U.S. patent application number 10/828659 was filed with the patent office on 2004-10-07 for processes for the preparation of oligonucleotides.
This patent application is currently assigned to ISIS Pharmaceuticals, Inc.. Invention is credited to Sanghvi, Yogesh S., Song, Quanlai.
Application Number | 20040198972 10/828659 |
Document ID | / |
Family ID | 24567595 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040198972 |
Kind Code |
A1 |
Sanghvi, Yogesh S. ; et
al. |
October 7, 2004 |
Processes for the preparation of oligonucleotides
Abstract
The present invention discloses methods for synthesizing
oligomeric compounds. The methods include a modified
phosphoramidite protocol wherein the oxidation and capping steps
are combined into a single step. The methods result in increased
efficiency and are especially amenable to the large scale synthesis
of oligomeric compounds.
Inventors: |
Sanghvi, Yogesh S.;
(Encinitas, CA) ; Song, Quanlai; (San Marcos,
CA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Assignee: |
ISIS Pharmaceuticals, Inc.
|
Family ID: |
24567595 |
Appl. No.: |
10/828659 |
Filed: |
April 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10828659 |
Apr 21, 2004 |
|
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09640279 |
Aug 16, 2000 |
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Current U.S.
Class: |
536/25.34 |
Current CPC
Class: |
C07H 19/04 20130101;
C07H 21/00 20130101 |
Class at
Publication: |
536/025.34 |
International
Class: |
C07H 021/04; C07H
021/02 |
Claims
1. A method of preparing an oligomeric compound having at least one
moiety of formula: 56wherein: X.sub.2 is O or S; X.sub.1 is
Pg--O--, Pg--S--, C.sub.1-C.sub.10 straight or branched chain
alkyl, CH.sub.3(CH.sub.2).sub.n--O--, R.sub.2R.sub.3N-- or a group
remaining from coupling a chiral auxiliary; nn is from 0 to 10; Pg
is CH.sub.3, --CH.sub.2CH.sub.2CN,
--C(CH.sub.3)(CH.sub.3)--CCl.sub.3, --CH.sub.2--CCl.sub.3,
--CH.sub.2CH.dbd.CH.sub.2, CH.sub.2CH.sub.2SiCH.su- b.3, 2-yl-ethyl
phenylsulfonate, .delta.-cyanobutenyl, cyano p-xylyl,
diphenylsilylethyl, 4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl
sulfonate, methyl-N-trifluoroacetyl ethyl, acetoxy phenoxy ethyl,
or a blocking group; each R.sub.2 and R.sub.3 is, independently,
hydrogen, C.sub.1-C.sub.10 alkyl, cycloalkyl or aryl; or
optionally, R.sub.2 and R.sub.3, together with the nitrogen atom to
which they are attached form a cyclic moiety; each Bx is,
independently, a heterocyclic base moiety; and each R.sub.1 is,
independently, H, a blocked hydroxyl group, or a sugar substituent
group; comprising the steps of: (a) providing a 5'-O-protected
compound of the formula: 57wherein: T.sub.1 is a hydroxylprotecting
group; and T.sub.2 is a covalent attachment to a support media, a
nucleoside bound to a support media, a nucleotide, an
oligonucleoside or an oligonucleotide; (b) treating said
5'-O-protected compound with a deprotecting reagent for a time and
under conditions effective to form a 5'-O-deprotected compound; (c)
coupling said 5'-O-deprotected compound with an activated
phosphorus composition of the formula: 58wherein: T.sub.3 is a
hydroxylprotecting group, a nucleoside, a nucleotide, an
oligonucleoside or an oligonucleotide; R.sub.4 is
N(L.sub.1)L.sub.2; each L.sub.1 and L.sub.2 is, independently,
C.sub.1-6 straight or branched alkyl, or a C.sub.5-7 cyclic
aliphatic ring system; or L.sub.1 and L.sub.2 are joined together
to form a 4- to 13-membered heterocyclic ring system including the
nitrogen atom to which L.sub.1 and L.sub.2 are attached; and
R.sub.5 is X.sub.1; or R.sub.4 and R.sub.5 together with the
phosphorus atom to which R.sub.4 and R.sub.5 are attached form a
chiral auxiliary; for a time and under conditions effective to form
an extended compound having the formula: 59(d) treating said
extended compound with a mixture comprising an oxidizing reagent
and a capping reagent for a time and under conditions effective to
form said oligomeric compound.
2. The method of claim 1 further comprising treating said
oligomeric compound with a reagent for a time and under conditions
effective to remove said blocking groups thereby forming a
deblocked oligomeric compound.
3. The method of claim 2 wherein said reagent is effective to
cleave the oligomeric compound from the support media.
4. The method of claim 3 wherein said reagent is aqueous ammonium
hydroxide.
5. The method of claim 2 further comprising treating said
oligomeric compound with a further reagent for a time and under
conditions effective to cleave the oligomeric compound from the
support media.
6. The method of claim 1 further comprising treating said
oligomeric compound with a deprotecting reagent for a time and
under conditions effective to deprotect the T.sub.3
hydroxylprotecting group.
7. The method of claim 1 wherein said mixture comprises from 0.02M
to 0.2M oxidizing reagent.
8. The method of claim 7 wherein said mixture comprises from 0.1M
to 0.2M oxidizing reagent.
9. The method of claim 1 wherein said oxidizing reagent transfers
an oxygen atom.
10. The method of claim 9 wherein said oxidizing reagent is iodine,
m-chloroperbenzoic acid, iodobenzene diacetate,
tetra-n-butylammonium periodate, tert-butyl hydroperoxide,
di-tert-butyl hydroperoxide, cumene hydroperoxide, hydrogen
peroxide; bis-trimethylsilyl peroxide; dinitrogen tetroxide, oxone,
molecular oxygen, (1S)-(+)-(10-camphorsulfonyl)oxazirid- ine or a
peracid.
11. The method of claim 10 wherein said oxidizing reagent is
iodine, m-chloroperbenzoic acid, iodobenzene diacetate, tert-butyl
hydroperoxide, di-tert-butyl hydroperoxide, hydrogen peroxide,
oxone, molecular oxygen or a peracid.
12. The method of claim 1 wherein said oxidizing reagent transfers
a sulfur atom.
13. The method of claim 12 wherein said oxidizing reagent is
3-amino-1,2,4-dithiazole-5-thione;
3-ethoxy-1,2,4-dithiazoline-5-one; 1,2,4-dithiazolidine-3,5-dione;
3-methyl-1,2,4-dithiazolin-5-one; or dimethylthiuram disulfide.
14. The method of claim 13 wherein said oxidizing reagent is
dimethylthiuram disulfide.
15. The method of claim 1 wherein said capping reagent comprises
about one part by volume of either acetic anhydride in acetonitrile
or tetrahydrofuran; or chloroacetic anhydride in acetonitrile or
tetrahydrofuran; added to about one part by volume of either
N-methylimidazole and pyridine in acetonitrile or tetrahydrofuran;
or t-butylphenoxyacetic anhydride in acetonitrile or
tetrahydrofuran.
16. The method of claim 15 wherein said capping reagent comprises
about one part by volume of 20% acetic anhydride in acetonitrile
mixed with about one part by volume of a solution having 20%
N-methylimidazole, 30% pyridine and 50% acetonitrile.
17. The method of claim 1 wherein said mixture comprises
dimethylthiuram disulfide, acetic anhydride, acetonitrile, N-methyl
imidazole and pyridine.
18. The method of claim 1 wherein said mixture comprises from about
0.05M to 0.2M dimethylthiuram disulfide, about 10% acetic
anhydride, about 10% N-methyl imidazole and about 15% pyridine in a
suitable solvent.
19. The method of claim 18 wherein said solvent is acetonitrile,
toluene, ethyl acetate, tetrahydrofuran, dichloromethane,
dichloroethane, dioxane, dimethylacetamide and
dimethylformamide.
20. The method of claim 1 wherein said coupling of the
5'-O-deprotected compound with the activated phosphorus composition
is performed in the presence of an activating agent.
21-78. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to methods for
synthesizing oligonucleotides and analogs thereof. In one aspect of
the invention the methods combine oxidation and capping into a
single step to improve the efficiency of synthesis. The overall
synthesis preferably is completed in less time with a reduction in
bulk reagents required. More specific objectives and advantages of
the invention will hereinafter be made clear or become apparent to
those skilled in the art during the course of explanation of
preferred embodiments of the invention.
BACKGROUND OF THE INVENTION
[0002] Modified oligonucleotides are of great value in molecular
biological research and in applications such as anti-viral therapy.
Modified oligonucleotides which can block RNA translation, and are
nuclease resistant, are useful as antisense reagents. In addition
to oligonucleotides that have phosphodiester internucleotide
linkages, sulfurized oligonucleotides which contain, for example,
phosphorothioate linkages are also of interest in these areas.
Because of their chirality (Rp and Sp) phosphorothioate containing
oligonucleotides are useful in determining stereochemical pathways
of certain enzymes which recognize nucleic acids.
[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 function, contribute in major
proportion to many diseases and regulatory functions in animals and
humans. 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 otherwise 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, A., et. al., Science, 1990, 250, 997-1000; and Wu, H.,
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 to 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
oligomers 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 synthesis of oligonucleotides has classically involved
obtaining a desired product on a small scale. The synthesis of
oligonucleotides has more recently evolved to the point that
routine syntheses are being performed on kilogram scale. Moving
forward the next step is the synthesis of oligonucleotides and
analogs on ton scale to supply large quantities to meet demands for
ongoing pharmaceutical sales and clinical trials. The evolution of
oligonucleotide synthetic techniques toward larger scale is
requiring a reevaluation of each aspect of the synthetic process.
There is an ongoing need in the art of oligomer synthesis to
improve the efficiency of synthesis.
[0011] The chemical literature discloses numerous protocols for
coupling nucleosides through phosphorous-containing covalent
linkages to produce oligonucleotides of defined sequence. One of
the most routinely used protocols is the phosphoramidite protocol
(see, e.g., Advances in the Synthesis of Oligonucleotides by the
Phosphoramidite Approach, Beaucage, S. L.; Iyer, R. P.,
Tetrahedron, 1992, 48, 2223-2311 and references cited therein; and
The synthesis of Modified Oligonucleotides by the Phosphoramidite
Approach and their applications, Beaucage, S. L.; Iyer, R. P.,
Tetrahedron, 1993, 49, 6123-6194 and references cited therein),
wherein a nucleoside or oligonucleotide having a free hydroxyl
group is reacted with a protected phosphoramidite monomer in the
presence of a weak acid to form a phosphite-linked structure.
Oxidation of the phosphite linkage with a suitable reagent effects
conversion of a P.sup.III internucleoside linkage to a pV
internucleoside linkage. For the purpose of this application, such
reagents include oxygen transfer reagents and sulfur transfer
reagents. Subsequent hydrolysis of the cyanoethyl group yields the
desired phosphodiester or phosphorothioate linkage.
[0012] Phosphoramidites are commercially available from a variety
of commercial sources (included are: Glen Research, Sterling, Va.;
Amersham Pharmacia Biotech Inc., Piscataway, N.J.; Cruachem Inc.,
Aston, Pa.; Chemgenes Corporation, Waltham, Mass.; Proligo LLC,
Boulder, Colo.; PE Biosystems, Foster City Calif.; Beckman Coulter
Inc., Fullerton, Calif.).
[0013] An efficient sulfur transfer reagent is important to the
success of obtaining high quality phosphorothioate product with a
low percentage of phosphate linkages. A number of sulfur transfer
reagents have been reported, including Beaucage reagent,
3-ethoxy-1,2,4-dithiozloine-5-one (EDITH),
1,2,4-dithiozoline-3,5-dione, 3-methyl-ethoxy-1,2,4-dithiozloine-
-5-one (MEDITH), phenylacetyl disulfide (PADS), tetraethylthiuram
disulfide (TETD) and others. The cost of sulfur transfer reagents
and their efficiency has to be taken into account when performing
large-scale manufacturing. Beaucage reagent is expensive (about
$5.00 per gram) and is being replaced by other cheaper reagents
like PADS. PADS was first introduced by van Boom but did not show
adequate sulfur transfer efficiency under the original conditions,
which did not use a base during the sulfurization step (Roclen et
al., Recl. Trav. Chim. Pays-Bas, 1991, 110, 325-331).
[0014] The use of elemental sulfur in olignucleotide synthesis
presents problems and is not suitable for automation because of
sulfur's insolubility in most organic solvents. Furthermore, carbon
disulfide, a preferred source of sulfur, has undesirable volatility
and an undesirably low flash point. Unwanted side products are
often observed with the use of dibenzoyl tetrasulfide. Beaucage
reagent, while a relatively efficient sulfurization reagent, is
difficult to synthesize and not particularly stable. Furthermore,
use of Beaucage reagent forms a secondary reaction product which is
a potent oxidizing agent. (R. P. Iyer et al., J. Am. Chem. Soc.
112, pp. 1253-1254 (1990); R. P. Iyer et al., J. Org. Chem. 55,
4693-4699 (1990)). This can further lead to unwanted side products
which can be difficult to separate from the desired reaction
product. Tetraethylthiuram disulfide while relatively inexpensive
and stable, has a sulfurization reaction rate which can be
undesirable slow.
[0015] A method of preparing phosphorothioate oligonucleotides
using tetraethylthiuram disulfide is disclosed in U.S. Pat. No.
5,166,387. Although the use of tetraethylthiuram disulfide as a
sulfur transfer reagent has been described since 1992, it has not
been used for commercial scale production of phosphorothioate
oligonucleotides. Beaucage reagent and phenylacetyl disulfide
(PADS) are the only reagents that have been used commercially with
good results. Our experiments with tetraethylthiuram disulfide
indicate that there is a significant amount of side product
formation (see Example 8) along with the desired phosphorothioate
product. Although we do not wish to be bound by theory it is
believed that tetraethylthiuram disulfide is an overly stable
sulfur transfer reagent that is difficult to dissociate after
reaction with the p.sup.III species.
[0016] Thus, there remains a need for improved methods and reagents
for preparing oligomeric compounds. The present invention is
directed to these, as well as other, important ends.
SUMMARY OF THE INVENTION The present invention provides methods of
preparing oligomeric compounds having at least one moiety of
formula:
[0017] 1
[0018] wherein:
[0019] X.sub.2 is O or S;
[0020] X.sub.1 is Pg--O--, Pg--S--, C.sub.1-C.sub.10 straight or
branched chain alkyl, CH.sub.3(CH.sub.2).sub.nn--O--,
R.sub.2R.sub.3N-- or a group remaining from coupling a chiral
auxiliary;
[0021] nn is from 0 to 10;
[0022] Pg is CH.sub.3, --CH.sub.2CH.sub.2CN,
--C(CH.sub.3)(CH.sub.3)--CCl.- sub.3, --CH.sub.2--CCl.sub.3,
--CH.sub.2CH.dbd.CH.sub.2, CH.sub.2CH.sub.2SiCH.sub.3, 2-yl-ethyl
phenylsulfonate, .delta.-cyano-butenyl, cyano p-xylyl,
diphenylsilylethyl, 4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl
sulfonate, methyl-N-trifluoroacetyl ethyl, acetoxy phenoxy ethyl,
or a blocking group;
[0023] each R.sub.2 and R.sub.3 is, independently, hydrogen,
C.sub.1-C.sub.10 alkyl, cycloalkyl or aryl;
[0024] or optionally, R.sub.2 and R.sub.3, together with the
nitrogen atom to which they are attached form a cyclic moiety that
may include an additional heteroatom selected from O, S and N;
[0025] each Bx is, independently, a heterocyclic base moiety;
and
[0026] each R.sub.1 is, independently, H, a blocked hydroxyl group,
or a sugar substituent group; comprising the steps of:
[0027] (a) providing a 5'-O-protected compound of the formula:
2
[0028] wherein:
[0029] T.sub.1 is a hydroxylprotecting group; and
[0030] T.sub.2 is a covalent attachment to a support media, a
nucleoside bound to a support media, a nucleotide, an
oligonucleoside or an oligonucleotide;
[0031] (b) treating said 5'-O-protected compound with a
deprotecting reagent for a time and under conditions effective to
form a 5'-O-deprotected compound;
[0032] (c) coupling said 5'-O-deprotected compound with an
activated phosphorus composition of the formula: 3
[0033] wherein:
[0034] T.sub.3 is a hydroxylprotecting group, a nucleoside, a
nucleotide, an oligonucleoside or an oligonucleotide;
[0035] R.sub.4 is N(L.sub.1)L.sub.2;
[0036] each L.sub.1 and L.sub.2 is, independently, C.sub.1-6
straight or branched alkyl, or a C.sub.5-7 cyclic aliphatic ring
system;
[0037] or L.sub.1 and L.sub.2 are joined together to form a 4- to
13-membered heterocyclic ring system including the nitrogen atom to
which L.sub.1 and L.sub.2 are attached, wherein said ring system
optionally includes at least one additional heteroatom selected
from O, N and S; and
[0038] R.sub.5 is X.sub.1;
[0039] or R.sub.4 and R.sub.5 together with the phosphorus atom to
which R.sub.4 and R.sub.5 are attached form a chiral auxiliary;
[0040] for a time and under conditions effective to form an
extended compound having the formula: 4
[0041] (d) treating said extended compound with a mixture
comprising an oxidizing reagent and a capping reagent for a time
and under conditions effective to form said oligomeric
compound.
[0042] The methods can include further treatment of the oligomeric
compound with a reagent for a time and under conditions effective
to remove the blocking groups, thereby forming a deblocked
oligomeric compound. This reagent can also be effective to cleave
the oligomeric compound from the support media. One such reagent is
aqueous ammonium hydroxide. Alternatively, the deblocking can be
effected with one reagent, and a further reagent can effect
cleavage from the support media. Also included is treatment with a
deprotecting reagent for a time and under conditions effective to
deprotect the T.sub.3 hydroxylprotecting group.
[0043] In one embodiment of the invention, the mixture comprises
from 0.02M to 0.2M oxidizing reagent with a preferred range is from
0.1M to 0.2M.
[0044] The oxidizing reagent can be one that transfers an oxygen
atom. Effective oxidizing reagents for oxygen transfer are iodine,
m-chloroperbenzoic acid, iodobenzene diacetate,
tetra-n-butylammonium periodate, tert-butyl hydroperoxide,
di-tert-butyl hydroperoxide, cumene hydroperoxide, hydrogen
peroxide; bis-trimethylsilyl peroxide; dinitrogen tetroxide, oxone,
molecular oxygen, (1S)-(+)-(10-camphorsulfonyl)oxazirid- ine or a
peracid with iodine, m-chloroperbenzoic acid, iodobenzene
diacetate, tert-butyl hydroperoxide, di-tert-butyl hydroperoxide,
hydrogen peroxide; oxone, molecular oxygen or a peracid being
preferred.
[0045] The oxidizing reagent can also be one that transfers a
sulfur atom. Effective oxidizing reagents for sulfur transfer are
3-amino-1,2,4-dithiazole-5-thione;
3-ethoxy-1,2,4-dithiazoline-5-one; 1,2,4-dithiazolidine-3,5-dione;
3-methyl-1,2,4-dithiazolin-5-one; or dimethylthiuram disulfide with
dimethylthiuram disulfide being preferred.
[0046] In one embodiment, the capping reagent is prepared by mixing
equal volumes of two components prior to the capping step. The
first component is a mixture of an acylating agent such as
N-methylimidazole or 4-dimethylaminopyridine and a base such as
pyridine or 2,6-lutidine in acetonitrile or tetrahydrofuran. The
second component is a solution of an acid anhydride such as acetic
anhydride, chloroacetic anhydride, t-butylphenoxyacetic anhydride
in acetonitrile or tetrahydrofuran.
[0047] One preferred capping reagent comprises about equal volumes
of a first component containing from 5% to about 25%
N-methylimidazole, from about 20% to about 50% pyridine and from
about 20% to about 50% acetonitrile, added to a second component
containing from about 20% to about 50% acetic anhydride in
acetonitrile.
[0048] Another preferred capping reagent comprises about equal
volumes of a first component containing about 5% to about 25%
4-dimethylaminopyridine, from about 20% to about 50% 2,6-lutidine
in acetonitrile, added to a second component containing from about
20% to about 50% acetic anhydride in acetonitrile.
[0049] The mixture of oxidizing reagent and capping reagent can
comprise, for example, dimethylthiuram disulfide, acetic anhydride,
acetonitrile, N-methyl imidazole, or pyridine. A preferred mixture
comprises from about 0.05M to 0.2M dimethylthiuram disulfide, about
10% acetic anhydride, about 10% N-methyl imidazole and about 15%
pyridine in a suitable solvent. Suitable solvents include
acetonitrile, toluene, ethyl acetate, tetrahydrofuran,
dichloromethane, dichloroethane, dioxane, dimethylacetamide and
dimethyl-formamide.
[0050] In another embodiment of the invention the coupling of the
5'-O-deprotected compound with the activated phosphorus composition
is performed in the presence of an activating agent that renders
the phosphorous atom more susceptible to nucleophilic attack.
Preferred activating agents include 1-H-tetrazole and
4,5-dicyanoimidazole.
[0051] Exemplary cyclic moieties according to the invention include
morpholino or phthalimido moieties.
[0052] Each L.sub.1 and L.sub.2 can be, independently, C.sub.1-6
alkyl with isopropyl being a preferred alkyl group for L.sub.1 and
L.sub.2. L.sub.1 and L.sub.2 can also be joined together to form a
heterocyclic ring system including the nitrogen atom to which said
L.sub.1 and L.sub.2 are attached, the ring system optionally
includes at least one additional heteroatom selected from O, N and
S. A preferred heterocyclic ring system is morpholino.
[0053] Representative substituent groups according to the invention
include, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl,
C.sub.2-C.sub.20 alkynyl, C.sub.5-C.sub.20 aryl, O-alkyl,
O-alkenyl, O-alkynyl, O-aryl, O-aralkyl, O-alkylamino,
O-alkylaminoalkyl (O-alkyl-N(H)alkyl), O-alkylaminodialkyl
(O-alkyl-N-(alkyl).sub.2), O-alkylalkoxy (O-alkyl-O-alkyl),
O-alkyl-(N-imidazole), thiol, S-alkyl, S-alkenyl, S-alkynyl,
NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, S-aryl, NH-aryl,
S-aralkyl, NH-aralkyl, N-phthalimido, halogen keto, carboxyl,
nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy,
N-imidazole, azido, hydrazino, hydroxylamino, isocyanato,
sulfoxide, sulfone, sulfide, disulfide, silyl, heterocycle,
carbocycle, polyamine, polyamide, polyalkylene glycol, or
polyether;
[0054] Alternatively, one or more substituent groups has one of
formula I or II: 5
[0055] wherein:
[0056] Z.sub.0 is O, S or NH;
[0057] J is a single bond, O or C(.dbd.O);
[0058] E is C.sub.1-C.sub.10 alkyl, N(R.sub.1)(R.sub.2),
N(R.sub.1)(R.sub.5), N.dbd.C(R.sub.1)(R.sub.2),
N.dbd.C(R.sub.1)(R.sub.5) or has one of formula III or IV; 6
[0059] each R.sub.6, R.sub.7, R.sub.8, R.sub.9 and R.sub.10 is,
independently, hydrogen, C(O)R.sub.11, substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical
functional group or a conjugate group, wherein the substituent
groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,
phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and
alkynyl;
[0060] or optionally, R.sub.7 and R.sub.8, together form a
phthalimido moiety with the nitrogen atom to which they are
attached;
[0061] or optionally, R.sub.9 and R.sub.10, together form a
phthalimido moiety with the nitrogen atom to which they are
attached;
[0062] each R.sub.11 is, independently, substituted or
unsubstituted C.sub.1-C.sub.10 alkyl, trifluoromethyl,
cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy,
9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy,
2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or
aryl;
[0063] R.sub.5 is T-L,
[0064] T is a bond or a linking moiety;
[0065] L is a chemical functional group, a conjugate group or a
support media;
[0066] each R.sub.1 and R.sub.2 is, independently, H, a nitrogen
protecting group, substituted or unsubstituted C.sub.1-C.sub.10
alkyl, substituted or unsubstituted C.sub.2-C.sub.10 alkenyl,
substituted or unsubstituted C.sub.2-C.sub.10 alkynyl, wherein said
substitution is OR.sub.3, SR.sub.3, NH.sub.3.sup.+,
N(R.sub.3)(R.sub.4), guanidino or acyl where said acyl is an acid
amide or an ester;
[0067] or R.sub.1 and R.sub.2, together, are a nitrogen protecting
group or are joined in a ring structure that optionally includes an
additional heteroatom selected from N and 0;
[0068] or R.sub.1, T and L, together, are a chemical functional
group;
[0069] each R.sub.3 and R.sub.4 is, independently, H,
C.sub.1-C.sub.10 alkyl, a nitrogen protecting group, or R.sub.3 and
R.sub.4, together, are a nitrogen protecting group;
[0070] or R.sub.3 and R.sub.4 are joined in a ring structure that
optionally includes an additional heteroatom selected from N and
O;
[0071] Z.sub.4 is OX, SX, or N(X).sub.2;
[0072] each X is, independently, H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 haloalkyl, C(.dbd.NH)N(H)R.sub.5,
C(.dbd.O)N(H)R.sub.5 or OC(.dbd.O)N(H)R.sub.5;
[0073] R.sub.5 is H or C.sub.1-C.sub.8 alkyl;
[0074] Z.sub.1, Z.sub.2 and Z.sub.3 comprise a ring system having
from about 4 to about 7 carbon atoms or having from about 3 to
about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero
atoms are selected from oxygen, nitrogen and sulfur and wherein
said ring system is aliphatic, unsaturated aliphatic, aromatic, or
saturated or unsaturated heterocyclic;
[0075] Z.sub.5 is alkyl or haloalkyl having 1 to about 10 carbon
atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2
to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms,
N(R.sub.1)(R.sub.2) OR.sub.1, halo, SR.sub.1 or CN;
[0076] each q.sub.1 is, independently, an integer from 1 to 10;
[0077] each q.sub.2 is, independently, 0 or 1;
[0078] q.sub.3 is 0 or an integer from 1 to 10;
[0079] q.sub.4 is an integer from 1 to 10;
[0080] q.sub.5 is from 0, 1 or 2; and provided that when q.sub.3 is
0, q.sub.4 is greater than 1.
[0081] In one embodiment of the present invention X.sub.1 is
Pg-O--, Pg--S--, --CH.sub.3, CH.sub.3--O--, morpholino or
--NR.sub.2R.sub.3 where each R.sub.2 and R.sub.3 is, independently,
hydrogen or C.sub.1-C.sub.10alkyl. Where Pg is CH.sub.2CH.sub.2CN,
diphenylsilylethyl, .delta.-cyanobutenyl, cyano p-xylyl,
methyl-N-trifluoroacetyl ethyl or acetoxy phenoxy ethyl.
[0082] Representative heterocyclic bases include adenine,
N.sup.6-benzoyladenine, cytosine, N.sup.4-benzoylcytosine,
5-methylcytosine, N.sup.4-benzoyl-5-methylcytosine, thymine,
uracil, guanine, N.sup.2-isobutyrylguanine and 2-aminoadenine, as
well as protected (or "blocked") forms thereof.
[0083] In certain embodiments of the present invention, the support
media bound nucleoside, nucleotide, oligonucleoside or
oligonucleotide is blocked at reactive sites. In one embodiment the
blocking groups are acid stable. In another embodiment the blocking
groups are base labile.
[0084] Deprotecting reagents can be acidic, neutral or basic.
Preferred deprotecting reagents are dichloroacetic acid,
trichloroacetic acid, zinc bromide, AlCl.sub.3, TiCl.sub.4,
(Et)AlCl, (1-Bu).sub.2AlCl, ceric ammonium nitrate,
1,1,1,3,3,3-hexafluoro-2-propanol, and diethyloxomalonate. A
particularly preferred deprotecting reagent is 2-5% dichloroacetic
acid in dichloromethane or dichloroethane. In a further embodiment
the deprotecting reagent is a fluoride moiety where a preferred
fluoride moiety is BF.sub.3-etherate.
[0085] The oligomeric compounds of the invention typically
comprises from 5 to about 50 nucleosides, with from 8 to about 30
nucleosides being preferred and from 15 to about 25 nucleosides
being more preferred.
[0086] The present invention also provides methods for the
preparation of oligomeric compounds having at least one moiety of
formula: 7
[0087] wherein:
[0088] X.sub.1 is Pg--O--, Pg--S--, C.sub.1-C.sub.10 straight or
branched chain alkyl, CH.sub.3(CH.sub.2).sub.nn--O--,
R.sub.2R.sub.3N-- or a group remaining from coupling a chiral
auxiliary;
[0089] nn is from 0 to 10;
[0090] Pg is CH.sub.3, --CH.sub.2CH.sub.2CN,
--C(CH.sub.3)(CH.sub.3)--CCl.- sub.31--CH.sub.2--CCl.sub.3,
--CH.sub.2CH.dbd.CH.sub.2, CH.sub.2CH.sub.2SiCH.sub.3, 2-yl-ethyl
phenylsulfonate, .delta.-cyanobutenyl, cyano p-xylyl,
diphenylsilylethyl, 4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl
sulfonate, methyl-N-trifluoroacetyl ethyl, acetoxy phenoxy ethyl,
or a blocking group;
[0091] each R.sub.2 and R.sub.3 is, independently, hydrogen,
C.sub.1-C.sub.10 alkyl, cycloalkyl or aryl;
[0092] or optionally, R.sub.2 and R.sub.3, together with the
nitrogen atom to which they are attached form a cyclic moiety that
may include an additional heteroatom selected from O, S and N;
[0093] each Bx is, independently, a heterocyclic base moiety or a
blocked heterocyclic base moiety; and
[0094] each R.sub.1 is, independently, H, a blocked hydroxyl group,
a sugar substituent group or a blocked sugar substituent group;
[0095] comprising the steps of:
[0096] (a) providing a 5'-O-protected compound of the formula:
8
[0097] wherein:
[0098] T.sub.1 is a hydroxylprotecting group; and
[0099] T.sub.2 is a covalent attachment to a support media, or a
support media bound nucleoside, nucleotide, oligonucleoside or
oligonucleotide;
[0100] (b) treating said 5'-O-protected compound with a
deprotecting reagent for a time and under conditions effective to
form a 5'-O-deprotected compound;
[0101] (c) coupling said 5'-O-deprotected compound with an
activated phosphorus composition of the formula: 9
[0102] wherein:
[0103] T.sub.3 is a hydroxylprotecting group, a nucleoside, a
nucleotide, an oligonucleoside or an oligonucleotide;
[0104] R.sub.4 is N(L.sub.1)L.sub.2;
[0105] each L.sub.1 and L.sub.2 is, independently, C.sub.1-6
straight or branched alkyl, or a C.sub.5-7 cyclic aliphatic ring
system;
[0106] or L.sub.1 and L.sub.2 are joined together to form a 4- to
13-membered heterocyclic ring system including the nitrogen atom to
which L.sub.1 and L.sub.2 are attached, wherein said ring system
optionally includes at least one additional heteroatom selected
from O, N and S; and
[0107] R.sub.5 is X.sub.1;
[0108] or R.sub.4 and R.sub.5 together with the phosphorus atom to
which R.sub.4 and R.sub.5 are attached form a chiral auxiliary;
[0109] for a time and under conditions effective to form an
extended compound having the formula: 10
[0110] (d) treating said extended compound with dimethylthiuram
disulfide in a solvent thereby forming a sulfurized compound having
the formula: 11
[0111] (e) treating said sulfurized compound with a capping reagent
for a time and under conditions effective to form said oligomeric
compound.
[0112] In one embodiment the dimethylthiuram disulfide is from
about 0.02M to about 0.2M in said solvent. In a preferred
embodiment the dimethylthiuram disulfide is from about 0.1M to
about 0.2M in said solvent.
[0113] The present invention also provides methods of preparing
oligomeric compounds having at least one moiety having one of the
formulas: 12
[0114] wherein:
[0115] X.sub.2 is O or S;
[0116] X.sub.1 is Pg--O--, Pg--S--, C.sub.1-C.sub.10 straight or
branched chain alkyl, CH.sub.3(CH.sub.2).sub.nn--O--,
R.sub.2R.sub.3N-- or a group remaining from coupling a chiral
auxiliary;
[0117] nn is from 0 to 10;
[0118] Pg is CH.sub.3, --CH.sub.2CH.sub.2CN,
--C(CH.sub.3)(CH.sub.3)--CCl.- sub.3, --CH.sub.2--CCl.sub.3,
--CH.sub.2CH.dbd.CH.sub.2, CH.sub.2CH.sub.2SiCH.sub.3, 2-yl-ethyl
phenylsulfonate, b-cyano-butenyl, cyano p-xylyl,
diphenylsilylethyl, 4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl
sulfonate, methyl-N-tri-fluoroacetyl ethyl, acetoxy phenoxy ethyl,
or a blocking group;
[0119] each R.sub.2 and R.sub.3 is, independently, hydrogen,
C.sub.1-C.sub.10 alkyl, cycloalkyl or aryl;
[0120] or optionally, R.sub.2 and R.sub.3, together with the
nitrogen atom to which they are attached form a cyclic moiety that
may include an additional heteroatom selected from O, S and N;
[0121] each Bx is, independently, a heterocyclic base moiety or a
blocked heterocyclic base moiety; and
[0122] each R.sub.1 is, independently, H, a blocked hydroxyl group,
a sugar substituent group or a blocked sugar substituent group;
[0123] comprising the steps of:
[0124] (a) providing a 5'-O-protected compound having one of the
formulas: 13
[0125] wherein:
[0126] T.sub.1 is a hydroxylprotecting group; and
[0127] T.sub.2 is a covalent attachment to a support media, or a
support media bound nucleoside, nucleotide, oligonucleoside or
oligonucleotide;
[0128] (b) treating said 5'-O-protected compound with a
deprotecting reagent for a time and under conditions effective to
form a 5'-O-deprotected compound;
[0129] (c) coupling said 5'-O-deprotected compound with an
activated phosphorus composition of the formula: 14
[0130] wherein:
[0131] T.sub.3 is a hydroxylprotecting group, a nucleoside, a
nucleotide, an oligonucleoside or an oligonucleotide;
[0132] R.sub.4 is N(L.sub.1)L.sub.2;
[0133] each L.sub.1 and L.sub.2 is, independently, C.sub.1-6
straight or branched alkyl, or a C.sub.5-7 cyclic aliphatic ring
system;
[0134] or L.sub.1 and L.sub.2 are joined together to form a 4- to
13-membered heterocyclic ring system including the nitrogen atom to
which L.sub.1 and L.sub.2 are attached, wherein said ring system
optionally includes at least one additional heteroatom selected
from O, N and S; and
[0135] R.sub.5 is X.sub.1;
[0136] or R.sub.4 and R.sub.5 together with the phosphorus atom to
which R.sub.4 and R.sub.5 are attached form a chiral auxiliary;
[0137] for a time and under conditions effective to form an
extended compound having one of the formulas: 15
[0138] and
[0139] (d) treating said extended compound with a mixture
comprising an oxidizing reagent and a capping reagent for a time
and under conditions effective to form said oligomeric
compound.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0140] The present invention is directed to methods for
synthesizing oligonucleotides and analogs thereof having at least
one moiety of one of the formulas: 16
[0141] wherein:
[0142] X.sub.2 is O or S;
[0143] X.sub.1 is Pg--O--, Pg--S--, C.sub.1-C.sub.10 straight or
branched chain alkyl, CH.sub.3(CH.sub.2).sub.nn--O--,
R.sub.2R.sub.3N-- or a group remaining from coupling a chiral
auxiliary;
[0144] nn is from 0 to 10;
[0145] Pg is CH.sub.3, --CH.sub.2CH.sub.2CN,
--C(CH.sub.3)(CH.sub.3)--CCl.-
sub.31--CH.sub.2--CCl.sub.31--CH.sub.2CH.dbd.CH.sub.2
CH.sub.2CH.sub.2SiCH.sub.3, 2-yl-ethyl phenylsulfonate,
5-cyanobutenyl, cyano p-xylyl, diphenylsilylethyl,
4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl sulfonate,
methyl-N-trifluoroacetyl ethyl, acetoxy phenoxy ethyl, or a
blocking group;
[0146] each R.sub.2 and R.sub.3 is, independently, hydrogen,
C.sub.1-C.sub.10 alkyl, cycloalkyl or aryl;
[0147] or optionally, R.sub.2 and R.sub.3, together with the
nitrogen atom to which they are attached form a cyclic moiety that
may include an additional heteroatom selected from O, S and N;
[0148] each Bx is, independently, a heterocyclic base moiety or a
blocked heterocyclic base moiety; and
[0149] each R.sub.1 is, independently, H, a blocked hydroxyl group,
a sugar substituent group, or a blocked substituent group;
[0150] The present methods can further include deblocking,
deprotecting and cleaving the resulting oligomeric compound. The
most common deblocked forms of X.sub.1 include --OH, --SH,
--N(R.sub.2)(R.sub.3), alkyl and alkoxy groups. Purification can be
performed at various stages, but is routinely performed with the
terminal protecting group attached such as a trityl on purification
including protected or deprotected.
[0151] The most widely used method for the large scale synthesis of
oligomeric compounds uses phosphoramidite chemistry on a support
media. In general the method requires 4 separate and distinct steps
per cycle: detritylation, coupling, oxidation and capping. Each
step requires automated synthesis equipment time and significant
quantities of reagents. In one aspect of the present invention, a
method is disclosed that employs a mixture comprising an oxidizing
reagent and a capping reagent to simultaneously effect oxidation of
internucleoside linkages and capping of unreacted hydroxyl groups.
This combined oxidation and capping step is amenable to otherwise
standard synthetic methods for the synthesis of oligomeric
compounds.
[0152] In general, a synthon bound to a support media, e.g. a
nucleoside or nucleosidic oligomer, is extended by addition of a
further synthon using standard chemistries to form a phosphite or
other P.sup.III intermediate linkage. Treatment of this P.sup.III
intermediate linkage in a single step with a mixture containing an
oxidizing reagent and a capping reagent will give an extended
compound having the desired P.sup.V oxidized linkage with unreacted
hydroxyl groups capped. Advantages of the present methods can
include a faster synthesis time and a reduction in cost due in part
to the deletion of one of the reagent consuming steps and
associated wash and rinse cycles. A further advantage can be gained
by selecting an inexpensive oxidizing reagent or one that is easily
prepared from inexpensive reagents.
[0153] Preferred mixtures of oxidizing and capping reagents include
those that are stable and include components that are mutually
soluble with each other. Oxidizing reagents amenable to the present
invention should be soluble in the solution comprising the
oxidizing reagent at concentrations of 0.02M, or greater, preferred
about 0.1M or more, more preferred from 0.1 to 0.2M. A preferred
mixture is an oxidizing reagent dissolved in cap A (20% acetic
anhydride in acetonitrile) mixed with an equal volume of cap B
(N-methylimidazole-pyridine-acetonitrile, 2:3:5, v/v/v). Oxidizing
reagents that are soluble and stable with an equal volume mixture
of cap A and cap B are preferred within the scope of the present
invention.
[0154] Oxidizing reagents that are effective to transfer an oxygen
atom (thereby converting a P.sup.III linkage to a P.sup.V linkage)
include without limitation m-chloroperbenzoic acid; iodobenzene
diacetate, tetra-n-butylammonium periodate; tert-butyl
hydroperoxide; di-tert-butyl hydroperoxide; cumene hydroperoxide;
hydrogen peroxide; bis-trimethylsilyl peroxide; and catalytic
amounts of trimethylsilyl triflate; dinitrogen tetroxide and
molecular oxygen in the presence of
2,2'-azobis(2-methylpropionitrile) under thermal or photo-chemical
conditions; and (1S)-(+)-(10-camphorsulfonyl)-oxaziridine;
iodine/tetrahydrofuran/water/pyridine; hydrogen peroxide/water;
tert-butyl hydroperoxide; and a peracid like m-chloroperbenzoic
acid (see review article Beaucage et al., Current Protocols in
Nucleic Acid Chemistry, 2000, 3.3.1-3.3.20). In the case of
oxidation to a sulfur species (sulfurization), the reaction is
generally performed under anhydrous conditions with an exclusion of
air, e.g. oxygen. In the case of oxidation the reaction can be
performed under aqueous conditions.
[0155] Oxidizing reagents that transfer a sulfur atom (sulfurizing
reagents) are used to form phosphorothioate or other sulfurized
internucleoside linkages such as, for example, phosphorodithioate
internucleoside linkages. Sulfurizing reagents amenable to the
present invention include those that are partially or completely
soluble in a selected capping reagent or reagents. In addition the
sulfurizing reagent should be compatible e.g. stable and
non-reactive with the capping reagents. Preferred sulfurizing
reagents are commercially available in bulk for considerably less
cost than most traditional sulfurizing agents that are currently in
use. Alternatively, a sulfurizing reagent is selected because of
its ease of synthesis from inexpensive bulk reagents.
[0156] One important criteria for a preferred sulfurizing reagent
is its ability incorporate sulfur and exclude incorporation of
oxygen. Analysis of an oxidized oligomer, using .sup.31P NMR, will
give the percentages of sulfurized and oxygenized internucleoside
linkages.
[0157] Preferred sulfurized linkages include those that are
prepared by methods known in the art to give chirally enhanced or
chirally pure sulfurized linkages for those linkages that are not
achiral. Preferred sulfurized linkages that are prepared by the
present methods include:
[0158] phosphorothioate (--O--P(S)(O)--O--);
[0159] phosphorodithioate (--O--P(S)(S)--O--);
[0160] phosphorthioamidate (--O--P(S)(NJ)-O--);
[0161] phosphonothioate (--O--P(J)(S)--O--);
[0162] boranothiophosphate (--O--P(S)(BJ.sub.3)-J-);
[0163] wherein "J" denotes a substituent group which is commonly
hydrogen or an alkyl group, but which can be a more complicated
group that varies from one type of linkage to another but is well
known to the art skilled.
[0164] Representative United States Patents that teach the
preparation of the above phosphorus atom containing linkages
include, but are not limited to, U.S. Pat. Nos. 3,687,808;
4,469,863; 4,476,301; 5,023,243; 5,166,387; 5,177,196; 5,188,897;
5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676;
5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;
5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361;
5,625,050; and 5,697,248, certain of which are commonly owned by
the assignee of this application, and each of which is herein
incorporated by reference.
[0165] Positional modifications, also known in the art, involve the
linking of nucleosides in a non-naturally occurring motif. As used
herein the term "positional modification" is meant to include
without limitation 2',5'-internucleoside linkages. Combining
modifications e.g. using modified chemistries and positional
modifications of selected internucleoside linkages is also amenable
to the present invention where for example a 2',5'-phosphoramidate
internucleoside linkage is employed. The 2'-5'-linkage has been
used at the termini of oligomeric compounds to enhance the nuclease
resistance (as described in U.S. application Ser. No. 09/435,806,
filed Nov. 8, 1999).
[0166] A representative list of substituent groups amenable to the
present invention include C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, C.sub.5-C.sub.20 aryl, O-alkyl,
O-alkenyl, O-alkynyl, O-alkylamino, (O-alkyl-N(H)alkyl),
O-alkylaminodialkyl (O-alkyl-N-(alkyl).sub.2), O-alkylalkoxy
(O-alkyl-O-alkyl), O-alkyl-(N-imidazole), S-alkenyl, S-alkynyl,
NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, O-aryl, S-aryl,
NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, halogen
(particularly fluoro), keto, carboxyl, nitro, nitroso, nitrile,
trifluoromethyl, trifluoromethoxy, imidazole, azido, hydrazino,
hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,
silyl, heterocycle, carbocycle, polyamine, polyamide, polyalkylene
glycol, 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 is
herein incorporated by reference in its entirety. Further sugar
modifications are disclosed in Cook, P. D., Anti-Cancer Drug
Design, 1991, 6, 585-607. 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, hereby incorporated by
reference in its entirety.
[0167] Additional substituent groups amenable to the present
invention include --SR and --NR.sub.2 groups, wherein each R is,
independently, hydrogen, a protecting group or substituted or
unsubstituted alkyl, alkenyl, or alkynyl. 2'-SR nucleosides are
disclosed in U.S. Pat. No. 5,670,633, issued Sep. 23, 1997, hereby
incorporated by reference in its entirety. The incorporation of
2'-SR monomer synthons are disclosed by Hamm et al., J. Org. Chem.,
1997, 62, 3415-3420. 2'-NR.sub.2 nucleosides are disclosed by
Goettingen, M., J. Org. Chem., 1996, 61, 6273-6281; and Polushin et
al., Tetrahedron Lett., 1996, 37, 3227-3230.
[0168] Further substituent groups have one of formula I or II:
17
[0169] wherein:
[0170] Z.sub.0 is O, S or NH;
[0171] J is a single bond, O or C(.dbd.O);
[0172] E is C.sub.1-C.sub.10 alkyl, N(R.sub.1)(R.sub.2),
N(R.sub.1)(R.sub.5), N.dbd.C(R.sub.1)(R.sub.2),
N.dbd.C(R.sub.1)(R.sub.5) or has one of formula III or IV; 18
[0173] each R.sub.6, R.sub.7, R.sub.8, R.sub.9 and R.sub.10 is,
independently, hydrogen, C(O)R.sub.11, substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical
functional group or a conjugate group, wherein the substituent
groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,
phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and
alkynyl;
[0174] or optionally, R.sub.7 and R.sub.8, together form a
phthalimido moiety with the nitrogen atom to which they are
attached;
[0175] or optionally, R.sub.9 and R.sub.10, together form a
phthalimido moiety with the nitrogen atom to which they are
attached;
[0176] each R.sub.11 is, independently, substituted or
unsubstituted C.sub.1-C.sub.10 alkyl, trifluoromethyl,
cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy,
9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy,
2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or
aryl;
[0177] R.sub.5 is T-L,
[0178] T is a bond or a linking moiety;
[0179] L is a chemical functional group, a conjugate group or a
solid support material;
[0180] each R.sub.1 and R.sub.2 is, independently, H, a nitrogen
protecting group, substituted or unsubstituted C.sub.1-C.sub.10
alkyl, substituted or unsubstituted C.sub.2-C.sub.10 alkenyl,
substituted or unsubstituted C.sub.2-C.sub.10 alkynyl, wherein said
substitution is OR.sub.3, SR.sub.3, NH.sub.3.sup.+,
N(R.sub.3)(R.sub.4), guanidino or acyl where said acyl is an acid
amide or an ester;
[0181] or R.sub.1 and R.sub.2, together, are a nitrogen protecting
group or are joined in a ring structure that optionally includes an
additional heteroatom selected from N and O;
[0182] or R.sub.1, T and L, together, are a chemical functional
group;
[0183] each R.sub.3 and R.sub.4 is, independently, H,
C.sub.1-C.sub.10 alkyl, a nitrogen protecting group, or R.sub.3 and
R.sub.4, together, are a nitrogen protecting group;
[0184] or R.sub.3 and R.sub.4 are joined in a ring structure that
optionally includes an additional heteroatom selected from N and
O;
[0185] Z.sub.4 is OX, SX, or N(X).sub.2;
[0186] each X is, independently, H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 haloalkyl, C(.dbd.NH)N(H)R.sub.5,
C(.dbd.O)N(H)R.sub.5 or OC(.dbd.O)N(H)R.sub.5;
[0187] R.sub.5 is H or C.sub.1-C.sub.8 alkyl;
[0188] Z.sub.1, Z.sub.2 and Z.sub.3 comprise a ring system having
from about 4 to about 7 carbon atoms or having from about 3 to
about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero
atoms are selected from oxygen, nitrogen and sulfur and wherein
said ring system is aliphatic, unsaturated aliphatic, aromatic, or
saturated or unsaturated heterocyclic;
[0189] Z.sub.5 is alkyl or haloalkyl having 1 to about 10 carbon
atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2
to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms,
N(R.sub.1)(R.sub.2) OR.sub.1, halo, SR.sub.1 or CN;
[0190] each q.sub.5 is, independently, an integer from 1 to 10;
[0191] each q.sub.2 is, independently, 0 or 1;
[0192] q.sub.3 is 0 or an integer from 1 to 10;
[0193] q.sub.4 is an integer from 1 to 10;
[0194] q.sub.5 is from 0, 1 or 2; and provided that when q.sub.3 is
0, q.sub.4 is greater than 1.
[0195] Representative substituent groups of Formula I are disclosed
in U.S. patent application Ser. No. 09/130,973, filed Aug. 7, 1998,
entitled "Capped 2'-Oxyethoxy Oligonucleotides," hereby
incorporated by reference in its entirety.
[0196] Representative cyclic substituent groups of Formula II are
disclosed in U.S. patent application Ser. No. 09/123,108, filed
Jul. 27, 1998, entitled "RNA Targeted 2'-Modified Oligonucleotides
that are Conformationally Preorganized," hereby incorporated by
reference in its entirety.
[0197] Particularly preferred substituent groups include
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2,
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)- ].sub.2 (where n and
m are from 1 to about 10), C.sub.1 to C.sub.10 lower alkyl,
substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,
SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino and substituted silyl. Another particularly
preferred modification includes 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3 or 2'-MOE, Martin et al., Helv.
Chim. Acta, 1995, 78, 486). A further preferred substituent group
is 2'-dimethylamino-oxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3- ).sub.2 group, also known as
2'-DMAOE. Representative aminooxy substituent groups are described
in co-owned U.S. patent application Ser. No. 09/344,260, filed Jun.
25, 1999, entitled "Aminooxy-Functionalized Oligomers"; and U.S.
patent application Ser. No. 09/370,541, filed Aug. 9, 1999, also
identified by attorney docket number ISIS-3993, entitled
Aminooxy-Functionalized Oligomers and Methods for Making Same;
hereby incorporated by reference in their entirety.
[0198] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F).
Similar modifications may also be made at other positions on
nucleosides and oligomers, particularly the 3' position of the
sugar on the 3' terminal nucleoside or at a 3'-position of a
nucleoside that has a linkage from the 2'-position such as a 2'-5'
linked oligomer and at the 5'-position at a 5'-terminus. Oligomers
may also have sugar mimetics such as cyclobutyl moieties in place
of the pentofuranosyl sugar. Representative United States patents
that teach the preparation of such modified sugars structures
include, but are not limited to, U.S. Pat. Nos. 4,981,957;
5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;
5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;
5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;
and 5,700,920, certain of which are commonly owned, and each of
which is herein incorporated by reference, and commonly owned U.S.
patent application Ser. No. 08/468,037, filed on Jun. 5, 1995, also
herein incorporated by reference.
[0199] Representative guanidino substituent groups that are shown
in formula III and IV are disclosed in co-owned U.S. patent
application Ser. No. 09/349,040, entitled "Functionalized
Oligomers", filed Jul. 7, 1999, hereby incorporated by reference in
its entirety.
[0200] Representative acetamido substituent groups are disclosed in
U.S. patent application Ser. No. 09/378,568, entitled
"2'-O-Acetamido Modified Monomers and Oligomers", filed Aug. 19,
1999, also identified by attorney docket number ISIS-4071, hereby
incorporated by reference in its entirety.
[0201] Representative dimethylaminoethyloxyethyl substituent groups
are disclosed in International Patent Application PCT/US99/17895,
entitled "2'-O-Dimethylaminoethyloxyethyl-Modified
Oligonucleotides", filed Aug. 6, 1999, also identified by attorney
docket number ISIS-4045, hereby incorporated by reference in its
entirety.
[0202] The use of mixed modifications in the terminal regions of an
oligonucleotide to impart nuclease resistance is also within the
scope of the present invention. For example an oligomeric compound
of the invention can have enhanced nuclease resistance resulting
from one or more modified internucleoside linkages at the 5' end
and one or more substituent groups at the 3' end. Another type of a
mixed modification includes having a modified internucleoside
linkage and a substituent group at the same end of a selected
oligomeric compound. Other examples include substituent groups or
modified linkages used in conjunction with a non-standard linkage
such as a 2',5'-internucleoside linkage.
[0203] Oligomeric compounds according to the present invention
preferably comprise from about 5 to about 50 nucleosides. It is
more preferred that such compounds comprise from about 8 to about
30 nucleosides, with 15 to 25 nucleosides being particularly
preferred.
[0204] In general, the term "hetero" denotes an atom other than
carbon, preferably but not exclusively N, O, or S. Accordingly, the
term "heterocyclic ring" denotes an alkyl ring system having one or
more heteroatoms (i.e., non-carbon atoms). Heterocyclic ring
structures of the present invention can be fully saturated,
partially saturated, unsaturated or with a polycyclic heterocyclic
ring each of the rings may be in any of the available states of
saturation. Heterocyclic ring structures of the present invention
also include heteroaryl, which includes fused systems including
systems where one or more of the fused rings contain no
heteroatoms. Heterocycles, including nitrogen heterocycles,
according to the present invention include, but are not limited to,
imidazole, pyrrole, pyrazole, indole, 1H-indazole,
.alpha.-carboline, carbazole, phenothiazine, phenoxazine,
tetrazole, triazole, pyrrolidine, piperidine, piperazine and
morpholine groups. A more preferred group of nitrogen heterocycles
includes imidazole, pyrrole, indole, and carbazole groups.
[0205] A heterocyclic base moiety (often referred to in the art
simply as a "base" or a "nucleobase") amenable to the present
invention includes both naturally and non-naturally occurring
nucleobases. The heterocyclic base moiety further may be protected
wherein one or more functionalities of the base bears a protecting
group. As used herein, "unmodified" or "natural" nucleobases
include the purine bases adenine and guanine, and the pyrimidine
bases thymine, cytosine and uracil. Modified nucleobases include
other synthetic and natural nucleobases such as 5-methylcytosine
(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and
guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,
5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo
uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and
other 8-substituted adenines and guanines, 5-halo particularly
5-bromo, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine. Further nucleobases include those disclosed in
U.S. Pat. No. 3,687,808, those disclosed in the Concise
Encyclopedia Of Polymer Science And Engineering, pages 858-859,
Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed
by Englisch et al., Angewandte Chemie, International Edition, 1991,
30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15,
Antisense Research and Applications, pages 289-302, Crooke, S. T.
and Lebleu, B., ed., CRC Press, 1993.
[0206] Certain nucleobases are particularly useful for increasing
the binding affinity of oligomeric compounds. These include
5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6
substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. (Id., pages 276-278) and are
presently preferred base substitutions, even more particularly when
combined with 2'-methoxyethyl sugar modifications.
[0207] Representative United States patents that teach the
preparation of modified nucleobases include, but are not limited
to, U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066;
5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;
5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;
5,614,617; and 5,681,941, certain of which are commonly owned, and
each of which is herein incorporated by reference, and commonly
owned U.S. patent application Ser. No. 08/762,488, filed on Dec.
10, 1996, also herein incorporated by reference.
[0208] The attachment of conjugate groups to oligomers is well
documented in the prior art. The present methods include
preparation of oligomeric compounds that include conjugate groups
covalently bound to functional groups such as primary or secondary
hydroxyl groups. Conjugate groups of the invention include
intercalators, reporter molecules, polyamines, polyamides,
polyethylene glycols, polyethers, groups that enhance the
pharmacodynamic properties of oligomers, and groups that enhance
the pharmacokinetic properties of oligomers. Typical conjugates
groups include cholesterols, phospholipids, biotin, phenazine,
phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,
coumarins, and dyes. Groups that enhance the pharmacodynamic
properties, in the context of this invention, include groups that
improve oligomer uptake, enhance oligomer resistance to
degradation, and/or strengthen sequence-specific hybridization with
RNA. Groups that enhance the pharmacokinetic properties, in the
context of this invention, include groups that improve oligomer
uptake, distribution, metabolism or excretion. Representative
conjugate groups are disclosed in International Patent Application
PCT/US92/09196, filed Oct. 23, 1992, U.S. Pat. No. 5,578,718,
issued Jul. 1, 1997, and U.S. Pat. No. 5,218,105. Each of the
foregoing is commonly assigned with this application. The entire
disclosure of each is incorporated herein by reference.
[0209] Preferred conjugate groups amenable to the present invention
include lipid moieties such as a cholesterol moiety (Letsinger et
al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid
(Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a
thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y.
Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem.
Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl.
Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol
or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10,
111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al.,
Biochimie, 1993, 75, 49), a phospholipid, e.g.,
di-hexadecyl-rac-glycerol or
triethylammonium-1,2-di-O-hexadecyl-rac-glyc- ero-3-H-phosphonate
(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al.,
Nucl. Acids Res., 1990, 18, 3777), a polyamine or a polyethylene
glycol chain (Manoharan et al., Nucleosides & Nucleotides,
1995, 14, 969), adamantane acetic acid (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et
al., Biochim. Biophys. Acta, 1995, 1264, 229), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923).
[0210] Other groups that can be attached to oligomeric compounds to
modify antisense properties include RNA cleaving complexes,
pyrenes, metal chelators, porphyrins, alkylators, hybrid
intercalator/ligands and photo-crosslinking agents. RNA cleavers
include o-phenanthroline/Cu complexes and
Ru(bipyridine).sub.3.sup.2+ complexes. The Ru(bpy).sub.3.sup.2+
complexes are believed to interact with nucleic acids and cleave
nucleic acids photochemically. Metal chelators include EDTA, DTPA,
and o-phenanthroline. Alkylators include compounds such as
iodoacetamide. Porphyrins include porphine, its substituted forms,
and metal complexes. Pyrenes include pyrene and other pyrene-based
carboxylic acids that could be conjugated using the similar
protocols.
[0211] As used herein, "polyamine" refers to a moiety containing a
plurality of amine or substituted amine functionalities. Polyamines
according to the present invention have at least two amine
functionalities. "Polypeptide" refers to a polymer comprising a
plurality of amino acids linked by peptide linkages, and includes
dipeptides and tripeptides. The amino acids may be
naturally-occurring or non-naturally-occurring amino acids.
Polypeptides according to the present invention comprise at least
two amino acids.
[0212] The methods of the present invention can employ activated
phosphorus compositions in coupling reactions. As used herein, the
term activated phosphorus composition includes activated phosphorus
containing monomers or oligomers that are reactive with a hydroxyl
group of another monomeric or oligomeric compound to form a
phosphorus-containing internucleotide linkage. Such activated
phosphorus groups contain activated phosphorus atoms in P.sup.III
valence state. Such activated phosphorus atoms are known in the art
and include, but are not limited to, phosphoramidite and chiral
auxiliary moieties. A preferred synthesis utilizes phosphoramidites
as activated phosphorus groups. Additional activated phosphites are
disclosed in Tetrahedron Report Number 309 (Beaucage and Iyer,
Tetrahedron, 1992, 48, 2223-2311).
[0213] Representative activated phosphorus containing monomers or
oligomers include those having the formula: 19
[0214] wherein
[0215] each Bx is, independently, a heterocyclic base moiety or a
blocked heterocyclic base moiety; and
[0216] each R.sub.1 is, independently, H, a blocked hydroxyl group,
a sugar substituent group, or a blocked substituent group;
[0217] T.sub.3 is an hydroxylprotecting group, a nucleoside, a
nucleotide, an oligonucleoside or an oligonucleotide;
[0218] R.sub.4 is N(L.sub.1)L.sub.2;
[0219] each L.sub.1 and L.sub.2 is, independently, C.sub.1-6
straight or branched alkyl, or a C.sub.5-7 cyclic aliphatic ring
system;
[0220] or L.sub.1 and L.sub.2 are joined together to form a 4- to
13-membered heterocyclic ring system including the nitrogen atom to
which L.sub.1 and L.sub.2 are attached, wherein said ring system
optionally includes at least one additional heteroatom selected
from O, N and S; and
[0221] R.sub.5 is X.sub.1;
[0222] X.sub.1 is Pg--O--, Pg--S--, C.sub.1-C.sub.10 straight or
branched chain alkyl, CH.sub.3(CH.sub.2).sub.n--O--,
R.sub.2R.sub.3N-- or a group remaining from coupling a chiral
auxiliary;
[0223] nn is from 0 to 10;
[0224] Pg is CH.sub.3, --CH.sub.2CH.sub.2CN,
--C(CH.sub.3)(CH.sub.3)--CCl.- sub.3, --CH.sub.2--CCl.sub.3,
--CH.sub.2CH.dbd.CH.sub.2 CH.sub.2CH.sub.2SiCH.sub.3, 2-yl-ethyl
phenylsulfonate, .delta.-cyanobutenyl, cyano p-xylyl,
diphenylsilylethyl, 4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl
sulfonate, methyl-N-trifluoroacetyl ethyl, acetoxy phenoxy ethyl,
or a blocking group;
[0225] each R.sub.2 and R.sub.3 is, independently, hydrogen,
C.sub.1-C.sub.10 alkyl, cycloalkyl or aryl;
[0226] or optionally, R.sub.2 and R.sub.3, together with the
nitrogen atom to which they are attached form a cyclic moiety that
may include an additional heteroatom selected from O, S and N;
or
[0227] R.sub.4 and R.sub.5 together with the phosphorus atom to
which R.sub.4 and R.sub.5 are attached form a chiral auxiliary.
[0228] Groups that are attached to the phosphorus atom of
internucleotide linkages before and after oxidation (R.sub.4 and
R.sub.5) can include nitrogen containing cyclic moieties such as
morpholine. Such oxidized internucleoside linkages include a
phosphoromorpholidothioate linkage (Wilk et al., Nucleosides and
nucleotides, 1991, 10, 319-322). Further cyclic moieties amenable
to the present invention include mono-, bi- or tricyclic ring
moieties which may be substituted with groups such as oxo, acyl,
alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido,
azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo,
haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro,
sulfide, sulfone, sulfonamide, thiol and thioalkoxy. A preferred
bicyclic ring structure that includes nitrogen is phthalimido. Some
representative examples of R.sub.4 and R.sub.5 groups which are
known to the art skilled and are amenable to the present invention
are shown below:
1 R.sub.4 R.sub.5 R.sub.4 R.sub.5 20 --O--CH.sub.3
--N--(CH.sub.3).sub.2 21 22 --O--CH.sub.3
--N--(CH.sub.2CH.sub.3).sub.2 23 24 --O--CH.sub.3 25 26 27 28 29 30
31 --O--CH.sub.2CH.sub.2SiCH.sub.3 --N--(CH.sub.3).sub.2
--O--CH.sub.2--CCl.sub.3 32 33 34 --O--CH.sub.2CH.dbd.CH.-
sub.2
[0229] further examples include:
2 R.sub.4 R.sub.5 R.sub.4 R.sub.5 --N--(CH.sub.3).sub.2 35 36
--O--CH.sub.3 37 38 39 --O--CH.sub.3 40 41 42 --O--CH.sub.3 43 44
45 --O--CH.sub.3 46 47 48 --O--CH.sub.3 49 50 51 --O--CH.sub.3
[0230] Functional groups including substituent groups discussed
above which may be located on heterocyclic base and sugar moieties
are routinely blocked with protecting (blocking groups) during
synthesis and subsequently deblocked. In general, a blocking group
renders a chemical functionality of a molecule inert to specific
reaction conditions and can later be removed from such
functionality in a molecule without substantially damaging the
remainder of the molecule. See, Green and Wuts, Protective Groups
in Organic Synthesis, 2d edition, John Wiley & Sons, New York,
1991. For example, amino groups can be blocked with nitrogen
protecting groups such as phthalimido, 9-fluorenylmethoxycarbony- l
(FMOC), triphenylmethylsulfenyl, t-BOC or benzyl groups. Carboxyl
groups can be protected as acetyl groups. Representative
hydroxylprotecting groups are described by Beaucage et al.,
Tetrahedron 1992, 48, 2223. Preferred hydroxylprotecting groups are
acid-labile groups, such as the trityl, monomethoxytrityl,
dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and
9-(p-methoxyphenyl)xanthin-9-yl (MOX). Chemical functional groups
can also be "blocked" by including them in a precursor form. Thus
an azido group can be considered a "blocked" form of an amine as
the azido group is easily converted to the amine. Further
representative protecting groups utilized in oligonucleotide
synthesis are discussed in Agrawal et al., Protocols for
Oligonucleotide Conjugates, Eds., Humana Press, New Jersey, 1994,
Vol. 26, pp. 1-72.
[0231] Standard oligonucleotide synthesis using phosphite
(P.sup.III) chemistry involves treatment of the growing oligomer
with a deprotecting reagent to create a free hydroxyl position that
is available for a further coupling reaction. Hydroxyl protecting
groups are preferably removed using a weak acid. Dependant on the
choice of protecting group the deprotecting reagent can be acidic,
basic, neutral or fluoride mediated. A representative list of
deprotecting reagents amenable to the present methods includes
without limitation protic acids used for removing acid labile
protecting groups such as dichloro- and trichloroacetic acids,
Lewis acids such as BF.sub.3-etherate, zinc bromide, AlCl.sub.3,
TiCl.sub.4, (Et)AlCl, (I-Bu).sub.2AlCl and other reagents such as
ceric ammonium nitrate, 1,1,1,3,3,3-hexafluoro-2-propano- l, and
diethyloxomalonate. A preferred deprotecting reagent that is used
routinely for example for the removal of various trityl protecting
groups is 2-5% dichloroacetic acid in either dichloromethane or
dichloroethane.
[0232] The use of blocking groups is common practice to protect or
block reactive or functional groups that are typically located on
or linked to nucleobases, internucleotide linkages and sugars.
Generally, blocking groups are removed using conditions that are
stronger than those encountered during the iterative elongation
steps in oligomer synthesis. This allows for deprotection of
hydroxyl groups and coupling without effecting blocked positions.
As used herein, the term "blocking group" describes a group that is
stable to the conditions that are used to deprotect groups such as
hydroxyl groups during the iterative elongation steps of oligomeric
compound synthesis. Blocking groups are generally removed after the
desired length has been synthesized. Standard phosphoramidite
chemistry frequently uses acid labile protecting groups on
hydroxyls that are used for coupling steps and strong base labile
blocking groups to block other reactive positions not used in
coupling steps. Many examples of protecting and blocking groups are
collectively described in for example Green and Wuts ibid.
Preferred blocking groups are removed by treatment with base. Some
representative base labile protecting groups include without
limitation, Fmoc (E. Atherton and R. C. Sheppard in The Peptides,
S. Udenfriend, J. Meienhofer, Eds., Academic Press, Orlando, 1987,
volume 9, p.1), and various substituted sulfonylethyl carbamates
exemplified by the Nsc group (Samukov et al., Tetrahedron Lett,
1994, 35:7821; Verhart and Tesser, Rec. Trav. Chim. Pays-Bas, 1987,
107:621).
[0233] After synthesis the resulting oligomeric compound generally
is cleaved from the solid support to obtain the free oligomer. The
step of deprotecting the blocked 5'-O-hydroxyl is usually performed
separately as this is generally accomplished using an acidic
deblocking reagent. This step is routinely performed after
deprotection, cleavage and purification has been performed to
enhance the purification process by keeping the terminal hydroxyl
blocked. The deprotection and cleavage steps can be separated into
separate steps or combined into a single step depending on the
particular protecting groups, solid support linking groups and the
choice of reagent or reagents used. In a preferred embodiment the
simultaneous deprotection and cleavage of the final oligomeric
compound following synthesis is accomplished in one step using a
solution of ammonium hydroxide (NH.sub.4OH (30%) for 15 hours at
60.degree. C., filtered, rinsed with ethanol/water (1/1, v/v), the
combined solutions are evaporated to dryness under vacuum).
[0234] The purification of oligomeric compounds is generally by
reversed phase high performance liquid chromatography (RP-HPLC)
performed on a Waters Nova-Pak C18 column (3.9.times.300 mm) using
a Waters HPLC system (600E System Controller, 996 Photodiode Array
Detector, 717 Autosampler). For analysis an acetonitrile (A)/0.1M
triethylammonium acetate gradient is used: 5% to 35% A from 0 to 10
min, then 35% to 40% A from 10 to 20 min, then 40% to 95% A from 20
to 25 min, flow rate=10 mL/min/50% A from 8 to 9 min, 9 to 26 min
at 50%, flow rate=1.0 mL/min, tR(DMT-off) 10-11 min, tR(DMT-on)
14-16 min. The DMT-on fractions are collected and are evaporated in
vacuum, redissolved in water and the DMT group removed as described
below.
[0235] Removal of the final hydroxylprotecting group from the
5'-hydroxyl group is generally performed by treatment with an
acidic solution such as acetic acid. The oligomeric compound is
treated with the acidic solution for about 30 minutes at room
temperature. The mixture is further treated with sodium acetate and
cold ethanol followed by vortexing and cooling with dry ice. The
precipitate is centrifuged, separated, washed and dried to give the
final deprotected product.
[0236] The term "nucleoside" as used in connection with this
invention refers to a monomeric unit made up of a heterocyclic base
moiety joined to a sugar moiety or sugar mimetic through a glycosyl
linkage. The term "nucleotide" refers to a nucleoside having a
phosphate group on its 3' or 5' sugar hydroxyl group.
[0237] In the context of this invention, the terms "oligomer" and
"oligomeric compound" refer to a plurality of naturally-occurring
or non-naturally-occurring nucleosides joined together in a
specific sequence. The terms "oligomer" and "oligomeric compound"
include oligonucleotides, oligonucleotide analogs, oligonucleosides
and chimeric oligomeric compounds where there are more than one
type of internucleoside linkages dividing the oligomeric compound
into regions. Whereas the term "oligonucleotide" has a well defined
meaning in the art, the term "oligomeric compound" or "oligomer" is
intended to be broader, inclusive of oligomers having all manner of
modifications known in the art. Gapped or chimeric compounds are
disclosed in for example, U.S. Pat. No. 5,623,065, issued Apr. 22,
1997, the contents of which are incorporated herein by
reference.
[0238] As used herein, the term "oligonucleoside" includes
oligomers or polymers containing two or more nucleoside subunits
having a non-phosphodiester linking moiety. Oligonucleosides
according to the invention have a ribofuranose moiety attached to a
nucleobase through a glycosyl bond.
[0239] Gapmer technology has been developed to incorporate
modifications at the ends ("wings") of oligomeric compounds,
leaving a phosphorothioate gap in the middle for RNase H activation
(Cook, P. D., Anti-Cancer Drug Des., 1991, 6, 585-607; Monia et
al., J. Biol. Chem., 1993, 268, 14514-14522). In a recent report,
the activities of a series of uniformly 2'-O modified 20 mer RNase
H-independent oligonucleotides that were antisense to the 5'-cap
region of human ICAM-1 transcript in HUVEC cells, were compared to
the parent 2'-deoxy phosphorothioate oligonucleotide (Baker et al.,
J. Bio. Chem., 1997, 272, 11994-12000). The 2'-MOE/P.dbd.O oligomer
demonstrated the greatest activity with an IC.sub.50 of 2.1 nM
(T.sub.m=87.1.degree. C.), while the parent P.dbd.S oligonucleotide
analog had an IC.sub.50 of 6.5 nM (T.sub.m=79.2.degree. C.).
Correlation of activity with binding affinity is not always
observed as the 2'-F/P.dbd.S (T.sub.m=87.9.degree. C.) was less
active than the 2'-MOE/P.dbd.S (T.sub.m=79.2.degree. C.) by four
fold. The RNase H competent 2'-deoxy P.dbd.S parent oligonucleotide
exhibited an IC.sub.50=41 nM.
[0240] As used herein the term "chiral auxiliary" is meant to
include groups that function to provide chirality to
internucleoside phosphorus linkages during synthesis. Chiral
auxiliaries amenable to the present invention include those that
form a P.sup.III intermediate capable of being oxidized. Chiral
auxiliaries will give either Sp or Rp chirality for the respective
internucleoside linkage in the final oligomeric compound.
Accordingly, chiral auxiliaries are allowed to remain on the
growing chain, and are removed at the end of the iterative
synthetic regime. Removal of chiral auxiliaries can be conveniently
accomplished in a single treatment after the completion of the
iterative synthesis. Chiral auxiliaries and methods of their
incorporation using standard protocols are disclosed in commonly
owned U.S. patent application Ser. No. 09/438,989, filed on Nov.
12, 1999, incorporated herein by reference. Further chiral
auxiliaries have been previously reported for use in the
preparation of oligomeric phosphorothioates (see Iyer et al.,
Tetrahedron letters, 1998, 39, 2491-2494 and Wilk et al., J. Am.
Chem. Soc., 2000, 122, 2149-2156). Representative chiral
auxiliaries include, without limitation those having the following
formulas: 52
[0241] As used herein, the term "alkyl" includes, but is not
limited to, straight chain, branched 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. Substituent groups
include, but are not limited to, alkyl, alkenyl, alkynyl, aryl,
hydroxyl, alkoxy, alcohol, benzyl, phenyl, nitro, thiol,
thioalkoxy, thioalkyl, trifluoromethyl, halo, nitrile,
trifluoromethoxy and azido. As used herein, the term "lower alkyl"
is intended to mean an alkyl group having 10 or fewer carbons.
[0242] Alkenyl groups according to the invention are to straight
chain, branch chain, and cyclic hydrocarbon groups containing at
least one carbon-carbon double bond, and alkynyl groups are to
straight chain, branch chain, and cyclic hydrocarbon groups
containing at least one carbon-carbon triply bond. Alkenyl and
alkynyl groups of the present invention can be substituted.
[0243] Aryl groups are substituted and unsubstituted aromatic
cyclic moieties including but not limited to phenyl, naphthyl,
anthracyl, phenanthryl, pyrenyl, and xylyl groups. Alkaryl groups
are those in which an aryl moiety links an alkyl moiety to a core
structure, and aralkyl groups are those in which an alkyl moiety
links an aryl moiety to a core structure.
[0244] 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. As used herein, the term "aryl" denotes
aromatic cyclic groups including, but not limited to, phenyl,
naphthyl, anthracyl, phenanthryl and pyrenyl. Preferred aryl and
aralkyl groups include, but are not limited to, phenyl, benzyl,
xylyl, naphthyl, toluyl, pyrenyl, anthracyl, azulyl, phenethyl,
cinnamyl, benzhydryl, and mesityl. Typical substituents for
substitution include, but are not limited to, hydroxyl, alkoxy,
alcohol, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, or
alkyl, aryl, alkenyl, or alkynyl groups.
[0245] As used herein, the term "alkanoyl" has its accustomed
meaning as a group of formula --C(.dbd.O)-alkyl. A preferred
alkanoyl group is the acetyl group.
[0246] In one aspect of the present invention oligomeric compounds
are prepared using support bound methodologies wherein the
oxidation and capping steps are combined into a single step. A
first modified or unmodified nucleoside is attached to a support
media preferably via a linkage to the 3'-position. The nucleoside
could alternatively be attached to a support media through the
2'-position as when preparing positionally modified internucleoside
linkages. Alternatively, the support media with the desired
nucleoside attached can be purchased from a number of commercial
sources. In a traditional synthesis this nucleoside will ultimately
become the nucleoside at the 3'-end of the final oligomeric
compound. The support media with the nucleoside attached is placed
in a reaction vessel such as a glass reactor. One of the hydroxyl
groups (preferably the 5'-hydroxyl group) is deprotected and
treated with a second nucleoside having a group reactive with the
hydroxyl group such as an activated phosphorus group or a chiral
auxiliary. This coupling step is preferably performed in the
presence of an activating agent such as DBU or 1-H-tetrazole. The
linkage thus formed is treated with a mixture containing reagents
for oxidizing and capping. A preferred mixture for incorporating a
sulfur atom is (0.3M) dimethylthiuram disulfide in cap A (20%
acetic anhydride in acetonitrile) mixed with and equal volume of
cap B (20% N-methylimidazole, 30% pyridine and 50% aetonitrile, by
volume). The cycle is optionally repeated to add additional
nucleosides until the desired oligomeric compound is completed.
[0247] As used herein, the term "sulfurizing reagent" includes
without limitation, dimethylthiuram disulfide (Cummings et al.,
Ind. Eng. Chem., 1928, 20, 1173); 1,2,4-dithiazolidine-3,5-dione
(DTSNH, see Xu et al., Nucleic Acids Research, 1996, 24,
1602-1607); 3-methyl-1,2,4-dithiazolin-- 5-one (MEDITH, see Zang et
al., Tetrahedron Lett., 1999, 40, 2095-20980); phenylacetyl
disulfide (PADS, see Kamer et al., Tetrahedron Lett., 1989, 30,
6757-6760; Cheruvallath et al., Organic Process Research &
Development, 2000, 4, 199-204); tetraethylthiuram disulfide (TETD,
see Vu et al., Tetrahedron Lett., 1991, 32, 3005-3008);
3H-1,2-benzodithiol-3-on- e-1,1-dioxide (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); benzyltriethylammonium tetrathiomolybdate (BTTM, see
e.g., Rao, M. V., et.al., Tetrahedron Lett., 1994, 35, 6741-6744);
di(phenylacetyl)disulfide (see e.g., Kamer, P. C. J., Tetrahedron
Lett., 1989, 30, 6757-6760); Bis(O,O-diisopropoxy
phosphinothioyl)disulfides (see Stec et al., Tetrahedron Lett.,
1993, 34, 5317-5320); 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH, see
Xu et al., 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); bis(ethoxythiocarbonyl)-tetrasulfide (see Zang
et al., Tetrahedron Lett., 1998, 39, 2467-2470);
bis(p-toluenesulfonyl)disulfide (Efimov et al., Nucleic Acids Res.,
1995, 23, 4029-4033); 3-amino-1,2,4-dithiazole-5-thione (see Org.
Process Res. Dev., 2000, 4, 194-198); ethylthiuram disulfide CAS #
3082-38-0; 5,6-dihydro-3H-imidazo[2,1-C]-1,2,4-dithiazole-3-thione
CAS # 33813-20-6;
4-methyl-5-(methylimino)-1,2,4-dithiazolidine-3-thione CAS #
20042-85-7; sulfur, sulfur in combination with ligands like
triaryl, trialkyl, triaralkyl, or trialkaryl phosphines. The
foregoing references are hereby incorporated by reference in their
entirety.
[0248] A preferred list of sulfurizing reagents includes:
3-amino-1,2,4-dithiazole-5-thione;
3-ethoxy-1,2,4-dithiazoline-5-one; 1,2,4-dithiazolidine-3,5-dione;
3-methyl-1,2,4-dithiazolin-5-one; and dimethylthiuram
disulfide.
[0249] A representative list of capping reagents useful in the
methods of the present invention include without limitation, acetic
anhydride, t-butylphenoxyacetic anhydride, phosphite monoesters,
and selected acid chlorides preferably delivered concurrently with
a nucleophilic catalyst (e.g. a strong base) such as for example
N-methylimidazole or triethylamine. Generally capping reagents
comprise a mixture of Cap A and Cap B. Representative mixtures
include without limitation:
[0250] Cap A: acetic anhydride in acetonitrile or
tetrahydrofuran;
[0251] chloroacetic anhydride in acetonitrile or
tetrahydrofuran;
[0252] Cap B: N-methylimidazole and pyridine in acetonitrile or
tetrahydrofuran;
[0253] 4-dimethylaminopyridine (DMAP) and pyridine in acetonitrile
or tetrahydrofuran;
[0254] 2,6-lutidine and N-methylimidazole in acetonitrile or
tetrahydrofuran.
[0255] A more detailed description capping reagents is discussed in
U.S. Pat. No. 4,816,571, issued Mar. 28, 1989 which is incorporated
herein by reference. A preferred capping reagent is acetic
anhydride routinely used as a mixture of cap A and cap B.
[0256] During the coupling step one compound having an active
phosphate is coupled to a second compound having a free hydroxyl
group. An activating agent is not believed to be essential for this
step but one is generally used to increase the reaction efficiency.
A list of activators and references for each can be found in
Eleueri et al., Organic Process Research & Development, 2000,
4, 182-189. Preferred activators include without limitation:
1H-tetrazole, 5-(2-nitrophenyl)-1H-tetrazole,
5-(p-nitrophenyl)-1H-tetrazole, 5-trifluoromethyl-1H-tetrazole,
5-ethylthio-1H-tetrazole, 5-benzyltio-1H-tetrazole,
2,4,5-tribromoimidazole, 2-nitroimidazole, 4,5-dichloroimidazole,
2-bromo-4,5-dicyanoimidazole, 4,5-dicyanoimidazole,
N-methylimidazole hydrochloride, 1-hydroxybenzotriazole,
5-chlorobenzotriazole, chlorotrimethylsilane, benzimidazolium
triflate, imidazolium triflate, pyridinium hydrochloride/imidazole,
pyridinium tetrafluoroborate, pyridinium chloride, pyridinium
bromide, pyridinium 4-methylbenzinesulfonate, N-methylimidazolium
trifluroborate, N-methylanilinium trichloroacetate,
N-methylanilinium trifluoroacetate, 1H-tetrazole/DMAP,
1H-tetrazole/N-methylimidazole, and N-methylimidazolium
trifluoromethanesulfonate (see see review article Beaucage et al.,
Current Protocols in Nucleic Acid Chemistry, 2000,
3.3.1-3.3.20).
[0257] The current method of choice for the preparation of
oligomeric compounds uses support media. Support media is used to
attach a first nucleoside or larger nucleosidic synthon which is
then iteratively elongated to give a final oligomeric compound.
Support media can be selected to be insoluble or have variable
solubility in different solvents to allow the growing oligomer to
be kept out of or in solution as desired. Traditional solid
supports are insoluble and are routinely placed in a reaction
vessel while reagents and solvents react and or wash the growing
chain until cleavage frees the final oligomer. More recent
approaches have introduced soluble supports including soluble
polymer supports to allow precipitating and dissolving the bound
oligomer at desired points in the synthesis (Gravert et al., Chem.
Rev., 1997, 97, 489-510). Representative support media that are
amenable to the methods of the present invention include without
limitation: controlled pore glass (CPG); oxalyl-controlled pore
glass (see, e.g., Alul, et al., Nucleic Acids Research 1991, 19,
1527); TENTAGEL Support, (see, e.g., Wright, et al., Tetrahedron
Letters 1993, 34, 3373); or POROS, a copolymer of
polystyrene/divinylbenzene available from Perceptive Biosystems.
The use of a soluble support media, poly(ethylene glycol), with
molecular weights between 5 and 20 kDa, for large-scale synthesis
of phosphorothioate oligonucleotides is described in, Bonora et
al., Organic Process Research & Development, 2000, 4,
225-231.
[0258] It was previously reported (Cummings A. D. et al. Ind. Eng.
Chem., 1928, 20, 1173) that dimethylthiuram disulfide was not a
stable compound and decomposes slowly on standing. The
dimethylthiuram disulfide, after standing for 1 month, failed to
show a melting point of 102.degree. C. The decomposition products
were identified as hydrogen sulfide, elemental sulfur and methyl
isothiocyanate. The lack of long shelf life for dimethylthiuram
disulfide has been attributed to the dithiocabamate derivative of
methylamine, which is a primary amine. The present invention
provides a new and improved procedure for the synthesis of
dimethylthiuram disulfide (see Example 7) which utilizes an acid
wash at the end of the synthesis. The primary amine is protonated
which stabilizes the dithiocarbamate structure from degradation. A
further improvement was realized by oxidizing the intermediate with
hydrogen peroxide as opposed to iodine. This led to the preparation
of white crystalline product instead of the yellow unstable product
previously reported. Dimethylthiuram disulfide made by this
protocol is very stable even after six months of storage at room
temperature.
[0259] Oligomeric compounds prepared by the methods of the present
invention can be used in diagnostics, therapeutics and as research
reagents and kits. They can also be used in pharmaceutical
compositions by including a suitable pharmaceutically acceptable
diluent or carrier. They can further be used for treating organisms
having a disease characterized by the undesired production of a
protein. For this purpose, the organism is contacted with an
oligomer having a sequence that is capable of specifically
hybridizing with a strand of nucleic acid encoding 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 organisms that can be treated
with therapeutic or diagnostic oligomeric compounds of the
invention.
[0260] 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.
EXAMPLE 1
[0261] 5c-Methyl-2t[(1-methyl-1-methylamino)ethyl]-cyclohexan-1r-ol
(Compound 1)
[0262] The title compound is synthesized according to a literature
procedure using (+)-pulegone (He et al., J. Org. Chem., 1990, 55,
2114-2119) by first preparing 5c-Methyl-2t
[(1-methyl-1-benzylamino)ethyl- ]-cyclohexan-1r-ol. This compound
is subjected to hydrogenolysis by Pd/H.sub.2 to give the
corresponding amino alcohol (removal of benzyl group). The amino
alcohol is then treated with 1 equivalent of HCHO followed by
NaCNBH.sub.3 reduction to give the title Compound. This isomer is
used to prepare Rp phosphorothioate linkages.
[0263] The isomer of the title compound (Compound 2) is prepared
from the naturally occuring (-)-pulegone (available from Fluka),
Compound 2 is obtained as a Chiral Adjuvant following a literature
procedure (He et al., Tetrahedron, 1987, 43, 4979-4987). This
isomer is used to prepare Sp phosphorothioate linkages.
Example 2
[0264] Preparation of Sp Monomer (Compound 3) 53
[0265] Compound 2 is treated with PCl.sub.3 (1 equivalent) with
excess of Hunig base in THF solvent at -5.degree. C. for 10
minutes. The resulting chloro compound is treated with a selected
2'-deoxy-5-O-DMT-nucleoside having a free 3'-OH group
(2'-O-deoxy-5'-O-DMT-6-N-benzoyl adenosine,
2'-O-deoxy-5'-O-DMT-4-N-benzoyl cytodine,
2'-O-deoxy-5'-O-DMT-2-N-butyryl guanosine,
2'-O-deoxy-5'-O-DMT-thymidine or modified optionally protected
5-O-DMT-nucleoside). TLC and .sup.13C NMR analysis is used to
reveal the formation of a single diastereomer. The crude material
is washed with saturated sodium bicarbonate and dried over
anhydrous sodium sulfate. The resulting material is purified either
by crystallization or by silica gel column chromatography.
EXAMPLE 3
[0266] Protected Dimer, (Compound 4)
[0267] Purified compound 3 is condensed with a 5'-HO-T-CPG (Example
5), or other solid support bound 5'-OH-nucleoside (such as
2'-O-deoxy-6-N-benzoyl adenosine, 2'-O-deoxy-4-N-benzoyl cytidine,
2'-O-deoxy-2-N-isobutyryl guanosine or other modified optionally
protected 5'-OH'-3'-CPG-nucleoside), for 2 hours using tetrazole as
the coupling agent. The capping and sulfurization is completed in
one step using (0.3M) dimethylthiuram disulfide in cap A (20%
acetic anhydride in acetonitrile) mixed with and equal volume of
cap B (20% N-methylimidazole, 30% pyridine and 50% aetonitrile, by
volume) giving the protected phosphorothioate dimer attached to
solid support. The protected dimer is cleaved from the solid
support, deprotected by treatment with concentrated ammonium
hydroxide (30%, 12 hours), and purified purified by HPLC. The
nucleoside dimer is treated with 80% aqueous acetic acid to remove
the 5'-triyl group. The Sp configuration is assigned as described
below in the procedures.
EXAMPLE 4
[0268] Preparation of Rp Monomer (Compound 5) 54
[0269] The Rp monomer is prepared following the procedures
illustrated for the Sp dimer in example 2 using Compound 1.
EXAMPLE 5
[0270] Preparation of Rp Dimer (Compound 6)
[0271] The Rp dimer is prepared following the procedures
illustrated for the Sp dimer in example 3 using Compound 5.
EXAMPLE 6
[0272] Synthesis of Chirally Pure
5'-T.sub.SpT.sub.RpT.sub.RpT.sub.RpT.sub-
.RpT.sub.SpT-3'Phosphorothioate Heptamer
[0273] 50 milligram (2 .mu.mole) of 5'-O-dimethoxytritylthymidine
bound to CPG (controlled pore glass) through an ester linkage is
taken up in a glass reactor, and a toluene solution of 3%
dichloroacetic acid (v/v) is added to deprotect the 5'-hydroxyl
group. The product is washed with acetonitrile and a 0.2 M solution
of Compound 3 (B=T) in acetonitrile (25 fold excess) and a 0.5 M
solution of DBU in acetonitrile (200 fold excess) is added and
allowed to react at room temperature for 15 minutes. The product is
washed with acetonitrile followed by the addition of a solution of
(0.3M) dimethylthiuram disulfide in cap A (20% acetic anhydride in
acetonitrile) mixed with and equal volume of cap B (20%
N-methylimidazole, 30% pyridine and 50% aetonitrile, by volume)
with reaction allowed to progress at room temperature for 5
minutes. The product is washed with acetonitrile.
[0274] In the next cycle Compound 5 (B=T) is used as the incoming
monomer and the cycle is repeated. This complete cycle is repeated
four more times to introduce the Rp linkages. In the final cycle
Compound 3 is used as the incoming monomer which introduces the
terminal Sp linkage. The solid support containing the heptamer is
treated with 30% aqueous ammonium hydroxide solution for 90 minutes
at room temperature. The aqueous solution is filtered, and
concentrated under reduced pressure to give the chirally pure
phosphorothioate heptamer.
EXAMPLE 7
[0275] Preparation of Dimethylthiuram Disulfide
[0276] In a 4-litter bottle equipped with a mechanical stirrer,
sodium hydroxide (80 g, 2 mol) was dissolved in water (500 mL) and
the solution was cooled to 0.degree. C. (ice-water bath). THF (200
mL), methylamine (40% in water, 170 mL, 2 mol) and carbon disulfide
(120 mL, 2 mol) were added and the mixture was stirred at 0.degree.
C. for 30 minutes. Crushed ice (1.5 kg) was added, followed by
glacial acetic acid (300 mL). Hydrogen peroxide (30%, 100 mL, 1
mol) was added dropwise over 10 minutes with temperature maintained
below 5.degree. C. by adding ice with stirring. Hexanes (or
heptane)(800 mL) was added and the mixture was stirred for another
30 minutes in an ice-water bath. The mixture was filtered and
washed with aqueous trichloroacetic acid (2%, 5.times.200 mL) and
hexanes (or heptane) (2.times.200 mL). The product was dried in air
for 1 day to a constant weight to give an off-white solid (205.8,
yield: 97%). M.p. 98-100.degree. C. (dec.)
3 HPLC analysis: Column: YMC ODS-AQ S3 120A, 4.6 .times. 100 mm
Flow rate: 1.0 mL/min Detector: UV at 244 nm Sample: inject 10
.mu.L (1-2 mg in 1 mL of glacial acetic acid) Retention time: 5.4
min
[0277]
4 Linear gradient Time (Min) Acetonitrile 0.2% Acetic acid 0 50 50
15 90 10 25 90 10 30 50 50 35 50 50
[0278] The crude product (200 g) was recrystallized by dissolution
in THF (1L) containing trichloroacetic acid (10 g). Water (200 mL)
was added followed by slow addition of hexanes (5 L) over 30
minutes. After stirring at 0.degree. C. for 1 hour, filtering,
washing with 2% aqueous trichloroacetic acid (3.times.200 mL)
followed by drying the recrystallized dimethylthiuram disulfide was
obtained.
EXAMPLE 8
[0279] Sulfurization of Triethyl Phosphite with Various Sulfurizing
Reagents
[0280] Small scale sulfurization reactions were performed in NMR
tubes using CD.sub.3CN to measure the efficiency of various
sulfurization reagents. The basic reaction scheme is shown
below:
5 55 Reagent (.dbd.S) % P.dbd.O % product % product (w/tetrazole)
Beaucage 0.7 100 -- Phenylacetyl 0.7 100 -- disulfide
Tetraethylthiuram 2 82 100 disulfide Tetramethylthiuram 4 2 93
disulfide Morpholino thiocarbonyl 1.7 50 93 disulfide
Dimethylthiuram 0 100 100 disulfide
[0281] The experimental conditions varied dependent on the
sulfurizing reagent used. The conditions are detailed below:
[0282] 1. 0.5 M beaucage in CD.sub.3CN (1 mL) and triethyl
phosphite (17 .mu.L) for 5 min.
[0283] 2. 0.2 M PADS in 3-picoline-CD.sub.3CN (1:1, 1 mL) and
triethyl phosphite (17 .mu.L) for 5 min.
[0284] 3. 0.2 M tetraethylthiuram disulfide in CD.sub.3CN (1 mL)
and triethyl phosphite (17 .mu.L) for 5 min.
[0285] 4. 0.2 M tetraethylthiuram disulfide and 0.3 M tetrazole in
CD.sub.3CN (1 mL) and triethyl phosphite (17 .mu.L) for 5 min.
Example 9
[0286] Solid Phase Synthesis of Full Phosphorothioate
5'-TTTTTTTC-3' Using Tetraethylthiuram Disufide
[0287] The above sequence was synthesized following standard
phosphoramidite protocols. The 4-step procedure was performed on
via automated synthesis using a 6.3 mL column on an OligoPilot II
(Armersham Pharmacia). The reagents and the amounts used are as
follows:
[0288] 1. Detritylation: 3% dichloroacetic acid in
dichloromethane.
[0289] 2. Coupling: 2 equivalents of phosphoramidite for 5 min.
[0290] 3. Thiolation: 1 column volume (CV) 0.5 M tetraethyl-thiuram
disulfide and 0.45 M tetrazole in acetonitrile for 10 min.
[0291] 4. Capping: 0.5 CV of cap A (20% acetic anhydride in
acetonitrile) and cap B (N-methylimidazole-pyridine-acetonitrile,
2:3:5, v/v/v) for 1 min.
[0292] After synthesis, the solid support was heated with
concentrated aqueous ammonia solution (50 mL) at 58.degree. C.
overnight and the filtered. The filtrate was concentrated under
reduced pressure and dried. The .sup.31P NMR study showed 3.7%
P.dbd.O and 96.3% P.dbd.S.
Example 10
[0293] Solid Phase Synthesis of Full Phosphorothioate
5'-TTTTTTTC-3' Using Morpholino Thiocarbonyl Disulfide
[0294] The above sequence was synthesized following standard
phosphoramidite protocols. The 4-step procedure was performed on
via automated synthesis using a 6.3 mL column on an OligoPilot II
(Armersham Pharmacia). The reagents and the amounts used are as
follows:
[0295] 1. Detritylation: 3% dichloroacetic acid in
dichloromethane.
[0296] 2. Coupling: 2 equivalents of phosphoramidite for 5 min.
[0297] 3. Thiolation: 1 column volume (CV) 0.3M morpholino
thiocarbonyl disulfide and 0.23M tetrazole in
dichloromethane-acetonitrile (1:1 v/v) for 10 min.
[0298] 4. Capping: 0.5 CV of cap A (20% acetic anhydride in
acetonitrile) and cap B (N-methylimidazole-pyridine-acetonitrile,
2:3:5, v/v/v) for 1 min.
[0299] After synthesis, the solid support was heated with
concentrated aqueous ammonia solution (50 mL) at 58.degree. C.
overnight and the filtered. The filtrate was concentrated under
reduced pressure and dried. .sup.31P NMR showed 3.3% P.dbd.O and
96.7% P.dbd.S.
EXAMPLE 11
[0300] Solid Phase Synthesis of Full Phosphorothioate 20mer, SEQ ID
NO. 1 (5'-GTGCTCATGG TGCACGGTCT-3'; all C's are 5-Me-C's)
[0301] A. Using Phenylacetyl Disulfide (PADS)
[0302] The above sequence (SEQ ID NO. 1) was synthesized following
standard phosphoramidite protocols. The 4-step procedure was
performed on via automated synthesis using a 6.3 mL column on an
OligoPilot II (Armersham Pharmacia). The reagents and the amounts
used are as follows:
[0303] 1. Detritylation: 3% dichloroacetic acid in
dichloromethane.
[0304] 2. Coupling: 2 equivalents of phosphoramidite for 5 min.
[0305] 3. Thiolation: 1 column volume (CV) 0.2 M phenylacetyl
disulfide (PADS) in 3-picoline-acetonitrile (1:1 v/v) for 10
min.
[0306] 4. Capping: 0.5 CV of cap A (20% acetic anhydride in
acetonitrile) and cap B (N-methylimidazole-pyridine-acetonitrile,
2:3:5, v/v/v) for 1 min.
[0307] After synthesis, the solid support was heated with
concentrated aqueous ammonia solution (50 mL) at 58.degree. C.
overnight and the filtered. The filtrate was concentrated under
reduced pressure and dried. Purity and .sup.31P NMR data are shown
below.
[0308] B. Using Dimethylthiuram Disulfide
[0309] The above sequence (SEQ ID NO. 1) was synthesized following
standard phosphoramidite protocols. The 3-step procedure was
performed on via automated synthesis using a 6.3 mL column on an
OligoPilot II (Armersham Pharmacia).
[0310] 1. Detritylation: 3% dichloroacetic acid in
dichloromethane.
[0311] 2. Coupling: 2 equivalents of phosphoramidite for 5 min.
[0312] 3. Thiolation-capping: 1 column volume (CV) 0.3 M
dimethylthiuram disulfide (DMDS) in cap A (20% acetic anhydride in
acetonitrile) and 1CV cap B
(N-methylimidazole-pyridine-acetonitrile, 2:3:5, v/v/v) for 1
min.
[0313] After synthesis, the solid support was heated with
concentrated aqueous ammonia solution (50 mL) at 58.degree. C.
overnight and the filtered. The filtrate was concentrated under
reduced pressure and dried. Purity and .sup.31P NMR data are shown
below.
6 O.D./ % % % Rgt Loading Crude O.D. mmol Trityl-on CGE P = O PADS
173 25625 148 74.7 78.1 0.40 DMDS 169 26265 149 76.2 81.5 0.22.
EXAMPLE 12
[0314] Preparation of thymidine 8-mer having phosphodiester
internucleotide linkages using a single step combining oxidation
and capping (5'-TTTTTTTT-3') A thymidine 8-mer was prepared
following the procedures illustrated above (see example 11). The
synthesis was performed as per the 3-step procedure (combined
oxidation and capping steps) using a 6.3 mL column on an OligoPilot
II (Armersham Pharmacia) and 1.89 g Primer Support T (93
.mu.mol/g).
[0315] 1. Detritylation: 3% dichloroacetic acid in toluene.
[0316] 2. Coupling: 3 equivalents of phosphoramidite for 5 min.
[0317] 3. Oxidation-capping: 1 CV of 0.1 M iodine in cap A (20%
acetic anhydride in acetonitrile) and 1 CV of cap B
(N-methylimidazole-pyridine-- acetonitrile, 2:3:5, v/v/v) for 2
min.
[0318] After synthesis, the solid support was heated with
concentrated aqueous ammonia solution (50 mL) at 58.degree. C.
overnight and the filtered. The filtrate was concentrated under
reduced pressure prior to analysis.
[0319] Crude yield: 7564 O.D.; full length percentage: 90.1%;
.sup.31P NMR: -0.383, -0.276 and -0.215 ppm.
* * * * *