in diagnostic or therapeutic contexts that entail targeting of
neoplastic or virus-infected cells. Thus, conjugated molecules of
the invention can be employed to implement nano-scale information
processing systems, suitable for solving computational problems in
a test tube or in a cell. See U.S. patent application Ser. No.
13/072,438. As noted above, such nano-scale systems can be
compatible with biological environments and have the potential for
use in the diagnosis and treatment of complex diseases, among other
applications.
[0364] Use of conjugated molecules of the invention in the manner
described here typically will entail transport of the molecules
into cells. Any methodology suitable for such intracellular
delivery can be employed. Illustrative of reagents that promote
delivery into cells of macromolecules, such as the conjugated
molecules of the invention, are transfecting agents, liposomes,
dendrimers, cholesterol, see Percot et al., Int'l J. Pharm. 278:
143-63 (2004), and delivery peptides and proteins, including
polyamines such as carboxy spermine, poly-Arginine, poly-Lysine and
the RGD peptide.
[0365] Thus, transport across a cell membrane could be achieved by
coupling an inventive oligo covalently to a peptide that contains a
cell internalization signal, a nuclear or sub-cellular localization
signal, and/or a cell targeting and endocytosis-triggering signal.
Such a peptide can be naturally-occurring or derived from synthetic
or engineered proteins; e.g., the peptide can be a synthetic
functional equivalent of naturally-occurring peptide. See Luo et
al., Nature Biotechnology 18: 33-37 (2000), and U.S. Pat. No.
7,087,770.
[0366] Cell delivery peptides and proteins can be covalently linked
to the inventive oligo by a disulfide likage. Typically, the cell
delivery peptide containing a cysteine residue will be allowed to
contact a free sulhydryl containing oligo under oxidative
conditions to facilitate the formation of the disulfide linkage.
The progress of the conjugation reaction and eventual purification
of the conjugated product can be performed by HPLC, using a C-18
reverse-phase column, and mass spectral analysis for identification
of the product.
[0367] According to one delivery strategy, a cell delivery peptide
or protein will be first bound to the inventive oligo. The peptide-
or protein-oligo complex then will be admixed with a transfection
agent or mixture of agents, and the resulting mixture will be
employed to deliver the oligo into cells. Suitable transfection
agents include cationic lipid compositions, particularly monovalent
and polyvalent cationic lipid compositions, more particularly
LIPOFECTIN.RTM., LIPOFECTAMINE.RTM., CELLFECTIN.RTM., DMRIE-C.RTM.,
DOTAP.RTM., and DOSPER.RTM., and dendrimer compositions,
particularly G5-G10 dendrimers, including dense star dendrimers,
poly(amidoamine) dendrimers (PAMAM.RTM.), grafted dendrimers, and
dendrimers known as dendrigrafts and SuperFect.RTM..
General Synthetic Methods
[0368] The starting materials for preparing conjugated molecules
according to the present invention include but are limited to,
oligonucleotides, nucleotide monomers, peptides, proteins, amino
acids, peptide nucleic acids and peptide nucleic acid derivatives.
The starting materials can be purchased from commercial vendors or
synthesized prior to use. The inventive conjugation components are
prepared by functionalizing a polymer within the category oligo to
contain one or two conjugation functionalities.
[0369] In the following examples preferred process conditions are
given, i.e., reaction temperatures, times, mole ratios of
reactants, solvents used and pressures. However, other process
conditions can also be used unless otherwise stated. Optimum
reaction conditions may vary depending on the reactants or solvents
used, but such conditions can be determined by one skilled in the
art by routine optimization procedures.
[0370] In addition to the protected groups described here, other
conventional protecting groups may be necessary to prevent certain
functional groups from undergoing undesired reactions. Suitable
protecting groups for various functional groups as well as suitable
conditions for protecting and deprotecting particular functional
groups are well known in the art. For instance, numerous protecting
groups are described in T. W. Greene and G. M. Wuts, PROTECTING
GROUPS IN ORGANIC SYNTHESIS, Third Edition (Wiley, New York, 1999),
and publications cited there.
[0371] Furthermore, various oligos may contain one or more chiral
centers. Accordingly, conjugated molecules can be prepared or
isolated as pure stereoisomers, i.e., as individual enantiomers or
diastereomers, or as mixtures of stereoisomer, such racemic
mixtures. All such stereoisomers and mixtures of stereoisomers are
within the scope of this invention. Such compounds can be prepared
using optically active starting materials, for instance, or
stereoselective reagents. Alternatively, a mixture of two or more
stereoisomers can be resolved using chiral column chromatography or
chiral resolving agents.
[0372] Oligos that are oligonucleotides may be prepared by
conventional solid-phase synthesis methodology, such as the
phosphoramidite technique, which uses phosphoramidite building
blocks derived from protected 2'-deoxynucleosides (dA, dC, dG, and
T) and ribonucleosides (A, C, G, and U). To obtain a desired
oligonucleotide, the building blocks are sequentially coupled to
the growing oligonucleotide chain in the order required by the
sequence of the product. Examples of such methods are described in
Reese, Organic & Biomolecular Chemistry 3: 3851 (2005), Iyer et
al., "Oligonucleotide synthesis" in COMPREHENSIVE NATURAL PRODUCTS
CHEMISTRY, Vol. 7: DNA and Aspects of Molecular Biology; and
Ogilvie; J. Amer. Chem. Soc. 99: 7741-43 (1997).
[0373] Peptides can be prepared from amino acids under amide
coupling conditions, such as suing an amide coupling reagent. For
example, the peptides can be prepared using a solid-phase
methodology as those described in F. F. Nord and R. B. Merrifield,
SOLID-PHASE PEPTIDE SYNTHESIS (Wiley 2006).
[0374] Peptide nucleic acids can be prepared according to
conventional methods as described, for instance, in U.S. Pat. Nos.
5,539,082 and 6,395,474.
[0375] The various compounds described herein, such as conjugation
components, intermediates, and conjugated molecules, may be
isolated and purified where appropriate using conventional
techniques such as precipitation, filtration, crystallization,
evaporation, distillation, and chromatography. Characterization of
these compounds may be performed using conventional methods such as
by melting point, mass spectrum, nuclear magnetic resonance, and
various other spectroscopic analyses.
EXAMPLES
[0376] The examples that follow are provided to illustrate certain
aspects of the present invention and to aid those of skill in the
art in practicing the invention. The examples are not to be
considered to limit the scope of the invention.
Example 1. A PNA Conjugation Component Conjugated with a DNA
Conjugation Component
##STR00208##
[0378] Scheme 4 above illustrates a general protocol for
conjugating a DNA to a peptide nucleic acid to obtain a
heteropolymer. In this scheme the 5'-terminal (5'-end) of an
appropriately functionalized DNA is permitted to contact a reactive
group on the C-terminus of a PNA moiety to synthesize a DNA-PNA
heteropolymer (Scheme 4 path (A), 5' to N-terminal
conjugation).
[0379] In one embodiment, the conjugation reaction proceeds by
providing a nucleophilic group, for example, a thiol, amine, or
hydroxide at the 5-terminus of the DNA and a suitable leaving group
such as halogen, mesylate, or tosylate at the N-terminus of the
PNA. Alternatively, conjugation can proceed by functionalizing the
5'-terminal of the Da to bear a suitable leaving group and allowing
such a DNA moiety to react with a nucleophile attached to the
N-terminus of a PNA. Pursuant to another aspect of the invention,
conjugation of the DNA to the PNA can proceed via the formation of
a disulfide bond or the formation of an amide (peptide) bond.
Alternatively, an alkyne functionalized DNA can be contacted with
an azide functionalized PNA to form a traizole ring that connects
the 5-terminus of the DNA to the N-terminus of the PNA. Protocols
for conjugating a DNA to a PNA via an amide linkage, thioether
linkage, or triazine moiety are further described below.
[0380] The DNA-PNA heteropolymers thus obtained can be cleaved from
the solid supports using a variety of cleaving reagent. Depending
on the type of solid support used and therefore, the nature of the
bond conjugating the DNA or the PNA to a support, strong and weak
acids such as hydrogen fluoride, trifluoroacetic acid,
trichloroacetic acid, bases such as ammonia, amines, or other
reagents such as hydrazine, uv light, and hydidres can be used to
cleave the heteropolymer from the supports. Typically, the cleaving
reagent is used along with scavengers to prevent racemization of
the product.
[0381] In the specific example illustrated below, the conjugation
product (X) is cleaved from the support through an elimination
reaction under basic conditions (concentrated ammonium hydroxide).
The specific support used in this case is a 1000 angstrom
Controlled Pore Glass (CPG) bead functionalized with the UnySupport
linker commercially available from GlenResearch.
##STR00209##
[0382] As described above the synthesis of a DNA-PNA heteropolymer
having a 5' to N-terminal connectivity requires the 5'-end of the
DNA to be suitable functionalized to facilitate the conjugation
reaction with the N-terminal of a PNA moiety. Illustrative of
groups that are suitable for functionalizing the 5'-terminal of DNA
are those shown in Scheme 5 below where variable "A" represents the
5'-terminal group of a DNA.
##STR00210## ##STR00211##
[0383] Methodologies for introducing functional groups illustrated
in Scheme 5 above, at the 5'-terminal of a DNA are well know in the
art See Isaac S. Marks et al., Bioconjugate Chemistry, (2011),
22(7), pp 1259-1263; Neal K. Devaraj et al., J Am Chem Soc.,
(2005), 127(24), pp 8600-8601; Joseph G. Harrison and Shankar
Balasubramanian, Nucl. Acids Res., (1998) 26 (13), pp 3136-3145;
and Podyminogin M A et al., Nucleic Acids Res., (2001), 29(24), pp
5090-8. Alternatively, a DNA-PNA heteropolymer that has a 3' to
C-terminal connectivity can be synthesized using a protocol similar
to the one illustrated above in Scheme 4, path (B). The synthesis
such a heteropolymer proceeds by introducing a functional group
selected from one of the groups that define variable "A" in Scheme
5 above at the 3'-end of a DNA and contacting the functionalized
DNA with a PNA whose C-terminus is separately functionalized to
include a group that can react with the 3'-end of the DNA.
[0384] The PNa moiety that takes part in the conjugation reaction
cam be functionalized at the C-terminal or the N-terminal of the
PNA molecule. FIGS. 3 and 4 illustrate reagents for N-terminal and
C-terminal modifications of PNA's. Protocols for modifying the N-
and C-terminii of PNA's using these reagents are well known in the
chemical art. See Andriy A. Mokhir et al., Bioconjugate Chem.,
(2003), 14 (5), pp 877-883; Brian D. Gildea et al., Tetrahedron
Letters, (1998), Volume 39, Issue 40, pp 7255-7258
[0385] Schemes 6-8 illustrate exemplary strategies for synthesizing
a 3'-functionalized DNA. The product DNA's illustrated in each of
Scheme 6-8 can be used to form the inventive DNA-PNA heteropolymer
as further described below.
##STR00212## ##STR00213##
##STR00214## ##STR00215##
##STR00216## ##STR00217##
Example 2. Amide Conjugate Linker Formation (1)
[0386] Scheme 9 illustrates the synthesis of a DNA-PNA
heteropolymer having a 5' to C-terminal connectivity.
##STR00218##
[0387] As illustrated above, the synthesis of the inventive
conjugated molecule, that is, a DNA-PNA heteropolymer proceeds by
contacting the amino group at 5'-end of the DNA (component 2-1) to
the carboxyl group of a PNA (component 2-2). The product 2-3 is
obtained through the formation of an amide bond using reaction
conditions similar to those used for performing conventional
amide-couplings. The numerical value of subscript "s" dictates the
distance separating the PNA from the DNA in the final product and
may influence the physical and/or biochemical properties of the
heteropolymer.
[0388] Suitable coupling reagents for forming an amide bond include
carbodiimides, such as N--N'-dicyclohexylcarbodiimide (DCC),
N--N'-diisopropylcarbodiimide (DIPCDI), and
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (EDCI). The
carbodiimides may be used alone or along with additives that
promote and improve coupling efficiency. Such coupling additives
include without limitation dimethylaminopyridine (DMAP) or
compounds belonging to the class benzotriazoles, e.g.,
7-aza-1-hydroxybenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt),
and 6-chloro- 1-hydroxybenzotriazole (Cl-HOBt).
[0389] Amide coupling reagents also include amininum and
phosphonium based reagents. Aminium salts include
N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-meth-
ylmethanaminium hexafluorophosphate N-oxide (HATU),
N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium
hexafluorophosphate N-oxide (HBTU),
N-[(1H-6-chlorobenzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethan-
aminium hexafluorophosphate N-oxide (HCTU),
N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium
tetrafluoroborate N-oxide (TBTU), and
N-[(1H-6-chlorobenzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethan-
aminium tetrafluoroborate N-oxide (TCTU). Phosphonium salts include
7-azabenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium
hexafluorophosphate (PyAOP) and
benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium
hexafluorophosphate (PyBOP). An amide formation step may be
conducted in a polar solvent such as dimethylformamide (DMF), where
the solvent also may include an organic base such as
diisopropylethylamine (DIEA) or dimethylaminopyridine (DMAP).
Example 3. Amide Conjugate Linker Formation (2)
[0390] Heteropolymers having an amide linkage between the DNA and
PNA units also are synthesized by permitting a PNA modified to have
a free amino group at its C-terminus to contact a 5'-carboxyl DNA
as illustrated below in Scheme 10.
##STR00219##
[0391] Briefly, the final product having an amide conjugate linker
is formed by contacting the amino group PNA conjugation component
2-2 with a (2,5-dioxopyrrolidin-1-yl)oxycarbonyl conjugation
functionality of at te 5'-end of DNA conjugation component 3-1.
Example 4. Triazole Conjugate Linker Formation (1)
[0392] Heteropolymers having a triazole linkage, such as component
4-3 in Scheme 11 below are synthesized by contacting a 3'-alkynyl
modified DNA (component 4-1) with a N-terminal azide modified PNA
(component 4-2).
##STR00220##
[0393] Alternatively, the DNA can be functionalized to have an
alkynyl group at its 5'-end and an azide at the C-terminal end of a
PNA. Briefly, synthesis proceeds as follows. The 5'-functionalized
DNA is dissolved in a solution of water and alcohol. Typically the
ratio of water to alcohol is in the range from 3:1 to 1:3. The
choice of alcohol depends on the amount and chemical nature of the
DNA being used. Exemplary alcohols include without limitation,
ethanol, propanol, n-butanol, t-butanol and i-propanol. Other
organic solvents such as dimethylformamide (DMF) can also be added
to the aqueous-alcoholic DNA solution to enhance dissolution of
DNA. A copper catalyst, such as copper sulfate or CuI is added to
the DNA solution, followed by the addition of a buffered solution
of the PNA azide. To aid the conjugation, cofactors such as
tris-hydroxypropyltriazole can be added to the reaction mixture.
The mole ratio of DNA to PNA in the reaction mixture can range from
1:1 to 1:3. The amount of DNA in the reaction mixture can be from
about 1.0 nmoles to about 20 moles, for example about 50 nmoles,
100 nmoles, 150 nmoles, 200 nmoles, 250 nmoles, 300 nmoles, 350
nmoles, 400 nmoles, 500 nmoles, 600 nmoles, 700 nmoles, 800 nmoles,
900 nmoles, 1 .mu.mole, 2 .mu.moles, 3 .mu.moles, 4 .mu.moles, 5
.mu.moles, 6 .mu.moles, 7 .mu.moles, 8 .mu.moles, 10 .mu.moles, 12
.mu.moles, 14 .mu.moles, 16 .mu.moles, 18 .mu.moles, or 20
.mu.moles. The amount of PNA is from about 1 nmoles to about 60
.mu.moles such as about 1.0 nmoles, 3 nmoles, 6 nmoles, 9 nmoles,
12 nmoles, 15 nmoles, 18 nmoles, 21 nmoles, 24 nmoles, 27 nmoles,
30 nmoles, 40 nmoles, 50 nmoles, 100 nmoles, 150 nmoles, 200
nmoles, 250 nmoles, 300 nmoles, 350 nmoles, 400 nmoles, 500 nmoles,
600 nmoles, 700 nmoles, 800 nmoles, 900 nmoles, 1 .mu.mole, 2
.mu.moles, 3 .mu.moles, 4 .mu.moles, 5 .mu.moles, 6 moles, 7
.mu.moles, 8 .mu.moles, 10 .mu.moles, 12 .mu.moles, 14 .mu.moles,
16 .mu.moles, 18 .mu.moles, 20 moles, 30 .mu.moles, 40 .mu.moles,
50 .mu.moles, or 60 .mu.moles. Depending on the scale of the
reaction, the final volume of the reaction mixture can range from
about 10 .mu.l to about 1 mL.
[0394] In a variation of the above described synthetic protocol,
the starting DNA or the PNA may be tethered to to solid support.
Solvents such as dichloromethane or DMF that hydrate and swell
solid supports can be added to enhance conjugation. In this case
the final conjugated molecule (i.e., DNA-PNA heteropolymer) is
cleaved from the support following synthesis.
Example 5. Triazole Conjugate Linker Formation (2)
[0395] Scheme 12 illustrates the synthesis of a DNA-PNA
heteropolymer (component 5-3) obtained by contacting a PNA having
an alkynyl functionality at the C-teminal (component 5-2) with a
5'-azide functionalized DNA (component 5-1).
##STR00221##
Example 6. Symmetric Conjugation Strategy Using Triazole Conjugate
Linkers
[0396] An exemplary synthesis of the inventive conjugate compounds
using a symmetric conjugation strategy based on the formation of
triazoles as the conjugate linkers is illustrated below in Scheme
13.
##STR00222##
[0397] The synthesis proceeds by contacting, a solid support 6-1
having an appropriate surface functional group, such as a bromo, a
chloro, a tosylate, a mesylate, or any other suitable leaving group
(a protected conjugation functionality) with an azide, (e.g.,
NaN.sub.3), to form the azide functionalized solid support 6-2. The
group "CL" conjugated to the solid support in the Scheme above can
be a bicyclic molecule capable of undergoing a dephosphorylation
reaction. Illustrative of a `CL` moiety having a bicyclic core is
the group illustrated below, where the indicates point to
attachment to a solid support such as controlled pore glass.
##STR00223##
This support is then contacted with a conjugation component 6-3
having the alkynyl conjugation functionality at one end and a bromo
group at its other end to obtain a solid support conjugated
component 6-4. After converting the bromo group of component 6-4 to
an azide, conjugation functionality 6-5 can further react with a
second conjugation component 6-3 to form a solid supported
conjugated molecule which can be cleaved from the solid support to
yield conjugated molecule 6-6. Depending on the desired size of the
final product, the solid supported conjugated molecule 6-5 can
react with required number of conjugation components 6-3 prior to
cleavge of the product from the solid support. The conjugation
components 6-3 may be the same or different, but each of these
conjugation components must have an alkynyl conjugation
functionality and a bromo group at their respective terminii.
Example 7. Thioether Conjugate Linker Formation (1)
[0398] DNA-PNA heteropoymers having a 5' to C-terminus connectivity
via a thioether conjugate linker are synthesized by contacting a
5'-bromo modified DNA (component 7-1) with a C-terminal thiol
modified PNA (component 7-2) as shown below in Scheme 14. The
product is a conjugated molecule 7-3 that has an alkylthioether
linkage.
##STR00224##
Example 8. Thioether Conjugate Linker Formation (2)
##STR00225##
[0400] In this example a thioether conjugate linker can be formed
by contacting a thiol conjugation functionality of a DNA
conjugation component 8-1 with a bromo conjugation functionality of
a PNA conjugation component 8-2 under conditions to give conjugated
molecule 8-3.
[0401] In a variation of the protocol illustrated above the
disulfide of a 3'-thiol functionalized DNA and a PNA having a
N-terminal maleimide group can be used for synthesizing a DNA-PNA
heteropolymer. Briefly, the 3'-functionalized DNA is dissolved in a
buffer and a reductant such as DTT, TCEP, [N,N'-dimethyl-N, N'-bis
(mercaptoacetyl)hydrazine (DMH), bis(2-mercaptoethyl)sulfone (BMS)
or meso-2,5-dimercapto-N, N, N',N'-tetramethyladipamide (DTA) is
added to the buffer solution. The amount of reductant added is
greater than the amout of DNA present in the buffer solution and
the reaction mixture is stirred at room temperature for about 1-2
hours. A solution of PNA in DMF is then added to the buffered
solution of DNA and reductant. After stirring for 6-16 hours, the
crude product is lyophilized, prior to purification using
reverse-phase high performance liquid chromatography.
[0402] The mole ratio of DNA to reductant in the buffer solution
can range from 1:1.2 to 1:3. The mole ratio of DNA to PNA in the
reaction mixture can range from 1:0.2 to 1:5. The amount of DNA in
the reaction mixture can be in the range from about 5.0 nmoles to
about 20 .mu.moles both numbers inclusive, while the amount of PNA
is from about 1 nmoles to about 100 .mu.moles both numbers
inclusive. As described above, depending on the scale of the
reaction, the final volume of the reaction mixture can range from
about 10 .mu.l to about 1 mL. The conjugation reaction can be
performed using a solid support bound DNA or a solid support bound
PNA. In this case the final conjugated molecule (i.e., DNA-PNA
heteropolymer) is cleaved from the support following synthesis.
[0403] While particular embodiments of the subject invention have
been discussed, they are illustrative only and not restrictive of
the invention. A review of this specification will make many
variations of the invention apparent to those skilled in the field
of the invention. The full scope of the invention should be
determined by reference both to the claims below, along with their
full range of equivalents, and to the specification, with such
variations.
* * * * *