U.S. patent application number 09/756354 was filed with the patent office on 2001-06-07 for method of synthesizing phosphorothioate oligonucleotides.
Invention is credited to Cole, Douglas L., Ravikumar, Vasulinga.
Application Number | 20010003132 09/756354 |
Document ID | / |
Family ID | 25103044 |
Filed Date | 2001-06-07 |
United States Patent
Application |
20010003132 |
Kind Code |
A1 |
Cole, Douglas L. ; et
al. |
June 7, 2001 |
Method of synthesizing phosphorothioate oligonucleotides
Abstract
This invention presents novel methods for synthesizing
phosphorothioate oligonucleotides, using support-bound
phosphoramidites. Novel intermediates useful in the methods are
also provided.
Inventors: |
Cole, Douglas L.; (San
Diego, CA) ; Ravikumar, Vasulinga; (Carlsbad,
CA) |
Correspondence
Address: |
WOODCOCK WASHBURN KURTZ MACKIEWICZ & NORRIS LLP
46th Floor
One Liberty Place
Philadelphia
PA
19103
US
|
Family ID: |
25103044 |
Appl. No.: |
09/756354 |
Filed: |
January 8, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09756354 |
Jan 8, 2001 |
|
|
|
08775011 |
Dec 27, 1996 |
|
|
|
6172217 |
|
|
|
|
Current U.S.
Class: |
536/22.1 |
Current CPC
Class: |
C07H 21/00 20130101 |
Class at
Publication: |
536/22.1 |
International
Class: |
C07H 021/00 |
Claims
What is claimed is:
1. A method for synthesizing a phosphorothioate oligonucleotides on
a solid phase support comprising the steps of: reacting a
phosphordiamidite of formula: 10wherein: R.sub.1 is a protecting
group; R.sub.2 and R.sub.3 are dialkylamino or morpholino; B is a
nucleosidic base; R.sub.6 is halogen, O-alkyl, O-alkylamino,
O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl
imidazole, or a polyether of the formula (O-alkyl).sub.m, where m
is 1 to about 10; with a support-bound synthon of formula:
11wherein: R.sub.4 is a linker connected to a solid support;
R.sub.5 is a phosphoryl protecting group; n is 0 to 100; to form a
support-bound phosphoramidite of formula: 12protecting the
support-bound phosphoramidite, thereby forming a support-bound
phosphite of formula: 13sulfurizing the support-bound phosphite to
form a protected phosphorothioate; and deprotecting the protected
phosphorothioate to form a further support-bound synthon wherein n
is increased by 1.
2. The method of claim 1 wherein the the support-bound
phosphoramidite is protected by reaction with an alcohol of formula
R.sup.5--OH, where R.sup.5 is a phosphoryl protecting group.
3. The method of claim 1 wherein at least one of R.sub.2 and
R.sub.3 is diisopropylamino.
4. The method of claim 1 wherein R.sub.2 and R.sub.3 are
diisopropylamino.
5. The method of claim 1 wherein R.sub.5 is 2-cyanoethyl.
6. The method of claim 1 wherein R.sub.5is 4-cyano-2-butenyl or
diphenylmethylsilylethyl.
7. The method of claim 1 wherein the reaction of the phosphite
compound with the support-bound synthon is preformed in the
presence of an organic base.
8. The method of claim 7 wherein the organic base is a
tetrazole.
9. The method of claim 1 wherein the sulfurization reagent is
Beaucage reagent, tetraethylthiuram disulfide, dibenzoyl
tetrasulfide, phenacetyl disilfide, 1,2,4-dithiuazoline-5-one,
3-ethoxy-1,2,4-dithiuazoline-5-one, a disulfide of a sulfonic acid,
sulfur, or sulfur in combination with a ligand.
10. The method of claim 1 wherein the sulfurization reagent is
Beaucage reagent, tetraethylthiuram disulfide, dibenzoyl
tetrasulfide, phenacetyl disilfide, 1,2,4-dithiuazoline-5-one, or
3-ethoxy-1,2,4-dithiuazoline-5-o- ne.
11. The method of claim 1 wherein the nucleosidic base is adenine,
guanine, cytosine, thymine, or uracil.
12. The method of claim 1 wherein n is from 10 to about 30
nucleotide units.
13. The method of claim 1 wherein n is from 15 to about 25
nucleotide units.
14. A compound of formula: 14wherein: B is a nucleosidic base;
R.sub.1 is a protecting group; R.sub.2 is dialkylamino or
morpholino; R.sub.4 is a linker connected to a solid support;
R.sub.5 is a phosphoryl protecting group; R.sub.6 is halogen,
O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino,
O-alkylaminoalkyl, O-alkyl imidazole, or a polyether of the formula
(O-alkyl).sub.m, where m is 1 to about 10; and n is 1 to about
100.
15. The compound of claim 14 wherein R.sub.2 is diisopropylamino or
morpholino.
16. The compound of claim 15 wherein the phosphoryl protecting
group is cyanoethyl.
17. The compound of claim 14 wherein the phosphoryl protecting
group is 4-cyano-2-butenyl or diphenylmethylsilylethyl.
18. The compound of claim 14 wherein n is 1 to 30.
19. The compound of claim 14 wherein n is from 15 to 25.
Description
FIELD OF THE INVENTION
[0001] This invention is directed to methods for synthesis of
phosphorothioate oligonucleotides and to novel synthetic
intermediates useful in the methods. The methods comprise the
in-situ generation of amidites on a solid support. The methods are
useful, inter alia, for the preparation of phosphorothioate
oligonucleotides which, in turn, are useful as diagnostic reagents,
research reagents and therapeutics agents.
BACKGROUND OF THE INVENTION
[0002] It is well known that most of the bodily states in mammals,
including most disease states, are affected by proteins. Such
proteins, either acting directly or through their enzymatic
functions, contribute in major proportion to many diseases in
animals and man. Classical therapeutics has generally focused on
interactions with such proteins in efforts to moderate their
disease causing or disease potentiating functions. Recently,
however, attempts have been made to moderate the actual production
of such proteins by interactions with molecules that direct their
synthesis, such as intracellular RNA. By interfering with the
production of proteins, it has been hoped to affect therapeutic
results with maximum effect and minimal side effects. It is the
general object of such therapeutic approaches to interfere with or
otherwise modulate gene expression leading to undesired protein
formation.
[0003] One method for inhibiting specific gene expression is the
use of oligonucleotides and oligonucleotide analogs as "antisense"
agents. The oligonucleotides or oligonucleotide analogs
complimentary to a specific, target, messenger RNA (mRNA) sequence
are used. Antisense methodology is often directed to the
complementary hybridization of relatively short oligonucleotides
and oligonucleotide analogs to single-stranded mRNA or
single-stranded DNA such that the normal, essential functions of
these intracellular nucleic acids are disrupted. Hybridization is
the sequence specific hydrogen bonding of oligonucleotides or
oligonucleotide analogs to Watson-Crick base pairs of RNA or
single-stranded DNA. Such base pairs are said to be complementary
to one another.
[0004] Prior attempts at antisense therapy have provided
oligonucleotides or oligonucleotide analogs that are designed to
bind in a specific fashion to a specific MRNA by hybridization
(i.e., oligonucleotides that are specifically hybridizable with a
target mRNA). Such oligonucleotides and oligonucleotide analogs are
intended to inhibit the activity of the selected MRNA by any of a
number of mechanisms, i.e., to interfere with translation reactions
by which proteins coded by the MRNA are produced. The inhibition of
the formation of the specific proteins that are coded for by the
MRNA sequences interfered with have been hoped to lead to
therapeutic benefits; however there are still problems to be
solved. See generally, Cook, P. D. Anti-Cancer Drug Design 1991,
6,585; Cook, P. D. Medicinal Chemistry Strategies for Antisense
Research, in Antisense Research & Applications, Crooke, et al.,
CRC Press, Inc.; Boca Raton, Fla., 1993; Uhlmann, et al., A. Chem.
Rev. 1990, 90, 543.
[0005] Oligonucleotides and oligonucleotide analogs are now
accepted as therapeutic agents holding great promise for
therapeutics and diagnostics methods. But applications of
oligonucleotides and oligonucleotide analogs as antisense agents
for therapeutic purposes, diagnostic purposes, and research
reagents often require that the oligonucleotides or oligonucleotide
analogs be synthesized in large quantities, be transported across
cell membranes or taken up by cells, appropriately hybridize to
targeted RNA or DNA, and subsequently terminate or disrupt nucleic
acid function. These critical functions depend on the initial
stability of oligonucleotides and oligonucleotide analogs toward
nuclease degradation.
[0006] A serious deficiency of unmodified oligonucleotides for
these purposes, particularly antisense therapeutics, is the
enzymatic degradation of the administered oligonucleotides by a
variety of intracellular and extracellular ubiquitous nucleolytic
enzymes.
[0007] A number of chemical modifications have been introduced into
antisense agents (i.e., oligonucleotides and oligonucleotide
analogs) to increase their therapeutic activity. Such modifications
are designed to increase cell penetration of the antisense agents,
to stabilize the antisense agents from nucleases and other enzymes
that degrade or interfere with their structure or activity in the
body, to enhance the antisense agents' binding to targeted RNA, to
provide a mode of disruption (terminating event) once the antisense
agents are sequence-specifically bound to targeted RNA, and to
improve the antisense agents' pharmacokinetic and pharmacodynamic
properties. It is unlikely that unmodified, "wild type,"
oligonucleotides will be useful therapeutic agents because they are
rapidly degraded by nucleases.
[0008] Oligonucleotides which have been modified to contain
phosphorothioate linkages are capable of terminating RNA by
activation of RNase H upon hybridization to RNA. These
oligonucleotide analogs have been demonstrated to be sequence
specific regulators of gene expression in eukaryotic and
procaryotic systems, and are the most promising candidates to date
for practical application as "antisense" therapeutic agents. See
Eckstein, Oligonucleotide and Analogs, A Practical Approach, 1991,
IRL Press, pp. 87-103.
[0009] Potential applications of phosphorothioate oligonucleotides
as drugs have created a new challenges in the large-scale synthesis
of these compounds. Thus, there remains a need for improved methods
of synthesizing phosphorothioate oligonucleotides. The present
invention addresses these, as well as other needs.
SUMMARY OF THE INVENTION
[0010] The present invention is directed to novel methods for the
preparation of oligomeric compounds having phosphorothioate
linkages. The present invention discloses solid support
oligonucleotide synthetic methods which involve the generation of
support-bound phosphoramidites. In preferred embodiments, the
methods comprise the steps of:
[0011] reacting a phosphordiamidite of formula: 1
[0012] wherein:
[0013] R.sub.1 is a protecting group;
[0014] R.sub.2 and R.sub.3 are dialkylamino or morpholino;
[0015] B is a nucleosidic base; and
[0016] R.sub.6 is halogen, O-alkyl, O-alkylamino, O-alkylalkoxy,
protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, or a
polyether of the formula (O-alkyl).sub.m, where m is 1 to about
10;
[0017] with a support-bound synthon of formula: 2
[0018] wherein:
[0019] R.sub.4 is a linker connected to a solid support;
[0020] R.sub.5 is a phosphoryl protecting group; and
[0021] n is 0 to 100;
[0022] to form a support-bound phosphoramidite. The support-bound
phosphoramidite has the formula: 3
[0023] The support-bound phosphoramidite is protected, preferably
by reaction with a reagent of formula R.sup.5--OH, to form a
support-bound phosphite of formula: 4
[0024] The support-bound phosphite is then sulfurized to form a
protected phosphorothioate, and then the protected phosphorothioate
is deprotected to form a further support-bound synthon wherein n is
increased by 1.
[0025] Throughout, it is understood that variable substituents
R.sub.1-6 may be the same or different in differing oligomeric
subunits.
[0026] In some preferred embodiments, R.sub.2 and R.sub.3 are
diisopropylamino. In other preferred embodiments R.sup.5 is
2-cyanoethyl, 4-cyano-2-butenyl, or diphenylmethylsilylethyl. In
further preferred embodiments the reaction of the phosphite
compound with the support-bound synthon is preformed in the
presence of an organic base, preferably tetrazole. In other
preferred embodiments the support-bound phosphite is oxidized with
a sulfurization reagent such as Beaucage reagent, tetraethylthiuram
disulfide, dibenzoyl tetrasulfide, phenacetyl disulfide,
1,2,4-dithiuazoline-5-one, 3-ethoxy-1,2,4-dithiuazoline-5-one, a
disulfide of a sulfonic acid, sulfur, or sulfur in combination with
a ligand such as triaryl, trialkyl, triaralkyl, or trialkaryl
phosphines.
[0027] The present methods provide for the synthesis of
oligonucleotides consisting of a wide variety of nucleosidic bases,
including naturally occurring nucleosidic bases such as adenine,
guanine, thymine, cytosine, and uracil, as well as nonnaturally
occurring nucleobases.
[0028] In preferred embodiments n is from 0 to about 100,
preferably 1 to about 30 and more preferably 15 to about 25.
[0029] The invention also provides compounds of the formula: 5
[0030] wherein:
[0031] B, R.sub.1, R.sub.2, R.sub.4, R.sub.5, and R.sub.6 are as
defined above, and n is 1 to about 100.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0032] The present invention presents novel methods for the
synthesis of phosphorothioate oligonucleotides, which comprise the
"in-situ" generation of a phosphoramidite bound to a solid support.
In preferred embodiments of the invention a phosphordiamidite
compound of formula: 6
[0033] wherein:
[0034] R.sub.1 is a protecting group;
[0035] B is a nucleosidic base;
[0036] R.sub.2 and R.sub.3 are dialkylamino or morpholino; and
[0037] R.sub.6 is halogen, O-alkyl, O-alkylamino, O-alkylalkoxy,
protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, or a
polyether of the formula (O-alkyl).sub.m,
[0038] where m is 1 to about 10;
[0039] is reacted with a support-bound synthon of formula: 7
[0040] wherein:
[0041] R.sub.4 is a linker connected to a solid support;
[0042] R.sub.5 is a phosphoryl protecting group;
[0043] n is 0 to about 100;
[0044] to form a support-bound phosphoramidite of formula: 8
[0045] Phosphorodiamidites can be prepared, for example, by the
condensation of a bis(dialkylamino)chlorophosphine with a
5'-protected nucleoside according to the procedure of Uznanski et
al., Tetrahedron Letters 1989 30 (5) 543-546.
[0046] In preferred embodiments of the methods of the present
invention, the initial support-bound synthon is prepared by the
covalent attachment of an appropriately protected nucleoside to a
solid support through the nucleoside 3'-oxygen, preferably through
a linker molecule, according to known procedures. See, for example,
Eckstein, supra. Procedures for the protection of nucleoside
5'-hydroxyls, exocyclic amine groups, and other functionalities can
be found, for example, in Greene and Wuts, Protective Groups in
Organic Synthesis, 2d ed, John Wiley & Sons, New York, 1991.
The 5'-O-protecting group of the support linked nucleoside is
typically removed by treatment with dilute acid and washed (rinsed)
from the support with a solvent, preferably anhydrous acetonitrile.
The reaction of phosphordiamidite and support-bound synthon is then
performed in a solvent such as acetonitrile, preferably in the
presence of an activating agent which is typically an organic base
such as, for example, tetrazole. The resulting support-bound
amidite is protected by the addition of a phosphoryl protecting
group. Phosphoryl protecting groups are known in the art as
protecting groups suitable for use in oligonucleotide synthetic
regimes. Representative phosphoryl protecting groups are the
2-cyanoethyl (see U.S. Pat. Nos. 4,725,677 and Re. 34,069 to Koster
et al.), methyl, 4-cyano-2-butenyl, and diphenylmethylsilylethyl
(DPSE) groups.
[0047] The phosphoryl protecting group is typically bound to the
phosphoryl group to be protected by the addition of a reagent of
formula HO-R.sub.5 and with the release of a diaklylamino,
morpholino, or similar amino group, to form a support-bound
phosphite having the formula: 9
[0048] The group --OR.sub.5 is preferably attached to the
phosphorus in the presence of an activating agent, which is
preferably an organic base such as tetrazole. The support-bound
phosphite is then sulfurized, and then deprotected at the
5'-position to form a further support-bound synthon where n is
increased by 1. The cycle is repeated in iterative fashion until
the desired phosphorothioate is achieved. The completed
oligonucleotide phosphorothioate is then cleaved from the solid
support, typically with a strong base such as ammonium hydroxide.
During cleavage, the phosphorus protecting groups are cleaved as
well as the link to the solid support. Thus, the cleavage step,
which can precede or follow deprotection of protected functional
groups, will yield a phosphorothioate free of all protecting
groups.
[0049] Sulfurizing agents used during oxidation to form
phosphorothioate linkages include Beaucage reagent (see e.g. Iyer,
R. P., et.al., J. Chem. Soc., 1990, 112, 1253-1254, and Iyer, R.
P., et.al., J. Org. Chem., 1990, 55, 4693-4699); tetraethylthiuram
disulfide (see e.g., Vu, H., Hirschbein, B. L., Tetrahedron Lett.,
1991, 32, 3005-3008); dibenzoyl tetrasulfide (see e.g., Rao, M. V.,
et.al., Tetrahedron Lett., 1992, 33, 4839-4842);
di(phenylacetyl)disulfide (see e.g., Kamer, P. C. J., Tetrahedron
Lett., 1989, 30, 6757-6760); 1,2,4-dithiuazoline-5-one (DtsNH) and
3-ethoxy-1,2,4-dithiuazoline-5-one (EDITH) and (see Xu et al.,
Nucleic Acids Research, 1996, 24, 3643-3644 and Xu et al., Nucleic
Acids Research, 1996, 24, 1602-1607); thiophosphorus compounds such
as those disclosed in U.S. Pat. No. 5,292,875 to Stec et al., and
U.S. Pat. No. 5,151,510 to Stec et al., disulfides of sulfonic
acids, such as those disclosed in Efimov et al., Nucleic Acids
Research, 1995, 23, 4029-4033, sulfur, sulfur in combination with
ligands like triaryl, trialkyl, triaralkyl, or trialkaryl
phosphines.
[0050] In the context of the present invention, the term
"oligonucleotide" refers to a plurality of joined nucleotide units
formed in a specific sequence. The term nucleotide has its
accustomed meaning as the phosphoryl ester of a nucleoside. The
term "nucleoside" also has its accustomed meaning as a
pentofuranosyl sugar which is bound to a nucleosidic base (i.e, a
nitrogenous heterocyclic base or "nucleobase").
[0051] The methods of the present invention can be used for the
synthesis of phosphorothioate oligomers having both naturally
occurring and non-naturally occurring constituent groups. For
example, the present invention can be used to synthesize
phosphorothioate oligomers having naturally occurring pentose sugar
components such as ribose and deoxyribose, and their substituted
derivatives, as well as other sugars known to substitute therefor
in oligonucleotide analogs.
[0052] The methods of the invention are used for the preparation of
phosphorothioate oligonucleotides. The constituent sugars and
nucleosidic bases of the phosphorothioate oligonucleotides can be
naturally occurring or non-naturally occurring. Non-naturally
occurring sugars and nucleosidic bases are typically structurally
distinguishable from, yet functionally interchangeable with,
naturally occurring sugars (e.g. ribose and deoxyribose) and
nucleosidic bases (e.g., adenine, guanine, cytosine, thymine).
Thus, non-naturally occurring nucleobases and sugars include all
such structures which mimic the structure and/or function of
naturally occurring species, and which aid in the binding of the
phosphorothioate to a target, or which otherwise advantageously
contribute to the properties of the phosphorothioate oligomer.
[0053] The methods of the invention are amenable to the synthesis
of phoshorothioate oligomers having a variety of substituents
attached to their 2'-positions. These include, for example,
halogens, O-alkyl, O-alkylamino, O-alkylalkoxy, protected
O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, and polyethers
of the formula (O-alkyl).sub.m, where m is 1 to about 10. Preferred
among these polyethers are linear and cyclic polyethylene glycols
(PEGs), and (PEG)-containing groups, such as crown ethers and those
which are disclosed by Ouchi, et al., Drug Design and Discovery
1992, 9, 93, Ravasio, et al., J. Org. Chem. 1991, 56, 4329, and
Delgardo et. al., Critical Reviews in Therapeutic Drug Carrier
Systems 1992, 9, 249. Further sugar modifications are disclosed in
Cook, P. D., supra. Fluoro, O-alkyl, O-alkylamino, O-alkyl
imidazole, O-alkylaminoalkyl, and alkyl amino substitution is
described in U.S. patent application Ser. No. 08/398,901, filed
Mar. 6, 1995, entitled Oligomeric Compounds having Pyrimidine
Nucleotide(s) with 2' and 5' Substitutions, the disclosure of which
is hereby incorporated by reference.
[0054] Sugars having O-substitutions on the ribosyl ring are also
amenable to the present invention. Representative substitutions for
ring O include S, CH.sub.2, CHF, and CF.sub.2, see, e.g., Secrist,
et al., Abstract 21, Program & Abstracts, Tenth International
Roundtable, Nucleosides, Nucleotides and their Biological
Applications, Park City, Utah, Sep. 16-20, 1992.
[0055] Representative nucleobases suitable for use in the methods
of the invention include adenine, guanine, cytosine, uridine, and
thymine, as well as other non-naturally occurring and natural
nucleobases such as xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine, 5-halo
uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudo uracil), 4-thiouracil, 8-halo, oxa, amino, thiol,
thioalkyl, hydroxyl and other 8-substituted adenines and guanines,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine. Further naturally and non naturally occurring
nucleobases include those disclosed in U.S. Pat. No. 3,687,808
(Merigan, et al.), in chapter 15 by Sanghvi, in Antisense Research
and Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993,
in Englisch et al., Angewandte Chemie, International Edition, 1991,
30, 613-722 (see especially pages 622 and 623, and in the Concise
Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz
Ed., John Wiley & Sons, 1990, pages 858-859, Cook, P. D.,
Anti-Cancer Drug Design, 1991, 6, 585-607. The terms "nucleosidic
base" and "nucleobase" are further intended to include heterocyclic
compounds that can serve as nucleosidic bases, including certain
`universal bases` that are not nucleosidic bases in the most
classical sense, but function similarly to nucleosidic bases. One
representative example of such a universal base is
3-nitropyrrole.
[0056] The methods of the present invention use labile protecting
groups to protect various functional moieties during synthesis.
Protecting groups are used in the oligonucleotide synthetic methods
of the invention for protection of several different types of
functionality. In general, protecting groups render chemical
functionality inert to specific reaction conditions and can be
appended to and removed from such functionality in a molecule
without substantially damaging the remainder of the molecule. See,
e.g., Green and Wuts, Protective Groups in Organic Synthesis, 2d
edition, John Wiley & Sons, New York, 1991. Representative
hydroxyl protecting groups used for nucleic acid chemistry are
described by Beaucage, et al., Tetrahedron 1992, 48, 2223.
Representative protecting groups useful to protect nucleotides
during phosphorothioate synthesis include base labile protecting
groups and acid labile protecting groups. Base labile protecting
groups are used to protect the exocyclic amino groups of the
heterocyclic nucleobases. This type of protection is generally
achieved by acylation. Two commonly used acylating groups are
benzoylchloride and isobutyrylchloride. These protecting groups are
stable to the reaction conditions used during oligonucleotide
synthesis and are cleaved at approximately equal rates during the
base treatment at the end of synthesis. The second type of
protection used in the phosphorothioate synthetic methods of the
invention is an acid labile protecting group, which is used to
protect the nucleotide 5' hydroxyl during synthesis.
[0057] The amino moiety of the phosphordiamidites of the invention
can be selected from various amines presently used for
phosphoramidites in standard oligonucleotide synthesis. These
include both aliphatic and heteroalkyl amines. One preferred amino
group is diisopropylamino. Other examples of suitable amines as are
described in various United States patents, principally those to M.
Caruthers and associates. These include U.S. Pat. Nos. 4,668,777;
4,458,066; 4,415,732; and 4,500,707; all of which are herein
incorporated by reference.
[0058] In some preferred embodiments of the invention the
phosphordiamidite is activated to nucleophilic attack by the 5'
hydroxyl by use of an activating agent. It is believed that the
activating agent displaces one of the amino groups from the
phosphordiamidite, thereby rendering the phosphorus of the
phosphordiamidite more susceptible to nucleophilic attack by the 5'
hydroxyl group of the growing nucleotide chain. Any activating
agent that can activate the phosphorous to nucleophilic attack
without interacting with the growing nucleotide chain may be
suitable for use with the present invention. One preferred
activating agent is tetrazole. Some commonly used commercially
available activating agents are thiotetrazole, nitrotetrazole, and
N,N-diisopropylaminohydrotetrazolide. Other suitable activating
agents are also disclosed in the above incorporated patents as well
as in U.S. Pat. No. 4,725,677 and in Berner, S., Muhlegger, K., and
Seliger, H., Nucleic Acids Research 1989, 17:853; Dahl, B. H.,
Nielsen, J. and Dahl, O., Nucleic Acids Research 1987, 15:1729; and
Nielson, J. Marugg, J. E., Van Boom, J. H., Honnens, J., Taagaard,
M. and Dahl, O., J. Chem. Research 1986, 26, all of which are
herein incorporated by reference.
[0059] It is generally preferable to perform a capping step, either
prior to or after sulfurization of the support-bound phosphite.
Such a capping step is generally known to provide benefits in the
prevention of shortened oligomer chains, by blocking chains that
have not reacted in the coupling cycle. One representative reagent
used for capping is acetic anhydride. Other suitable capping
reagents and methodologies can be found in U.S. Pat. No.
4,816,571.
[0060] As used herein, the term "alkyl" includes but is not limited
to straight chain, branch chain, and alicyclic hydrocarbon groups.
Alkyl groups of the present invention may be substituted.
Representative alkyl substituents are disclosed in U.S. Pat. No.
5,212,295, at column 12, lines 41-50.
[0061] As used herein, the term "aralkyl" denotes alkyl groups
which bear aryl groups, for example, benzyl groups. The term
"alkaryl" denotes aryl groups which bear alkyl groups, for example,
methylphenyl groups. "Aryl" groups are aromatic cyclic compounds
including but not limited to phenyl, naphthyl, anthracyl,
phenanthryl, pyrenyl, and xylyl.
[0062] As used herein, the term O-alkylamino denotes a group of
formula O-alkyl-NH.sub.2. The term O-alkylalkoxy denotes a group of
formula -O-alkyl-O-alkyl. The term O-alkylaminoalkyl denotes an
O-alkylamino group wherein the amino moiety bears one or more
additional alkyl groups. The The term O-akylimidazole means a group
of formula O-alkylimidazole.
[0063] As used herein, the term "heterocycloalkyl" denotes an alkyl
ring system having one or more heteroatoms (i.e., non-carbon
atoms). Preferred heterocycloalkyl groups include, for example,
morpholino groups. As used herein, the term "heterocycloalkenyl"
denotes a ring system having one or more double bonds, and one or
more heteroatoms. Preferred heterocycloalkenyl groups include, for
example, pyrrolidino groups.
[0064] In some preferred embodiments of the invention R.sub.4 is a
linker connected to a solid support. Solid supports are substrates
which are capable of serving as the solid phase in solid phase
synthetic methodologies, such as those described in Caruthers U.S.
Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679;
and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and Re. 34,069.
Linkers are known in the art as short molecules which serve to
connect a solid support to functional groups (e.g., hydroxyl
groups) of initial synthon molecules in solid phase synthetic
techniques. Suitable linkers are disclosed in, for example,
Oligonucleotides And Analogues A Practical Approach, Ekstein, F.
Ed., IRL Press, N.Y, 1991, Chapter 1, pages 1-23.
[0065] Solid supports according to the invention include those
generally known in the art to be suitable for use in solid phase
methodologies, including, for example, controlled pore glass (CPG),
oxalyl-controlled pore glass (see, e.g., Alul, et al., Nucleic
Acids Research 1991, 19, 1527), TentaGel Support (an
aminopolyethyleneglycol derivatized support (see, e.g., Wright, et
al., Tetrahedron Letters 1993, 34, 3373)) and Poros (a copolymer of
polystyrene/divinylbenzene).
[0066] In some preferred embodiments of the invention R.sub.1 or
R.sub.4 can be a hydroxyl protecting group. A wide variety of
hydroxyl protecting groups can be employed in the methods of the
invention. Preferably, the protecting group is stable under basic
conditions but can be removed under acidic conditions. In general,
protecting groups render chemical functionalities inert to specific
reaction conditions, and can be appended to and removed from such
functionalities in a molecule without substantially damaging the
remainder of the molecule. Representative hydroxyl protecting
groups are disclosed by Beaucage, et al., Tetrahedron 1992, 48,
2223-2311, and also in Greene and Wuts, supra, at Chapter 2.
Preferred protecting groups used for R.sub.2, R.sub.3 and R.sub.3a
include dimethoxytrityl (DMT), monomethoxytrityl,
9-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl
(Mox). The R.sub.2 or R.sub.3 group can be removed from oligomeric
compounds of the invention by techniques well known in the art to
form the free hydroxyl. For example, dimethoxytrityl protecting
groups can be removed by protic acids such as formic acid,
dichloroacetic acid, trichloroacetic acid, p-toluene sulphonic acid
or with Lewis acids such as for example zinc bromide. See for
example, Greene and Wuts, supra.
[0067] In some preferred embodiments of the invention amino groups
are appended to alkyl or other groups, such as, for example,
2'-alkoxy groups (e.g., where R.sub.1 is alkoxy). Such amino groups
are also commonly present in naturally occurring and non-naturally
occurring nucleobases. It is generally preferred that these amino
groups be in protected form during the synthesis of oligomeric
compounds of the invention. Representative amino protecting groups
suitable for these purposes are discussed in Greene and Wuts,
Protective Groups in Organic Synthesis, Chapter 7, 2d ed, John
Wiley & Sons, New York, 1991. Generally, as used herein, the
term "protected" when used in connection with a molecular moiety
such as "nucleobase" indicates that the molecular moiety contains
one or more functionalities protected by protecting groups.
[0068] Phosphorothioates produced by the methods of the invention
will preferably be hybridizable to a specific target
oligonucleotide. Preferably, the phosphorothioates produced by the
methods of the invention comprise from about 1 to about 100 monomer
subunits. It is more preferred that such compounds comprise from
about 10 to about 30 monomer subunits, with 15 to 25 monomer
subunits being particularly preferred.
[0069] In one aspect of the invention, the compounds of the
invention are used to modulate RNA or DNA, which code for a protein
whose formation or activity it is desired to modulate. The
targeting portion of the composition to be employed is, thus,
selected to be complementary to the preselected portion of DNA or
RNA, that is to be hybridizable to that portion.
[0070] The oligomeric compounds of the invention can be used in
diagnostics, therapeutics and as research reagents and kits. They
can be used in pharmaceutical compositions by including a suitable
pharmaceutically acceptable diluent or carrier. They further can be
used for treating organisms having a disease characterized by the
undesired production of a protein. The organism should be contacted
with an oligonucleotide having a sequence that is capable of
specifically hybridizing with a strand of nucleic acid coding for
the undesirable protein. Treatments of this type can be practiced
on a variety of organisms ranging from unicellular prokaryotic and
eukaryotic organisms to multicellular eukaryotic organisms. Any
organism that utilizes DNA-RNA transcription or RNA-protein
translation as a fundamental part of its hereditary, metabolic or
cellular control is susceptible to therapeutic and/or prophylactic
treatment in accordance with the invention. Seemingly diverse
organisms such as bacteria, yeast, protozoa, algae, all plants and
all higher animal forms, including warm-blooded animals, can be
treated. Further, each cell of multicellular eukaryotes can be
treated, as they include both DNA-RNA transcription and RNA-protein
translation as integral parts of their cellular activity.
Furthermore, many of the organelles (e.g., mitochondria and
chloroplasts) of eukaryotic cells also include transcription and
translation mechanisms. Thus, single cells, cellular populations or
organelles can also be included within the definition of organists
that can be treated with therapeutic or diagnostic
oligonucleotides.
[0071] Additional advantages and novel features of this invention
will become apparent to those skilled in the art upon examination
of the examples thereof provided below, which should not be
construed as limiting the appended claims.
EXAMPLE 1
[0072] Preparation of 5'-O-dimethoxytrityl-base protected
nucleoside 3'-O-phosphorbismorpholidites.
[0073] These compounds were synthesized according to the procedure
of Uznanski, B. et al., Tetrahedron Letters, 1987, 28,
3401-3404.
EXAMPLE 2
[0074] Preparation of 5'-O-dimethoxytrityl-base protected
nucleoside 3'-O-phosphorbisdiethylamidites.
[0075] These compounds were synthesized according to the procedure
of Yamana, K. et al., Tetrahedron, 1989, 45, 4135-4140.
EXAMPLE 3
[0076] Synthesis of T-T phosphorothioate dimer:
[0077] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-phosphorbismorpholi- dite
in acetonitrile and a 0.4 M solution of 1H-tetrazole in
acetonitrile is added, and allowed to react at room temperature for
5 minutes. The product is washed with acetonitrile, and then a 0.8M
solution of 3-hydroxypropionitrile in acetonitrile and a 0.4 M
solution of 1H-tetrazole in acetonitrile are added and allowed to
react at room temperature for 5 minutes. The product is washed with
acetonitrile and this step is repeated one more time. Then the
product is washed with acetonitrile and a 0.05 M solution of
Beaucage reagent in acetonitrile is added and allowed to react at
room temperature for 5 minutes. This sulfurization step is repeated
one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap the unallowed
to 5'-hydroxyl group. The product is washed with acetonitrile.
[0078] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes. The aqueous
solution is filtered, and concentrated under reduced pressure to
give a phosphorothioate dimer of T-T.
EXAMPLE 4
[0079] Synthesis of C-T phosphorothioate dimer:
[0080] 100 milligrams (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. A 0.2 M solution of
N.sup.4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine--
3'-O-phosphorbismorpholidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile are added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of 3-hydroxypropionitrile in
acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile
are added and allowed to react at room temperature for 5 minutes.
The product is washed with acetonitrile, and this step is repeated
one more time. The product is then washed with acetonitrile, and a
0.05 M solution of Beaucage reagent in acetonitrile is added and
allowed to react at room temperature for 5 minutes. This
sulfurization step is repeated one more time for 5 minutes. The
support is washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added
to cap any unreacted 5'-hydroxyl groups. The product is washed with
acetonitrile.
[0081] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dC-T.
EXAMPLE 5
[0082] Synthesis of dG-T phosphorothioate dimer:
[0083] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanos- in
e-3'-O-phosphorbismorpholidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of 3-hydroxypropionitrile in
acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile
are added and allowed to react at room temperature for 5 minutes.
The product is washed with acetonitrile and this step is repeated
one more time. Then the product is washed with acetonitrile and a
0.05 M solution of Beaucage reagent in acetonitrile is added and
allowed to react at room temperature for 5 minutes. This
sulfurization step is repeated one more time for 5 minutes. The
support is washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added
to cap any unreacted 5'-hydroxyl groups. The product is washed with
acetonitrile.
[0084] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dG-T.
EXAMPLE 6
[0085] Synthesis of dA-T phosphorothioate dimer:
[0086] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenos-
ine-3'-O-phosphorbismorpholidite in acetonitrile and a 0.4 M
solution of 1H-tetrazole in acetonitrile is added, and allowed to
react at room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of 3-hydroxypropionitrile in
acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile
are added and allowed to react at room temperature for 5 minutes.
The product is washed with acetonitrile and this step is repeated
one more time. Then the product is washed with acetonitrile and a
0.05 M solution of Beaucage reagent in acetonitrile is added and
allowed to react at room temperature for 5 minutes. This
sulfurization step is repeated one more time for 5 minutes. The
support is washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added
to cap the any unreacted 5'-hydroxyl groups. The product is washed
with acetonitrile.
[0087] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dA-T.
EXAMPLE 7
[0088] Synthesis of T-T phosphorothioate dimer:
[0089] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-phosphorbismorpholi- dite
in acetonitrile and a 0.4 M solution of 1H-tetrazole in
acetonitrile is added, and allowed to react at room temperature for
5 minutes. The product is washed with acetonitrile, and then a 0.8M
solution of 2-diphenylmethylsilylethanol in acetonitrile and a 0.4
M solution of 1H-tetrazole in acetonitrile are added and allowed to
react at room temperature for 5 minutes. The product is washed with
acetonitrile and this step is repeated one more time. Then the
product is washed with acetonitrile and a 0.05 M solution of
Beaucage reagent in acetonitrile is added and allowed to react at
room temperature for 5 minutes. This sulfurization step is repeated
one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap any unreacted
5'-hydroxyl groups. The product is washed with acetonitrile.
[0090] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes. The aqueous
solution is filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of T-T.
EXAMPLE 8
[0091] Synthesis of C-T phosphorothioate dimer:
[0092] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine--
3'-O-phosphorbismorpholidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of
2-diphenylmethylsilylethanol in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile are added and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile and this step is repeated one more time. Then the
product is washed with acetonitrile and a 0.05 M solution of
Beaucage reagent in acetonitrile is added and allowed to react at
room temperature for 5 minutes. This sulfurization step is repeated
one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap any unreacted
5'-hydroxyl groups. The product is washed with acetonitrile.
[0093] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dC-T.
EXAMPLE 9
[0094] Synthesis of dG-T phosphorothioate dimer:
[0095] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanos- in
e-3'-O-phosphorbismorpholidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of
2-diphenylmethylsilylethanol in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile are added and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile and this step is repeated one more time. Then the
product is washed with acetonitrile and a 0.05 M solution of
Beaucage reagent in acetonitrile is added and allowed to react at
room temperature for 5 minutes. This sulfurization step is repeated
one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap the any
unreacted 5'-hydroxyl groups to 5'-hydroxyl group. The product is
washed with acetonitrile.
[0096] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dG-T.
EXAMPLE 10
[0097] Synthesis of dA-T phosphorothioate dimer:
[0098] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenos- in
e-3'-O-phosphorbismorpholidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of
2-diphenylmethylsilylethanol in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile are added and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile and this step is repeated one more time. Then the
product is washed with acetonitrile and a 0.05 M solution of
Beaucage reagent in acetonitrile is added and allowed to react at
room temperature for 5 minutes. This sulfurization step is repeated
one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap any unreacted
5'-hydroxyl groups. The product is washed with acetonitrile.
[0099] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dA-T.
EXAMPLE 11
[0100] Synthesis of T-T phosphorothioate dimer:
[0101] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2'
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-phosphorbismorpholi- dite
in acetonitrile and a 0.4 M solution of 1H-tetrazole in
acetonitrile is added, and allowed to react at room temperature for
5 minutes. The product is washed with acetonitrile, and then a 0.8M
solution of 4-cyano-2-butene-1-ol in acetonitrile and a 0.4 M
solution of 1H-tetrazole in acetonitrile are added and allowed to
react at room temperature for 5 minutes. The product is washed with
acetonitrile and this step is repeated one more time. Then the
product is washed with acetonitrile and a 0.05 M solution of
Beaucage reagent in acetonitrile is added and allowed to react at
room temperature for 5 minutes. This sulfurization step is repeated
one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap any unreacted
5'-hydroxyl groups. The product is washed with acetonitrile.
[0102] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes. The aqueous
solution is filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of T-T.
EXAMPLE 12
[0103] Synthesis of C-T phosphorothioate dimer:
[0104] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine--
3'-O-phosphorbismorpholidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of 4-cyano-2-butene-1-ol in
acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile
are added and allowed to react at room temperature for 5 minutes.
The product is washed with acetonitrile and this step is repeated
one more time. Then the product is washed with acetonitrile and a
0.05 M solution of Beaucage reagent in acetonitrile is added and
allowed to react at room temperature for 5 minutes. This
sulfurization step is repeated one more time for 5 minutes. The
support is washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added
to cap any unreacted 5'-hydroxyl groups. The product is washed with
acetonitrile.
[0105] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dC-T.
EXAMPLE 13
[0106] Synthesis of dG-T phosphorothioate dimer:
[0107] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanos- in
e-3'-O-phosphorbismorpholidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of 4-cyano-2-butene-1-ol in
acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile
are added and allowed to react at room temperature for 5 minutes.
The product is washed with acetonitrile and this step is repeated
one more time. Then the product is washed with acetonitrile and a
0.05 M solution of Beaucage reagent in acetonitrile is added and
allowed to react at room temperature for 5 minutes. This
sulfurization step is repeated one more time for 5 minutes. The
support is washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added
to cap any unreacted 5'-hydroxyl groups. The product is washed with
acetonitrile.
[0108] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dG-T.
EXAMPLE 14
[0109] Synthesis of dA-T phosphorothioate dimer:
[0110] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenos- in
e-3'-O-phosphorbismorpholidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of 4-cyano-2-butene-1-ol in
acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile
are added and allowed to react at room temperature for 5 minutes.
The product is washed with acetonitrile and this step is repeated
one more time. Then the product is washed with acetonitrile and a
0.05 M solution of Beaucage reagent in acetonitrile is added and
allowed to react at room temperature for 5 minutes. This
sulfurization step is repeated one more time for 5 minutes. The
support is washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added
to cap any unreacted 5'-hydroxyl groups. The product is washed with
acetonitrile.
[0111] The carrier containing the compound is treated with
30'-aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dA-T.
EXAMPLE 15
[0112] Synthesis of T-T phosphorothioate dimer:
[0113] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-phosphorbisdiethyl
amidite in acetonitrile and a 0.4 M solution of 1H-tetrazole in
acetonitrile is added, and allowed to react at room temperature for
5 minutes. The product is washed with acetonitrile, and then a 0.8M
solution of 3-hydroxypropionitrile in acetonitrile and a 0.4 M
solution of 1H-tetrazole in acetonitrile are added and allowed to
react at room temperature for 5 minutes. The product is washed with
acetonitrile and this step is repeated one more time. Then the
product is washed with acetonitrile and a 0.05 M solution of
Beaucage reagent in acetonitrile is added and allowed to react at
room temperature for 5 minutes. This sulfurization step is repeated
one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap any unreacted
5'-hydroxyl groups. The product is washed with acetonitrile.
[0114] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes. The aqueous
solution is filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of T-T.
EXAMPLE 16
[0115] Synthesis of C-T phosphorothioate dimer:
[0116] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine--
3'-O-phosphorbisethylamidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of 3-hydroxypropionitrile in
acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile
are added and allowed to react at room temperature for 5 minutes.
The product is washed with acetonitrile and this step is repeated
one more time. Then the product is washed with acetonitrile and a
0.05 M solution of Beaucage reagent in acetonitrile is added and
allowed to react at room temperature for 5 minutes. This
sulfurization step is repeated one more time for 5 minutes. The
support is washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added
to cap any unreacted 5'-hydroxyl groups. The product is washed with
acetonitrile.
[0117] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dC-T.
EXAMPLE 17
[0118] Synthesis of dG-T phosphorothioate dimer:
[0119] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanos- in
e-3'-O-phosphorbisethylamidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of 3-hydroxypropionitrile in
acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile
are added and allowed to react at room temperature for 5 minutes.
The product is washed with acetonitrile and this step is repeated
one more time. Then the product is washed with acetonitrile and a
0.05 M solution of Beaucage reagent in acetonitrile is added and
allowed to react at room temperature for 5 minutes. This
sulfurization step is repeated one more time for 5 minutes. The
support is washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added
to cap any unreacted 5'-hydroxyl groups. The product is washed with
acetonitrile.
[0120] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dG-T.
EXAMPLE 18
[0121] Synthesis of dA-T phosphorothioate dimer:
[0122] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenos- in
e-3'-O-phosphorbisethylamidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of 3-hydroxypropionitrile in
acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile
are added and allowed to react at room temperature for 5 minutes.
The product is washed with acetonitrile and this step is repeated
one more time. Then the product is washed with acetonitrile and a
0.05 M solution of Beaucage reagent in acetonitrile is added and
allowed to react at room temperature for 5 minutes. This
sulfurization step is repeated one more time for 5 minutes. The
support is washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added
to cap any unreacted 5'-hydroxyl groups. The product is washed with
acetonitrile.
[0123] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dA-T.
EXAMPLE 19
[0124] Synthesis of T-T phosphorothioate dimer:
[0125] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-phosphorbisethylami- dite
in acetonitrile and a 0.4 M solution of 1H-tetrazole in
acetonitrile is added, and allowed to react at room temperature for
5 minutes. The product is washed with acetonitrile, and then a 0.8M
solution of 2-diphenylmethylsilylethanol in acetonitrile and a 0.4
M solution of 1H-tetrazole in acetonitrile are added and allowed to
react at room temperature for 5 minutes. The product is washed with
acetonitrile and this step is repeated one more time. Then the
product is washed with acetonitrile and a 0.05 M solution of
Beaucage reagent in acetonitrile is added and allowed to react at
room temperature for 5 minutes. This sulfurization step is repeated
one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap any unreacted
5'-hydroxyl groups. The product is washed with acetonitrile.
[0126] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes. The aqueous
solution is filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of T-T.
EXAMPLE 20
[0127] Synthesis of C-T phosphorothioate dimer:
[0128] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine--
3'-O-phosphorbisethylamidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of
2-diphenylmethylsilylethanol in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile are added and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile and this step is repeated one more time. Then the
product is washed with acetonitrile and a 0.05 M solution of
Beaucage reagent in acetonitrile is added and allowed to react at
room temperature for 5 minutes. This sulfurization step is repeated
one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap any unreacted
5'-hydroxyl groups. The product is washed with acetonitrile.
[0129] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dC-T.
EXAMPLE 21
[0130] Synthesis of dG-T phosphorothioate dimer:
[0131] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanos- in
e-3'-O-phosphorbisethylamidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of
2-diphenylmethylsilylethanol in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile are added and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile and this step is repeated one more time. Then the
product is washed with acetonitrile and a 0.05 M solution of
Beaucage reagent in acetonitrile is added and allowed to react at
room temperature for 5 minutes. This sulfurization step is repeated
one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap any unreacted
5'-hydroxyl groups. The product is washed with acetonitrile.
[0132] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dG-T.
EXAMPLE 22
[0133] Synthesis of dA-T phosphorothioate dimer:
[0134] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenos- in
e-3'-O-phosphorbisethylamidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of
2-diphenylmethylsilylethanol in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile are added and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile and this step is repeated one more time. Then the
product is washed with acetonitrile and a 0.05 M solution of
Beaucage reagent in acetonitrile is added and allowed to react at
room temperature for 5 minutes. This sulfurization step is repeated
one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap any unreacted
5'-hydroxyl groups. The product is washed with acetonitrile.
[0135] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dA-T.
EXAMPLE 23
[0136] Synthesis of T-T phosphorothioate dimer:
[0137] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-phosphorbisethylami- dite
in acetonitrile and a 0.4 M solution of 1H-tetrazole in
acetonitrile is added, and allowed to react at room temperature for
5 minutes. The product is washed with acetonitrile, and then a 0.8M
solution of 4-cyano-2-butene-1-ol in acetonitrile and a 0.4 M
solution of 1H-tetrazole in acetonitrile are added and allowed to
react at room temperature for 5 minutes. The product is washed with
acetonitrile and this step is repeated one more time. Then the
product is washed with acetonitrile and a 0.05 M solution of
Beaucage reagent in acetonitrile is added and allowed to react at
room temperature for 5 minutes. This sulfurization step is repeated
one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap any unreacted
5'-hydroxyl groups. The product is washed with acetonitrile.
[0138] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes. The aqueous
solution is filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of T-T.
EXAMPLE 24
[0139] Synthesis of C-T phosphorothioate dimer:
[0140] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine--
3'-O-phosphorbisethylamidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of 4-cyano-2-butene-1-ol in
acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile
are added and allowed to react at room temperature for 5 minutes.
The product is washed with acetonitrile and this step is repeated
one more time. Then the product is washed with acetonitrile and a
0.05 M solution of Beaucage reagent in acetonitrile is added and
allowed to react at room temperature for 5 minutes. This
sulfurization step is repeated one more time for 5 minutes. The
support is washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added
to cap any unreacted 5'-hydroxyl groups. The product is washed with
acetonitrile.
[0141] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dC-T.
EXAMPLE 25
[0142] Synthesis of dG-T phosphorothioate dimer:
[0143] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanos- in
e-3'-O-phosphorbisethylamidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of 4-cyano-2-butene-1-ol in
acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile
are added and allowed to react at room temperature for 5 minutes.
The product is washed with acetonitrile and this step is repeated
one more time. Then the product is washed with acetonitrile and a
0.05 M solution of Beaucage reagent in acetonitrile is added and
allowed to react at room temperature for 5 minutes. This
sulfurization step is repeated one more time for 5 minutes. The
support is washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added
to cap any unreacted 5'-hydroxyl groups. The product is washed with
acetonitrile.
[0144] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dG-T.
EXAMPLE 26
[0145] Synthesis of dA-T phosphorothioate dimer:
[0146] 100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with dichloromethane and
then with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenos- in
e-3'-O-phosphorbisethylamidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to react at
room temperature for 5 minutes. The product is washed with
acetonitrile, and then a 0.8M solution of 4-cyano-2-butene-1-ol in
acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile
are added and allowed to react at room temperature for 5 minutes.
The product is washed with acetonitrile and this step is repeated
one more time. Then the product is washed with acetonitrile and a
0.05 M solution of Beaucage reagent in acetonitrile is added and
allowed to react at room temperature for 5 minutes. This
sulfurization step is repeated one more time for 5 minutes. The
support is washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added
to cap any unreacted 5'-hydroxyl groups. The product is washed with
acetonitrile.
[0147] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes and then
incubated at 55.degree. C. for 12 hours. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate dimer of dA-T.
EXAMPLE 27
[0148] Synthesis of 5'-TTTT-3' phosphorothioate heptamer:
[0149] 50 milligram (2 mmole) of 5'-O-Dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with acetonitrile. Then, a
0.2 M solution of
5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-phosphorbisethylamidite
in acetonitrile and a 0.4 M solution of 1H-tetrazole in
acetonitrile is added, and allowed to at room temperature for 5
minutes. The product is washed with acetonitrile, and then a 0.8M
solution of 4-cyano-2-butene-1-ol in acetonitrile and a 0.4 M
solution of 1H-tetrazole in acetonitrile are added and allowed to
react at room temperature for 5 minutes. The product is washed with
acetonitrile, and this step is repeated one more time. Then a 0.05
M solution of Beaucage reagent in acetonitrile is added and allowed
to at room temperature for 5 minutes. This sulfurization step is
repeated one more time for 5 minutes. The support is washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methyl imidazole/THF is added to cap any unreacted
5'-hydroxyl groups. The product is washed with acetonitrile.
[0150] This complete cycle is repeated five more times to get the
completely protected thymidine heptamer. The carrier containing the
compound is treated with 30% aqueous ammonium hydroxide solution
for 90 minutes at room temperature. The aqueous solution is
filtered, concentrated under reduced pressure to give a
phosphorothioate heptamer of TTTTTTT.
EXAMPLE 28
[0151] Synthesis of 5'-d(GACT)-3' phosphorothioate tetramer:
[0152] 50 milligram (2 mmole) of 5'-O-Dimethoxytritylthymidine
bonded to CPG (controlled pore glass) through an ester linkage is
taken in a glass reactor, and a dichloromethane solution of 2%
dichloroacetic acid (volume/volume) is added to deprotect the
5'-hydroxyl group. The product is washed with acetonitrile. Then, a
0.2 M solution of
5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-phosphorbisethylamidite
in acetonitrile and a 0.4 M solution of 1H-tetrazole in
acetonitrile is added, and allowed to at room temperature for 5
minutes. The product is washed with acetonitrile, and then a 0.8M
solution of 4-cyano-2-butene-1-ol in acetonitrile and a 0.4 M
solution of 1H-tetrazole in acetonitrile are added and allowed to
react at room temperature for 5 minutes. The product is washed with
acetonitrile, and this step is repeated one more time. The product
is washed with acetonitrile, and then a 0.05 M solution of Beaucage
reagent in acetonitrile is added and allowed to at room temperature
for 5 minutes. This sulfurization step is repeated one more time
for 5 minutes. The support is washed with acetonitrile and then a
solution of acetic anhydride/lutidine/THF (1:1:8), and N-methyl
imidazole/THF is added to cap any unreacted 5'-hydroxyl groups. The
product is washed with acetonitrile.
[0153] A dichloromethane solution of 2% dichloroacetic acid
(volume/volume) is added to deprotect the 5'-hydroxyl group. The
product is washed with acetonitrile. Then, a 0.2 M solution of
N.sup.4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-phospho-
rbisethylamidite in acetonitrile and a 0.4 M solution of
1H-tetrazole in acetonitrile is added, and allowed to at room
temperature for 5 minutes. The product is washed with acetonitrile,
and then a 0.8M solution of 4-cyano-2-butene-1-ol in acetonitrile
and a 0.4 M solution of 1H-tetrazole in acetonitrile are added and
allowed to react at room temperature for 5 minutes. The product is
washed with acetonitrile, and this step is repeated one more time.
The product is washed with acetonitrile, and then a 0.05 M solution
of Beaucage reagent in acetonitrile is added and allowed to at room
temperature for 5 minutes. This sulfurization step is repeated one
more time for 5 minutes. The support is washed with acetonitrile
and then a solution of acetic anhydride/lutidine/THF (1:1:8), and
N-methyl imidazole/THF is added to cap any unreacted 5'-hydroxyl
groups. The product is washed with acetonitrile.
[0154] A dichloromethane solution of 2% dichloroacetic acid
(volume/volume) is added to deprotect the 5'-hydroxyl group. The
product is washed with acetonitrile. Then, a 0.2 M solution of
N.sup.6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine-3'-O-phosph-
orbisethylamidite in anhydrous acetonitrile and a 0.4 M solution of
1H-tetrazole in acetonitrile is added, and allowed to at room
temperature for 5 minutes. The product is washed with acetonitrile,
and then a 0.8M solution of 4-cyano-2-butene-1-ol in acetonitrile
and a 0.4 M solution of 1H-tetrazole in acetonitrile are added and
allowed to react at room temperature for 5 minutes. The product is
washed with acetonitrile, and this step is repeated one more time.
The product is washed with acetonitrile, and then a 0.05 M solution
of Beaucage reagent in acetonitrile is added and allowed to at room
temperature for 5 minutes. This sulfurization step is repeated one
more time for 5 minutes. The support is washed with acetonitrile
and then a solution of acetic anhydride/lutidine/THF (1:1:8), and
N-methyl imidazole/THF is added to cap any unreacted 5'-hydroxyl
groups. The product is washed with acetonitrile.
[0155] A dichloromethane solution of 2% dichloroacetic acid
(volume/volume) is added to deprotect the 5'-hydroxyl group. The
product is washed with acetonitrile. Then, a 0.2 M solution of
N.sup.2-isobutyryl-5'-0(4,4'-dimethoxytrityl) -2'-deoxyguanosin
e-3'-O-phosphorbisethylamidite in acetonitrile and a 0.4 M solution
of 1H-tetrazole in acetonitrile is added, and allowed to at room
temperature for 5 minutes. The product is washed with acetonitrile,
and then a 0.8M solution of 4-cyano-2-butene-1-ol in acetonitrile
and a 0.4 M solution of 1H-tetrazole in acetonitrile are added and
allowed to react at room temperature for 5 minutes. The product is
washed with acetonitrile, and this step is repeated one more time.
The product is washed with acetonitrile, and then a 0.05 M solution
of Beaucage reagent in acetonitrile is added and allowed to at room
temperature for 5 minutes. This sulfurization step is repeated one
more time for 5 minutes. The support is washed with acetonitrile
and then a solution of acetic anhydride/lutidine/THF (1:1:8), and
N-methyl imidazole/THF is added to cap any unreacted 5'-hydroxyl
groups. The product is washed with acetonitrile.
[0156] The carrier containing the compound is treated with 30%
aqueous ammonium hydroxide solution for 90 minutes at room
temperature and then incubated at 55.degree. C. for 24 hour. The
aqueous solution is filtered, concentrated under reduced pressure
to give a phosphorothioate tetramer of 5'-dG-dA-dC-T-3'.
[0157] It is intended that each of the patents, applications, and
printed publications mentioned or referred to in this specification
be herein incorporated by reference in their entirety.
[0158] As those skilled in the art will appreciate, numerous
changes and modifications may be made to the preferred embodiments
of the invention without departing from the spirit of the
invention. It is intended that all such variations fall within the
scope of the invention.
* * * * *