U.S. patent application number 10/336200 was filed with the patent office on 2003-08-14 for processes for the synthesis of oligomeric compounds.
Invention is credited to Guzaev, Andrei, Manoharan, Muthiah.
Application Number | 20030153743 10/336200 |
Document ID | / |
Family ID | 22070755 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030153743 |
Kind Code |
A1 |
Manoharan, Muthiah ; et
al. |
August 14, 2003 |
Processes for the synthesis of oligomeric compounds
Abstract
Methods for the preparation of oligonucleotides having
bioreversible phosphate blocking groups are disclosed. The
oligonucleotides are prepared utilizing amidite type chemistry
wherein the bioreversible phosphorus protecting group is formed as
an integral part of the amidite reagent.
Inventors: |
Manoharan, Muthiah;
(Carlsbad, CA) ; Guzaev, Andrei; (Carlsbad,
CA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Family ID: |
22070755 |
Appl. No.: |
10/336200 |
Filed: |
January 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10336200 |
Jan 3, 2003 |
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09066638 |
Apr 24, 1998 |
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6531590 |
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Current U.S.
Class: |
536/25.34 ;
435/91.2 |
Current CPC
Class: |
C07B 2200/11 20130101;
Y02P 20/55 20151101; C07H 21/00 20130101 |
Class at
Publication: |
536/25.34 ;
435/91.2 |
International
Class: |
C07H 021/02; C07H
021/04; C12P 019/34 |
Claims
What is claimed is:
1. A method for preparing an oligomeric compound comprising a
moiety having the Formula I: 32wherein: Z is aryl having 6 to about
14 carbon atoms or alkyl having from one to about six carbon atoms;
Y.sub.1 is O or S; Y.sub.2 is O or S; Y.sub.3 is C(.dbd.O) or S; q
is 2 to about 4; R.sub.1 is H, OH, F, or a group of formula
R.sub.7--(R.sub.8).sub.n; R.sub.7 is C.sub.3-C.sub.20 alkyl,
C.sub.4-C.sub.20 alkenyl, C.sub.2-C.sub.20; alkynyl,
C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkenyloxy, or
C.sub.2-C.sub.20 alkynyloxy; R.sub.8 is hydrogen, amino, protected
amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso,
nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl,
NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,
NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino,
hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,
silyl, aryl, heterocycle, carbocycle, intercalator, reporter
molecule, conjugate, polyamine, polyamide, polyalkylene glycol,
polyether, a group that enhances the pharmacodynamic properties of
oligonucleotides, a group that enhances the pharmacokinetic
properties of oligonucleotides, or a group of formula
(--O--X.sub.1).sub.p, where p is 2 to about 10 and X.sub.3 is alkyl
having from one to about 10 carbons; B is a naturally occurring or
non-naturally occurring nucleobase that is optionally protected and
optionally radiolabeled; comprising the steps of: providing a
compound having the Formula II: 33wherein: R.sub.3 is hydrogen, a
hydroxyl protecting group, or a linker connected to a solid
support; M is an optionally protected internucleotide linkage; each
B, independently is a naturally occurring or non-naturally
occurring nucleobase that is optionally protected and optionally
radiolabeled; n is 0 to about 50; R.sub.5 is --N(R).sub.6,.sub.2 or
a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to
7 atoms and up to 3 heteroatoms selected from nitrogen, sulfur, and
oxygen; R.sub.6 is straight or branched chain alkyl having from 1
to 10 carbons; and reacting the compound of Formula II with a
compound having Formula III: 34wherein: R.sub.3a is hydrogen; m is
0 to about 50; R.sub.2 is a hydroxyl protecting group, or a linker
connected to a solid support, provided that R.sub.2 and R.sub.3 are
not both simultaneously a linker connected to a solid support;
thereby forming the oligomeric compound.
2. The method of claim 1 further comprising the step of oxidizing
or sulfurizing the oligomeric compound to form a compound having
Formula III, wherein R.sub.3a is hydrogen, a hydroxyl protecting
group, or a linker connected to a solid support; and where m is
increased by n+1.
3. The method of claim 2 further comprising a capping step.
4. The method of claim 3 wherein the capping step is performed
prior to oxidation.
5. The method of claim 3 further comprising the step of cleaving
the oligomeric compound to produce a compound having the Formula
IV: 35wherein R.sub.2 is H.
6. The method of claim 5 wherein the cleaving step occurs
enzymatically.
7. The method of claim 5 wherein the cleaving step occurs in
vivo.
8. The method of claim 1 wherein q is 2; and Y.sub.3 is
C(.dbd.O).
9. The method of claim 1 wherein Z is methyl, phenyl or
t-butyl.
10. The method of claim 9 wherein Z is t-butyl.
11. The method of claim 8 wherein n is 0.
12. The method of claim 8 wherein R.sub.2 is a linker to a solid
support.
13. The method of claim 8 wherein Y.sub.1 and Y.sub.2 are each
0.
14. The method of claim 8 wherein Y.sub.1 and Y.sub.2 are each
S.
15. The method of claim 8 wherein Y.sub.1 is O and Y.sub.2 is
S.
16. The method of claim 8 wherein each R.sub.6 is isopropyl.
17. The method of claim 8 wherein n is 0; R.sub.3 is H, R.sub.5 is
diisopropylamino; Y.sub.1 is O; Y.sub.2 is S; and Z is methyl or
t-butyl.
18. The method of claim 17 wherein Z is t-butyl.
19. The method of claim 1 wherein B is a radiolabeled
nucleobase.
20. The method of claim 19 wherein the radiolabeled nucleobase has
the formula: 36wherein * denotes a .sup.14C atom.
21. The method of claim 17 wherein B is a radiolabeled nucleobase
of formula: 37wherein * denotes a .sup.14C atom.
22. The method of claim 1 wherein the compound of Formula II is
formed by reaction of a compound having Formula V: 38with a
compound having the Formula VI: 39in the presence of an acid.
23. The method of claim 1 wherein the compound of Formula II is
obtained by reaction of a compound having Formula V: 40with a
chlorophosphine compound of formula ClP[i-Pr.sub.2N].sub.2,
followed by reaction with a compound of Formula XX: 41in the
presence of an acid.
24. The method of claim 1 wherein M is an optionally protected
phosphodiester, phosphorothioate, phosphorodithioate, or alkyl
phosphonate internucleotide linkage.
25. A method for preparing a phosphoramidite of Formula: 42wherein:
R.sub.3 is hydrogen, a hydroxyl protecting group, or a linker
connected to a solid support; Z is aryl having 6 to about 14 carbon
atoms or alkyl having from one to about six carbon atoms; Y.sub.1
is O or S; Y.sub.2 is O or S; Y.sub.3 is C(.dbd.O) or S; q is 2 to
about 4; M is an optionally protected internucleotide linkage; each
B, independently is a naturally occurring or non-naturally
occurring nucleobase that is optionally protected and optionally
radiolabeled; n is 0 to about 50; R.sub.1 is H, OH, F, or a group
of formula R.sub.7--(R.sub.8).sub.n; R.sub.7 is C.sub.3-C.sub.20
alkyl, C.sub.4-C.sub.2, alkenyl, C.sub.2-C.sub.20 alkynyl,
C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkenyloxy, or
C.sub.2-C.sub.20 alkynyloxy; R.sub.8 is hydrogen, amino, protected
amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso,
nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl,
NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,
NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino,
hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,
silyl, aryl, heterocycle, carbocycle, intercalator, reporter
molecule, conjugate, polyamine, polyamide, polyalkylene glycol,
polyether, a group that enhances the pharmacodynamic properties of
oligonucleotides, a group that enhances the pharmacokinetic
properties of oligonucleotides, or a group of formula
(--O--X.sub.3).sub.p, where p is 1 to about 10 and X.sub.3 is alkyl
having from one to about 10 carbons; R.sub.5 is --N(R.sub.6).sub.2,
or a heterocycloalkyl or heterocycloalkenyl ring containing from 4
to 7 atoms and up to 3 heteroatoms selected from nitrogen, sulfur,
and oxygen; R.sub.6 is straight or branched chain alkyl having from
1 to 10 carbons; comprising the steps of: providing a compound
Formula: 43 and reacting the compound with a diaminohalophosphine
of Formula: 44wherein X is halogen; thereby providing a
phosphordiamidite of Formula: 45and contacting the nucleoside
phosphordiamidite with a reagent of Formula XX: 46to produce the
phosphoramidite.
26. The method of claim 25 wherein q is 2 and Y.sub.3 is
C(.dbd.O).
27. The method of claim 26 wherein n is 0.
28. The method of claim 26 wherein each R.sub.6 is alkyl.
29. The method of claim 26 wherein each R.sub.6 is isopropyl.
30. The method of claim 26 wherein Y.sub.1 and Y.sub.2 are each
O.
31. The method of claim 26 wherein Y.sub.1 and Y.sub.2 are each
S.
32. The method of claim 26 wherein Y.sub.1 is O and Y.sub.2 is
S.
33. The method of claim 26 wherein Z is methyl, phenyl or
t-butyl.
34. The method of claim 33 wherein Z is t-butyl.
35. The method of claim 26 wherein Z is methyl, phenyl or t-butyl,
n is 0, Y.sub.1 is O and Y.sub.2 is S.
36. The method of claim 26 wherein M is a optionally protected
phosphodiester, phosphorothioate, phosphorodithioate, or alkyl
phosphonate internucleotide linkage.
37. The method of claim 26 wherein X is chlorine.
38. The method of claim 25 wherein B is a radiolabeled
nucleobase.
39. The method of claim 35 wherein B is a radiolabeled
nucleobase.
40. The method of claim 25 or claim 35 wherein the radiolabeled
nucleobase has the Formula: 47wherein * denotes a .sup.14C
atom.
41. A method for preparing a phosphoramidite of Formula: 48wherein:
R.sub.3 is hydrogen, a hydroxyl protecting group, or a linker
connected to a solid support; Z is aryl having 6 to about 14 carbon
atoms or alkyl having from one to about six carbon atoms; Y.sub.1
is O or S; Y.sub.2 is O or S; Y.sub.3 is C(.dbd.O) or S; M is an
optionally protected internucleotide linkage; each B, independently
is a naturally occurring or non-naturally occurring nucleobase that
is optionally protected and optionally radiolabeled; n is 0 to
about 50; R.sub.1 is H, OH, F, or a group of formula
R.sub.7--(R.sub.8).sub.n; R.sub.7 is C.sub.3-C.sub.10 alkyl,
C.sub.4-C.sub.20 alkenyl, C.sub.2-C.sub.29 alkynyl,
C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20 alkenyloxy, or
C.sub.2-C.sub.20 alkynyloxy; R.sub.8 is hydrogen, amino, protected
amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso,
nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl,
NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,
NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino,
hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,
silyl, aryl, heterocycle, carbocycle, intercalator, reporter
molecule, conjugate, polyamine, polyamide, polyalkylene glycol,
polyether, a group that enhances the pharmacodynamic properties of
oligonucleotides, a group that enhances the pharmacokinetic
properties of oligonucleotides, or a group of formula
(--O--X.sub.3).sub.p, where p is 1 to about 10 and X.sub.3 is alkyl
having from one to about 10 carbons; R.sub.5 is --N(R.sub.6).sub.2,
or a heterocycloalkyl or heterocycloalkenyl ring containing from 4
to 7 atoms and up to 3 heteroatoms selected from nitrogen, sulfur,
and oxygen; R.sub.6 is straight or branched chain alkyl having from
1 to 10 carbons; comprising the steps of: providing a compound
Formula: 49and reacting the compound with a compound of Formula:
50wherein: R.sub.5 is --N(R).sub.6,.sub.2 or a heterocycloalkyl or
heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3
heteroatoms selected from nitrogen, sulfur, and oxygen; R.sub.6 is
straight or branched chain alkyl having from 1 to 10 carbons;
thereby producing the phosphordiamidite.
42. The method of claim 41 wherein q is 2 and Y.sub.3 is
C(.dbd.O).
43. The method of claim 42 wherein n is 0.
44. The method of claim 42 wherein each R.sub.6 is alkyl.
45. The method of claim 42 wherein each R.sub.6 is isopropyl.
46. The method of claim 42 wherein Y.sub.1 and Y.sub.2 are each
O.
47. The method of claim 42 wherein Y.sub.1 and Y.sub.2 are each
S.
48. The method of claim 42 wherein Y.sub.1 is O and Y.sub.2 is
S.
49. The method of claim 42 wherein Z is methyl, phenyl or
t-butyl.
50. The method of claim 49 wherein Z is t-butyl.
51. The method of claim 42 wherein Z is methyl, phenyl or t-butyl,
n is 0, Y.sub.1 is O and Y.sub.2 is S.
52. The method of claim 42 wherein M is a optionally protected
phosphodiester, phosphorothioate, phosphorodithioate, or alkyl
phosphonate internucleotide linkage.
53. The method of claim 41 wherein B is a radiolabeled
nucleobase.
54. The method of claim 42 wherein B is a radiolabeled
nucleobase.
55. The method of claim 41 wherein the radiolabeled nucleobase has
the Formula: 51wherein * denotes a .sup.14C atom.
56. The method of claim 41 wherein the compound of Formula: 52is
formed by the reaction of a compound of Formula: 53with a compound
of Formula: 54in the presence of an acid.
Description
FIELD OF THE INVENTION
[0001] This invention is directed to methods for the preparation of
protected forms of oligonucleotides wherein at least one of the
phosphate moieties of the oligonucleotide is protected with a
protecting group that is removable by intracellular enzymes. The
invention is further directed to methods for preparing such
oligonucleotides that contain radioactive labels. The invention
also is directed to the preparation of amidite reagents for
preparing these oligonucleotides. The methods of the invention can
be used to prepare prodrug forms of oligonucleotides and chimeric
oligonucleotides that are modified with certain functional groups
that are cleavable by intercellular enzymes to release the
oligonucleotide from its prodrug form. The oligonucleotides
prepared by the methods of the invention can be of any known
sequence, preferably one that is complementary to a target strand
of a mRNA. The compounds produced by the methods of the invention
are useful for therapeutics, diagnostics, and as research
reagents.
BACKGROUND OF THE INVENTION
[0002] Oligonucleotides and their analogs have been developed and
used in molecular biology in a variety of procedures as probes,
primers, linkers, adapters, and gene fragments. Modifications to
oligonucleotides used in these procedures include labeling with
nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline
phosphatase, or other reporter molecules. Other modifications have
been made to the ribose phosphate backbone to increase the nuclease
stability of the resulting analog. Examples of such modifications
include incorporation of methyl phosphonate, phosphorothioate, or
phosphorodithioate linkages, and 2'-O-methyl ribose sugar units.
Further modifications include those made to modulate uptake and
cellular distribution. With the success of these compounds for both
diagnostic and therapeutic uses, there exists an ongoing demand for
improved oligonucleotides and their analogs.
[0003] It is well known that most of the bodily states in
multicellular organisms, including most disease states, are
effected by proteins. Such proteins, either acting directly or
through their enzymatic or other functions, contribute in major
proportion to many diseases and regulatory functions in animals and
man. For disease states, classical therapeutics has generally
focused upon interactions with such proteins in efforts to moderate
their disease-causing or disease-potentiating functions. In newer
therapeutic approaches, modulation of the actual production of such
proteins is desired. By interfering with the production of
proteins, the maximum therapeutic effect may be obtained with
minimal side effects. It is therefore a general object of such
therapeutic approaches to interfere with or other-wise modulate
gene expression, which would lead to undesired protein
formation.
[0004] One method for inhibiting specific gene expression is with
the use of oligonucleotides, especially oligonucleotides which are
complementary to a specific target messenger RNA (mRNA) sequence.
Several oligonucleotides are currently undergoing clinical trials
for such use. Phosphorothioate oligonucleotides are presently being
used as such antisense agents in human clinical trials for various
disease states, including use as antiviral agents.
[0005] Transcription factors interact with double-stranded DNA
during regulation of transcription. Oligonucleotides can serve as
competitive inhibitors of transcription factors to modulate their
action. Several recent reports describe such interactions (see
Bielinska, et. al., Science 1990, 250, 997-1000; and Wu, et. al.,
Gene 1990, 89, 203-209).
[0006] In addition to such use as both indirect and direct
regulators of proteins, oligonucleotides and their analogs also
have found use in diagnostic tests. Such diagnostic tests can be
performed using biological fluids, tissues, intact cells or
isolated cellular components. As with gene expression inhibition,
diagnostic applications utilize the ability of oligonucleotides and
their analogs two hybridize with a complementary strand of nucleic
acid. Hybridization is the sequence specific hydrogen bonding of
oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs
to RNA or DNA. The bases of such base pairs are said to be
complementary to one another.
[0007] Oligonucleotides and their analogs are also widely used as
research reagents. They are useful for understanding the function
of many other biological molecules as well as in the preparation of
other biological molecules. For example, the use of
oligonucleotides and their analogs as primers in PCR reactions has
given rise to an expanding commercial industry. PCR has become a
mainstay of commercial and research laboratories, and applications
of PCR have multiplied. For example, PCR technology now finds use
in the fields of forensics, paleontology, evolutionary studies and
genetic counseling. Commercialization has led to the development of
kits which assist non-molecular biology-trained personnel in
applying PCR. Oligonucleotides and their analogs, both natural and
synthetic, are employed as primers in such PCR technology.
[0008] Oligonucleotides and their analogs are also used in other
laboratory procedures. Several of these uses are described in
common laboratory manuals such as Molecular Cloning, A Laboratory
Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor
Laboratory Press, 1989; and Current Protocols In Molecular Biology,
F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses
include as synthetic oligonucleotide probes, in screening
expression libraries with antibodies and oligomeric compounds, DNA
sequencing, in vitro amplification of DNA by the polymerase chain
reaction, and in site-directed mutagenesis of cloned DNA. See Book
2 of Molecular Cloning, A Laboratory Manual, supra. See also
"DNA-protein interactions and The Polymerase Chain Reaction" in
Vol. 2 of Current Protocols In Molecular Biology, supra.
[0009] Oligonucleotides and their analogs can be synthesized to
have customized properties that can be tailored for desired uses.
Thus a number of chemical modifications have been introduced into
oligomeric compounds to increase their usefulness in diagnostics,
as research reagents and as therapeutic entities. Such
modifications include those designed to increase binding to a
target strand (i.e. increase their melting temperatures, Tm, to
assist in identification of the oligonucleotide or an
oligonucleotide-target complex, to increase cell penetration, to
stabilize against nucleases and other enzymes that degrade or
interfere with the structure or activity of the oligonucleotides
and their analogs, to provide a mode of disruption (terminating
event) once sequence-specifically bound to a target, and to improve
the pharmacokinetic properties of the oligonucleotide.
[0010] The complementarity of oligonucleotides has been used for
inhibition of a number of cellular targets. Such complementary
oligonucleotides are commonly described as being antisense
oligonucleotides. Various reviews describing the results of these
studies have been published including Progress In Antisense
Oligonucleotide Therapeutics, Crooke, S. T. and Bennett, C. F.,
Annu. Rev. Pharmacol. Toxicol., 1996, 36, 107-129. These
oligonucleotides have proven to be very powerful research tools and
diagnostic agents. Further, certain oligonucleotides that have been
shown to be efficacious are currently in human clinical trials.
[0011] Antisense therapy involves the use of oligonucleotides
having complementary sequences to target RNA or DNA. Upon binding
to a target RNA or DNA, an antisense oligonucleotide can
selectively inhibit the genetic expression of these nucleic acids
or can induce other events such as destruction of a targeted RNA or
DNA or activation of gene expression.
[0012] Destruction of targeted RNA can be effected by activation of
RNase H. RNase H is an endonuclease that cleaves the RNA strand of
DNA:RNA duplexes. This enzyme, thought to play a role in DNA
replication, has been shown to be capable of cleaving the RNA
component of the DNA:RNA duplexes in cell free systems as well as
in Xenopus oocytes.
[0013] RNase H is very sensitive to structural alterations in
antisense oligonucleotides. To activate RNase H, a DNA:RNA
structure must be formed. Therefore for an antisense
oligonucleotide to activate RNase H, at least a part of the
oligonucleotide must be DNA like. To be DNA like requires that the
sugars of the nucleotides of the oligonucleotide have a 2'-deoxy
structure and the phosphate linkages of the oligonucleotide have
negative charges. Chemical modifications of the DNA portion of
oligonucleotide at either of these two positions resulted in
oligonucleotides that are no longer substrates for RNase H.
[0014] However, 2'-deoxy nucleotides have weaker binding affinity
to their counterpart ribonucleotides than like ribonucleotides
would, i.e., RNA:RNA binding is stronger than DNA:RNA binding, and
the presence of the negative charges has been thought to contribute
to reduced cellular uptake of the antisense oligonucleotide.
Therefore, to circumvent the limitations of DNA like
oligonucleotides, chimeric oligonucleotides have been synthesized
wherein a DNA like central portion having 2'-deoxy nucleotides and
negative charged phosphate linkages is included as the center of a
large oligonucleotide that has other types of nucleotides on either
side of the DNA like center portion. The center portion must be of
a certain size in order to activate RNase H upon binding of the
oligonucleotide to a target RNA.
[0015] There remains a continuing long-felt need for modified
antisense compounds that incorporate chemical modifications for
improving characteristics such as compound stability, cellular
uptake and detectability, but are also available for regulation of
target RNA through each of the known mechanisms of action of
antisense compounds. Such regulation of target RNA would be useful
for therapeutic purposes both in vivo and ex vivo and, as well as,
for diagnostic reagents and as research reagents including reagents
for the study of both cellular and in vitro events.
[0016] Labeling with radioactive isotopes provides an efficient
tool for studying pharmacological properties of antisense
oligonucleotides. As with other classes of drug compounds, this
novel class of therapeutics requires high sensitivity
radiodetection for evaluation of distribution of antisense agents
in tissues and assessment of their metabolic fate. Several methods
to introduce .sup.35S at the internucleosidic thiophosphate of
.sup.3H or .sup.14C at the base moiety of synthetic
oligonucleotides have been reported. Among these labels, .sup.14C
offers the highest specific activity and the longest half-life.
Considering catabolism of nucleic acids, labeling with .sup.14C at
the C-2 position of thymidine results in formation of
.sup.14CO.sub.2 which is cumbersome to trap and analyze. On the
other hand, labeling at either the C-4 or C-6 position leads to
.beta.-aminoisobutyric acid as the metabolite which is much more
convenient to analyze.
[0017] There remains a need for methods of preparing labeled
antisense oligonucleotides that overcome the foregoing
difficulties. The present invention is directed to the foregoing
important ends.
SUMMARY OF THE INVENTION
[0018] The present invention is directed to methods for the
preparation of oligonucleotides having at least one bioreversible
protecting group that confers enhanced chemical and biophysical
properties. The bioreversible protecting groups further lend
nuclease resistance to the oligonucleotides. The bioreversible
protecting groups are removed in a cell, in the cell cytosol, or in
vitro in cytosol extract, by endogenous enzymes. In certain
preferred oligonucleotides of the invention the bioreversible
protecting groups are designed for cleavage by carboxyesterases to
yield unprotected oligonucleotides.
[0019] In one aspect of the present invention, methods are provided
for the preparation of oligomeric compounds having at least one
moiety having the Formula I: 1
[0020] wherein:
[0021] Z is aryl having 6 to about 14 carbon atoms or alkyl having
from one to about six carbon atoms;
[0022] Y.sub.1 is O or S;
[0023] Y.sub.2 is O or S;
[0024] Y.sub.3 is C(.dbd.O) or S;
[0025] q is 2 to about 4;
[0026] R.sub.1 is H, OH, F, or a group of formula
R.sub.7--(R.sub.8).sub.n- ;
[0027] R.sub.7 is C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl,
C.sub.2-C.sub.2, alkynyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20
alkenyloxy, or C.sub.2-C.sub.2 alkynyloxy;
[0028] R.sub.8 is hydrogen, amino, protected amino, halogen,
hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile,
trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,
N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,
NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino,
hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,
silyl, aryl, heterocycle, carbocycle, intercalator, reporter
molecule, conjugate, polyamine, polyamide, polyalkylene glycol,
polyether, a group that enhances the pharmacodynamic properties of
oligonucleotides, a group that enhances the pharmacokinetic
properties of oligonucleotides, or a group of formula
(--O--X.sub.3).sub.p, where p is 1 to about 10 and X.sub.3 is alkyl
having from one to about 10 carbons;
[0029] B is a naturally occurring or non-naturally occurring
nucleobase that is optionally protected and optionally
radiolabeled.
[0030] These methods comprise the steps of providing a compound
having the Formula II: 2
[0031] wherein:
[0032] R.sub.3 is hydrogen, a hydroxyl protecting group, or a
linker connected to a solid support;
[0033] M is an optionally protected internucleotide linkage;
[0034] each B, independently is a naturally occurring or
non-naturally occurring nucleobase that is optionally protected and
optionally radiolabeled;
[0035] n is 0 to about 50;
[0036] R.sub.5 is --N(R.sub.6).sub.2, or a heterocycloalkyl or
heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3
heteroatoms selected from nitrogen, sulfur, and oxygen;
[0037] R.sub.6 is straight or branched chain alkyl having from 1 to
10 carbons.
[0038] Compounds of Formula II are then reacted with compounds
having Formula III: 3
[0039] (wherein R.sub.3a is hydrogen; m is 0 to about 50; and
R.sub.2 is a hydroxyl protecting group, or a linker connected to a
solid support, provided that R.sub.2 and R.sub.3 are not both
simultaneously a linker connected to a solid support), thereby
forming the oligomeric compound.
[0040] In some preferred embodiments, the methods of the invention
further comprise oxidizing or sulfurizing the oligomeric compound
to form a further compound having Formula III, wherein R.sub.3 is
hydrogen, a hydroxyl protecting group, or a linker connected to a
solid support, and where m is increased by n+1. In further
preferred embodiments, the methods of the invention include a
capping step, which can be performed prior to or subsequent to
oxidation or sulfurization.
[0041] In preferred embodiments, the methods of the invention
further comprise cleaving the oligomeric compound to produce a
compound having the Formula IV: 4
[0042] In some particularly preferred embodiments, the cleaving
step occurs enzymatically, more preferably in vivo.
[0043] In some preferred embodiments, the compound of Formula II is
formed by reaction of a compound having Formula V: 5
[0044] with a compound having the Formula VI: 6
[0045] in the presence of an acid.
[0046] In further preferred embodiments, the compound of Formula II
is obtained by reaction of a compound having Formula V with a
chlorophosphine compound of formula ClP[i-Pr.sub.2N].sub.2,
followed by reaction with a compound of Formula XX: 7
[0047] in the presence of an acid.
[0048] Also provided in accordance with the present invention are
methods for the preparation of a phosphoramidite of Formula II:
8
[0049] wherein:
[0050] R.sub.3 is hydrogen, a hydroxyl protecting group, or a
linker connected to a solid support;
[0051] z is aryl having 6 to about 14 carbon atoms or alkyl having
from one to about six carbon atoms;
[0052] Y.sub.1 is O or S;
[0053] Y.sub.2 is O or S;
[0054] Y.sub.3 is C(.dbd.O) or S;
[0055] q is 2 to about 4;
[0056] M is an optionally protected internucleotide linkage;
[0057] each B, independently is a naturally occurring or
non-naturally occurring nucleobase that is optionally protected and
optionally radiolabeled;
[0058] n is 0 to about 50;
[0059] R.sub.1 is H, OH, F, or a group of formula
R.sub.7--(R.sub.8).sub.n- ;
[0060] R.sub.7 is C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl,
C.sub.2-C.sub.20 alkynyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20
alkenyloxy, or C.sub.2-C.sub.20 alkynyloxy;
[0061] R.sub.8 is hydrogen, amino, protected amino, halogen,
hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile,
trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,
N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,
NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino,
hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,
silyl, aryl, heterocycle, carbocycle, intercalator, reporter
molecule, conjugate, polyamine, polyamide, polyalkylene glycol,
polyether, a group that enhances the pharmacodynamic properties of
oligonucleotides, a group that enhances the pharmacokinetic
properties of oligonucleotides, or a group of formula
(--O--X.sub.3).sub.p, where p is 1 to about 10 and X.sub.3 is alkyl
having from one to about 10 carbons;
[0062] R.sub.5 is --N(R.sub.6).sub.2, or a heterocycloalkyl or
heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3
heteroatoms selected from nitrogen, sulfur, and oxygen;
[0063] R.sub.6 is straight or branched chain alkyl having from 1 to
10 carbons.
[0064] Such methods comprise the steps of providing a compound
Formula V: 9
[0065] and reacting the compound with a diaminohalophosphine of
Formula: 10
[0066] (wherein X is halogen, with chlorine being preferred),
thereby producing a phosphordiamidite of Formula: 11
[0067] The phosphordiamidite is then contacted with a reagent of
Formula XX: 12
[0068] to produce the phosphoramidite.
[0069] Also provided in accordance with the present invention are
methods for the preparation of a phosphoramidite of Formula II:
13
[0070] wherein:
[0071] R.sub.3 is hydrogen, a hydroxyl protecting group, or a
linker connected to a solid support;
[0072] Z is alkyl having from one to about six carbon atoms;
[0073] Y.sub.1 is O or S;
[0074] Y.sub.2 is O or S;
[0075] Y.sub.3 is O or S;
[0076] q is 2 to about 4;
[0077] M is an optionally protected internucleotide linkage;
[0078] each B, independently is a naturally occurring or
non-naturally occurring nucleobase that is optionally protected and
optionally radiolabeled;
[0079] n is 0 to about 50;
[0080] R.sub.1 is H, OH, F, or a group of formula
R.sub.7--(R.sub.8).sub.n- ;
[0081] R.sub.7 is C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl,
C.sub.2-C.sub.20 alkynyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20
alkenyloxy, or C.sub.2-C.sub.21 alkynyloxy;
[0082] R.sub.8 is hydrogen, amino, protected amino, halogen,
hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile,
trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,
N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,
NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino,
hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,
silyl, aryl, heterocycle, carbocycle, intercalator, reporter
molecule, conjugate, polyamine, polyamide, polyalkylene glycol,
polyether, a group that enhances the pharmacodynamic properties of
oligonucleotides, a group that enhances the pharmacokinetic
properties of oligonucleotides, or a group of formula
(--O--X.sub.3) p, where p is 1 to about 10 and X.sub.3 is alkyl
having from one to about 10 carbons;
[0083] R.sub.5 is --N(R.sub.6).sub.2, or a heterocycloalkyl or
heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3
heteroatoms selected from nitrogen, sulfur, and oxygen;
[0084] R.sub.6 is straight or branched chain alkyl having from 1 to
10 carbons.
[0085] These methods comprise the steps of providing a compound
Formula V: 14
[0086] and reacting the compound with a compound of Formula: 15
[0087] wherein:
[0088] Z is alkyl having from one to about six carbon atoms;
[0089] Y.sub.1 is O or S;
[0090] Y.sub.2 is O or S;
[0091] Y.sub.3 is C(.dbd.O) or S;
[0092] q is 2 to about 4;
[0093] R.sub.5 is --N(R.sub.6).sub.2, or a heterocycloalkyl or
heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3
heteroatoms selected from nitrogen, sulfur, and oxygen;
[0094] R.sub.6 is straight or branched chain alkyl having from 1 to
10 carbons;
[0095] thereby producing the phosphordiamidite.
[0096] In some preferred embodiments, the compound of Formula:
16
[0097] is formed by the reaction of a compound of Formula: 17
[0098] with a compound of Formula: 18
[0099] in the presence of an acid.
[0100] In preferred embodiments of the foregoing methods, Z is
methyl, t-butyl or phenyl, with t-butyl being preferred. In some
particularly preferred embodiments, n is 0.
[0101] In some preferred embodiments of the foregoing methods,
R.sub.2 is a linker to a solid support.
[0102] In preferred embodiments of the foregoing methods, Y.sub.1
and Y.sub.2 are each O and Y.sub.3 is C(.dbd.O), or Y.sub.1 and
Y.sub.2 are each S and Y.sub.3 is C(.dbd.O), or Y.sub.1 is S and
Y.sub.2 is 0 and Y.sub.3 is C(.dbd.O). In especially preferred
embodiments, Y.sub.1 is 0 and Y.sub.2 is S and Y.sub.3 is
C(.dbd.O).
[0103] In some preferred embodiments of the foregoing methods, each
R.sub.6 is isopropyl. In some especially preferred embodiments, n
is 0; R.sub.3 is H, R.sub.5 is diisopropylamino; Y.sub.1 is O;
Y.sub.2 is S; Y.sub.3 is C(.dbd.O); and Z is methyl, phenyl or
t-butyl, with t-butyl being preferred.
[0104] In some preferred embodiments of the foregoing methods, B
radiolabeled nucleobase. In more preferred embodiments, the
radiolabeled nucleobase has the formula: 19
[0105] wherein * denotes a .sup.14C atom.
[0106] In preferred embodiments of the foregoing methods, M is an
optionally protected phosphite, phosphodiester, phosphotriester,
phosphorothioate, phosphorodithioate, or alkyl phosphonate
internucleotide linkage. In especially phosphotriester,
phosphorothioate, phosphorodithioate, or alkyl phosphonate
internucleotide linkage protected with protecting group of formula
--Y.sub.1--(CH.sub.2).sub.q--Y- .sub.2--Y.sub.3-Z.
BRIEF DESCRIPTION OF THE DRAWINGS
[0107] The numerous objects and advantages of the present invention
may be better understood by those skilled in the art by reference
to the accompanying non-scale figures, in which:
[0108] FIG. 1 shows compounds 15-19.
[0109] FIG. 2 is a synthetic scheme for compounds 5, 7, 11, and
12.
[0110] FIG. 3 is a synthetic scheme compound 22.
[0111] FIG. 4 is a synthetic scheme compounds 23 and 24
DETAILED DESCRIPTION OF THE INVENTION
[0112] The present invention relates to methods for the preparation
of oligonucleotides having at least one bioreversible protecting
group. The bioreversible protecting groups contribute to certain
enhanced chemical and biophysical properties of the
oligonucleotides including resistance to exo- and endonuclease
degradation.
[0113] Oligonucleotides represent a new class of compounds that
specifically inhibit gene expression by Watson-Crick base pair
formation with their targets which usually are known mRNA
sequences. After binding to the mRNA, down regulation of gene
product expression occurs. Crooke, S. T., Nucleic Acid Therapeutics
In Pharmaceutical Manufacturing International, Sterling, London, 89
(1992). Use of the first synthesized oligonucleotides, i.e.,
phosphodiester linked oligonucleotides, was limited by the lack
nuclease resistance of these compounds. Nuclease resistance has
mainly been resolved by the use of modified oligonucleotides.
Milligan, et al., J. Med. Chem. 1991, 36, 1923; Varma, Synlett
1991, 621; Uhlmann, et al., Chem. Rev. 1990, 90, 534.
[0114] It has been reported that phosphodiester and
phosphorothioate oligonucleotides, both which have a polyanionic
character, enter the cell by an active process (adsorptive
endocytosis and/or fluid phase endocytosis) and this uptake varies
with different cell types. It has been reported that the neutral
methylphosphonodiester oligonucleotides enter cells by a different
mechanism that is also energy dependent. Spiller, et al.,
Anti-Cancer Drug Design 1991, 7, 115. Certain increases in
penetration of the oligonucleotides into cell has been achieved by
derivatizing oligonucleotides with poly L-lysine, cholesterol or
other like moieties or by encapsulation into liposomes.
[0115] In one aspect, the present invention is directed to a
further approach to assist cellular uptake of oligonucleotides. In
this approach a prodrug strategy is utilized wherein a
prooligonucleotide is formed that is believed to temporarily mask
the negative charges of phosphodiester, phosphorothioate and
phosphorodithioate oligonucleotides by the introduction of a
bioreversible group on at least some of phosphate groups of these
oligomers. The resulting neutral prooligonucleotides have been
found to be enzymatically stable against degradative enzymes. While
we do not wish to be bound by theory, we believe this will help
oligonucleotides to escape from the endosomes should they become
embedded therein and will present a completely different
bioavailability pattern in relation with their route of
administration. A perceived prerequisite of this approach is that
bioreversible groups should be selected that have stability in
culture medium and that have selective intracellularly hydrolysis
after uptake, due to the existence of a greater enzymatic activity
in cytosol than in biological fluids.
[0116] The present invention is directed to methods for the
preparation of oligonucleotides having at least one bioreversible
protecting group that have enhanced chemical and biophysical
properties for cellular membrane penetration as well as resistance
to exo- and endonuclease degradation in vivo. In certain preferred
embodiments of the invention, the bioreversible protecting groups
are removed in the cell cytosol by endogenous carboxyesterases to
yield biologically active oligonucleotide compounds that are
capable of hybridizing to and/or having an affinity for specific
nucleic acid or peptide sequences thus interacting with endogenous
and/or pathogenic biomolecules.
[0117] In one aspect of the present invention, methods are provided
for the preparation of oligomeric compounds comprising a moiety
having at least one moiety of Formula I: 20
[0118] wherein:
[0119] Z is aryl having 6 to about 14 carbon atoms or alkyl having
from one to about six carbon atoms;
[0120] Y.sub.1 is O or S;
[0121] Y.sub.2 is O or S;
[0122] Y.sub.3 is C(.dbd.O) or S;
[0123] q is 2 to about 4;
[0124] R.sub.1 is H, OH, F, or a group of formula
R.sub.7--(R.sub.9).sub.n- ;
[0125] R.sub.7 is C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl,
C.sub.2-C.sub.20 alkynyl, C.sub.1-C.sub.20 alkoxy, C.sub.2-C.sub.20
alkenyloxy, or C.sub.2-C.sub.20 alkynyloxy;
[0126] R.sub.8 is hydrogen, amino, protected amino, halogen,
hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile,
trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,
N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,
NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino,
hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,
silyl, aryl, heterocycle, carbocycle, intercalator, reporter
molecule, conjugate, polyamine, polyamide, polyalkylene glycol,
polyether, a group that enhances the pharmacodynamic properties of
oligonucleotides, a group that enhances the pharmacokinetic
properties of oligonucleotides, or a group of formula
(--O--X.sub.3) p, where p is 1 to about 10 and X.sub.3 is alkyl
having from one to about 10 carbons;
[0127] B is a naturally occurring or non-naturally occurring
nucleobase that is optionally protected and optionally
radiolabeled;
[0128] comprising the steps of:
[0129] providing a compound having the Formula II: 21
[0130] wherein:
[0131] R.sub.3 is hydrogen, a hydroxyl protecting group, or a
linker connected to a solid support;
[0132] M is an optionally protected internucleotide linkage;
[0133] each B, independently is a naturally occurring or
non-naturally occurring nucleobase that is optionally protected and
optionally radiolabeled;
[0134] n is 0 to about 50;
[0135] R.sub.5 is --N(R.sub.6).sub.2, or a heterocycloalkyl or
heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3
heteroatoms selected from nitrogen, sulfur, and oxygen;
[0136] R.sub.6 is straight or branched chain alkyl having from 1 to
10 carbons; and
[0137] reacting the compound of Formula II with a compound having
Formula III: 22
[0138] wherein:
[0139] R.sub.3a is hydrogen;
[0140] m is 0 to about 50;
[0141] R.sub.2 is a hydroxyl protecting group, or a linker
connected to a solid support, provided that R.sub.2 and R.sub.3 are
not both simultaneously a linker connected to a solid support;
[0142] to form the oligomeric compound.
[0143] The methods of the present invention are useful for the
preparation of oligomeric compounds containing monomeric subunits
that are joined by a variety of linkages, including phosphite,
phosphodiester, phosphotrieter, phosphorothioate, aklyl phosphonate
and/or phosphorodithioate linkages. As used herein, the terms
"oligomer" and "oligomeric compound" are used to refer to compounds
containing a plurality of monomer subunits that are joined by such
internucleotide linkages. The term "oligomeric compound" therefore
includes naturally occurring oligonucleotides, synthetic
oligonucleotides, and their analogs.
[0144] In some preferred embodiments of the methods of the
invention, a phosphoramidite of Formula II is reacted with a
growing nucleotide chain to produce a phosphite compound containing
the linkage of Formula I. Preferably, capping, and/or oxidation or
sulfurization steps are performed, and the iterative cycle is
repeated until the desired nucleobase sequence is attained. This is
followed by cleavage to produce a compound of Formula IV.
[0145] Methods for coupling compounds of Formula II and Formula III
include both solution phase and solid phase chemistries.
Representative solution phase techniques are described in U.S. Pat.
No. 5,210,264, which is assigned to the assignee of the present
invention. In preferred embodiments, the methods of the present
invention are employed for use in iterative solid phase
oligonucleotide synthetic regimes. Representative solid phase
techniques are those typically employed for DNA and RNA synthesis
utilizing standard phosphoramidite chemistry, (see, e.g., Protocols
For Oligonucleotides And Analogs, Agrawal, S., ed., Humana Press,
Totowa, N.J., 1993, hereby incorporated by reference in its
entirety). A preferred synthetic solid phase synthesis utilizes
phosphoramidites as activated phosphate compounds. In this
technique, a phosphoramidite monomer is reacted with a free
hydroxyl on the growing oligomer chain to produce an intermediate
phosphite compound, which is subsequently oxidized to the pV state
using standard methods. This technique is commonly used for the
synthesis of several types of linkages including phosphodiester,
phosphorothioate, and phosphorodithioate linkages.
[0146] Typically, the first step in such a process is attachment of
a first monomer or higher order subunit containing a protected
5'-hydroxyl to a solid support, usually through a linker, using
standard methods and procedures known in the art. See for example,
Oligonucleotides And Analogues A Practical Approach, Ekstein, F.
Ed., IRL Press, N.Y., 1991. The support-bound monomer or higher
order first synthon is then treated to remove the 5'-protecting
group, to form a compound of Formula III wherein R.sub.2 is a
linker connected to a solid support. Typically, this is
accomplished by treatment with. acid. The solid support bound
monomer is then reacted with a phosphoramidite of Formula II to
form a phosphite linkage of Formula I. In some preferred
embodiments, synthons of Formula II and Formula III are reacted
under anhydrous conditions in the presence of an activating agent
such as,: for example, 1H-tetrazole,
5-(4-nitrophenyl)-1H-tetrazole, or diisopropylamino
tetrazolide.
[0147] In preferred embodiments, phosphite or thiophosphite
compounds containing a linkage of Formula I are oxidized or
sulfurized to produce compounds having a desired internucleotide
linkage. The choice of oxidizing or sulfurizing agent will
determine whether the linkage of Formula I will be oxidized or
sulfurized to, for example, a phosphotriester, thiophosphotriester,
or a dithiophosphotriester linkage.
[0148] It is generally preferable to perform a capping step either
prior to or after oxidation or sulfurization of the phosphite
triester, thiophosphite triester, or dithiophosphite triester. As
understood by those skilled in the art, the capping step involves
attachment of a "cap" moiety to oligonucleotide chains that have
not reacted in a given coupling cycle. The cap moiety preferably is
reactive with the terminal portion of oligonucleotides that did not
participate in the coupling cycle but is not reactive with
oligonucleotides that did participate and, moreover, is not itself
reactive with the coupling reagents. Such a capping step is
generally known to be beneficial by preventing 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, issued Mar. 28, 1989, hereby
incorporated by reference in its entirety.
[0149] Further treatment of the oxidized or sulfurized oligomer
with an acid removes the 5'-hydroxyl protecting group, and thus
transforms the solid support bound oligomer into a further compound
of Formula III wherein R.sub.3a is hydrogen. This species is then
reacted with a further compound of Formula II to begin the next
synthetic iteration. This process is repeated until an oligomer of
desired length is produced.
[0150] The completed oligomer is then cleaved from the solid
support. The cleavage step, which can precede or follow
deprotection of protected functional groups, will in preferred
embodiments yield a compound having Formula IV wherein R.sub.2 is
hydrogen. In some preferred embodiments, cleavage of the
oligonucleotide from the solid support also removes protecting
groups of the internucleotide linkages. Thus, in some preferred
embodiments, the linkages between monomeric subunits are converted
during cleavage from phosphotriester, thiophosphotriester, or
dithiophosphotriester linkages to phosphodiester, phosophorothiote,
or phosphorodithioate linkages.
[0151] In other preferred embodiments of the invention, cleavage of
the oligonucleotide from the solid support does not effect the
removal of protecting groups of the internucleotide linkages. In
such embodiments, the result of cleavage is a compound of formula
IV where R.sub.2 is H, and each internucleotide linkage formed by
the methods of the invention bear a protecting group of formula
--Y.sub.1--(CH.sub.2).sub.q--Y.sub.2--- Y.sub.3-Z.
[0152] The methods of the present invention are applicable to the
synthesis of a wide variety of oligomeric compounds which contain,
for example, phosphite, phosphodiester, phosphotriester,
phosphorothioate, phosphorodithioate and/or alkylphosphonate
internucleotide linkages.
[0153] In preferred embodiments, the methods of the invention are
used for the preparation of oligonucleotides and their analogs. As
used herein, the term "oligonucleotide" is intended to include both
naturally occurring and non-naturally occurring (i.e., "synthetic")
oligonucleotides. Naturally occurring oligonucleotides are those
which occur in nature; for example ribose and deoxyribose
phosphodiester oligonucleotides having adenine, guanine, cytosine,
thymine and uracil nucleobases. As used herein, non-naturally
occurring oligonucleotides are oligonucleotides that contain
modified sugar, internucleoside linkage and/or nucleobase moieties.
Such oligonucleotide analogs are typically structurally
distinguishable from, yet functionally interchangeable with,
naturally occurring or synthetic wild type oligonucleotides. Thus,
non-naturally occurring oligonucleotides include all such
structures which function effectively to mimic the structure and/or
function of a desired RNA or DNA strand, for example, by
hybridizing to a target.
[0154] Representative nucleobases 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, Anti-Cancer Drug Design 1991, 6, 585-607, each of which are
hereby incorporated by reference in their entirety). The term
"nucleosidic base" is further intended to include heterocyclic
compounds that can serve as like nucleosidic bases including
certain "universal bases" that are not nucleosidic bases in the
most classical sense but serve as nucleosidic bases. Especially
mentioned as a universal base is 3-nitropyrrole.
[0155] Representative 2' sugar modifications (moiety R.sub.1 in the
formulas described herein) amenable to the present invention
include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected
O-alkylamino, O-alkylaminoalkyl, O-alkylimidazole, 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, each of which are hereby incorporated by
reference in their entirety. Further sugar modifications are
disclosed in Cook, P. D., supra. Fluoro, O-alkyl, O-alkylamino,
O-alkylimidazole, 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, hereby incorporated by
reference in its entirety.
[0156] 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, Sept. 16-20, 1992, hereby
incorporated by reference in its entirety.
[0157] 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, hereby incorporated by
reference in its entirety.
[0158] "Aryl" groups are aromatic cyclic compounds including but
not limited to phenyl, naphthyl, anthracyl, phenanthryl, pyrenyl,
and xylyl.
[0159] In general, the term "hetero" denotes an atom other than
carbon, preferably but not exclusively N, O, or S. Accordingly, 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.
[0160] In some preferred embodiments of the invention R.sub.2,
R.sub.3 or R.sub.3a can be 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, hereby incorporated by reference in its
entirety.
[0161] Preferred linkers for use in linking the growing
oligonucleotide chain to the solid support in some preferred
embodiments of the methods of the invention will be cleaved by
reagents that do not result in removal of the
--Y.sub.1--(CH.sub.2).sub.q--Y.sub.2--Y.sub.3-Z protecting group.
One such linker is the oxalyl linker (Alul, R. H., et al., Nucl.
Acids Res. 1991, 19, 1527) between a LCAA-CPG solid support and the
oligomer. Other photolabile supports have been reported (Holmes et
al., J. Org. Chem. 1997, 62, 2370-2380; Greenberg et al.,
Tetrahedron Lett. 1993, 34, 251-254). The o-nitrobenzyl
functionalized solid support has been previously reported
(Dell'Aquila et al., Tetrahedron Lett. 1997, 38, 5289-5292).
Another preferred method of cleavage without removal of
internucleoside protecting groups is by irradiation with
ultraviolet light in aqueous acetonitrile.
[0162] 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, hereby incorporated by reference in
its entirety), TentaGel Support, an aminopolyethyleneglycol
derivatized support (see, e.g., Wright, et al., Tetrahedron Letters
1993, 34, 3373, hereby incorporated by reference in its entirety)
and Poros, a copolymer of polystyrene/divinylbenzene.
[0163] In some preferred embodiments of the invention R.sub.2,
R.sub.3 or R.sub.3a 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, Protective Groups in Organic Synthesis, Chapter,
2d ed, John Wiley & Sons, New York, 1991, each of which are
hereby incorporated by reference in their entirety. 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.1 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.
[0164] 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.
[0165] Sulfurizing agents used during oxidation to form
phosphorothioate and phosphorodithioate linkages include Beaucage
reagent (see e.g. Iyer, et.al., J. Chem. Soc. 1990, 112, 1253-1254,
and Iyer, et.al., J. Org. Chem. 1990, 55, 4693-4699);
tetraethylthiuram disulfide (see e.g., Vu, et al., Tetrahedron
Lett. 1991, 32, 3005-3008); dibenzoyl tetrasulfide (see e.g., Rao,
et.al., Tetrahedron Lett. 1992, 33, 4839-4842);
di(phenylacetyl)disulfide (see e.g., Kamer, Tetrahedron Lett. 1989,
30, 6757-6760); Bis(O,O-diisopropoxy phosphinothioyl)disulfide (see
Stec et al., Tetrahedron Lett. 1993, 34, 5317-5320);
3-ethoxy-1,2,4-dithiazoline-- 5-one (see Nucleic Acids Research,
1996 24, 1602-1607, and Nucleic Acids Research, 1996 24,
3643-3644); Bis(p-chlorobenzenesulfonyl)disulfide (see Nucleic
Acids Research, 1995 23, 4029-4033); sulfur, sulfur in combination
with ligands like triaryl, trialkyl, triaralkyl, or trialkaryl
phosphines. The foregoing references are hereby incorporated by
reference in their entirety.
[0166] Useful oxidizing agents used to form the phosphodiester or
phosphorothioate linkages include
iodine/tetrahydrofuran/water/pyridine or hydrogen peroxide/water or
tert-butyl hydroperoxide or any peracid like m-chloroperbenzoic
acid. In the case of sulfurization the reaction is performed under
anhydrous conditions with the exclusion of air, in particular
oxygen whereas in the case of oxidation the reaction can be
performed under aqueous conditions.
[0167] Oligonucleotides or oligonucleotide analogs according to the
present invention hybridizable to a specific target preferably
comprise from about 5 to about 50 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. When used as "building blocks" in assembling larger
oligomeric compounds (i.e., as synthons of Formula II), smaller
oligomeric compounds are preferred. Libraries of dimeric, trimeric,
or higher order compounds of general Formula II can be prepared for
use as synthons in the methods of the invention. The use of small
sequences synthesized via solution phase chemistries in automated
synthesis of larger oligonucleotides enhances the coupling
efficiency and the purity of the final oligonucloetides. See for
example: Miura, et al., Chem. Pharm. Bull. 1987, 35, 833-836;
Kumar, et al., J. Org. Chem. 1984, 49, 4905-4912; Bannwarth,
Helvetica Chimica Acta 1985, 68, 1907-1913; Wolter, et al.,
Nucleosides and Nucleotides 1986, 5, 65-77, each of which are
hereby incorporated by reference in their entirety.
[0168] The oligonucleotides produced by preferred embodiments of
the methods of the invention having bioreversible protecting groups
are also referred to in this specification as pro-oligonucleotides.
Such pro-oligonucleotides are capable of improved cellular lipid
bilayers penetrating potential as well as resistance to exo- and
endonuclease degradation in vivo. In cells, the bioreversible
protecting groups are removed in the cell cytosol by endogenous
carboxyesterases to yield biologically active oligonucleotide
compounds that are capable of hybridizing to and/or having an
affinity for specific nucleic acid.
[0169] The compounds produced by the methods of the invention
mitigate one potential problem with the therapeutic use of
oligonucleotides of natural composition, i.e., phosphodiester
oligonucleotides; specifically 1) their very short biological
half-lives due to degradation by nucleases which tend to be
ubiquitous, and 2) their inherent negative charge and hydrophilic
nature which makes it very difficult biophysically for
oligonucleotides to pass through lipid cellular membranes.
[0170] The methods of the invention can be used to prepare
antisense pro-oligonucleotides to synthetic DNA or RNA or mixed
molecules of complementary sequences to a target sequence belonging
to a gene or to an RNA messenger whose expression they are
specifically designed to block or down-regulate. The methods of the
invention can be used to prepare antisense oligonucleotides that
can be directed against a target messenger RNA sequence or,
alternatively against a target DNA sequence, and hybridize to the
nucleic acid to which they are complementary. Accordingly, the
compounds produced by the methods of the invention effectively
block or down-regulate gene expression.
[0171] The pro-oligonucleotides produced according to the methods
of the invention can also be directed against certain bicatenary
DNA regions (homopurine/homopyrimidine sequences or sequences rich
in purines/pyrimidines) and thus form triple helices. The formation
of a triple helix, at a particular sequence, can block the
interaction of protein factors which regulate or otherwise control
gene expression and/or may facilitate irreversible damage to be
introduced to a specific nucleic acid site if the resulting
oligonucleotide is made to possess a reactive functional group.
[0172] As used herein, a target nucleic acid shall mean any nucleic
acid that can hybridize with a complementary nucleic acid like
compound. Further in the context of this invention, "hybridization"
shall mean hydrogen bonding, which may be Watson-Crick, Hoogsteen
or reversed Hoogsteen hydrogen bonding, between complementary
nucleobases. "Complementary" as used herein, refers to the capacity
for precise pairing between two nucleobases. For example, adenine
and thymine are complementary nucleobases which pair through the
formation of hydrogen bonds. "Complementary" and "specifically
hybridizable," as used herein, refer to precise pairing or sequence
complementarity between a first and a second nucleic acid-like
oligomers containing nucleoside subunits. For example, if a
nucleobase at a certain position of the first nucleic acid is
capable of hydrogen bonding with a nucleobase at the same position
of the second nucleic acid, then the first nucleic acid and the
second nucleic acid are considered to be complementary to each
other at that position. The first and second nucleic acids are
complementary to each other when a sufficient number of
corresponding positions in each molecule are occupied by
nucleobases which can hydrogen bond with each other. Thus,
"specifically hybridizable" and "complementary" are terms which are
used to indicate a sufficient degree of complementarity such that
stable and specific binding occurs between a compound of the
invention and a target RNA molecule. It is understood that an
oligomeric compound of the invention need not be 100% complementary
to its target RNA sequence to be specifically hybridizable. An
oligomeric compound is specifically hybridizable when binding of
the oligomeric compound to the target RNA molecule interferes with
the normal function of the target RNA to cause a loss of utility,
and there is a sufficient degree of complementarity to avoid
non-specific binding of the oligomeric compound to non-target
sequences under conditions in which specific binding is desired,
i.e. under physiological conditions in the case of in vivo assays
or therapeutic treatment, or in the case of in vitro assays, under
conditions in which the assays are performed.
[0173] In some preferred embodiments of the methods of the
invention, compounds of Formula II are prepared by reaction of a
protected nucleoside having Formula V: 23
[0174] with a compound having the Formula VI: 24
[0175] in the presence of an acid. Suitable acids include those
known in the art to be useful for coupling of phosphoramidites,
including, for example, diisopropylammonium tetrazolide.
[0176] Compounds of Formula VI are preferably prepared by reacting
a compound of formula HO--Y.sub.1--(CH.sub.2).sub.q--Y.sub.2--Y3-Z
with a compound of formula X--P(R.sub.5).sub.2, preferably where
R.sub.5 is diisopropylamino, and X is halogen, preferably
chlorine.
[0177] Thus, the present invention also provides methods for the
preparation of nucleotide phosphoramidites of Formula II-comprising
providing a compound of formula: 25
[0178] and reacting the compound with a compound of Formula VI:
26
[0179] to produce the phosphordiamidite.
[0180] In further preferred embodiments, methods are provided for
the preparation of nucleotide phosphoramidites of Formula II
comprising providing a compound Formula: 27
[0181] and reacting the compound with a diaminohalophosphine of
Formula: 28
[0182] wherein X is halogen, preferably chlorine, to produce a
phosphordiamidite of Formula: 29
[0183] and contacting the nucleoside phosphordiamidite with a
reagent of Formula XX: 30
[0184] to produce the phosphoramidite.
[0185] As used herein, the term "contacting" means the placement
together of moieties, directly or indirectly, such that they become
physically associated with each other. Thus, "contacting" includes,
inter alia, placement together in a container.
[0186] In some preferred embodiments, the foregoing methods can be
performed in a single vessel; i.e., in a "one pot" system, without
isolation of the intermediate phosphordiamidite.
[0187] Preferably, the diaminohalophosphine of Formula
X--P(R.sub.5).sub.2 is prepared by reaction of PX.sub.3, where X is
preferably chlorine, with at least two equivalents of an amine
having the formula (R.sub.6).sub.2N, in which each of the R.sub.6
groups can be the same or different, and are preferably alkyl
having 1 to about 10 carbon atoms, more preferably 1 to 6 carbon
atoms, with 3 carbon atoms, with isopropyl groups, being especially
preferred.
[0188] In the compounds and methods of the present invention, M can
be a choral phosphorus linkage, for example a phosphorothioate. See
Stec, et al., in Methods in Molecular Biology Vol. 20: Protocols
for Oligonucleotides and Analogs, S. Agrawal, Ed., Humana Press,
Totowa, N.J. (1993), at Chapter 14. See also Stec, W. J. et al.,
Nucleic Acids Research, Vol. 19, No. 21, 5883-5888 (1991); and
European Patent Application EP 0 506 242 A1, each of which are
hereby incorporated by reference in their entirety.
[0189] The oligomeric products of the methods 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 and translation mechanisms. Thus, single cells, cellular
populations or organelles can also be included within the
definition of organisms that can be treated with therapeutic or
diagnostic oligonucleotides.
[0190] The present invention also provides novel methods for
.sup.14C labeling of synthetic oligonucleotides. In some preferred
embodiments, the methods of the invention include methods for the
preparation of mono- or polynucleotide phosphoramidites having
Formula I above wherein the nucleobase is radiolabeled, and is
preferably [4,6-di-.sup.14C]thymidine. Thus, in some preferred
embodiments, the radiolabeled nucleobase has the formula: 31
[0191] wherein * denotes a .sup.14C atom.
[0192] The radiolabeled nucleotide phosphoramidites prepared by the
methods of the invention can be used in the preparation of labeled
oligonucleotides according to the methods of the invention, or
according to standard oligonucleotide synthetic regimes.
S-Acylthioethyl ("SATE") 4,6-di-.sup.14C thymidine phosphoramidite
can also be used for the synthesis of normal oligonucleotides. For
example this amidite has been used to synthesize T.sub.12 oligomer
phosphorothioate having the .sup.14C labels at the 5' end (see
Example 24 below).
[0193] As will be recognized, the steps of the methods of the
present invention need not be performed any particular number of
times or in any particular sequence. Additional objects,
advantages, and novel features of this invention will become
apparent to those skilled in the art upon examination of the
following examples thereof, which are intended to be illustrative
and not intended to be limiting.
EXAMPLES
[0194] General Procedures
[0195] All reagents and solvents were purchased from Aldrich
Chemical Co. Flash chromatography was performed on silica gel
(Baker 40 .mu.m). Thin layer chromatography was performed on
Kieselgel 60 F-254 glass plates from E. Merck and compounds were
visualized with UV light and sulfuric acid-methanol spray followed
by charring. Solvent systems used for thin-layer chromatography and
flash chromatography were: A; ethyl acetate-hexanes 1:1. B; ethyl
acetate-hexanes-TEA 2:3:0.5. .sup.1H and .sup.31P spectra were
recorded using a Gemini 200 Varian spectrometer. All reactions were
performed under an argon atmosphere and solutions rotary evaporated
at 35-45.degree. C. in vacuo using a vacuum pump-vacuum controller
combination.
Example 1
[0196] 2'-Methoxyethyl-5'-O-(4,4'-dimethoxytrityl)-5-methyluridine
(S-pivaloyl-2-thioethyl) bis[N,N-diisopropylphosphoramidite]
(2)
[0197] To a stirring and cooled solution of
2'-methoxyethyl-5'-O-(4,4'-dim- ethoxytrityl)-5-methyluridine (1)
(log, 16 mmoles) and diisopropylethylamine (2.7 g, 21 mmoles) in
dry dichloromethane (200 ml) in an ice bath was added dropwise a
solution of N,N-(diisopropylamino)chl- orophosphine (5.2 g, 19
mmoles) in dry dichloromethane. The resulting mixture was stirred
at room temperature for 55 minutes. Tetrazole was added (0.6 g, 8.0
mmoles) and a solution of S-(2-hydroxyethyl)thiopivaloa- te (3.4 g,
21 mmoles) in dry dichloromethane was added dropwise in a periods
of 15 minutes. The reaction mixture was stirred for 20 hours at
room temperature. At the end of this time, the mixture was diluted
with dry CH.sub.2Cl.sub.2 (100 ml) and washed with NaHCO3 (80 ml)
and brine 3 times (100 ml) each, dried over MgSO.sub.4 and
evaporated to a solid yellow foam. Flash chromatography using 1:1
(Hexanes:EtOAc) and containing 0.5% triethylamine yielded 10.3 g
(70%) of 2. TLC (Hexanes:EtOAc 1:1) R.sub.f=0..sup.35. .sup.31P-NMR
(CD.sub.3CN): 150.31, 150.54
Example 2
[0198]
2'-Methoxyethyl-5'-O-(4,4'-dimethoxytrityl)-5-methyluridine(s-aceta-
te-2-thioethyl) bis[N,N-diisopropylphosphoramidite] (3)
[0199] A solution of (1) (log, 16 mmoles) and diisopropylethylamine
(2.7 g, 21 mmoles) in dry dichloromethane (200 ml) was cooled in an
ice bath and stirred for 15 minutes. A solution of
N,N-(diisopropylamino)chloropho- sphine (5.2 g, 19 mmoles) in dry
CH.sub.2Cl.sub.2 was added dropwise. The resulting mixture was
stirred at room temperature for 45 minutes. Tetrazole was added
(0.6 g, 8.0 mmoles) and a solution of freshly prepared
S-(2-hydroxyethyl)-thioacetate (2.6 g, 21 mmoles) in dry
CH.sub.2Cl.sub.2 was added in a period of 10 minutes. The reaction
mixture was further stirred for 18 hours at room temperature. At
the end of this time, the mixture was diluted with dry
CH.sub.2Cl.sub.2 (100 ml) and washed with NaHCO.sub.3 (60 ml) and
brine 3 times (80 ml) each and dried over MgSO.sub.4 and evaporated
to a solid light yellow foam. Purification by flash chromatography
using 1:1 (Hexanes:EtOAc) and containing 0.5% triethylamine yielded
8.4 g (60%) of (3). TLC (Hexanes:EtOAc 1:1) R.sub.f=0.27,
.sup.31P-NMR (CD.sub.3CN): 150.47, 150.63.
Example 3
[0200] 6-Benzoylamino-2'-deoxy-5'-O-dimethoxytrityl-adenosine-(S-pi
valoyl-2-thioethyl) bis[N,N-diisopropylphosphoramidite] (5).
[0201] To a cooled solution of
6-benzoylamino-2'-deoxy-5'-O-dimethyltrityl- -adenosine (4) (5 g,
7.3 mmoles) and diisopropylamine (1.22 g, 9.5 mmoles) in dry
dichloromethane (100 ml) stirred in an ice bath, was added a
solution of N,N-(diisopropylamino)chlorophosphine (2.33 g, 8.76
mmoles) dropwise in dry CH.sub.2Cl.sub.2. The resulting mixture was
stirred at room temperature for 45 minutes. A solution of
S-(2-hydroxyethyl) thiopivaloate (1.42 g, 8.76 mmoles) and
tetrazole (0.255 g, 3.65 mmoles) in dry CH.sub.2Cl.sub.2 was added
in a period of 10 minutes. The reaction mixture was stirred for 22
hours at room temperature. The mixture was diluted with
CH.sub.2Cl.sub.2 (50 ml) and washed with NaHCO.sub.3 (15 ml) and
brine (25 ml) dried over MgSO.sub.4, filtered and evaporated the
solvent to a light yellow foam. Purification by flash
chromatography using Hexanes:EtOAc (1:3) and containing 0.5%
triethyamine, yielded 5.2 g (70%) of 5. TLC (Hexanes:EtOAc 1:1)
R.sub.f0.31. .sup.31P-NMR (CD.sub.3CN): 148.97, 149.13.
Example 4
[0202] 6-Benzoylamino-2'-deoxy-5'-O-dimethyltrityl-cytidine-(S-piva
loyl-2-thioethyl) bis[N,N-diisopropylphosphoramidite] (7).
[0203] The title compound was prepared according to the
phosphitylation procedure used in the synthesis of 5. Yield of 7
was 70%. .sup.31P-NMR (CD.sub.3CN): 148.95, 149.25.
Example 5
[0204] 6-Benzoylamino-2'-deoxy-5'-O-dimethyltrityl-cytidine-(S-benz
oyl-2-thioethyl) bis[N,N-diisopropylphosphoramidite] (8).
[0205] The title compound was prepared according to the
phosphitylation procedure used in the synthesis of 5. Yield of 8
was 60%. .sup.31P-NMR (CD.sub.3CN):148.94, 149.40.
Example 6
[0206] 2-Isobutyryl-2'-deoxy-5'-O-dimethyltrityl-guanosine-(s-pival
oyl-2-thioethyl) bis[N,N-diisopropylphosphoramidite] (10).
[0207] The title compound was prepared according to the
phosphitylation procedure used in the synthesis of 5. The purity of
10 was 30% by .sup.31P-NMR.
Example 7
[0208] 6-Benzoylamino-2'-deoxy-5'-O-dimethoxytrityl-adenosine-(S-ac
etate-2-thioethyl) bis[N,N-diisopropylphosphoramidite] (11).
[0209] The title compound was prepared according to the
phosphitylation procedure used in the synthesis of 5.
Example 8
[0210] 6-Benzoylamino-2'-deoxy-5'-O-dimethoxytrityl-cytidine-(S-ace
tate-2-thioethyl) bis[N,N-diisopropylphosphoramidite] (12).
[0211] The title compound was prepared according to the
phosphitylation procedure used in the synthesis of 5.
Example 9
[0212] Radiolabeling of Synthetic Oligonucleotides with the Aid of
(S-Pivaloyl 2-Mercaptoethyl)
[4,6-Di-.sup.14c]-3'-O-[(5'-O-(4,4'-Dimethox- ytrityl)Thymidyl]
N,N-Diisopropylphosphoramidite (S-Pivaloyl 2-mercaptoethyl)
[4,6-di-.sup.14C]-3'-O-[(5'-0-(4,4'-dimethoxytrityl)thym- idyl]
N,N-diisopropylphosphoramidite (15).
[0213] Bis(N,N-diisopropylamino)phosphorochloridite (267 mg, 1
mmol) in CH.sub.2Cl.sub.2 (2.5 mL)) was added to a magnetically
stirred solution of S-pivaloyl 2-mercaptoethanol (162 mg, 1 mmol)
and ethyldiisopropylamine (142 mg, 1.1 mmol) in CH.sub.2Cl.sub.2 (1
mL for 5 minutes) at -30.degree. C. The mixture was allowed to warm
to room temperature and was stirred for 30 minutes to give 13. The
volume of solution was adjusted to 4.0 mL, an aliquot (320 .mu.L)
was taken and added to dry
[4,6-di-.sup.14C]-5'-O-(4,4'-dimethoxytrityl)thymidine 14 (21.7 mg,
40 (mol; specific activity 25 Ci mol .sup.1) Anhydrous 1H-tetrazole
(0.45 M in MeCN; 71 (L, 32 (mol) was added, and the mixture was
stirred for 40 minutes at room temperature. The reaction was
quenched with aqueous NaHCO.sub.3 (5%; 2 mL), diluted with
saturated NaCl (5 mL) and extracted with benzene (3.times.10 mL).
The extracts were dried over Na.sub.2SO.sub.4 and evaporated in
vacuo. The residue was dissolved in 50% aqueous MeCN and purified
by reversed phase HPLC on a DeltaPak 15 .mu.m C18 300 column
(7.8.times.300 mm). Isocratic elution with 50% aqueous MeCN for 10
min and with 75% aqueous MeCN for 25 minutes at a flow rate 5 mL
min.sup.-1 was applied. Fractions containing pure 15 (tR=25.5 min)
were collected, diluted with water (50 mL) and extracted with
benzene (5.times.10 mL). Extracts were dried over Na.sub.2SO.sub.4
and evaporated in vacuo to give S-pivaloyl 2-mercaptoethyl
[4,6-di-.sup.14C]-3'-O-(5'-O-(4,4'-dimethoxytrityl)thymidyl]
N,N-diisopropyl-phosphoramidite 15 (20.0 .mu.mol, 50.0.degree.;
specitic activity 21 Ci mol.sup.-1)
Example 10
[0214] Preparation of .sup.14C-Radiolabeled Oligonucleotides with
the aid of [4,6-di-.sup.14C]Thymidine Building Block (15)
[0215] A. Oligonucleotide Synthesis.
[0216] Non-radioactive 2-(pivaloylthio)ethyl- and
2-cyanoethyl-protected undecathymidylates 16 and 17 were assembled
on an ABI 380B DNA Synthesizer using either 2-cyanoethyl
3-(4,4'-dimethoxytrityloxy)-3-(2-ni- trophenyl) ethyl phosphate or
[[2-(4,4'-dimethoxytrityloxy)ethyl]sulfonyl]- ethyl succinyl
derivatized CPG (linkers A and B, correspondingly, FIG. 1,
phosphoramidite chemistry, and 3H-1,2-benzodithiol-3-one
1,1-dioxide (0.05 M in MeCN) as a sulfur-transfer reagent. For
preparation of 16, 5'-O-(4,4'-dimethoxytrityl) thymidyl
2-(pivaloylthio)ethyl N,N-diisopropylaminophosphite, 18, (FIG. 2)
was employed as a building block. Otherwise, commercial thymidine
CE phosphoramidite, 19, was used.
[0217] B. Radioactive Labeling.
[0218] The solid support-bound oligonucleotide 16 or 17 from
preceding step was placed into a 5 mL reaction funnel and dried on
an oil pump for 2 hours. To this, phosphoramidite 18 (8.9 mg, 10.7
(mol) in MeCN (54 (L) followed by 1H-tetrazole (22%, 315 (mol) in
MeCN (700 (L) was added, and the mixture was shaken for 3 minutes.
The liquid phase was filtered off, and phosphoramidite 15 (13.9 mg,
16.5 mmol, 1000 (Ci) in MeCN (100 AL) and 1H-tetrazole (5.0 mg) in
MeCN (160 .mu.L) were added. The coupling mixture was shaken for 15
minutes, the solution was filtered off, and
3H-1,2-benzodithiol-3-one 1,1-dioxide (0.05 M in MeCN; 4 mL) was
added. After gentle shaking for 5 minutes, the solution was
filtered off, and the solid support was washed with MeCN (5.times.5
mL) and briefly dried on an oil pump. The solid support was treated
with 18 (117 mg) in MeCN (700 AL) and 1H-tetrazole (22 mg) in MeCN
(700 (L) for 10 minutes, filtered, treated with
3H-1,2-benzodithiol-3-one 1,1-dioxide (0.05 M in MeCN; 4 mL) for 5
min, and extensively washed with MeCN. The product obtained by
labeling 16 was detritylated by treatment with dichloroacetic acid
in CH.sub.2Cl.sub.2 (3%, 15 mL), washed with MeCN (5.times.5 mL)
and dried on an oil pump to give support-bound oligonucleotide 20
(FIG. 3). Oligonucleotide 21 prepared by labeling 17 (FIG. 4) was
dried on an oil pump and used in 5'-DMT-protected form.
[0219] C. Releasing and Isolation of Oligonucleotide 22.
[0220] The solid support bound 20 (FIG. 3) was placed in Pyrex test
tubes (ca. 50 mg in each tube) and each portion was suspended in
80% aqueous MeCN (3 mL). The suspension was degassed, placed in
photochemical reactor, and irradiated for 30 minutes at room
temperature. The tube was centrifuged, and supernatant was
collected. A fresh portion of 80% aqueous MeCN was added. This
procedure was repeated for 5 times until less than 4 OD of
oligonucleotide material was released after irradiation for 30
minutes. The collected supernatants were diluted with water to get
a solution in 30% aqueous MeCN and filtered. Filtrates were applied
on an HPLC column (DeltaPak 15.mu. C18 300 .ANG., 3.9(300 mm; 60%
aq. MeCN as buffer A, MeCN as buffer B), and eluted in a linear
gradient from 10 to 100% B (30 minutes at a flow rate 5.0 mL
min.sup.-1). The main peak was collected and evaporated in vacuo to
afford 22 (659 OD, 32 mg, 100 (.mu.Ci). MALDI/TOF MS: found 5592.3;
calculated for C.sub.204H.sub.302N.sub.24O.sub.85P.sub.12S.sub.24
5592.0.
[0221] An aliquot of 22 (5 OD) was treated with concentrated
aqueous ammonia (2 mL) for 8 hours at room temperature, evaporated
to dryness, and re-dissolved in water (200 .mu.L). Analysis by
ion-exchange chromatography (Resource.TM. Q-1 mL column
(Pharmacia); 0.02 M aqueous Tris as buffer A, 5 M NaBr as buffer B;
a linear gradient from 0 to 30% B in 25 minutes) and capillary
electrophoresis (CE) revealed coelution with authentic sample of
dodecathymidyl thiophosphate.
[0222] D. Deprotection and Isolation of 24.
[0223] The solid support-bound 21 (FIG. 4) was treated with
concentrated ammonia (5 mL) for 2 hours at room temperature, the
solution was collected in a screw-cap vial and heated at 65.degree.
C. for 18 hours. After evaporation to dryness, the residue was
dissolved in water (3 mL) and filtered. The 5'-DMTr protected
oligonucleotide 23 was isolated by HPLC (DeltaPak 15.mu. C18 300
.ANG., 3.9(300 mm; 0.1 M NH.sub.4OAc as buffer A, 0.05 M
NH.sub.4OAc in 75% aqueous MeCN as buffer B; a linear gradient from
15 to 80% B in 30 minutes at a flow rate 5.0 mL min.sup.-1). The
collected fractions were evaporated, treated with 80% aqueous AcOH
for 20 min, and evaporated to dryness. The residue was desalted on
the same column eluting first with 0.1 M NaOAc (10 min), then with
water (10 min) and finally eluting 24 as a sodium salt with 50%
aqueous MeCN (20 min) at a flow rate 5.0 mL min.sup.-1. Evaporation
gave 634 OD (21.2 mg, 59.3 (Ci) of 24. Analysis by ion-exchange
chromatography (Resource.TM. Q-1 mL column (Pharmacia); 0.02 M
aqueous Tris as buffer A, 5 M NaBr as buffer B; a linear gradient
from 0 to 30% B in 25 min) and capillary electrophoresis (CE)
revealed coelution with authentic sample of dodecathymidyl
thiophosphate. MALDI/TOF MS: found 3861.19; calculated for
C.sub.120H.sub.158N.sub.24O.sub.73P.sub.12S.sub.12 3861.14.
Example 11
[0224] Dodeca[(2-pivaloylthio)ethyl
2'-O-(methoxyethyl)-5-methyluridyl phosphate] (25).
[0225] The title compound was assembled on an ABI 380B synthesizer
by using 0.1 M (2-pivaloylthio)ethyl
5'-O-(4,4'-dimethoxytrityl)-2'-O-(2-met- hoxyethyl)-5-methylur idil
N,N-diisopropylaminophosphite 2 in MeCN, 0.45 M 1H-tetrazole as an
activator, 0.05 M (1S)-(+)-(10-camphorsulfonyl) oxaziridine in MeCN
as an oxidizer, and 6 minute coupling time. Upon completion of the
chain assembly (DMTr-Off synthesis) the solid support was dried on
an oil pump, placed in a Pyrex test tube and suspended in 80%
aqueous MeCN (3 mL). The suspension was degassed, placed in
photochemical reactor, and irradiated for 30 minutes at room
temperature. The tube was centrifuged, and supernatant was
collected. A fresh portion of 80% aqueous MeCN was added. This
procedure was repeated for 5 times until less than 4 OD of
oligonucleotide material was released after irradiation for 30
minutes. The collected supernatants were diluted with water to
yield a solution in 30% aqueous MeCN, applied on an HPLC column
(DeltaPak 15.mu. C18 300 A, 3.9*300 mm), and chromatographed in a
linear gradient from 25 to 80% MeCN in water for 40 minutes. The
main peak was collected and evaporated in vacuo to afford the title
compound in 25% yield.
[0226] An aliquot (5 OD) of the obtained material was treated with
concentrated aqueous ammonia (2 mL) for 8 hours at room
temperature, evaporated to dryness, and re-dissolved in water (200
.mu.L). Analysis by capillary electrophoresis (CE) revealed
comigration with authentic sample of
dodeca[2'-O-(2-methoxyethyl)-5-methyluridyl phosphate]. ES MS:
found 4557.45, calculated for
C.sub.156H.sub.230N.sub.24O.sub.109P.sub.12 4557.26.
Example 12
[0227] Dodeca[(2-pivaloylthio)ethyl
2'-O-(2-methoxyethyl)-5-methyluridyl thiophosphate] (26).
[0228] The title compound was prepared as described above except
that 3H-1,2-benzodithiol-3-one 1,1-dioxide (0.05 M in MeCN) was
used in an oxidation step as a sulfur transfer reagent.
Chromatography on the same column in a linear gradient from 70 to
100% MeCN in water afforded the title compound in 15% yield. After
treatment with concentrated aqueous ammonia as above, analysis by
capillary electrophoresis (CE) revealed comigration with authentic
sample of dodeca[2'-O-(2-methoxyethyl)-5-methy- luridyl
thiophosphate]ES MS: found 4750.49, calculated for
C.sub.156H.sub.230N.sub.24O.sub.97P.sub.12S.sub.12 4750.10.
[0229] Analysis/Bioanalysis of SATE Oligomers
[0230] Mass analysis of SATE oligonucleotides was carried out using
a MALDI-TOF mass spectrometer. Reversed phase HPLC analysis was
also used to characterize the radiochemical purity of SATE
oligomers. Preliminary results indicated that the extent or SATE
removal can be analyzed using either reversed phase chromatography
or MALDI-TOF. An oligonucleotide containing from 12 to 0 SATE
groups was analyzed using a Zorbax C3 column using a buffer of 20
mM ammonium acetate. Elution was accomplished with an acetonitrile
gradient. Fractions were collected and subjected to MALDI analysis
confirming the mass identity of the peaks observed. Matrix
formulation was a 1:1 mixture of a saturated solution of
Tri-hydroxyacetophenone in 50/50 ACN/H.sub.2O, and 0.2 M
di-aminohydrogen citrate in 50/50 ACN/H.sub.2O. Sample (1 .mu.L)
was mixed with matrix (5 .mu.L) and this mixture (1 .mu.L) was used
for negative mode MALDI analysis.
[0231] Extraction methods for fully modified SATE oligomers and for
oligomers that have lost one or more SATE groups will be developed
for these analytical methods. These extraction methods will include
both solid phase and phenol chloroform extraction protocols.
Preliminary results indicate that a fully modified SATE oligomer
can be isolated in a 80%-90% yield from tissue homogenates.
[0232] Treatment of SATE oligomers with ammonium hydroxide results
in the loss of the SATE groups, leaving a phosphorothioate
internucleotide linkage. Using methods established in our
laboratory (See, Leeds, et al., Anal. Biochem. 1996, 235, 36;
Crooke, et al., J. Pharmacol. Exp. Ther. 1996, 277(2), 923),
analysis of these phosphorothioates will be accomplished using
either Capillary Gel Electrophoresis (CGE) with UV detection, or
using strong anion exchange chromatography with detection by UV or
by fraction collection and scintillation counting. Briefly,
oligonucleotides are isolated from homogenized tissue samples by
treatment with proteinase K, followed by phenol/chloroform
extraction, then solid phase extraction using anion exchange
purification followed by reversed phase desalting of the isolated
oligonucleotides and metabolites, followed by CGE analysis. For
anion exchange chromatography with scintillation counting, the
extracted oligonucleotides are analyzed after the phenol/chloroform
step. Both methods will allow discrimination between full-length
oligomers and metabolic degradation products.
[0233] It is intended that each of the patents, applications,
printed publications, and other published documents mentioned or
referred to in this specification be herein incorporated by
reference in their entirety.
[0234] Those skilled in the art will appreciate that numerous
changes and modifications may be made to the preferred embodiments
of the invention and that such changes and modifications may be
made without departing from the spirit of the invention. It is
therefore intended that the appended claims cover all such
equivalent variations as fall within the true spirit and scope of
the invention.
* * * * *