U.S. patent application number 11/099430 was filed with the patent office on 2005-12-01 for processes and reagents for oligonucleotide synthesis and purification.
Invention is credited to Jung, Michael E., Manoharan, Muthiah, Pandey, Rajendra K., Rajeev, Kallanthottathil G., Wang, Gang.
Application Number | 20050267300 11/099430 |
Document ID | / |
Family ID | 35056873 |
Filed Date | 2005-12-01 |
United States Patent
Application |
20050267300 |
Kind Code |
A1 |
Manoharan, Muthiah ; et
al. |
December 1, 2005 |
Processes and reagents for oligonucleotide synthesis and
purification
Abstract
The present invention relates to processes and reagents for
oligonucleotide synthesis and purification. One aspect of the
present invention relates to compounds useful for activating
phosphoramidites in oligonucleotide synthesis. Another aspect of
the present invention relates to a method of preparing
oligonucleotides via the phosphoramidite method using an activator
of the invention. Another aspect of the present invention relates
to sulfur-transfer agents. In a preferred embodiment, the
sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one.
Another aspect of the present invention relates to a method of
preparing a phosphorothioate by treating a phosphite with a
sulfur-transfer reagent of the invention. In a preferred
embodiment, the sulfur-transfer agent is a
3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the present
invention relates to compounds that scavenge acrylonitrile produced
during the deprotection of phosphate groups bearing ethylnitrile
protecting groups. In a preferred embodiment, the acrylonitrile
scavenger is a polymer-bound thiol. Another aspect of the present
invention relates to agents used to oxidize a phosphite to a
phosphate. In a preferred embodiment, the oxidizing agent is sodium
chlorite, chloroamine, or pyridine-N-oxide. Another aspect of the
present invention relates to methods of purifying an
oligonucleotide by annealing a first single-stranded
oligonucleotide and second single-stranded oligonucleotide to form
a double-stranded oligonucleotide; and subjecting the
double-stranded oligonucleotide to chromatographic purification. In
a preferred embodiment, the chromatographic purification is
high-performance liquid chromatography.
Inventors: |
Manoharan, Muthiah; (Weston,
MA) ; Jung, Michael E.; (Los Angeles, CA) ;
Rajeev, Kallanthottathil G.; (Cambridge, MA) ;
Pandey, Rajendra K.; (Framingham, MA) ; Wang,
Gang; (Piscataway, NJ) |
Correspondence
Address: |
FOLEY HOAG, LLP
PATENT GROUP, WORLD TRADE CENTER WEST
155 SEAPORT BLVD
BOSTON
MA
02110
US
|
Family ID: |
35056873 |
Appl. No.: |
11/099430 |
Filed: |
April 5, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60559782 |
Apr 5, 2004 |
|
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Current U.S.
Class: |
536/25.33 |
Current CPC
Class: |
Y02P 20/55 20151101;
C07H 21/04 20130101; C07H 21/00 20130101 |
Class at
Publication: |
536/025.33 |
International
Class: |
C07H 021/04 |
Claims
We claim:
1. A method of forming a phosphite compound, comprising the steps
of: admixing a phosphoramidite, alcohol, and activating agent to
form a phosphite compound, wherein said activating agent is
selected from the group consisting of 48wherein X is C(R.sup.6) or
N; R.sup.1, R.sup.2, R.sup.3, and R.sup.6 each independently
represent H, --NO.sub.2, --CN, --CF.sub.3, --SO.sub.2R.sup.8,
--SR.sup.8, halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,
alkoxyl, --OR.sup.7, --N(R.sup.7).sub.2, --N(R.sup.7)C(O)R.sup.8,
--C(O)R.sup.7, or --CO.sub.2R.sup.8; or an instance of R.sup.1 and
R.sup.6, R.sup.1 and R.sup.2, or R.sup.2 and R.sup.3 can be taken
together for form a 4-8 member ring containing 0-4 heteratoms
selected from the group consisting of O, N and S; R.sup.4 is absent
or represents independently for each occurrence
--(C(R.sup.9).sub.2).sub.nCH.sub.3.Y; R.sup.5 is H or
--(C(R.sup.9).sub.2).sub.nCH.sub.3; R.sup.7 represents
independently for each occurrence H, alkyl, aryl, or aralkyl;
R.sup.8 represents independently for each occurrence alkyl, aryl,
or aralkyl; R.sup.9 represents independently for each occurrence H
or alkyl; n represents independently for each occurrence 0 to 15
inclusive; and Y represents independently for each occurrence
halogen or R.sup.8CO.sub.2.sup.-; 49wherein R.sup.1 and R.sup.3
each represent independently H, --NO.sub.2, --CN, --CF.sub.3,
--SO.sub.2R.sup.6, --SR.sup.6, halogen, alkyl, alkenyl, alkynyl,
aryl, aralkyl, --N(R.sup.5)C(O)R.sup.6, --C(O)R.sup.5, or
--CO.sub.2R.sup.6; R.sup.2 is absent or represents independently
for each occurrence --(C(R.sup.7).sub.2).sub.nCH.sub.3.Y; R.sup.4
is H or --(C(R.sup.7).sub.2).sub.nCH.sub.3; R.sup.5 represents
independently for each occurrence H, alkyl, aryl, or aralkyl;
R.sup.6 represents independently for each occurrence alkyl aryl, or
aralkyl; R.sup.7 represents independently for each occurrence H or
alkyl; n represents independently for each occurrence 0 to 15
inclusive; and Y represents independently for each occurrence
halogen or R.sup.6CO.sub.2.sup.-; 50wherein R.sup.1 and R.sup.2
each represent independently H, --NO.sub.2, --CN, --CF.sub.3,
--SO.sub.2R.sup.6, --SR.sup.6, halogen, alkyl, alkenyl, alkynyl,
aryl, aralkyl, --N(R.sup.5)C(O)R.sup.6, --C(O)R.sup.5, or
--CO.sub.2R.sup.6; R.sup.3 is absent or represents independently
for each occurrence --(C(R.sup.7).sub.2).sub.nCH.sub.3.Y; R.sup.4
is H or --(C(R.sup.7).sub.2).sub.nCH.sub.3; R.sup.5 represents
independently for each occurrence H, alkyl, aryl, or aralkyl;
R.sup.6 represents independently for each occurrence alkyl, aryl,
or aralkyl; R.sup.7 represents independently for each occurrence H
or alkyl; n represents independently for each occurrence 0 to 15
inclusive; and Y represents independently for each occurrence
halogen or R.sup.6CO.sub.2.sup.-; 51wherein R.sup.1 is H,
--SR.sup.5, alkyl, aryl, --N(R.sup.4).sub.2,
--(C(R.sup.4).sub.2).sub.mCO.sub.2R.sup.5, --NO.sub.2, --CN,
--CF.sub.3, --SO.sub.2R.sup.5, --SR.sup.5, halogen, alkenyl,
alkynyl, aralkyl, --N(R.sup.4)C(O)R.sup.5, --C(O)R.sup.4, or
--CO.sub.2R.sup.5; R.sup.2 is absent or represents independently
for each occurrence --(C(R.sup.6).sub.2).sub.nCH.sub.3.Y; R.sup.3
is H or --(C(R.sup.6).sub.2).sub.nCH.sub.3; R.sup.4 represents
independently for each occurrence H, alkyl, aryl, or aralkyl;
R.sup.5 represents independently for each occurrence alkyl, aryl,
or aralkyl; R.sup.6 represents independently for each occurrence H
or alkyl; n represents independently for each occurrence 0 to 15
inclusive; m is 1, 2, 3, 4, 5, 6, 7, or 8; and Y represents
independently for each occurrence halogen or R.sup.5CO.sub.2.sup.-;
and 52wherein R.sup.1, R.sup.3, and R.sup.4 each represent
independently H, --NO.sub.2, --CN, --CF.sub.3, --SO.sub.2R.sup.7,
--SR.sup.7, halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,
--N(R.sup.6)C(O)R.sup.5, --C(O)R.sup.6, or --CO.sub.2R.sup.7;
R.sup.2 is absent or represents independently for each occurrence
--(C(R.sup.8).sub.2).sub.nCH.sub.3.Y; R.sup.5 is H or
--(C(R.sup.8).sub.2).sub.nCH.sub.3; R.sup.6 represents
independently for each occurrence H alkyl aryl, or aralkyl; R.sup.7
represents independently for each occurrence alkyl, aryl, or
aralkyl; R.sup.8 represents independently for each occurrence H or
alkyl; n represents independently for each occurrence 0 to 15
inclusive; and Y represents independently for each occurrence
halogen or R.sup.7CO.sub.2.sup.-.
2. The method of claim 1, wherein said phosphoramidite is a
3'-nucleoside phosphoramidite, 3'-nucleotide phosphoramidite, or
3'-oligonucleotide phosphoramidite.
3. The method of claim 1, wherein said phosphoramidite is
represented by formula A: 53wherein R.sub.1 is alkyl, aryl,
aralkyl, or --Si(R.sub.5).sub.3; wherein said alkyl, aryl, and
aralkyl group is optionally substituted with --CN, --NO.sub.2,
--CF.sub.3, halogen, --O.sub.2CR.sub.5, or --OSO.sub.2R.sub.5;
R.sub.2 is optionally substituted alkyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, or alkenyl; R.sub.3 and R.sub.4
each represent independently alkyl, cycloalkyl, heterocycloalkyl,
aryl, or aralkyl; or R.sub.3 and R.sub.4 taken together form a 3-8
member ring; and R.sub.5 is alkyl, cycloalkyl, heterocycloalkyl,
aryl, or aralkyl.
4. The method of claim 3, wherein R.sub.1 is
--CH.sub.2CH.sub.2CN.
5. The method of claim 3, wherein R.sub.2 is an optionally
substituted heterocycloalkyl.
6. The method of claim 3, wherein R.sub.2 is an optionally
substituted ribose.
7. The method of claim 3, wherein R.sub.2 is an optionally
substituted deoxyribose.
8. The method of claim 3, wherein R.sub.2 is a nucleoside or
nucleotide.
9. The method of claim 3, wherein R.sub.3 and R.sub.4 are
alkyl.
10. The method of claim 1, wherein said alcohol is an optionally
substituted ribose.
11. The method of claim 1, wherein said alcohol is an optionally
substituted deoxyribose.
12. The method of claim 1, wherein said alcohol is a nucleoside,
nucleotide, or oligonucleotide.
13. The method of claim 1, wherein said alcohol is represented by
R.sub.5--OH, wherein R.sub.5 is optionally substituted alkyl,
cycloalkyl, heterocycloalkyl, aryl, aralkyl, alkenyl, or
--(C(R.sub.6).sub.2).sub.phe- terocycloalkyl; R.sub.6 is H or
alkyl; and p is 1, 2, 3, 4, 5, 6, 7, or 8.
14. The method of claim 13, wherein R.sub.5 is
--(C(R.sub.6).sub.2).sub.ph- eterocycloalkyl.
15. The method of claim 1, further comprising the step of admixing
a proton-shuttle compound to the mixture comprising said
phosphoramidite, said alcohol, and said activating agent, wherein
the pKa of said proton-shuttle compound is greater than the pKa of
said activating agent, and the pKa of said proton-shuttle compound
is less than the pKa of said phosphoramidite.
16. The method of claim 15, wherein said proton-shuttle compound is
a primary, secondary, or tertiary amine.
17. The method of claim 15, wherein said proton-shuttle compound is
represented by N(R.sub.7)(R.sub.8)R.sub.9, wherein R.sub.7,
R.sub.8, and R.sub.9 each represent independently for each
occurrence H, alkyl, cycloalkyl, aryl, aralkyl, alkenyl; or R.sub.7
and R.sub.8 taken together form a 3-8 membered ring; and R.sub.9 is
H, alkyl, cycloalkyl, aryl, or aralkyl.
18. A compound represented by formula E: 54wherein X is O or S;
R.sup.1 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or
heteroaralkyl; R.sup.2 is H, alkyl, cycloalkyl, aryl, heteroaryl,
aralkyl, heteroaralkyl, --C(O)N(R.sup.3)R.sup.4,
--C(S)N(R.sup.3)R.sup.4, --C(S)N(R.sup.3).sub.2, --C(S)OR.sup.4,
--CO.sub.2R.sup.4, --C(O)R.sup.4, or --C(S)R.sup.4; R.sup.3 is H or
alkyl; and R.sup.4 is H, alkyl, cycloalkyl, aryl, heteroaryl,
aralkyl, or heteroaralkyl.
19. The compound of claim 18, wherein X is O.
20. The compound of claim 18, wherein R.sup.2 is H, alkyl, or
cycloalkyl.
21. The compound of claim 18, wherein R.sup.2 is aryl or
aralkyl.
22. The compound of claim 18, wherein R.sup.2 is
--C(O)N(R.sup.3)R.sup.4, --C(S)N(R.sup.3)R.sup.4,
--C(S)N(R.sup.3).sub.2, --C(S)OR.sup.4, --CO.sub.2R.sup.4,
--C(O)R.sup.4, or --C(S)R.sup.4.
23. The compound of claim 18, wherein R.sup.3 is H.
24. The compound of claim 18, wherein R.sup.4 is alkyl or aryl.
25. The compound of claim 18, wherein X is O, and R.sup.2 is H.
26. The compound formed by a process, comprising the steps of:
admixing about 1 equivalent of chlorocarbonyl sulfenyl chloride,
about 1 equivalent of thiourea, and about 1 equivalent of
triethylamine in a container cooled with a ice-bath at about
0.degree. C. under an atmosphere of argon, stirring the resultant
mixture for about 6 hours, filtering said mixture, concentrating
said mixture to give a residue, and recystallizing said residue
from dichloromethane-hexanes to give the compound.
27. A method of removing an ethylcyanide protecting group,
comprising the steps of: admixing a phosphate compound bearing a
ethylcyanide group with a base in the presence acrylonitrile
scavenger, wherein said acrylonitrile scavenger is a polymer-bound
thiol, 4-n-heptylphenylmethane- thiol, alkane thiol having at least
10 carbon atoms, heteroarylthiol, the sodium salt of an alkyl
thiol, 55wherein R.sup.1 is alkyl; and R.sup.2 is --SH, or
--CH.sub.2SH.
28. The method of claim 27, wherein said acrylonitrile scavenger is
56
29. The method of claim 27, wherein said phosphate compound is an
oligonucleotide.
30. The method of claim 27, wherein said phosphate compound is an
oligonucleotide containing at least one phosphorothioate group.
31. The method of claim 27, wherein said phosphate compound is an
oligomer of ribonucleotides.
32. The method of claim 27, wherein said phosphate is represented
by formula G: 57wherein R.sub.1 is optionally substituted alkyl,
cycloalkyl, heterocycloalkyl, aryl, aralkyl, or alkenyl; R.sub.2 is
optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl,
aralkyl, alkenyl, or --(C(R.sub.3).sub.2).sub.pheterocycloalkyl;
R.sub.3 is H or alkyl; and p is 1, 2, 3, 4, 5, 6, 7, or 8.
33. The method of claim 32, wherein R.sub.1 is an optionally
substituted heterocycloalkyl.
34. The method of claim 32, wherein R.sub.1 is an optionally
substituted ribose.
35. The method of claim 32, wherein R.sub.1 is an optionally
substituted deoxyribose.
36. The method of claim 32, wherein R.sub.1 is a nucleoside,
nucleotide, or oligonucleotide.
37. The method of claim 32, wherein R.sub.1 is 58wherein R'.sub.1
represents independently for each occurrence alkyl, aryl, aralkyl,
or --Si(R.sub.4).sub.3; wherein said alkyl, aryl, and aralkyl group
is optionally substituted with --CN, --NO.sub.2, --CF.sub.3, or
halogen; R.sub.4 is alkyl, aryl, or aralkyl; and n.sup.1 is 1 to 50
inclusive.
38. The method of claim 37, wherein n.sup.1 is 1 to 25
inclusive.
39. The method of claim 37, wherein n.sup.1 is 1 to 15
inclusive.
40. The method of claim 37, wherein n.sup.1 is 1 to 10
inclusive.
41. The method of claim 37, wherein n.sup.1 is 1 to 5
inclusive.
42. A method of removing an amide protecting group from an
oligonucleotide, comprising the steps of: admixing an
oligonucleotide bearing an amide protecting group with a polyamine,
PEHA, PEG-NH.sub.2, Short PEG-NH.sub.2, cycloalkyl amine,
hydroxycycloalkyl amine, hydroxyamine, K.sub.2CO.sub.3/MeOH
microwave, thioalkylamine, thiolated amine,
.beta.-amino-ethyl-sulfonic acid, or the sodium sulfate of
.beta.-amino-ethyl-sulfonic acid.
43. The method of claim 42, wherein said oligonucleotide is an
oligomer of ribonucleotides.
44. A method of purifying an oligonucleotide, comprising the steps
of: annealing a first oligonucleotide with a second oligonucleotide
to form a double-stranded oligonucleotide, subjecting said
double-stranded oligonucleotide to chromatographic
purification.
45. The method of claim 44, wherein said chromatographic
purification is liquid chromatography.
46. The method of claim 44, wherein said chromatographic
purification is high-performance liquid chromatography.
47. The method of claim 44, wherein said first oligonucleotide is
an oligomer of ribonucleotides.
48. The method of claim 44, wherein said second oligonucleotide is
an oligomer of ribonucleotides.
49. The method of claim 44, wherein said first oligonucleotide is
an oligomer of ribonucleotides, and said second oligonucleotide is
an oligomer of ribonucleotides.
50. A method of preparing an oligonucleotide comprising a
dinucleoside unit, comprising the steps of: synthesizing a
dinucleoside group via solution-phase chemistry, attaching said
dinucleoside group to a solid support to form a primer, adding
additional nucleotides to said primer using solid-phase synthesis
techniques.
51. The method of claim 50, wherein each nucleoside residue of said
dinucleoside group is independently a natural or unnatural
nucleoside.
52. The method of claim 50, wherein said dinucleoside group
comprises two nucleoside residues each independently comprising a
sugar and a nucleobase, wherein said sugar is a D-ribose or
D-deoxyribose, and said nucleobase is natural or unnatural.
53. The method of claim 50, wherein said dinucleoside group
comprises two nucleoside residues each independently comprising a
sugar and a nucleobase, wherein said sugar is a L-ribose or
L-deoxyribose, and said nucleobase is natural or unnatural.
54. The method of claim 50, wherein said dinucleoside group
comprises two thymidine residues.
55. The method of claim 50, wherein said dinucleoside group
comprises two deoxythymidine residues.
56. The method of claim 50, wherein said dinucleoside group
comprises two 2'-modified 5-methyl uridine or uridine residues,
wherein the 2'-modifications are 2'-OTBDMS, 2'-OMe, 2'-F,
2'-O--CH2-CH2-O-Me, 2'-O-alkylaminoderivatives.
57. The method of claim 50, wherein said dinucleoside group
comprises a phosphorothioate linkage, phosphorodithioate linkage,
alkyl phosphonate linkage, or boranophosphate linkage.
58. The method of claim 50, wherein said dinucleoside group
comprises a phosphorothioate linkage, alkyl phosphonate linkage, or
boranophosphate linkage; and said dinucleoside group is a single
stereoisomer at the phosphorus atom.
59. The method of claim 50, wherein the linkage between the
nucleoside residues of said dinucleoside group is a 3'-5'
linkage.
60. The method of claim 50, wherein the linkage between the
nucleoside residues of said dinucleoside group is a 2'-5'
linkage.
61. The method of claim 50, wherein said dinucleoside group
comprises two nucleoside residues each independently comprising a
sugar and a nucleobase, wherein said sugar is a D-ribose or
D-deoxyribose, and said nucleobase is natural or unnatural; and the
linkage between the nucleoside residues of said dinucleotide group
is unnatural and non-phosphate.
62. The method of claim 50, wherein said dinucleoside group
comprises two nucleoside residues each independently comprising a
sugar and a nucleobase, wherein said sugar is a L-ribose or
L-deoxyribose, and said nucleobase is natural or unnatural; and the
linkage between the nucleoside residues of said dinucleotide group
is MMI, amide linkage, or guanidinium linkage.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application Ser. No. 60/559,782, filed Apr. 5,
2004; the entirety of which is hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] The study of oligonucleotides is a key area of research for
many academic and industrial laboratories. See S. Agrawal Trends in
Biotechnology 1996, 14, 375-382; J. Marr Drug Discovery Today 1996,
1, 94-102; and W. Rush Science 1997, 276, 1192-1193. The
therapeutic and diagnostic potential of oligonucleotides has
sparked a substantial amount of research activity. One important
application of oligonucleotides is the ability to modulate gene and
protein function in a sequence-specific manner. However, many
research efforts are hampered by the small quantities of
oligonucleotides that are available for study. A method to produce
large quantities of oligonucleotide compounds having high purity
would greatly facilitate oligonucleotide research. Furthermore, it
would be highly useful to be able to prepare derivatives of certain
oligonucleotides. However, the synthesis of oligonucleotides and
their analogs is often a tedious and costly process.
[0003] RNA is generally synthesized and purified by methodologies
based on the following steps: phosphoramidite coupling using
tetrazole as the activating agent, oxidation of the phosphorus
linker to the diester, deprotection of exocyclic amino protecting
groups using NH.sub.4OH, removal of 2'-OH alkylsilyl protecting
groups using tetra-n-butylammonium fluoride (TBAF), and gel
purification and analysis of the deprotected RNA. Examples of
chemical synthesis, deprotection, purification and analysis
procedures are provided by Usman et al. in J. Am. Chem. Soc. 1987,
109, 7845; Scaringe et al. in Nucleic Acids Res. 1990, 18,
5433-5341; Perreault et al. in Biochemistry 1991, 30, 4020-4025;
and Slim and Gait in Nucleic Acids Res. 1991, 19, 1183-1188. Odai
and coworkers describe reverse-phase chromatographic purification
of RNA fragments used to form a ribozyme. See Odai et al. FEBS
Lett. 1990, 267, 150-152. Unfortunately, the aforementioned
chemical synthesis, deprotection, purification and analysis
procedures are time consuming (10-15 min. coupling times), subject
to inefficient activation of the RNA amidites by tetrazole,
incomplete deprotection of the exocyclic amino protecting groups by
NH.sub.4OH, limited by the low capacity of RNA purification using
gel electrophoresis, and further limited by low resolution analysis
of the RNA by gel electrophoresis. Therefore, the need exists for
improved synthetic processes for the synthesis of
oligonucleotides.
[0004] One important class of oligonucleotide analogues are
compounds that have a phosphorothioate in place of the
phosphodiester linkage. Phosphorothioate analogues are important
compounds in nucleic acid research and protein research. For
example, phosphorothioate-containing antisense oligonucleotides
have been used in vitro and in vivo as inhibitors of gene
expression. Site-specific attachment of reporter groups onto the
DNA or RNA backbone is facilitated by incorporation of single
phosphorothioate linkages. Phosphorothioates have also been
introduced into oligonucleotides for mechanistic studies on
DNA-protein and RNA-protein interactions, as well as catalytic
RNAs.
[0005] Introduction of phosphorothioate linkages into
oligonucleotides, assembled by solid-phase synthesis, can be
achieved using either an H-phosphonate approach or a
phosphoramidite approach. The H-phosphonate approach involves a
single sulfur-transfer step, carried out after the desired sequence
has been assembled, to convert all of the internucleotide linkages
to phosphorothioates. Alternatively, the phosphoramidite approach
features a choice at each synthetic cycle: a standard oxidation
provides the normal phosphodiester internucleotide linkage, whereas
a sulfurization step introduces a phosphorothioate at that specific
position in the sequence. An advantage of using phosphoramidite
chemistry is the capability to control the state of each linkage,
P.dbd.O vs. P.dbd.S, in a site-specific manner. The earliest
studies to create phosphorothioates used elemental sulfur, but the
success of the phosphoramidite approach is dependent on the
availability and application of more efficient, more soluble
sulfur-transfer reagents that are compatible with automated
synthesis. Therefore, the need exists for novel sulfur-transfer
reagents that are compatible with automated oligonucleotide
synthesis.
[0006] Another important class of oligonucleotides is
double-stranded RNA which can be used to initiate a type of gene
silencing known as RNA interference (RNAi). RNA interference is an
evolutionarily conserved gene-silencing mechanism, originally
discovered in studies of the nematode Caenorhabditis elegans (Lee
et al, Cell 75:843 (1993); Reinhart et al., Nature 403:901 (2000)).
It is triggered by introducing dsRNA into cells expressing the
appropriate molecular machinery, which then degrades the
corresponding endogenous mRNA. The mechanism involves conversion of
dsRNA into short RNAs that direct ribonucleases to homologous mRNA
targets (summarized, Ruvkun, Science 2294:797 (2001)). This process
is related to normal defenses against viruses and the mobilization
of transposons.
[0007] Double-stranded ribonucleic acids (dsRNAs) are naturally
rare and have been found only in certain microorganisms, such as
yeasts or viruses. Recent reports indicate that dsRNAs are involved
in phenomena of regulation of expression, as well as in the
initiation of the synthesis of interferon by cells (Declerq et al.,
Meth. Enzymol. 78:291 (1981); Wu-Li, Biol. Chem. 265:5470 (1990)).
In addition, dsRNA has been reported to have anti-proliferative
properties, which makes it possible also to envisage therapeutic
applications (Aubel et al., Proc. Natl. Acad. Sci., USA 88:906
(1991)). For example, synthetic dsRNA has been shown to inhibit
tumor growth in mice (Levy et al. Proc. Nat. Acad. Sci. USA,
62:357-361 (1969)), is active in the treatment of leukemic mice
(Zeleznick et al., Proc. Soc. Exp. Biol. Med. 130:126-128 (1969));
and inhibits chemically-induced tumorigenesis in mouse skin
(Gelboin et al., Science 167:205-207 (1970)).
[0008] Treatment with dsRNA has become an important method for
analyzing gene functions in invertebrate organisms. For example,
Dzitoveva et al. showed for the first time, that RNAi can be
induced in adult fruit flies by injecting dsRNA into the abdomen of
anesthetized Drosophila, and that this method can also target genes
expressed in the central nervous system (Mol. Psychiatry
6(6):665-670 (2001)). Both transgenes and endogenous genes were
successfully silenced in adult Drosophila by intra-abdominal
injection of their respective dsRNA. Moreover, Elbashir et al.,
provided evidence that the direction of dsRNA processing determines
whether sense or antisense target RNA can be cleaved by a small
interfering RNA (siRNA)-protein complex (Genes Dev. 15(2): 188-200
(2001)).
[0009] Two recent reports reveal that RNAi provides a rapid method
to test the function of genes in the nematode Caenorhabditis
elegans; and most of the genes on C. elegans chromosome I and III
have now been tested for RNAi phenotypes (Barstead, Curr. Opin.
Chem. Biol. 5(1):63-66 (2001); Tavemarakis, Nat. Genet.
24(2):180-183 (2000); Zamore, Nat. Struct. Biol. 8(9):746-750
(2001).). When used as a rapid approach to obtain loss-of-function
information, RNAi was used to analyze a random set of ovarian
transcripts and have identified 81 genes with essential roles in C.
elegans embryogenesis (Piano et al., Curr. Biol. 10(24):1619-1622
(2000). RNAi has also been used to disrupt the pupal hemocyte
protein of Sarcophaga (Nishikawa et al., Eur. J. Biochem.
268(20):5295-5299 (2001)).
[0010] Like RNAi in invertebrate animals, post-transcriptional gene
silencing (PTGS) in plants is an RNA-degradation mechanism. In
plants, this can occur at both the transcriptional and the
post-transcriptional levels; however, in invertebrates only
post-transcriptional RNAi has been reported to date (Bernstein et
al., Nature 409(6818):295-296 (2001). Indeed, both involve
double-stranded RNA (dsRNA), spread within the organism from a
localized initiating area, to correlate with the accumulation of
small interfering RNA (siRNA) and require putative RNA-dependent
RNA polymerases, RNA helicases and proteins of unknown functions
containing PAZ and Piwi domains.
[0011] Some differences are evident between RNAi and PTGS were
reported by Vaucheret et al., J. Cell Sci. 114(Pt 17):3083-3091
(2001). First, PTGS in plants requires at least two genes--SGS3
(which encodes a protein of unknown function containing a
coil-coiled domain) and MET1 (which encodes a
DNA-methyltransferase)--that are absent in C. elegans, and thus are
not required for RNAi. Second, all of the Arabidopsis mutants that
exhibit impaired PTGS are hyper-susceptible to infection by the
cucumovirus CMV, indicating that PTGS participates in a mechanism
for plant resistance to viruses. RNAi-mediated oncogene silencing
has also been reported to confer resistance to crown gall
tumorigenesis (Escobar et al., Proc. Natl. Acad. Sci. USA,
98(23):13437-13442 (2001)).
[0012] RNAi is mediated by RNA-induced silencing complex (RISC), a
sequence-specific, multicomponent nuclease that destroys messenger
RNAs homologous to the silencing trigger. RISC is known to contain
short RNAs (approximately 22 nucleotides) derived from the
double-stranded RNA trigger, but the protein components of this
activity remained unknown. Hammond et al. (Science
293(5532):1146-1150 (August 2001)) reported biochemical
purification of the RNAi effector nuclease from cultured Drosophila
cells, and protein microsequencing of a ribonucleoprotein complex
of the active fraction showed that one constituent of this complex
is a member of the Argonaute family of proteins, which are
essential for gene silencing in Caenorhabditis elegans, Neurospora,
and Arabidopsis. This observation suggests links between the
genetic analysis of RNAi from diverse organisms and the biochemical
model of RNAi that is emerging from Drosophila in vitro
systems.
[0013] Svoboda et al. reported in Development 127(19):4147-4156
(2000) that RNAi provides a suitable and robust approach to study
the function of dormant maternal mRNAs in mouse oocytes. Mos
(originally known as c-mos) and tissue plasminogen activator mRNAs
are dormant maternal mRNAs that are recruited during oocyte
maturation, and translation of Mos mRNA results in the activation
of MAP kinase. The dsRNA directed towards Mos or TPA mRNAs in mouse
oocytes specifically reduced the targeted mRNA in both a time- and
concentration-dependent manner, and inhibited the appearance of MAP
kinase activity. See also, Svoboda et al. Biochem. Biophys. Res.
Commun. 287(5):1099-1104 (2001).
[0014] The need exists for small interfering RNA (siRNA) conjugates
having improved pharmacologic properties. In particular, the
oligonucleotide sequences have poor serum solubility, poor cellular
distribution and uptake, and are rapidly excreted through the
kidneys. It is known that oligonucleotides bearing the native
phospodiester (P=O) backbone are susceptable to nuclease-mediated
degradation. See L. L. Cummins et al. Nucleic Acids Res. 1995, 23,
2019. The stability of oligonucleotides has been increased by
converting the P.dbd.O linkages to P.dbd.S linkages which are less
susceptible to degradation by nucleases in vivo. Alternatively, the
phosphate group can be converted to a phosphoramidate or alkyl
phosphonate, both of which are less prone to enzymatic degradation
than the native phosphate. See Uhlmann, E.; Peyman, A. Chem. Rev.
1990, 90, 544. Modifications to the sugar groups of the
oligonucleotide can confer stability to enzymatic degradation. For
example, oligonucleotides comprising ribonucleic acids are less
prone to nucleolytic degradation if the 2'-OH group of the sugar is
converted to a methoxyethoxy group. See M. Manoharan Chem Bio Chem.
2002, 3, 1257 and references therein.
[0015] Therefore, the need exists for improved synthetic processes
that facilitate the synthesis of oligonucleotides. Representative
examples of needed improvements are better activating agents for
phosphoramidite coupling of nucleotides, better sulfur-transfer
reagents for preparing phophorothioate-containing oligonucleotides,
and improved procedures for purifying oligonucleotides.
SUMMARY OF THE INVENTION
[0016] The present invention relates to processes and reagents for
oligonucleotide synthesis and purification. One aspect of the
present invention relates to compounds useful for activating
phosphoramidites in oligonucleotide synthesis. Another aspect of
the present invention relates to a method of preparing
oligonucleotides via the phosphoramidite method using an activator
of the invention. Another aspect of the present invention relates
to sulfur-transfer agents. In a preferred embodiment, the
sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one.
Another aspect of the present invention relates to a method of
preparing a phosphorothioate by treating a phosphite with a
sulfur-transfer reagent of the invention. In a preferred
embodiment, the sulfur-transfer agent is a
3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the present
invention relates to compounds that scavenge acrylonitrile produced
during the deprotection of phosphate groups bearing ethylnitrile
protecting groups. In a preferred embodiment, the acrylonitrile
scavenger is a polymer-bound thiol. Another aspect of the present
invention relates to agents used to oxidize a phosphite to a
phosphate. In a preferred embodiment, the oxidizing agent is sodium
chlorite, chloroamine, or pyridine-N-oxide. Another aspect of the
present invention relates to methods of purifying an
oligonucleotide by annealing a first single-stranded
oligonucleotide and second single-stranded oligonucleotide to form
a double-stranded oligonucleotide; and subjecting the
double-stranded oligonucleotide to chromatographic purification. In
a preferred embodiment, the chromatographic purification is
high-performance liquid chromatography.
BRIEF DESCRIPTION OF FIGURES
[0017] FIG. 1 depicts activator compounds useful in
phosphoramidite-mediated oligonucleotide synthesis.
[0018] FIG. 2 depicts activating agents useful in
phosphoramidite-mediated oligonucleotide synthesis.
[0019] FIG. 3 depicts activating agents useful in
phosphoramidite-mediated oligonucleotide synthesis.
[0020] FIG. 4 depicts sulfur-transfer agents useful in preparing
phosphorothioate linkages in oligonucleotides.
[0021] FIG. 5 depicts sulfur-transfer agents useful in preparing
phosphorothioate linkages in oligonucleotides.
[0022] FIG. 6 depicts the results of the synthesis of 25 and 26
with PADS or EDITH. Note that
25=5'-GsCsGGAUCAAACCUCACCAsAsdTsdT-3',
26=5'-UsUsGGUGAGGUUUGAUCCGsCsdTsdT-3', PADS (fresh) indicates that
less than 24 hours hads elapsed since dissolving, PADS (aged)
indicates that greater than 48 hours had elapsed since dissolving,
and the term "nd" indicates that the value was not determined. The
term "PADS" refers to the compound (benzylC(O)S).sub.2. The term
"EDITH" refers to 3-ethoxy-1,2,4-dithiazolidine-5-one.
[0023] FIG. 7 depicts desilylating reagents and assorted bases used
in oligonucleotide synthesis.
[0024] FIG. 8 depicts acrylonitrile quenching agents.
[0025] FIG. 9 depicts a flow chart for siRNA purification and QC.
Note: LC-MS indicates liquid-chromatography mass spectrophotometric
analysis; and CGE indicates capillary gel electrophoresis
analysis.
[0026] FIG. 10 depicts the structure of AL-4112, AL-4180,
AL-DP-4014, AL-2200, AL-2201, AL-DP-4127, AL-2299, AL-2300,
AL-DP-4139, AL-2281, AL-2282, and AL-DP-4140.
[0027] FIG. 11 depicts the first part of the two-strand approach to
purification of AL-DP-4014, the components of which are AL-4112 and
AL-4180.
[0028] FIG. 12 depicts the second part of the two-strand approach
to purification of AL-DP-4014, the components of which are AL-4112
and AL-4180. Note: RP HPLC indicates reverse phase high-performance
liquid chromatographic analysis. IEX HPLC indicates ion exchange
high-performance liquid chromatographic analysis.
[0029] FIG. 13 depicts a reverse phase HPLC chromatogram of
AL-DP-4014.
[0030] FIG. 14 depicts a LC-MS chromatogram of AL-DP-4014.
[0031] FIG. 15 depicts a mass spectrum of the peak at 9.913 minutes
in the LC chromatogram of AL-DP-4014 shown in FIG. 14.
[0032] FIG. 16 depicts a capillary gel electrophoresis chromatogram
of AL-DP-4014.
[0033] FIG. 17 depicts a reverse phase HPLC chromatogram of
AL-DP-4014.
[0034] FIG. 18 depicts an ion exchange chromatogram of
AL-DP-4014.
[0035] FIG. 19 depicts a LC-MS chromatogram of AL-DP-4127.
[0036] FIG. 20 depicts a mass spectrum of the peak at 10.616
minutes in the LC chromatogram of AL-DP-4127 shown in FIG. 19.
[0037] FIG. 21 depicts a mass spectrum of the peak at 12.921
minutes in the LC chromatogram of AL-DP-4127 shown in FIG. 19.
[0038] FIG. 22 depicts a mass spectrum of the peak at 16.556
minutes in the LC chromatogram of AL-DP-4127 shown in FIG. 19.
[0039] FIG. 23 depicts a LC-MS chromatogram of AL-DP-4127.
[0040] FIG. 24 depicts a mass spectrum of a minor contaminant which
appears as a peak at 13.397 minutes in the LC chromatogram of
AL-DP-4127 shown in FIG. 23.
[0041] FIG. 25 depicts a mass spectrum of a minor contaminant which
appears as a peak at 13.201 minutes in the LC chromatogram of
AL-DP-4127 shown in FIG. 23.
[0042] FIG. 26 depicts a capillary gel electrophoresis chromatogram
of AL-DP-4127.
[0043] FIG. 27 depicts a reverse phase HPLC chromatogram of
AL-DP-4127.
[0044] FIG. 28 depicts an ion exchange chromatogram of
AL-DP-4127.
[0045] FIG. 29 depicts a LC-MS chromatogram of AL-DP-4139.
[0046] FIG. 30 depicts a mass spectrum of the peak at 13.005
minutes in the LC chromatogram of AL-DP-4139 shown in FIG. 29.
[0047] FIG. 31 depicts a capillary gel electrophoresis chromatogram
of AL-DP-4139.
[0048] FIG. 32 depicts a reverse phase HPLC chromatogram of
AL-DP-4139.
[0049] FIG. 33 depicts an ion exchange chromatogram of
AL-DP-4139.
[0050] FIG. 34 depicts a LC-MS chromatogram of AL-DP-4140.
[0051] FIG. 35 depicts a mass spectrum of the peak at 13.965
minutes in the LC chromatogram of AL-DP-4140 shown in FIG. 34.
[0052] FIG. 36 depicts a mass spectrum of the peak at 17.696
minutes in the LC chromatogram of AL-DP-4140 shown in FIG. 34.
[0053] FIG. 37 depicts a capillary gel electrophoresis chromatogram
of AL-DP-4140.
[0054] FIG. 38 depicts a reverse phase HPLC chromatogram of
AL-DP-4140.
[0055] FIG. 39 depicts an ion exchange chromatogram of
AL-DP-4140.
[0056] FIG. 40 depicts alternative steps for the two-strand RNA
purification procedure.
[0057] FIG. 41 depicts alternative steps for the two-strand RNA
purification procedure.
[0058] FIG. 42 depicts alternative steps for the two-strand RNA
purification procedure.
[0059] FIG. 43 depicts alternative steps for the two-strand RNA
purification procedure.
[0060] FIG. 44 depicts nucleosides bearing various 2'-protecting
groups. Note: The term "B" indicates protected C, G, A, U, or
5-Me-U. The term "X" indicates CN, NO.sub.2, CF.sub.3, SO.sub.2R,
or CO.sub.2R. The term "X'" indicates CN, NO.sub.2, CF.sub.3, F, or
OMe. The term "Z" indicates H or alkyl. The term "R.sup.1"
indicates oxazole, thiazole, or azole.
[0061] FIG. 45 depicts nucleosides bearing various 2'-protecting
groups which can be removed by enzymatic cleavage. Note: The term
"B" indicates U, 5-Me-U, 5-Me-C, G, or A. The term "X" indicates H,
CN, NO.sub.2, CF.sub.3. The term "X'" indicates H, CN, NO.sub.2,
CF.sub.3, SO.sub.2R, or CO.sub.2R.
[0062] FIG. 46 depicts nucleosides bearing various base protecting
groups amenable to the present invention. Note R is H, OMe, F, MOE,
or TOM.
[0063] FIG. 47 depicts RNA building blocks amenable to the present
invention, wherein the nucleoside has a TOM protecting group.
[0064] FIG. 48 depicts 5'-silyl protected RNA suitable for the
silyl deprotection methods described herein. Note: Base is
N-benzoyladenine, N-acetylcytosine, N-isoputyrylguanine, or uracil.
R is cyclooctyl for guanosine and uridine. R is cyclododecyl for
adenosine and cytidine. See Scaringe, S. A.; Wincott, F, E and
Caruthers, M. H. J. Am. Chem. Soc. 1998, 120, 11820-21.
[0065] FIG. 49 depicts a general procedure for solid-phase RNA
synthesis.
[0066] FIG. 50 depicts sulfur-transfer agents useful in preparing
phosphorothioate linkages in oligonucleotides.
[0067] FIG. 51 depicts building blocks for conjugation of
cholesteryl- and aminoalkyl-hydroxyprolinol at the 5' and 3'-ends
of oligonucleotides. I and III are for 5'-conjugation, and II and
IV are for 3'-conjugation. See Example 8.
DETAILED DESCRIPTION OF THE INVENTION
[0068] The present invention relates to processes and reagents for
oligonucleotide synthesis and purification. Aspects of the
processes and reagents are described in the paragraphs below.
[0069] Activators for Phosphoramidite-Mediated Synthesis of
Oligonucleotides
[0070] The most commonly used process in oligonucleotide synthesis
using solid phase chemistry is the phosphoramidite approach. In a
typical procedure, a phosphoramidite is reacted with a
support-bound nucleotide, or oligonucleotide, in the presence of an
activator. The phosphoroamidite coupling-product is oxidized to
afford a protected phosphate. A variety of different
phosphoramidite derivatives are known to be compatible with this
procedure, and the most commonly used activator is 1H-tetrazole.
Similar processes have been described using a soluble support. See
Bonora et al. Nucleic Acids Res., 1993, 21, 1213-1217. The
phosphoramidite approach is also widely used in solution phase
chemistries for oligonucleotide synthesis. In addition,
deoxyribonucleoside phosphoramidite derivatives have been used in
the synthesis of oligonucleotides. See Beaucage et al. Tetrahedron
Lett. 1981, 22, 1859-1862.
[0071] Phosphoramidites derivatives from a variety of nucleosides
are commercially available. 3'-O-phosphoramidites are the most
widely used amidites, but the synthesis of oligonucleotides can
involve the use of 5'-O- and 2'-O-phosphoramidites. See Wagner et
al. Nuclosides & Nuclotides 1997, 17, 1657-1660 and Bhan et al.
Nuclosides & Nuclotides 1997, 17, 1195-1199. There are also
many phosphoramidites available that are not nucleosides (Cruachem
Inc., Dulles, Va.; Clontech, Palo Alto, Calif., Glen Research,
Sterling, Va., ChemGenes, Wilmington, Mass.).
[0072] Prior to performing the phosphoramidite coupling procedure
described above, the 3'-OH group of the 5'-O-protected nucleoside
has to be phosphityled. Additionally, exocyclic amino groups and
other functional groups present on nucleobase moieties are normally
protected prior to phosphitylation. Traditionally, phosphitylation
of nucleosides is performed by treatment of the protected
nucleosides with a phosphitylating reagent such as
chloro-(2-cyanoethoxy)-N,N-diisopropylami- nophosphine which is
very reactive and does not require an activator or
2-cyanoethyl-N,N,N',N'-tetraiso-propylphosphorodiamidite (bis
amidite reagent) which requires an activator. After preparation,
the nucleoside 3'-O-phosphoramidite is coupled to a 5'-OH group of
a nucleoside, nucleotide, oligonucleoside or oligonucleotide. The
activator most commonly used in phosphitylation reactions is
1H-tetrazole.
[0073] Despite the common usage of 1H-tetrazole in phosphoramidite
coupling and phosphitylation reactions, there are inherent problems
with the use of 1H-tetrazole, especially when performing larger
scale syntheses. For example, 1H-tetrazole is known to be
explosive. According to the material safety data sheet (MSDS)
1H-tetrazole (1H-tetrazole, 98%) can be harmful if inhaled,
ingested or absorbed through the skin. The MSDS also states that
1H-tetrazole can explode if heated above its melting temperature of
155.degree. C. and may form very sensitive explosive metallic
compounds. Hence, 1H-tetrazole requires special handling during its
storage, use, and disposal.
[0074] In addition to its toxicity and explosive nature,
1H-tetrazole is acidic and can cause deblocking of the
5'-O-protecting group and can also cause depurination during the
phosphitylation step of amidite synthesis. See Krotz et al.
Tetrahedron Lett. 1997, 38, 3875-3878. Inadvertent deblocking of
the 5'-O-protecting group is also a problem when
chloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine is used.
Recently, trimethylchlorosilane has been used as an activator in
the phosphitylation of 5'-O-DMT nucleosides with bis amidite
reagent, but this reagent is usually contaminated with HCl which
leads to deprotection and formation of undesired products. See W.
Dabkowski et al. Chem. Comm. 1997, 877. The results for this
phosphitylation are comparable to those for 1H-tetrazole.
Activators with a higher pKa (i.e., less acidic) than 1H-tetrazole
(pKa 4.9) such as 4,5-dicyanoimidazole (pKa 5.2) have been used in
the phosphitylation of 5'-O-DMT thymidine. See C. Vargeese Nucleic
Acids Res. 1998, 26, 1046-1050.
[0075] Another disadvantage to using 1H-tetrazole is the cost of
the reagent. The 2003 Aldrich Chemical Company catalog lists
1H-tetrazole at over seven dollars a gram. Furthermore, due to the
explosive nature of 1H-tetrazole it is only listed as a dilute
solution in acetonitrile. This reagent is used in excess of the
stoichiometric amount of nucleoside present in the reaction mixture
resulting in considerable cost, especially during large-scale
syntheses.
[0076] The solubility of 1H-tetrazole is also a factor in the
large-scale synthesis of phosphoramidites, oligonucleotides and
their analogs. The solubility of 1H-tetrazole is about 0.5 M in
acetonitrile. This low solubility is a limiting factor on the
volume of solvent that is necessary to run a phosphitylation
reaction. An activator having higher solubility would be preferred
in order to minimize the volume of solvents used in the reactions,
thereby lowering the cost and the production of waste effluents.
Furthermore, commonly used 1H-tetrazole (0.45 M solution) for
oligonucleotide synthesis precipitates 1H-tetrazole when the room
temperature drops below 20.degree. C. Inadvertent precipitation of
1H-tetrazole can block the lines on an automated synthesizer
leading to synthesis failure.
[0077] In response to the problems associated with the use of
1H-tetrazole, several activators for phosphoramidite coupling have
been reported. 5-Ethylthio-1H-tetrazole (Wincott, F., et al.
Nucleic Acids Res. 1995, 23, 2677) and
5-(4-nitrophenyl)-1H-tetrazole (Pon, R. T. Tetrahedron Lett. 1987,
28, 3643) have been used for the coupling of sterically crowded
ribonucleoside monomers e.g. for RNA-synthesis. The pKa's for
theses activators are 4.28 and 3.7 (1:1 ethanol:water),
respectively. The use of pyridine hydrochloride/imidazole (pKa 5.23
(water)) as an activator for coupling of monomers was demonstrated
by the synthesis of a dimer (Gryaznov, S. M.; Letsinger, L. M.
Nucleic Acids Res. 1992, 20, 1879). Benzimidazolium triflate (pKa
4.5 (1:1 ethanol:water)) (Hayakawa et al. J. Org. Chem. 1996, 61,
7996-7997) has been used as an activator for the synthesis of
oligonucleotides having bulky or sterically crowded phosphorus
protecting groups such as aryloxy groups. The use of imidazolium
triflate (pKa 6.9 (water)) was demonstrated for the synthesis of a
dimer in solution (Hayakawa, Y.; Kataoka, M. Nucleic Acids and
Related Macromolecules: Synthesis, Structure, Function and
Applications, Sep. 4-9, 1997, Ulm, Germany). The use of
4,5-dicyanoimidazole as an activator for the synthesis of
nucleoside phosphoramidite and several 2'-modified oligonucleotides
including phosphorothioates has also been reported.
[0078] Due to ongoing clinical demand, the synthesis of
oligonucleotides and their analogs is being performed on
increasingly larger scale reactions than in the past. See Crooke et
al. Biotechnology and G enetic Engineering Reviews 1998, 15,
121-157. T here exists a need for phosphoramidite activators that
pose fewer hazards, are less acidic, and less expensive than
activating agents that are currently being used, such as
1H-tetrazole. This invention is directed to this, as well as other,
important ends.
[0079] Activators of the Invention
[0080] The activator compounds of the invention have superior
properties for activating phosphoramidites used in oligonucleotide
synthesis. The activator compounds are generally less explosive and
more soluble in acetonitrile than 1H-tetrazole. In addition, the
activator compounds of the invention required shorter reaction
times in the synthesis of a decamer RNA molecule compared to
1H-tetrazole. See Example 1. In certain instances, the activator
compound of the invention has an electron-withdrawing group to
decrease the pKa of the compound. More acidic activator compounds
can increase the rate of the phosphoramidite coupling reaction in
certain instances. Importantly, shorter reaction times minimize the
opportunity for side reactions to occur, thereby providing the
desired product in higher purity. In addition, activator compounds
of the invention can be the free heterocyclic compound or a mixture
of the activator and its corresponding monoalkyl, dialkyl, or
trialkyl ammonium salt with varying salt to activator molar ratio.
Select preferred activator compounds of the invention are presented
in FIGS. 1, 2, and 3.
[0081] One aspect of the present invention relates to a compound
represented by formula I: 1
[0082] wherein
[0083] X is C(R.sup.6) or N;
[0084] R.sup.1, R.sup.2, R.sup.3, and R.sup.6 each independently
represent H, --NO.sub.2, --CN, --CF.sub.3, --SO.sub.2R.sup.8,
--SR.sup.8, halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,
alkoxyl, --OR.sup.7, --N(R.sup.7).sub.2, --N(R.sup.7)C(O)R.sup.8,
--C(O)R.sup.7, or --CO.sub.2R.sup.8; or an instance of R.sup.1 and
R.sup.2, or R.sup.2 and R.sup.3 can be taken together to form a 4-8
member ring containing 0-4 heteratoms selected from the group
consisting of O, N and S;
[0085] R.sup.4 is absent or represents independently for each
occurrence --(C(R.sup.9).sub.2).sub.nCH.sub.3.Y;
[0086] R.sup.5 is H or --(C(R.sup.9).sub.2).sub.nCH.sub.3;
[0087] R.sup.7 represents independently for each occurrence H,
alkyl, aryl, or aralkyl;
[0088] R.sup.8 represents independently for each occurrence alkyl,
aryl, or aralkyl;
[0089] R.sup.9 represents independently for each occurrence H or
alkyl;
[0090] n represents independently for each occurrence 0 to 15
inclusive; and
[0091] Y represents independently for each occurrence halogen or
R.sup.8CO.sub.2.sup.-.
[0092] In certain embodiments, the present invention relates to the
aforementioned compound, wherein X is C(R.sup.6).
[0093] In certain embodiments, the present invention relates to the
aforementioned compound, wherein X is N.
[0094] In certain embodiments, the present invention relates to the
aforementioned compound, wherein X is C(R.sup.6); R.sup.1, R.sup.2,
R.sup.3, and R.sup.6 each independently represent H, --NO.sub.2, or
--CN; R.sup.4 is absent; and R.sup.5 is H.
[0095] In certain embodiments, the present invention relates to the
aforementioned compound, wherein X is C(R.sup.6); R.sup.1, R.sup.2,
R.sup.3, and R.sup.6 are H; R.sup.4 is absent; and R.sup.5 is
H.
[0096] In certain embodiments, the present invention relates to the
aforementioned compound, wherein X is N; R.sup.1, R.sup.2, and
R.sup.3 are H; R.sup.4 is absent; and R.sup.5 is H.
[0097] Another aspect of the present invention relates to a
compound represented by formula II: 2
[0098] wherein
[0099] R.sup.1 and R.sup.3 each represent independently H,
--NO.sub.2, --CN, --CF.sub.3, --SO.sub.2R.sup.6, --SR.sup.6,
halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,
--N(R.sup.5)C(O)R.sup.6, --C(O).sup.5, or CO.sub.2R.sup.6;
[0100] R.sup.2 is absent or represents independently for each
occurrence --(C(R.sup.7).sub.2).sub.nCH.sub.3.Y;
[0101] R.sup.4 is H or --(C(R.sup.7).sub.2).sub.nCH.sub.3;
[0102] R.sup.5 represents independently for each occurrence H,
alkyl, aryl, or aralkyl;
[0103] R.sup.6 represents independently for each occurrence alkyl,
aryl, or aralkyl;
[0104] R.sup.7 represents independently for each occurrence H or
alkyl;
[0105] n represents independently for each occurrence 0 to 15
inclusive; and
[0106] Y represents independently for each occurrence halogen or
R.sup.6CO.sub.2.sup.-.
[0107] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.1 and R.sup.3 each represent
independently H, --NO.sub.2, or --CN; R.sup.2 is absent; and
R.sup.4 is H.
[0108] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.1 is H; R.sup.3 is
--NO.sub.2; R.sup.2 is absent; and R.sup.4 is H.
[0109] Another aspect of the present invention relates to a
compound represented by formula II: 3
[0110] wherein
[0111] R.sup.1 and R.sup.2 each represent independently H,
--NO.sub.2, --CN, --CF.sub.3, --SO.sub.2R.sup.6, --SR.sup.6,
halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,
--N(R.sup.5)C(O)R.sup.6, --C(O)R.sup.5, or --CO.sub.2R.sup.6;
[0112] R.sup.3 is absent or represents independently for each
occurrence --(C(R.sup.7).sub.2).sub.nCH.sub.3.Y;
[0113] R.sup.4 is H or --(C(R.sup.7).sub.2).sub.nCH.sub.3;
[0114] R.sup.5 represents independently for each occurrence H,
alkyl, aryl, or aralkyl;
[0115] R.sup.6 represents independently for each occurrence alkyl,
aryl, or aralkyl;
[0116] R.sup.7 represents independently for each occurrence H or
alkyl;
[0117] n represents independently for each occurrence 0 to 15
inclusive; and
[0118] Y represents independently for each occurrence halogen or
R.sup.6CO.sub.2.sup.-.
[0119] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.1 and R.sup.2 each represent
independently H, --NO.sub.2, or --CN; R.sup.4 is absent; and
R.sup.4 is H.
[0120] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.1 is H; R.sup.2 is
--NO.sub.2; R.sup.3 is absent; and R.sup.4 is H.
[0121] Another aspect of the present invention relates to a
compound represented by formula IV: 4
[0122] wherein
[0123] R.sup.1 is H, --SR.sup.5, alkyl, aryl, --N(R.sup.4).sub.2,
--(C(R.sup.4).sub.2).sub.mCO.sub.2R.sup.5, --NO.sub.2, --CN,
--CF.sub.3, --SO.sub.2R.sup.5, --SR.sup.5, halogen, alkenyl,
alkynyl, aralkyl, --N(R.sup.4)C(O)R.sup.5, --C(O)R.sup.4, or
--CO.sub.2R.sup.5;
[0124] R.sup.2 is absent or represents independently for each
occurrence --(C(R.sup.6).sub.2).sub.nCH.sub.3.Y;
[0125] R.sup.3 is H or --(C(R.sup.6).sub.2).sub.nCH.sub.3;
[0126] R.sup.4 represents independently for each occurrence H,
alkyl, aryl, or aralkyl;
[0127] R.sup.5 represents independently for each occurrence alkyl,
aryl, or aralkyl;
[0128] R.sup.6 represents independently for each occurrence H or
alkyl;
[0129] n represents independently for each occurrence 0 to 15
inclusive;
[0130] m is 1, 2, 3, 4, 5, 6, 7, or 8; and
[0131] Y represents independently for each occurrence halogen or
R.sup.5CO.sub.2.sup.-.
[0132] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.1 is --SR.sup.5, alkyl,
aryl, --N(R.sup.4).sub.2, or
--(C(R.sup.4).sub.2).sub.mCO.sub.2R.sup.5.
[0133] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.2 is absent, and R.sup.3 is
H.
[0134] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.1 is --SR.sup.5, alkyl,
aryl, --N(R.sup.4).sub.2, or
--(C(R.sup.4).sub.2).sub.mCO.sub.2R.sup.5; R.sup.2 is absent;
R.sup.3 is H; R.sup.4 is H; R.sup.5 is alkyl or aralkyl; and m is
1.
[0135] Another aspect of the present invention relates to a
compound represented by formula V: 5
[0136] wherein
[0137] R.sup.1, R.sup.3, and R.sup.4 each represent independently
H, --NO.sub.2, --CN, --CF.sub.3, --SO.sub.2R.sup.7, --SR.sup.7,
halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,
--N(R.sup.6)C(O)R.sup.5, --C(O)R.sup.6, or --CO.sub.2R.sup.7;
[0138] R.sup.2 is absent or represents independently for each
occurrence --(C(R.sup.8).sub.2).sub.nCH.sub.3.Y;
[0139] R.sup.5 is H or --(C(R.sup.8).sub.2).sub.nCH.sub.3;
[0140] R.sup.6 represents independently for each occurrence H,
alkyl, aryl, or aralkyl;
[0141] R.sup.7 represents independently for each occurrence alkyl,
aryl, or aralkyl;
[0142] R.sup.8 represents independently for each occurrence H or
alkyl;
[0143] n represents independently for each occurrence 0 to 15
inclusive; and
[0144] Y represents independently for each occurrence halogen or
R.sup.7CO.sub.2.sup.-.
[0145] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.2 is absent, and R.sup.5 is
H.
[0146] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.1 is H, R.sup.2 is absent,
R.sup.3 and R.sup.4 are --CN, and R.sup.5 is H.
[0147] Another aspect of the present invention relates to a method
of forming a phosphite compound, comprising the steps of:
[0148] admixing a phosphoramidite, alcohol, and activating agent to
form a phosphite compound, wherein said activating agent is
selected from the group consisting of 6
[0149] wherein
[0150] X is C(R.sup.6) or N;
[0151] R.sup.1, R.sup.2, R.sup.3, and R.sup.6 each independently
represent H, --NO.sub.2, --CN, --CF.sub.3, --SO.sub.2R.sup.8,
--SR.sup.8, halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,
alkoxyl, --OR.sup.7, --N(R.sup.7).sub.2, --N(R.sup.7)C(O)R.sup.8,
--C(O)R.sup.7, or --CO.sub.2R.sup.8; or an instance of R.sup.1 and
R.sup.6, R.sup.1 and R.sup.2, or R.sup.2 and R.sup.3 can be taken
together for form a 4-8 member ring containing 0-4 heteratoms
selected from the group consisting of O, N and S;
[0152] R.sup.4 is absent or represents independently for each
occurrence --(C(R.sup.9).sub.2).sub.nCH.sub.3.Y;
[0153] R.sup.5 is H or --(C(R.sup.9).sub.2).sub.nCH.sub.3;
[0154] R.sup.7 represents independently for each occurrence H,
alkyl, aryl, or aralkyl;
[0155] R.sup.8 represents independently for each occurrence alkyl,
aryl, or aralkyl;
[0156] R.sup.9 represents independently for each occurrence H or
alkyl;
[0157] n represents independently for each occurrence 0 to 15
inclusive; and
[0158] Y represents independently for each occurrence halogen or
R.sup.8CO.sub.2.sup.-; 7
[0159] wherein
[0160] R.sup.1 and R.sup.3 each represent independently H,
--NO.sub.2, --CN, --CF.sub.3, --SO.sub.2R.sup.6, --SR.sup.6,
halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,
--N(R.sup.5)C(O)R.sup.6, --C(O)R.sup.5, or --CO.sub.2R.sup.6;
[0161] R.sup.2 is absent or represents independently for each
occurrence --(C(R.sup.7).sub.2).sub.nCH.sub.3.Y;
[0162] R.sup.4 is H or --(C(R.sup.7).sub.2).sub.nCH.sub.3;
[0163] R.sup.5 represents independently for each occurrence H,
alkyl, aryl, or aralkyl;
[0164] R.sup.6 represents independently for each occurrence alkyl,
aryl, or aralkyl;
[0165] R.sup.7 represents independently for each occurrence H or
alkyl;
[0166] n represents independently for each occurrence 0 to 15
inclusive; and
[0167] Y represents independently for each occurrence halogen or
R.sub.6CO.sub.2.sup.-; 8
[0168] wherein
[0169] R.sup.1 and R.sup.2 each represent independently H,
--NO.sub.2, --CN, --CF.sub.3, --SO.sub.2R.sup.6, --SR.sup.6,
halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,
--N(R.sup.5)C(O)R.sup.6, --C(O)R.sup.5, or --CO.sub.2R.sup.6;
[0170] R.sup.3 is absent or represents independently for each
occurrence --(C(R.sup.7).sub.2).sub.nCH.sub.3.Y;
[0171] R.sup.4 is H or --(C(R.sup.7).sub.2).sub.nCH.sub.3;
[0172] R.sup.5 represents independently for each occurrence H,
alkyl, aryl, or aralkyl;
[0173] R.sup.6 represents independently for each occurrence alkyl,
aryl, or aralkyl;
[0174] R.sup.7 represents independently for each occurrence H or
alkyl;
[0175] n represents independently for each occurrence 0 to 15
inclusive; and
[0176] Y represents independently for each occurrence halogen or
R.sup.6CO.sub.2.sup.-; 9
[0177] wherein
[0178] R.sup.1 is H, --SR.sup.5, alkyl, aryl, --N(R.sup.4).sub.2,
--(C(R.sup.4).sub.2).sub.mCO.sub.2R.sup.5--NO.sub.2, --CN,
--CF.sub.3, --SO.sub.2R.sup.5, --SR.sup.5, halogen, alkenyl,
alkynyl, aralkyl, --N(R.sup.4)C(O)R.sup.5, --C(O)R.sup.4, or
--CO.sub.2R.sup.5;
[0179] R.sup.2 is absent or represents independently for each
occurrence --(C(R.sup.6).sub.2).sub.nCH.sub.3.Y;
[0180] R.sup.3 is H or --(C(R.sup.6).sub.2).sub.nCH.sub.3;
[0181] R.sup.4 represents independently for each occurrence H,
alkyl, aryl, or aralkyl;
[0182] R.sup.5 represents independently for each occurrence alkyl,
aryl, or aralkyl;
[0183] R.sup.6 represents independently for each occurrence H or
alkyl;
[0184] n represents independently for each occurrence 0 to 15
inclusive;
[0185] m is 1, 2, 3, 4, 5, 6, 7, or 8; and
[0186] Y represents independently for each occurrence halogen or
R.sup.5CO.sub.2.sup.-; and 10
[0187] wherein
[0188] R.sup.1, R.sup.3, and R.sup.4 each represent independently
H, --NO.sub.2, --CN, --CF.sub.3, --SO.sub.2R.sup.7, --SR.sup.7,
halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,
--N(R.sup.6)C(O)R.sup.5, --C(O)R.sup.6, or --CO.sub.2R.sup.7;
[0189] R.sup.2 is absent or represents independently for each
occurrence --(C(R.sup.8).sub.2).sub.nCH.sub.3.Y;
[0190] R.sup.5 is H or --(C(R.sup.8).sub.2).sub.nCH.sub.3;
[0191] R.sup.6 represents independently for each occurrence H,
alkyl aryl, or aralkyl;
[0192] R.sup.7 represents independently for each occurrence alkyl,
aryl, or aralkyl;
[0193] R.sup.8 represents independently for each occurrence H or
alkyl;
[0194] n represents independently for each occurrence 0 to 15
inclusive; and
[0195] Y represents independently for each occurrence halogen or
R.sup.7CO.sub.2.sup.-.
[0196] In certain embodiments, the present invention relates to the
aforementioned method, wherein said phosphoramidite is a
3'-nucleoside phosphoramidite, 3'-nucleotide phosphoramidite, or
3'-oligonucleotide phosphoramidite.
[0197] In certain embodiments, the present invention relates to the
aforementioned method, wherein said phosphoramidite is represented
by formula A: 11
[0198] wherein
[0199] R.sub.1 is alkyl, aryl, aralkyl, or --Si(R.sub.5).sub.3;
wherein said alkyl, aryl, and aralkyl group is optionally
substituted with --CN, --NO.sub.2, --CF.sub.3, halogen,
--O.sub.2CR.sub.5, or --OSO.sub.2R.sub.5;
[0200] R.sub.2 is optionally substituted alkyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, or alkenyl;
[0201] R.sub.3 and R.sub.4 each represent independently alkyl,
cycloalkyl, heterocycloalkyl, aryl, or aralkyl; or R.sub.3 and
R.sub.4 taken together form a 3-8 member ring; and
[0202] R.sub.5 is alkyl, cycloalkyl, heterocycloalkyl, aryl, or
aralkyl.
[0203] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is --CH.sub.2CH.sub.2CN.
[0204] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is an optionally substituted
heterocycloalkyl.
[0205] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is an optionally substituted
ribose.
[0206] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is an optionally substituted
deoxyribose.
[0207] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is a nucleoside or
nucleotide.
[0208] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.3 and R.sub.4 are alkyl.
[0209] In certain embodiments, the present invention relates to the
aforementioned method, wherein said alcohol is an optionally
substituted ribose.
[0210] In certain embodiments, the present invention relates to the
aforementioned method, wherein said alcohol is an optionally
substituted deoxyribose.
[0211] In certain embodiments, the present invention relates to the
aforementioned method, wherein alcohol is a nucleoside, nucleotide,
or oligonucleotide.
[0212] In certain embodiments, the present invention relates to the
aforementioned method, wherein said alcohol is represented by
R.sub.5--OH, wherein R.sub.5 is optionally substituted alkyl,
cycloalkyl, heterocycloalkyl, aryl, aralkyl, alkenyl, or
--(C(R.sub.6).sub.2).sub.phe- terocycloalkyl; R.sub.6 is H or
alkyl; and p is 1, 2, 3, 4, 5, 6, 7, or 8.
[0213] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.5 is
--(C(R.sub.6).sub.2).sub.pheter- ocycloalkyl.
[0214] In certain embodiments, the present invention relates to the
aforementioned method, further comprising the step of admixing a
proton-shuttle compound to the mixture comprising said
phosphoramidite, said alcohol, and said activating agent, wherein
the pKa of said proton-shuttle compound is greater than the pKa of
said activating agent, and the pKa of said proton-shuttle compound
is less than the pKa of said phosphoramidite.
[0215] In certain embodiments, the present invention relates to the
aforementioned method, wherein said proton-shuttle compound is a
primary, secondary, or tertiary amine.
[0216] In certain embodiments, the present invention relates to the
aforementioned method, wherein said proton-shuttle compound is
represented by N(R.sub.7)(R.sub.8)R.sub.9, wherein R.sub.7,
R.sub.8, and R.sub.9 each represent independently for each
occurrence H, alkyl, cycloalkyl, aryl, aralkyl, alkenyl; or R.sub.7
and R.sub.8 taken together form a 3-8 membered ring; and R.sub.9 is
H, alkyl, cycloalkyl, aryl, or aralkyl.
[0217] Sulfur-Transfer Reagents
[0218] 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. Sulfurized
oligonucleotides containing phosphorothioate (P.dbd.S) linkages are
of interest in these areas. Phosphorothioate-containing
oligonucleotides are also useful in determining the stereochemical
pathways of certain enzymes which recognize nucleic acids.
[0219] Standard techniques for sulfurization of
phosphorus-containing compounds have been applied to the synthesis
of sulfurized deoxyribonucleotides. Examples of sulfurization
reagents which have been used include elemental sulfur, dibenzoyl
tetrasulfide, 3-H-1,2-benzidithiol-3-one 1,1-dioxide (also known as
Beaucage reagent), tetraethylthiuram disulfide (TETD), and
bis(O,O-diisopropoxy phosphinothioyl) disulfide (known as Stec
reagent). Most of the known sulfurization reagents, however, have
one or more significant disadvantages.
[0220] Elemental sulfur presents problems and is not suitable for
automation because of its 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. The 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. See
R. P. Iyer et al. J. Am. Chem. Soc. 1990, 112, 1253-1254 and R. P.
Iyer et al. J. Org. Chem. 1990, 55, 4693-4699. This can 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 sulfinurization reaction
rate which can be undesirable slow.
[0221] A method for producing a phosphorothioate ester by reaction
of a phosphite ester with an acyl disulfide is disclosed in Dutch
patent application No. 8902521. The disclosed method is applied to
a purified phosphotriester dimer utilizing solution-phase
chemistry. The method is time and labor intensive in that it was
only shown to work in a complex scheme which involved carrying out
the first stage of synthesis (formation of a phosphite) in
acetonitrile, removing the acetonitrile, purifying the intermediate
phosphotriester, and proceeding with the sulfinurization in a
solvent mixture of dichloroethane (DCE) and 2,4,6-collidine.
Furthermore, the method was demonstrated only with a dinucleotide.
There was no suggestion that the Dutch method could be employed
with larger nucleic acid structures, that the same could employ a
common solvent throughout all steps of synthesis, that improved
yields could be obtained, or that the method could be adapted for
conventional automated synthesis without extensive modification of
the scheme of automation. Although acetonitrile is mentioned as one
of several possible solvents, utility of the method for carrying
out all steps of the synthesis in acetonitrile as a common solvent
was not demonstrated. While other publications (Kamer et al.
Tetrahedron Lett. 1989, 30(48), 6757-6760 and Roelen et al. Rech.
Trav. Chim. Pays-Bas 1991, 110, 325-331) show sulfurization of
oligomers having up to six nucleotides, the aforementioned
shortcomings are not overcome by the methods disclosed in these
references.
[0222] A thioanhydride derivative EDITH
(3-ethoxy-1,2,4-dithiazolidine-5-o- ne) is disclosed in U.S. Pat.
No. 5,852,168 (the '168 application). Herein we have established
that, contrary to expectations, this reagent can be used in the
synthesis of 2'-substituted RNA and chimeric RNA. Importantly, even
though these reaction conditions are basic they do not result in
elimination of the 2'-substitutent or other degredation of the
RNA.
[0223] Finally, PADS (phenylacetyl disulfide) is disclosed in U.S.
Pat. Nos. 6,242,591 and 6,114,519. These patents disclose a methof
of sulfurization carried out by contacting a deoxynucleic acid with
an acetyl disulfide for a time suffiient to effect formation of a
phosphorothioate functional group. However, these patents do not
provide examples of such a reaction in the syntheis of RNA
(including 2'-substituted RNA and chimeric RNA), as is demonstrated
herein. In addition, even though these reaction conditions are
basic they do not result in elimination of the 2'-substitutent or
other degredation of the RNA.
[0224] Thus, the need exists for improved methods and reagents for
preparing sulfur-containing phosphorous groups, such as
phosphorothioate linkages, in oligonucleotides and other organic
compounds. The present invention relates to sulfur-transfer
reagents and methods for the formation of phosphorothioates. The
methods are amenable to the formation of phosphorothioate linkages
in oligonucleotides or derivatives, without the need for complex
solvent mixtures, repeated washing, or solvent changes.
[0225] Certain preferred sulfur-transfer reagents of the invention
are presented in FIGS. 4, 5, and 50.
[0226] One aspect of the present invention relates to the compound
represented by formula D:
R.sup.1--XS.paren close-st..sub.nX--R.sup.2 D
[0227] wherein
[0228] X represents independently for each occurrence C(O), C(S),
SO.sub.2, CO.sub.2, CS.sub.2, or SO;
[0229] R.sup.1 and R.sup.2 represent independently for each
occurrence alkyl, cycloalkyl, aryl, heteroaryl; aralkyl,
heteroaralkyl, or --N(R.sup.3)R.sup.4; or R.sup.1 and R.sup.2 taken
together form an optionally subsituted aromatic ring;
[0230] R.sup.3 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
or heteroaralkyl;
[0231] R.sup.4 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
or heteroaralkyl;
[0232] n is 2, 3, or 4; and
[0233] provided that when X is C(O), R.sup.1 is not benzyl.
[0234] In certain embodiments, the present invention relates to the
aforementioned compound, wherein n is 2.
[0235] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.1 and R.sup.2 are phenyl,
benzyl, cyclohexyl, pyrrole, pyridine, or --CH.sub.2-pyridine.
[0236] In certain embodiments, the present invention relates to the
aforementioned compound, wherein X is C(O), R.sup.1 is phenyl, and
R.sup.2 is phenyl.
[0237] In certain embodiments, the present invention relates to the
aforementioned compound, wherein X is SO.sub.2, R.sup.1 is phenyl,
and R.sup.2 is phenyl.
[0238] In certain embodiments, the present invention relates to the
aforementioned compound, wherein X is C(O), R.sup.1 is pyrrole, and
R.sup.2 is pyrrole.
[0239] In certain embodiments, the present invention relates to the
aforementioned compound, wherein X is C(O), and R.sup.1 and R.sup.2
taken together form a phenyl ring.
[0240] Another aspect of the present invention relates to the
compound represented by formula D1:
XS.paren close-st..sub.nY D1
[0241] wherein
[0242] X is CN, P(OR.sup.2).sub.2, P(O)(OR.sup.2).sub.2,
C(O)R.sup.1, C(S)R.sup.1, SO.sub.2R.sup.1, CO.sub.2R.sup.1,
CS.sub.2R.sup.1, or SOR.sup.1;
[0243] Y is CN, P(OR.sup.2).sub.2, or P(O)(OR.sup.2).sub.2;
[0244] R.sup.1 represents independently for each occurrence alkyl,
cycloalkyl, aryl, heteroaryl; aralkyl, heteroaralkyl, or
--N(R.sup.3)R.sup.4;
[0245] R.sup.2 represents independently for each occurrence H,
alkyl, cycloalkyl, aryl, heteroaryl; aralkyl, heteroaralkyl, alkali
metal, or transition metal; or two instances of R.sup.2 taken
together form an alkaline earth metal or transitional metal with an
overall charge of +2.
[0246] R.sup.3 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
or heteroaralkyl;
[0247] R.sup.4 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
or heteroaralkyl; and
[0248] n is 2, 3, or 4.
[0249] In certain embodiments, the present invention relates to the
aforementioned compound, wherein n is 2.
[0250] In certain embodiments, the present invention relates to the
aforementioned compound, wherein Y is CN.
[0251] In certain embodiments, the present invention relates to the
aforementioned compound, wherein Y is P(OR.sup.2).sub.2.
[0252] Another aspect of the present invention relates to the
compound represented by formula E: 12
[0253] wherein X is O or S;
[0254] R.sup.1 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
or heteroaralkyl;
[0255] R.sup.2 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
heteroaralkyl, --C(O)N(R.sup.3)R.sup.4, --C(S)N(R.sup.3)R.sup.4,
--C(S)N(R.sup.3).sub.2, --C(S)OR.sup.4, --CO.sub.2R.sup.4,
--C(O)R.sup.4, or --C(S)R.sup.4;
[0256] R.sup.3 is H or alkyl; and
[0257] R.sup.4 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
or heteroaralkyl.
[0258] In certain embodiments, the present invention relates to the
aforementioned compound, wherein X is O.
[0259] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.2 is H, alkyl, or
cycloalkyl.
[0260] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.2 is aryl or aralkyl.
[0261] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.2 is
--C(O)N(R.sup.3)R.sup.4, --C(S)N(R.sup.3)R.sup.4,
--C(S)N(R.sup.3).sub.2, --C(S)OR.sup.4, --CO.sub.2R.sup.4,
--C(O)R.sup.4, or --C(S)R.sup.4.
[0262] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.3 is H.
[0263] In certain embodiments, the present invention relates to the
aforementioned compound, wherein R.sup.4 is alkyl or aryl.
[0264] In certain embodiments, the present invention relates to the
aforementioned compound, wherein X is O, and R.sup.2 is H.
[0265] Another aspect of the present invention relates to a
compound formed by the process, comprising the steps of:
[0266] admixing about 1 equivalent of chlorocarbonyl sulfenyl
chloride, about 1 equivalent of thiourea, and about 1 equivalent of
triethylamine in a container cooled with a ice-bath at about
0.degree. C. under an atmosphere of argon, stirring the resultant
mixture for about 6 hours, filtering said mixture, concentrating
said mixture to give a residue, and recrystallizing said residue
from dichloromethane-hexanes to give the compound.
[0267] Another aspect of the present invention relates to a method
of forming a phosphorothioate compound, comprising the steps
of:
[0268] admixing a phosphite and a sulfur transfer reagent to form a
phosphorothioate, wherein said sulfur transfer reagent is selected
from the group consisting of MoS.sub.4.Et.sub.3NCH.sub.2Ph, 13
[0269] wherein
[0270] X represents independently for each occurrence C(O), C(S),
SO.sub.2, CO.sub.2, CS.sub.2, or SO;
[0271] R.sup.1 and R.sup.2 represent independently for each
occurrence alkyl, cycloalkyl, aryl, heteroaryl; aralkyl,
heteroaralkyl, or --N(R.sup.3)R.sup.4; or R.sup.1 and R.sup.2 taken
together form an optionally substituted aromatic ring;
[0272] R.sup.3 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
or heteroaralkyl;
[0273] R.sup.4 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
or heteroaralkyl;
[0274] n is 2, 3, or 4; and
[0275] provided that when X is C(O), R.sup.1 is not benzyl;
CS.paren close-st..sub.nY D1
[0276] wherein
[0277] X is CN, P(OR.sup.2).sub.2, P(O)(OR.sup.2).sub.2,
C(O)R.sup.1, C(S)R.sup.1, SO.sub.2R.sup.1, CO.sub.2R.sup.1,
CS.sub.2R.sup.1, or SOR.sup.1;
[0278] Y is CN, P(OR.sup.2).sub.2, or P(O)(OR.sup.2).sub.2;
[0279] R.sup.1 represents independently for each occurrence alkyl,
cycloalkyl, aryl, heteroaryl; aralkyl, heteroaralkyl, or
--N(R.sup.3)R.sup.4;
[0280] R.sup.2 represents independently for each occurrence H,
alkyl, cycloalkyl, aryl, heteroaryl; aralkyl, heteroaralkyl, alkali
metal, or transition metal; or two instances of R.sup.2 taken
together form an alkaline earth metal or transitional metal with an
overall charge of +2.
[0281] R.sup.3 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
or heteroaralkyl;
[0282] R.sup.4 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
or heteroaralkyl; and
[0283] n is 2, 3, or 4; and 14
[0284] wherein
[0285] X is O or S;
[0286] R.sup.1 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
or heteroaralkyl;
[0287] R.sup.2 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
heteroaralkyl, --C(O)N(R.sup.3)R.sup.4, --,
--C(S)N(R.sup.3)R.sup.4, --C(S)N(R.sup.3).sub.2, --C(S)OR.sup.4,
--CO.sub.2R.sup.4, --C(O)R.sup.4, or --C(S)R.sup.4;
[0288] R.sup.3 is H or alkyl; and
[0289] R.sup.4 is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,
or heteroaralkyl.
[0290] In certain embodiments, the present invention relates to the
aforementioned method, wherein said phosphite is represented by
formula F: 15
[0291] wherein
[0292] R.sub.1 is alkyl, aryl, aralkyl, or --Si(R.sub.4).sub.3;
wherein said alkyl, aryl, and aralkyl group is optionally
substituted with --CN, --NO.sub.2, --CF.sub.3, halogen,
--O.sub.2CR.sub.5, or --OSO.sub.2R.sub.4;
[0293] R.sub.2 is optionally substituted alkyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, or alkenyl;
[0294] R.sub.3 is optionally substituted alkyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, alkenyl, or
--(C(R.sub.5).sub.2).sub.phe- terocycloalkyl;
[0295] R.sub.4 is alkyl, cycloalkyl, heterocycloalkyl, aryl, or
aralkyl;
[0296] R.sub.5 is H or alkyl; and
[0297] p is 1, 2, 3, 4, 5, 6, 7, or 8.
[0298] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is --CH.sub.2CH.sub.2CN.
[0299] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is an optionally substituted
heterocycloalkyl.
[0300] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is an optionally substituted
ribose.
[0301] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is an optionally substituted
deoxyribose.
[0302] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is a nucleoside, nucleotide,
or oligonucleotide.
[0303] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is 16
[0304] wherein R'.sub.1 represents independently for each
occurrence alkyl, aryl, aralkyl, or --Si(R.sub.4).sub.3; wherein
said alkyl, aryl, and aralkyl group is optionally substituted with
--CN, --NO.sub.2, --CF.sub.3, or halogen; and n.sup.1 is 1 to 50
inclusive.
[0305] In certain embodiments, the present invention relates to the
aforementioned method, wherein n.sup.1 is 1 to 25 inclusive.
[0306] In certain embodiments, the present invention relates to the
aforementioned method, wherein n.sup.1 is 1 to 15 inclusive.
[0307] In certain embodiments, the present invention relates to the
aforementioned method, wherein n.sup.1 is 1 to 10 inclusive.
[0308] In certain embodiments, the present invention relates to the
aforementioned method, wherein n.sup.1 is 1 to 5 inclusive.
[0309] Acrylonitrile Quenching Agents
[0310] Ethylnitrile is a common phosphate protecting group used in
oligonucleotide synthesis. One of the advantages of this protecting
group is that it can be easily removed by treating the protected
phosphate with a base. The overall transformation is illustrated
below. 17
[0311] However, the acrylonitrile generated from the deprotection
reaction is a good electrophile which can react with nucleophilic
functional groups on the desired nucleotide or oligonucleotide
product. This side-reaction reduces the yield of the desired
product and introduces impurities which can be difficult to remove.
Therefore, the need exists for a reagent that will react
selectively with the acrylonitrile produced during the deprotection
reaction. Representative examples of compounds that would serve as
acrylonitrile scavenging agents during the deprotection reaction
are polymer-bound thiols, alkane thiol having at least 10 carbon a
toms, heteroarylthiol, the sodium salt of an alkane thiol, and
thiols that have sufficiently low volitility so that they are
odorless, e.g., thiols that have a high molecular weight.
[0312] Odorless thiols have been described by K. Nishide and M.
Node in Green Chem. 2004, 6, 142. Some examples of odorless thiols
include dodecanethiol, 4-n-heptylphenylmethanethiol,
4-trimethylsilylphenylmethan- ethiol, and
4-trimethylsilylbenzenethiol. For additional examples see
Development of Odorless Thiols and Sulfides and Their Applications
to Organic Synthesis. Nishide, Kiyoharu; Ohsugi, Shin-ichi;
Miyamoto, Tetsuo; Kumar, Kamal; Node, Manabu. Kyoto Pharmaceutical
University, Misasagi, Yamashina, Kyoto, Japan. Monatshefte fuer
Chemie 2004, 135(2), 189-200. Benzene thiol and benzyl mercaptan
derivatives having only faint odors have been described by Nishide
and coworkers. Representative examples include: 4-RC.sub.6H.sub.4X,
3-RC.sub.6H.sub.4X and 2-C.sub.6H.sub.4X (R=Me.sub.3Si, Et.sub.3Si
or Pr.sub.3Si; X=SH or CH.sub.2SH) See Nishide, Kiyoharu; Miyamoto,
Tetsuo; Kumar, Kamal; Ohsugi, Shin-ichi; Node, Manabu of Kyoto
Pharmaceutical University, Misasagi, Yamashina, Kyoto, Japan. in
"Synthetic Equivalents of Benzenethiol and Benzyl Mercaptan Having
Faint Smell: Odor Reducing Effect of Trialkylsilyl Group."
Tetrahedron Lett. 2002, 43(47), 8569-8573. See Node and coworkers
for a description of odorless l-dodecanethiol. and
p-heptylphenylmethanethiol. Node, Manabu; Kumar, Kamal; Nishide,
Kiyoharu; Ohsugi, Shin-ichi; Miyamoto, Tetsuo. of Kyoto
Pharmaceutical University, Yamashina, Misasagi, Kyoto, Japan. in
"Odorless substitutes for foul-smelling thiols: syntheses and
applications." Tetrahedron Lett. 2001, 42(52), 9207-9210.
[0313] Representative examples of acrylonitrile quenching agents
are shown in FIG. 8.
[0314] One aspect of the present invention relates to a method of
removing an ethylcyanide protecting group, comprising the steps
of:
[0315] admixing a phosphate compound bearing a ethylcyanide group
with a base in the presence acrylonitrile scavenger, wherein said
acrylonitrile scavenger is a polymer-bound thiol,
4-n-heptylphenylmethanethiol, alkane thiol having at least 10
carbon atoms, heteroarylthiol, the sodium salt of an alkyl thiol,
18
[0316] wherein R.sup.1 is alkyl; and R is --SH, or
--CH.sub.2SH.
[0317] In certain embodiments, the present invention relates to the
aforementioned method, wherein said acrylonitrile scavenger is
19
[0318] In certain embodiments, the present invention relates to the
aforementioned method, wherein said phosphate compound is an
oligonucleotide.
[0319] In certain embodiments, the present invention relates to the
aforementioned method, wherein said phosphate compound is an
oligonucleotide containing at least one phosphorothioate group.
[0320] In certain embodiments, the present invention relates to the
aforementioned method, wherein said phosphate compound is an
oligomer of ribonucleotides.
[0321] In certain embodiments, the present invention relates to the
aforementioned method, wherein said phosphate is represented by
formula G: 20
[0322] wherein
[0323] R.sub.1 is optionally substituted alkyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, or alkenyl;
[0324] R.sub.2 is optionally substituted alky, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, alkenyl, or
--(C(R.sub.3).sub.2).sub.phe- terocycloalkyl;
[0325] R.sub.3 is H or alkyl; and
[0326] p is 1, 2, 3, 4, 5, 6, 7, or 8.
[0327] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is an optionally substituted
heterocycloalkyl.
[0328] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is an optionally substituted
ribose.
[0329] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is an optionally substituted
deoxyribose.
[0330] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is a nucleoside, nucleotide,
or oligonucleotide.
[0331] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is 21
[0332] wherein R'.sub.1 represents independently for each
occurrence alkyl, aryl, aralkyl, or --Si(R.sub.4).sub.3; wherein
said alkyl, aryl, and aralkyl group is optionally substituted with
--CN, --NO.sub.2, --CF.sub.3, or halogen; R.sub.4 is alkyl, aryl,
or aralkyl; and n.sup.1 is 1 to 50 inclusive.
[0333] In certain embodiments, the present invention relates to the
aforementioned method, wherein n.sup.1 is 1 to 25 inclusive.
[0334] In certain embodiments, the present invention relates to the
aforementioned method, wherein n.sup.1 is 1 to 15 inclusive.
[0335] In certain embodiments, the present invention relates to the
aforementioned method, wherein n.sup.1 is 1 to 10 inclusive.
[0336] In certain embodiments, the present invention relates to the
aforementioned method, wherein n.sup.1 is 1 to 5 inclusive.
[0337] Methods for Preserving P.dbd.S Bonds
[0338] The P.dbd.S bond of phosphorothioate nucleotides is
sensitive to oxidizing agents, resulting in conversion of the
P.dbd.S bond to a P.dbd.O bond. One aspect of the present invention
relates to methods of preventing unwanted oxidation of the P.dbd.S
bond. One method of preventing unwanted oxidation of the P.dbd.S
bond is to mix a compound which is more readily oxidized than the
P.dbd.S bond of a phosphothioate group with the
phosphorothioate-containing nucleotide. Examples of compounds that
are oxidized more readily than the P.dbd.S bond of a phosphothioate
group include 2-hydroxylethanethiol, EDTA, vitamin E, thiols
including odorless thiols, and vitamin C. Other such compounds can
be readily identified by one of ordinary skill in the art by
comparing the oxidation potential of the P.dbd.S bond of a
phosphorothioate to the antioxidant additive. The antioxidant
should be oxidized more easily than the P.dbd.S bond of the
phosphorothioate.
[0339] Oxidizing agents for Preparing P.dbd.O Bonds
[0340] As described above, oligonucleotides having a p
hosphorothioate linkage are promising therapeutic agents. In
certain instances, it is advantageous to prepare an oligonucleotide
having a mixture of phosphate and phosphorothioate linkages. One
procedure to prepare oligonucleotides having a mixture of phosphate
and phosphorothioate linkages involves attaching a first
oligonucleotide to a second oligonucleotide, wherein the first
oligonucleotide consists of nucleosides linked via phosphorothioate
groups, and the second oligonucleotide consists of nucleosides
linked by phosphite groups. Then, the phosphite groups are oxidized
to give the phosphate linkage. Alternatively, oligonucleotides can
be added sequentially to the first oligonucleotide using the
phosphoramide method. Then, the newly added nucleosides, which are
linked via phosphite groups, are oxidized to convert the phosphite
linkage to a phosphate linkage. One of the most commonly used
oxidizing agents for converting a phosphite to a phosphate is
I.sub.2/amine. Consequently, the I.sub.2/amine reagent is a very
strong oxidant which also oxidizes phosphorothioates to phosphates.
Hence, milder oxidizing agents are needed which will oxidize a
phosphite to a phosphate, but will not oxidize a phosphorothioate
group. Three examples of oxidizing agents that will oxidize a
phosphite to a phosphate, but will not oxidize a phosphorothioate
group, are NaClO.sub.2, chloroamine, and pyridine-N-oxide.
Additional oxidizing agents amenable to the present invention are
CCl.sub.4, CCl.sub.4/water/acetonitrile, CCl.sub.4/water/pyridine,
dimethyl carbonate, mixture of KNO.sub.3/TMSCl in CH.sub.2Cl.sub.2,
NBS, NCS, or a combination of oxidizing agent, an aprotic organic
solvent, a base and water.
[0341] One aspect of the present invention relates to a method of
oxidizing a phosphite to a phosphate, comprising the steps of:
[0342] admixing a phosphite with an oxidizing agent to produce a
phosphate, wherein said oxidizing agent is NaClO.sub.2,
chloroamine, pyridine-N-oxide, CCl.sub.4,
CCl.sub.4/water/acetonitrile, CCl.sub.4/water/pyridine, dimethyl
carbonate, mixture of KNO.sub.3/TMSCl in CH.sub.2Cl.sub.2, NBS, or
NCS.
[0343] In certain embodiments, the present invention relates to the
aforementioned method, wherein said oxidizing agent is NaClO.sub.2,
chloroamine, or pyridine-N-oxide.
[0344] In certain embodiments, the present invention relates to the
aforementioned method, wherein said phosphite is an oligomer of a
nucleoside linked via phosphite groups.
[0345] In certain embodiments, the present invention relates to the
aforementioned method, wherein said nucleoside is a
ribonucleoside.
[0346] In certain embodiments, the present invention relates to the
aforementioned method, wherein said phosphite is represented by
formula H: 22
[0347] wherein
[0348] R.sub.1 is alkyl, aryl, aralkyl, or --Si(R.sub.4).sub.3;
wherein said alkyl, aryl, and aralkyl group is optionally
substituted with --CN, --NO.sub.2, --CF.sub.3, halogen,
--O.sub.2CR.sub.5, or --OSO.sub.2R.sub.4;
[0349] R.sub.2 is optionally substituted alkyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, or alkenyl;
[0350] R.sub.3 is optionally substituted alkyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, alkenyl, or
--(C(R.sub.5).sub.2).sub.phe- terocycloalkyl;
[0351] R.sub.4 is alkyl, cycloalkyl, heterocycloalkyl, aryl, or
aralkyl;
[0352] R.sub.5 is H or alkyl; and
[0353] p is 1, 2, 3, 4, 5, 6, 7, or 8.
[0354] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is --CH.sub.2CH.sub.2CN.
[0355] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is an optionally substituted
heterocycloalkyl.
[0356] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is an optionally substituted
ribose.
[0357] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is an optionally substituted
deoxyribose.
[0358] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is a nucleoside, nucleotide,
or oligonucleotide.
[0359] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is 23
[0360] wherein R'.sub.1, represents independently for each
occurrence alkyl, aryl, aralkyl, or --Si(R.sub.4).sub.3; wherein
said alkyl, aryl, and aralkyl group is optionally substituted with
--CN, --NO.sub.2, --CF.sub.3, or halogen; and n.sup.1 is 1 to 50
inclusive.
[0361] In certain embodiments, the present invention relates to the
aforementioned method, wherein n.sup.1 is 1 to 25 inclusive.
[0362] In certain embodiments, the present invention relates to the
aforementioned method, wherein n.sup.1 is 1 to 15 inclusive.
[0363] In certain embodiments, the present invention relates to the
aforementioned method, wherein n.sup.1 is 1 to 10 inclusive.
[0364] In certain embodiments, the present invention relates to the
aforementioned method, wherein n.sup.1 is 1 to 5 inclusive.
[0365] Agents for the Deprotection/Cleavage of Protecting
Groups
[0366] RNA is often synthesized and purified by methodologies based
on: tetrazole to activate the RNA amidite, NH.sub.4OH to remove the
exocyclic amino protecting groups, n-tetrabutylammonium fluoride
(TBAF) to remove the 2'-OH alkylsilyl protecting groups, and gel
purification and analysis of the deprotected RNA. The RNA compounds
may be formed either chemically or using enzymatic methods.
[0367] One important component of oligonucleotide synthesis is the
installation and removal of protecting groups. Incomplete
installation or removal of a protecting group lowers the overall
yield of the synthesis and introduces impurities that are often
very difficult to remove from the final product. In order to obtain
a reasonable yield of a large RNA molecule (i.e., about 20 to 40
nucleotide bases), the protection of the amino functions of the
bases requires either amide or substituted amide protecting groups.
The amide or substituted amide protecting groups must be stable
enough to survive the conditions of synthesis, and yet removable at
the end of the synthesis. These requirements are met by the
following amide protecting groups: benzoyl for adenosine,
isobutyryl or benzoyl for cytidine, and isobutyryl for guanosine.
The amide protecting groups are often removed at the end of the
synthesis by incubating the RNA in NH.sub.3/EtOH or 40% aqueous
MeNH.sub.2. In the case of the phenoxyacetyl type protecting groups
on guanosine and adenosine and a cetyl protecting groups on
cytidine, an incubation in ethanolic ammonia for 4 h at 65.degree.
C. is used to obtain complete removal of these protecting groups.
However, deprotection procedures using mixtures of NH.sub.3 or
MeNH.sub.2 are complicated by the fact that both ammonia and
methylamine are corrosive gases. Therefore, handling the reagents
can be dangerous, particulary when the reaction is conducted at a
large scale, e.g, manufacturing scale. The volatile nature of
NH.sub.3 and MeNH.sub.2 also requires special procedures to capture
and neutralize any excess NH.sub.3 and MeNH.sub.2 once the
deprotection reaction is complete. Therefore, the need exists for
less volatile reagents that are capable of effecting the amide
deprotection reaction in high yield.
[0368] One aspect of the present invention relates to amino
compounds with relatively low volatility capable of effecting the
amide deprotection reaction. The classes of compounds with the
aforementioned desirable characteristics are listed below. In
certain instances, preferred embodiments within each class of
compounds are listed as well.
[0369] 1) Polyamines
[0370] The polyamine compound used in the invention relates to
polymers containing at least two amine functional groups, wherein
the amine functional group has at least one hydrogen atom. The
polymer can have a wide range of molecular weights. In certain
embodiment, the polyamine compound has a molecular weight of
greater than about 5000 g/mol. In other embodiments, the polyamine
compound compound has a molecular weight of greater than about
10,000; 20,000, or 30,000 g/mol.
[0371] 2) PEHA 24
[0372] 3) PEG-NH.sub.2
[0373] The PEG-NH.sub.2 compound used in the invention relates to
polyethylene glycol polymers comprising amine functional groups,
wherein the amine functional group has at least one hydrogen atom.
The polymer can have a wide range of molecular weights. In certain
embodiment, the PEG-NH.sub.2 compound has a molecular weight of
greater than about 5000 g/mol. In other embodiments, the
PEG-NH.sub.2 compound has a molecular weight of greater than about
10, 000; 20,000, or 30,000 g/mol.
[0374] 4) Short PEG-NH.sub.2
[0375] The short PEG-NH.sub.2 compounds used in the invention
relate to polyethylene glycol polymers comprising amine functional
groups, wherein the amine functional group has at least one
hydrogen atom. The polymer has a relatively low molecular weight
range.
[0376] 5) Cycloalkylamines and Hydroxycycloalkyl amines
[0377] The cycloalkylamines used in the invention relate to
cycloalkyl compounds comprising at least one amine functional
group, wherein the amine functional group has at least one hydrogen
atom. The hydroxycycloalkyl amines used in the invention relate to
cycloalkyl compounds comprising at least one amine functional group
and at least one hydroxyl functional group, wherein the amine
functional group has at least one hydrogen atom. Representative
examples are listed below. 25
[0378] 6) Hydroxyamines
[0379] The hydroxyamines used in the invention relate to alkyl,
aryl, and aralkyl compounds comprising at least one amine
functional group and at least one hydroxyl functional group,
wherein the amine functional group has at least one hydrogen atom.
Representative examples are 9-aminononanol, 4-aminophenol, and
4-hydroxybenzylamine.
[0380] 7) K.sub.2CO.sub.3/MeOH with or without Microwave
[0381] 8) Cysteamine (H.sub.2NCH.sub.2CH.sub.2SH) and Thiolated
Amines
[0382] 9) .beta.-Amino-ethyl-sulfonic acid, or the sodium sulfate
of .beta.-amino-ethyl-sulfonic acid
[0383] One aspect of the present invention relates to a method of
removing an amide protecting group from an oligonucleotide,
comprising the steps of:
[0384] admixing an oligonucleotide bearing an amide protecting
group with a polyamine, PEHA, PEG-NH.sub.2, Short PEG-NH.sub.2,
cycloalkyl amine, hydroxycycloalkyl amine, hydroxyamine,
K.sub.2CO.sub.3/MeOH microwave, thioalkylamine, thiolated amine,
.beta.-amino-ethyl-sulfonic acid, or the sodium sulfate of
.beta.-amino-ethyl-sulfonic acid.
[0385] In certain embodiments, the present invention relates to the
aforementioned method, wherein said oligonucleotide is an oligomer
of ribonucleotides.
[0386] Reagents for Deprotection of a Silyl Group
[0387] As described in the previous section, the use of protecting
groups is a critical component of oligonucleotide synthesis.
Furthermore, the installation and removal of protecting groups must
occur with high yield to minimize the introduction of impurities
into the final product. The Applicants have found that the
following reagents are superior for removing a silyl protecting
group during the synthesis of a oligonucleotide: pyridine-HF,
DMAP-HF, urea-HF, ammonia-HF, ammonium fluoride-HF, TSA-F, DAST,
and polyvinyl pyridine-HF. For example, see FIG. 7 and Example 5.
Other aryl amine-HF reagents useful in this invention include
compounds represented by AA: 26
[0388] wherein
[0389] R.sup.1 is alkyl, aryl, heteroaryl, aralkyl or
heteroaralkyl;
[0390] R.sup.2 is alkyl, aryl, heteroaryl, aralkyl or
heteroaralkyl; and
[0391] R.sup.3 is aryl or heteroaryl.
[0392] For example, aryl amines of the hydrofluoride salts are
selected from the group consisting of (dialkyl)arylamines,
(alkyl)diarylamines, (alkyl)(aralkyl)arylamines,
(diaralkyl)arylamines, (dialkyl)heteroarylamines,
(alkyl)diheteroarylamines, (alkyl)(heteroaryl)arylamines,
(alkyl)(heteroaralkyl)arylamines, (alkyl)(aralkyl)heteroarylamines,
(diaralkyl)heteroarylamines, (diheteoroaralkyl)heteroarylamines,
and (aralkyl)(heteroaralkyl)heteroary- lamines.
[0393] In addition, the aforementioned methods can be practised
with a hydrofluoride salt of a compound selected from the group
consisting of 2728
[0394] wherein, independently for each occurrence: X is O, S,
NR.sup.1 or CR.sub.2; Y is N or CR; R is hydrogen, halogen, alkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl,
heteroaralkyl, --C(.dbd.O)--, --C(.dbd.O)X--, --OR.sup.1,
--N(R.sup.1).sub.2, --SR.sup.1 or --(CH.sub.2).sub.m--R.sup.1;
R.sup.1 is hydrogen, halogen, alkyl, cycloalkyl, heterocycloalkyl,
aryl, heteroaryl, aralkyl or heteroaralkyl; and m is 0-10
inclusive.
[0395] In certain instances, the rate of the deprotection reaction
can be excelerated by conducting the deprotection reaction in the
presence of microwave radiation. As illustrated in Example 6, the
tert-butyldimethylsilyl groups on a 10-mer or 12-mer could be
removed in 2 minutes or 4 minutes, respectively, by treatment with
1 M TBAF in THF, Et.sub.3N--HF, or pyridine-HF/DBU in the presence
of microwave radiation (300 Watts, 2450 MHz).
[0396] One aspect of the present invention relates to a method
removing a silyl protecting group from a oligonucleotide,
comprising the steps of:
[0397] admixing an oligonucleotide bearing a silyl protecting group
with pyridine-HF, DMAP-HF, Urea-HF, TSA-F, DAST, polyvinyl
pyridine-HF, or an aryl amine-HF reagent of formula AA: 29
[0398] wherein
[0399] R.sup.1 is alkyl, aryl, heteroaryl, aralkyl or
heteroaralkyl;
[0400] R.sup.2 is alkyl, aryl, heteroaryl, aralkyl or
heteroaralkyl; and
[0401] R.sup.3 is aryl or heteroaryl.
[0402] In certain embodiments, the present invention relates to the
aforementioned method, wherein said oligonucleotide is an oligomer
of ribonucleotides.
[0403] In certain embodiments, the present invention relates to the
aforementioned method, wherein the reaction is carried out in the
presence of microwave radiation.
[0404] Solid Supports for Oligonucleotide Synthesis
[0405] Solid-phase oligonucleotide synthesis is often performed on
controlled pore glass. However, solid-phase oligonucleotide
synthesis can be carried out on:
[0406] 1) Fractosil
[0407] 2) Non CPG, but silica based solid supports not including
controlled pore glass
[0408] 3) Universal linker on polystyrene beads.
[0409] 4) Argogel
[0410] 5) Argopore
[0411] 6) AM Polystyrene
[0412] 7) Novagel
[0413] 8) PEGA; EM Merck poly(vinyl alcohol) (PVA); and Nitto Denko
polystyrene
[0414] Experiments conducted using ArgoGel (dT succinate loaded on
the support, loading=229.35 .mu.mole/g) revealed that Poly-T
synthesis was quite good. However, the material can be sticky
leading to difficulties when weighing and loading the column.
[0415] Experiments conducted using Argopore-1 (dT succinate loaded
on the support, loading=322.14 .mu.mole/g) revealed that the
material exhibited good flow through, and the material was not
sticky. However, the synthesis coupling efficiency was reduced
after 4-5 couplings.
[0416] Experiments conducted using Argopore-2 (dT succinate loaded
on the support, loading=194 .mu.mole/g) revealed that Poly-T
synthesis was quite good.
[0417] Linkers to Solid Supports
[0418] The oligonucleotide is generally attached to the solid
support via a linking group. Suitable linking groups are an oxalyl
linker, succinyl, dicarboxylic acid linkers, glycolyl linker, or
thioglycolyl linker. Silyl linkers can also be used. See, e.g.,
DiBlasi, C. M.; Macks, D. E.; Tan, D. S. "An Acid-Stable
tert-Butyldiarylsilyl (TBDAS) Linker for Solid-Phase Organic
Synthesis" Org. Lett. 2005; ASAP Web Release Date: 30-Mar.-2005;
(Letter) DOI: 10.1021/o1050370y. DiBlasi et al. describe a robust
tert-butyldiarylsilyl (TBDAS) linker for solid-phase organic
synthesis. Importantly, the TBDAS linker is stable to aqueous HF in
CH.sub.3CN, which allows for the use of orthogonal HF-labile
protecting groups in solid-phase synthetic schemes. In one
approach, they established that cleavage of the linker could be
achieved with tris(dimethylamino)-sulfonium
(trimethylsilyl)-difluoride (TAS-F).
[0419] Solvents
[0420] In response to the growing emphasis on conducting reactions
in solvents that are more environmentally friendly, we have found
that oligonucleotides can be prepared using non-halogenated
solvents. For example, oligonucleotides can be prepared using
toluene, tetrahydrofuran, or 1,4-dioxane as the solvent.
[0421] RNA Synthesis Via H-Phosphonate Coupling
[0422] Synthesis of RNA using the H-phosphonate coupling method
involves reacting a nucleoside substituted with an H-phosphonate
with the hydroxyl group of a second nucleoside in the presence of
an activating agent. One of the most commonly used activating
agents is pivaloyl chloride. However, pivaloyl chloride is not
ideal for large-scale preparations because it is flammable,
corrosive, volatile (bp 105-106.degree. C.), and has a relatively
low flashpoint (Fp 8.degree. C.). Therefore, the need exists for
new activating agents devoid of the aforementioned drawbacks.
[0423] There are currently many useful condensing reagents known to
the art skilled that are amenable to the H-phosphonate method of
oligonucleotide synthesis. See Wada et al. J. Am. Chem. Soc. 1997,
119, 12710-12721. Useful condensing reagents include acid
chlorides, chlorophosphates, carbonates, carbonium type compounds
and phosphonium type compounds. In a preferred embodiment the
condensing reagent is selected from a group consisting of pivaloyl
chloride, adamantyl chloride, 2,4,6-triisopropyl-benzenesulfonyl
chloride, 2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane,
diphenyl phosphorochloridate, bis(2-oxo-3-oxazolidinyl)phosphinic
chloride, bis(pentafluorophenyl)carbonate,
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetram- ethyluronium
hexafluorophosphate, O-(azabenzotriazol-1-yl)-1,1,3,3-tetrame- thyl
uronium hexafluorophosphate,
6-(trifluoromethyl)benzotriazol-1-yl-oxy-
-tris-pyrrolidino-phosphonium hexafluorophosphate,
bromo-tris-pyrrolidino-- phosphonium hexafluorophosphate,
benzotriazole-1-yl-oxy-tris-pyrrolidino-p- hosphonium
hexafluorophosphate and 2-(benzotriazol-1-yloxy)-1,3-dimethyl-2-
-pyrrolidin-1-yl-1,3,2-diazaphospho lidinium hexafluorophosphate.
Additionally, 2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinanane,
NEP-Cl/pyridine/MeCN system has been described. See U.S. Pat. No.
6,639,061.
[0424] The Applicants disclose herein other activating agents that
can be used in the H-phosphonate coupling method. Classes of
compound that are better activating agents include acid chlorides
of long-chain alkyl groups, acid chlorides of aromatic groups, acid
chlorides of alkyl groups substituted with aromatic groups, and
polymer bound acyl chlorides. Representative examples of activiting
agents are decanoyl chloride, dodecanoyl chloride, benzoyl
chloride, 1,2-dibenzyl ethanoyl chloride, naphthoyl chloride,
anthracenecarbonyl chloride, and fluorenecarbonyl chloride.
[0425] The Applicants disclose herein other oxidizing agents that
can be used in the H-phosphonate coupling method. One of the most
common oxidizing agents is iodine. However, iodine is a very strong
oxidizing agent that can lead to unwanted oxidation of sensitive
functional groups on the nucleotide or oligonucleotide.
Representative examples of oxidizing agents that can be used in the
H-phosphonate coupling method include: camphorylsulfonyloxazaridine
and N,O-bis(trimethylsilyl)-acetami- de in MeCN/pyridine,
CCl.sub.4/pyridine/water/MeCN, and DMAP in
pyridine/CCl.sub.4/water.
[0426] Another aspect of the present invention relates to a method
of forming a phosphodiester compound, comprising the steps of:
[0427] admixing a H-phosphonate, alcohol, and activating agent to
form a phosphodiester compound, wherein said activating agent is
selected from the group consisting of C.sub.8-C.sub.20
alkylcarbonyl chloride, arylcarbonyl chloride, and aralkylcarbonyl
chloride.
[0428] In certain embodiments, the present invention relates to the
aforementioned method, wherein said activating agent is decanoyl
chloride, dodecanoyl chloride, benzoyl chloride, 1,2-dibenzyl
ethanoyl chloride, naphthoyl chloride, anthracenecarbonyl chloride,
or fluorenecarbonyl chloride.
[0429] In certain embodiments, the present invention relates to the
aforementioned method, wherein said H-phosphonate is represented by
formula I: 30
[0430] wherein
[0431] R.sub.1 is optionally substituted alkyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, or alkenyl.
[0432] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is an optionally substituted
heterocycloalkyl.
[0433] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is an optionally substituted
ribose.
[0434] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is an optionally substituted
deoxyribose.
[0435] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is a nucleoside or
nucleotide.
[0436] In certain embodiments, the present invention relates to the
aforementioned method, wherein said alcohol is an optionally
substituted ribose.
[0437] In certain embodiments, the present invention relates to the
aforementioned method, wherein said alcohol is an optionally
substituted deoxyribose.
[0438] In certain embodiments, the present invention relates to the
aforementioned method, wherein said alcohol is a nucleoside,
nucleotide, or oligonucleotide.
[0439] In certain embodiments, the present invention relates to the
aforementioned method, wherein said alcohol is represented by
R.sub.5--OH, wherein R.sub.5 is optionally substituted alkyl,
cycloalkyl, heterocycloalkyl, aryl, aralkyl, alkenyl, or
--(C(R.sub.6).sub.2).sub.phe- terocycloalkyl; R.sub.6 is H or
alkyl; and p is 1, 2, 3, 4, 5, 6, 7, or 8.
[0440] In certain embodiments, the present invention relates to the
aforementioned method, wherein said phosphodiester is represented
by formula J: 31
[0441] wherein
[0442] R.sub.1 is optionally substituted alkyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, or alkenyl; and
[0443] R.sub.2 is optionally substituted alkyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, alkenyl, or
--(C(R.sub.6).sub.2).sub.phe- terocycloalkyl; R.sub.6 is H or
alkyl; and p is 1, 2, 3, 4, 5, 6, 7, or 8.
[0444] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is an optionally substituted
heterocycloalkyl.
[0445] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is an optionally substituted
ribose.
[0446] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is an optionally substituted
deoxyribose.
[0447] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.1 is a nucleoside or
nucleotide.
[0448] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is
(C(R.sub.6).sub.2).sub.pheteroc- ycloalkyl.
[0449] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is an optionally substituted
ribose.
[0450] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is an optionally substituted
deoxyribose.
[0451] In certain embodiments, the present invention relates to the
aforementioned method, wherein R.sub.2 is a nucleoside or
nucleotide.
[0452] Purification of Double-Stranded RNA
[0453] One common problem encountered in RNA preparation is
obtaining the desired oligonucleotide in high purity. In many
cases, reactions used to prepare the oligonucleotide do not achieve
100% conversion, or they generate side-products. Unfortunately, the
unreacted starting materials and side-products often have similar
chemical properties, making it very difficult to separate the
desired product from these impurities.
[0454] The most quantitative procedure for recovering a fully
deprotected RNA molecule is by either ethanol precipitation, or an
anion exchange cartridge desalting, as described in Scaringe et al.
Nucleic Acids Res. 1990, 18, 5433-5341. Purification of long RNA
sequences is often performed using a two-step chromatographic
procedure in which the molecule is first purified on a reverse
phase column with either the trityl group at the 5' position on or
off. This purification is carried out using an acetonitrile
gradient with triethylammonium or bicarbonate salts as the aqueous
phase. In the case where the trityl group is still attached to the
RNA during purification, the trityl group may be removed by the
addition of an acid and drying of the partially purified RNA
molecule. The final purification is carried out on an anion
exchange column, using alkali metal perchlorate salt gradients to
elute the fully purified RNA molecule as the appropriate metal
salts, e.g. Na.sup.+, Li.sup.+ etc. A final de-salting step on a
small reverse-phase cartridge completes the purification
procedure.
[0455] In certain instances, purification of long RNA molecules is
carried out using anion exchange chromatography, particularly in
conjunction with alkali perchlorate salts. This system is used to
purify very long RNA molecules. In p articular, it is advantageous
to use a Dionex NUCLEOPAK 100.RTM. or a Pharmacia MONO Q.RTM. anion
exchange column for the purification of RNA by the anion exchange
method. This anion exchange purification may be used following a
reverse-phase purification or prior to reverse-phase purification.
This method results in the formation of a sodium salt of the
ribozyme during the chromatography. Replacement of the sodium
alkali earth salt by other metal salts, e.g., lithium, magnesium or
calcium perchlorate, yields the corresponding salt of the RNA
molecule during the purification.
[0456] In the case of the two-step purification procedure wherein
the first step is a reverse-phase purification followed by an anion
exchange step, the reverse-phase purification is usually perfomed
using polymeric, e.g., polystyrene based, reverse-phase media using
either a 5'-trityl-on or 5'-trityl-off method. Either molecule may
be recovered using this reverse-phase method, and then, once
detritylated, the two fractions may be pooled and submitted to an
anion exchange purification step as described above.
[0457] However, many synthetic RNA products still contain
substantial quantities of impurities despite performing the arduous
purification steps, as described above. Therefore, the need exists
for a new purification procedure to provide RNA in a highly pure
form.
[0458] The Applicants have surprising discovered that impurities in
a composition of single-stranded RNA can be readily removed by HPLC
purification of a mixture of single-stranded RNA that has been
annealed to generate double-stranded RNA. A diagram illustrating
the overall procedure is presented in FIG. 9. The structure of
AL-4112, AL-4180, AL-DP-4014, AL-2200, AL-22-1, AL-DP-4127,
AL-2299, AL-2300, AL-DP-4139, AL-2281, AL-2282, and AL-DP-4140 is
presented in FIG. 10. The specific procedure for the purification
of AL-DP-4014, the components of which are AL-4112 and AL-4180, is
shown in FIGS. 11 and 12. AL-DP-4127, AL-DP-4139, and AL-DP-4140
were also purified using the procedures described in FIGS. 9, 11,
and 12. The results from the analyses are presented in FIGS.
19-39.
[0459] Alternative procedures of RNA purification using the
two-strand method are presented in FIGS. 40-43.
[0460] One aspect of the present invention relates to a method of
purifying an oligonucleotide, comprising the steps of:
[0461] annealing a first oligonucleotide with a second
oligonucleotide to form a double-stranded oligonucleotide,
subjecting said double-stranded oligonucleotide to chromatographic
purification.
[0462] In certain embodiments, the present invention relates to the
aforementioned method, wherein said annealing a first
oligonucleotide with a second oligonuclotide is done at a
temperature between a first temperature and a second temperature,
wherein said first temperature is about the T.sub.m of a
double-stranded oligonucleotide consisting of said first
oligonucleotide and a third oligonuclotide, wherein said third
oligonuclotide is the antisense sequence corresponding to the first
oligonuclotide, and said second temperature is about 5 degrees
below said first temperature.
[0463] In certain embodiments, the present invention relates to the
aforementioned method, wherein said chromatographic purification is
liquid chromatography.
[0464] In certain embodiments, the present invention relates to the
aforementioned method, wherein said chromatographic purification is
high-performance liquid chromatography.
[0465] In certain embodiments, the present invention relates to the
aforementioned method, wherein said first oligonucleotide is an
oligomer of ribonucleotides.
[0466] In certain embodiments, the present invention relates to the
aforementioned method, wherein said second oligonucleotide is an
oligomer of ribonucleotides.
[0467] In certain embodiments, the present invention relates to the
aforementioned method, wherein said first oligonucleotide is an
oligomer of ribonucleotides, and said second oligonucleotide is an
oligomer of ribonucleotides.
[0468] RNA HPLC Methods
[0469] As described above, high-peformance liquid chromatography
(HPLC) is an important method used in the purification of RNA
compounds. A large variety of columns, solvents, additives, and
conditions have been reported for purifying oligonucleotides.
However, current procedures for purifying RNA compounds are not
able to separate the RNA compound from significant amounts of
impurities. The Applicants report herein improvements to existing
HPLC procedures thereby providing the RNA compound with
substantially fewer impurities:
[0470] 1) Use tetrabutylammonium acetate as ion-pairing agent in
analytical HPLC separations of oligonucleotides. See M. Gilar for
use of tetrabutylammonium acetate in analytical HPLC separations.
M. Gilar Analytical Biochemistry 2001, 298, 196-206.
[0471] 2) HPLC purification in DMT-off mode with C-18 column or C-4
column for lipophilic conjugates of RNA compounds.
[0472] 3) HPLC purification of RNA compounds using ethanol or
acetonitrile as the solvent.
[0473] 2'-Protecting Groups for RNA Synthesis
[0474] As described above, protecting groups play a critical role
in RNA synthesis. The Applicants describe herein several new
protecting groups that can be used in RNA synthesis. One class of
2'-protecting groups that can be used in RNA synthesis is
carbonates. One preferred carbonate is propargyl carbonate shown
below. 32
[0475] The propargyl carbonate can be removed using
benzyltriethylammonium tetrathiomolybdate as described in Org.
Lett. 2002, 4, 4731.
[0476] Another class of 2'-protecting groups that can be used in
RNA synthesis is acetals. Acetal groups can be deprotected using
aqueous acid. Several representative acetal protecting groups are
shown below. See FIG. 44 for additional examples. 33
[0477] Other 2'-protecting groups that can be used in RNA synthesis
are shown below. 34
[0478] In addition, a bis-silyl strategy could be used in RNA
synthesis. This strategy involves protecting both the 2'-hydroxyl
group of the ribose and the phosphate attached to the 3'-position
of the ribose with a silyl group. A representative example is
presented below in FIG. 44.
[0479] Representative examples of the above-mentioned protecting
groups on various nucleosides are presented in FIG. 44.
[0480] Alternate 5'-Protecting Groups
[0481] In place of dimethoxytrityl (DMT), monomethoxytrityl (MMT),
9-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl
(Mox) and their analogs can be employed.
[0482] Alternate Base-Protecting Groups
[0483] 1) Nps and DNPS groups (Fukuyama)
[0484] 2) phenacetyl (removal by penicillin G acylase)
[0485] Enzymatic Methods for Removal of Protecting Groups
[0486] Another aspect of the present invention relates to
protecting groups which can be removed enzymatically. Aralkyl
esters represented by --O.sub.2CCH.sub.2R, wherein R is phenyl,
pyridinyl, aniline, quinoline, or isoquinoline can be removed from
the 2'-position of a nucleoside by enzymatic cleavage using
penicillin G acylase. Representative examples of nucleosides
bearing aralkyl ester protecting groups at the 2'-position of the
ribose ring are presented in FIG. 45. In addition, certain internal
amidites, including those shown in FIG. 45, can be removed by
enzymatic cleavage.
[0487] One aspect of the present invention relates to a method of
removing a protecting group, comprising the steps of:
[0488] admixing an optionally substituted ribose bearing a
protecting group at the C2 position with an enzyme to produce an
optionally substituted ribose bearing a hydroxyl group at the C2
position.
[0489] In certain embodiments, the present invention relates to the
aforementioned method, wherein said protecting group is an aralkyl
ester.
[0490] In certain embodiments, the present invention relates to the
aforementioned method, wherein said protecting group is represented
by the formula --O.sub.2CCH.sub.2R, wherein R is phenyl, pyridinyl,
aniline, quinoline, or isoquinoline.
[0491] In certain embodiments, the present invention relates to the
aforementioned method, wherein said enzyme is penicillin G
acylase.
[0492] In certain embodiments, the present invention relates to the
aforementioned method, wherein said ribose is a ribonucleotide
oligomer.
[0493] Synthesis of Oligonucleotides Containing a TT Unit
[0494] In certain embodiments, it is preferable to prepare an
oligonucleotide comprising two adjacent thymidine nucleotides. In a
more preferred embodiment, the thymidine nucleotides are located at
the 3' end of the oligonucleotide. The thymidine-thymidine (TT)
nucleotide unit can be prepared using solution-phase chemistry, and
then the TT unit is attached to a solid support. In certain
embodiments, the TT unit is linked via a phosphorothioate group. In
certain instances, the different stereoisomers of the
phosphorothioate TT unit may be separated prior to attachment of
the TT unit to the solid support. The remainder of the
oligonucleotide strand can be synthesized via standard solid-phase
synthesis techniques using the TT-support bound unit as a primer.
In certain instances, the thymidine-thymidine nucleotide unit is
made of deoxythymidine residues.
[0495] One aspect of the present invention relates to a method of
preparing an oligonucleotide comprising a dinucleoside unit,
comprising the steps of:
[0496] synthesizing a dinucleoside group via solution-phase
chemistry, attaching said dinucleoside group to a solid support to
form a primer, adding additional nucleotides to said primer using
solid-phase synthesis techniques.
[0497] In certain embodiments, the present invention relates to the
aforementioned method, wherein each nucleoside residue of said
dinucleoside group is independently a natural or unnatural
nucleoside.
[0498] In certain embodiments, the present invention relates to the
aforementioned method, wherein said dinucleoside group comprises
two nucleoside residues each independently comprising a sugar and a
nucleobase, wherein said sugar is a D-ribose or D-deoxyribose, and
said nucleobase is natural or unnatural.
[0499] In certain embodiments, the present invention relates to the
aforementioned method, wherein said dinucleoside group comprises
two nucleoside residues each independently comprising a sugar and a
nucleobase, wherein said sugar is an L-ribose or L-deoxyribose, and
said nucleobase is natural or unnatural.
[0500] In certain embodiments, the present invention relates to the
aforementioned method, wherein said dinucleoside group comprises
two thymidine residues.
[0501] In certain embodiments, the present invention relates to the
aforementioned method, wherein said dinucleoside group comprises
two deoxythymidine residues.
[0502] In certain embodiments, the present invention relates to the
aforementioned method, wherein said dinucleoside group comprises
two 2'-modified 5-methyl uridine or uridine residues, wherein the
2'-modifications are 2'-O-TBDMS, 2'-OMe, 2'-F, 2'-O--CH2--CH2-O-Me,
or 2'-O-alkylamino derivatives.
[0503] In certain embodiments, the present invention relates to the
aforementioned method, wherein said dinucleoside group comprises a
phosphorothioate linkage, phosphorodithioate linkage, alkyl
phosphonate linkage, or boranophosphate linkage.
[0504] In certain embodiments, the present invention relates to the
aforementioned method, wherein said dinucleoside group comprises a
phosphorothioate linkage, alkyl phosphonate linkage, or
boranophosphate linkage; and said dinucleoside group is a single
stereoisomer at the phosphorus atom.
[0505] In certain embodiments, the present invention relates to the
aforementioned method, wherein the linkage between the nucleoside
residues of said dinucleoside group is a 3'-5' linkage.
[0506] In certain embodiments, the present invention relates to the
aforementioned method, wherein the linkage between the nucleoside
residues of said dinucleoside group is a 2'-5' linkage.
[0507] In certain embodiments, the present invention relates to the
aforementioned method, wherein said dinucleoside group comprises
two nucleoside residues each independently comprising a sugar and a
nucleobase, wherein said sugar is a D-ribose or D-deoxyribose, and
said nucleobase is natural or unnatural; and the linkage between
the nucleoside residues of said dinucleotide group is unnatural and
non-phosphate.
[0508] In certain embodiments, the present invention relates to the
aforementioned method, wherein said dinucleoside group comprises
two nucleoside residues each independently comprising a sugar and a
nucleobase, wherein said sugar is an L-ribose or L-deoxyribose, and
said nucleobase is natural or unnatural; and the linkage between
the nucleoside residues of said dinucleotide group is MMI, amide
linkage, or guanidinium linkage.
[0509] Improved Procedures for the Synthesis of Nucleosides,
Nucleotides, and Oligonucleotides
[0510] Importantly, any one of the above-mentioned improvements can
be used alone with standard methods of preparing nucleosides,
nuclotides, and oligonucleotides, or more than one of the
above-mentioned improvements can be used together with standard
methods of preparing nucleosides, nuclotides, and oligonucleotides.
Furthermore, one of ordinary skill in the art can readily determine
the optimal conditions for each of the improvements described
above.
[0511] General Description of Oligonucleotides
[0512] As described above, the present invention relates to
processes and reagents for oligonucleotide synthesis and
purification. The following description is meant to briefly
describe some of the major types and structural features of
oligonucleotides. Importantly, the following section is only
representative and not meant to limit the scope of the present
invention.
[0513] Oligonucleotides can be made of ribonucleotides,
deoxyribonucleotides, or mixtures of ribonucleotides and
deoxyribonucleotides. The nucleotides can be natural or unnatural.
Oligonucleotides can be single stranded or double stranded. Various
modifications to the sugar, base, and phosphate components of
oligonucleotides are described below. As defined here,
oligonucleotides having modified backbones or internucleoside
linkages include those that retain a phosphorus atom in the
backbone and those that do not have a phosphorus atom in the
backbone. For the purposes the invention, modified oligonucleotides
that do not have a phosphorus atom in their intersugar backbone can
also be considered to be oligonucleosides.
[0514] Specific oligonucleotide chemical modifications are
described below. It is not necessary for all positions in a given
compound to be uniformly modified, and in fact more than one of the
following modifications may be incorporated in a single siRNA
compound or even in a single nucleotide thereof.
[0515] Preferred modified internucleoside linkages or backbones
include, for example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalklyphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free-acid forms are also
included.
[0516] 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; 5,177,196; 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, each of which is herein incorporated by
reference.
[0517] Preferred modified internucleoside linkages or backbones
that do not include a phosphorus atom therein (i.e.,
oligonucleosides) have backbones that are formed by short chain
alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl
or cycloalkyl intersugar linkages, or one or more short chain
heteroatomic or heterocyclic intersugar linkages. These include
those having morpholino linkages (formed in part from the sugar
portion of a nucleoside); siloxane backbones; sulfide, sulfoxide
and sulfone backbones; formacetyl and thioformacetyl backbones;
methylene formacetyl and thioformacetyl backbones; alkene
containing backbones; sulfamate backbones; methyleneimino and
methylenehydrazino backbones; sulfonate and sulfonamide backbones;
amide backbones; and others having mixed N, O, S and CH.sub.2
component parts.
[0518] Representative United States patents that teach the
preparation of the above oligonucleosides include, but are not
limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and
5,677,439, each of which is herein incorporated by reference.
[0519] In other preferred oligonucleotide mimetics, both the sugar
and the internucleoside linkage, i.e., the backbone, of the
nucleoside units are replaced with novel groups. The nucleobase
units are maintained for hybridization with an appropriate nucleic
acid target compound. One such oligonucleotide, an oligonucleotide
mimetic, that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide-containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to atoms of the amide portion of the
backbone. Representative United States patents that teach the
preparation of PNA compounds include, but are not limited to, U.S.
Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is
herein incorporated by reference. Further teaching of PNA compounds
can be found in Nielsen et al., Science, 1991, 254, 1497.
[0520] Some preferred embodiments of the present invention employ
oligonucleotides with phosphorothioate linkages and
oligonucleosides with heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2--[known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2--, and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0521] Oligonucleotides may additionally or alternatively comprise
nucleobase (often referred to in the art simply as "base")
modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases include the purine bases adenine (A) and
guanine (G), and the pyrimidine bases thymine (T), cytosine (C),
and uracil (U). 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.
[0522] 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. Certain of these nucleobases are particularly useful
for increasing the binding affinity of the oligonucleotides of the
invention. 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'-O-methoxyethyl sugar modifications.
[0523] Representative United States patents that teach the
preparation of certain of the above-noted modified nucleobases as
well as other modified nucleobases include, but are not limited to,
the above noted U.S. Pat. No. 687,808, as well as U.S. Pat. Nos.
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; 5,681,941; and
5,808,027; all of which are hereby incorporated by reference.
[0524] The oligonucleotides may additionally or alternatively
comprise one or more substituted sugar moieties. Preferred
oligonucleotides comprise one of the following at the 2' position:
OH; F; O-, S-, or N-alkyl, O-, S-, or N-alkenyl, or O, S- or
N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be
substituted or unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2
to C.sub.10 alkenyl and alkynyl. Particularly preferred are
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, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.su- b.3)].sub.2, where n and
m are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: 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.2 CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3,
NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. a preferred
modification includes 2'-methoxyethoxy
[2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE] (Martin et al. Helv. Chim. Acta
1995, 78, 486), i.e., an alkoxyalkoxy group. a further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3)- .sub.2 group, also known as
2'-DMAOE, as described in U.S. Pat. No. 6,127,533, filed on Jan.
30, 1998, the contents of which are incorporated by reference.
[0525] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-O-methoxyethyl, 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 the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides.
[0526] As used herein, the term "sugar substituent group" or
"2'-substituent group" includes groups attached to the 2'-position
of the ribofuranosyl moiety with or without an oxygen atom. Sugar
substituent groups include, but are not limited to, fluoro,
O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino,
O-alkylaminoalkyl, O-alkyl imidazole and polyethers of the formula
(O-alkyl).sub.m, wherein 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 hereby incorporated by reference in its entirety.
Further sugar modifications are disclosed by Cook (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. Pat. No. 6,166,197, entitled "Oligomeric
Compounds having Pyrimidine Nucleotide(s) with 2' and 5'
Substitutions," hereby incorporated by reference in its
entirety.
[0527] Additional sugar substituent groups amenable to the present
invention include 2'-SR and 2'-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 is disclosed by Hamm et al. (J. Org. Chem.
1997, 62, 3415-3420). 2'-NR nucleosides are disclosed by
Goettingen, M. J. Org. Chem., 1996, 61, 6273-6281; and Polushin et
al. Tetrahedron Lett. 1996, 37, 3227-3230. Further representative
2'-substituent groups amenable to the present invention include
those having one of formula I or II: 35
[0528] wherein,
[0529] E is C.sub.1-C.sub.10 alkyl, N(Q.sub.3)(Q.sub.4) or
N.dbd.C(Q.sub.3)(Q.sub.4); each Q.sub.3 and Q.sub.4 is,
independently, H, C.sub.1-C.sub.10 alkyl, dialkylaminoalkyl, a
nitrogen protecting group, a tethered or untethered conjugate
group, a linker to a solid support; or Q.sub.3 and Q.sub.4,
together, form a nitrogen protecting group or a ring structure
optionally including at least one additional heteroatom selected
from N and 0;
[0530] q.sub.1 is an integer from 1 to 10;
[0531] q.sub.2 is an integer from 1 to 10;
[0532] q.sub.3 is 0 or 1;
[0533] q.sub.4 is 0, 1 or 2;
[0534] each Z.sub.1, Z.sub.2 and Z.sub.3 is, independently,
C.sub.4-C.sub.7 cycloalkyl, C.sub.5-C.sub.14 aryl or
C.sub.3-C.sub.15 heterocyclyl, wherein the heteroatom in said
heterocyclyl group is selected from oxygen, nitrogen and
sulfur;
[0535] Z.sub.4 is OM.sub.1, SM.sub.1, or N(M.sub.1).sub.2; each
M.sub.1 is, independently, H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 haloalkyl, C(.dbd.NH)N(H)M.sub.2,
C(.dbd.O)N(H)M.sub.2 or OC(.dbd.O)N(H)M.sub.2; M.sub.2 is H or
C.sub.1-C.sub.8 alkyl; and
[0536] Z.sub.5 is C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10
haloalkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl,
C.sub.6-C.sub.14 aryl, N(Q.sub.3)(Q.sub.4), OQ.sub.3, halo,
SQ.sub.3 or CN.
[0537] Representative 2'-O-sugar substituent groups of formula I
are disclosed in U.S. Pat. No. 6,172,209, entitled "Capped
2'-Oxyethoxy Oligonucleotides," hereby incorporated by reference in
its entirety. Representative cyclic 2'-O-sugar substituent groups
of formula II are disclosed in U.S. Pat. No. 6,271,358, filed Jul.
27, 1998, entitled "RNA Targeted 2'-Modified Oligonucleotides that
are Conformationally Preorganized," hereby incorporated by
reference in its entirety.
[0538] Sugars having O-substitutions on the ribosyl ring are also
amenable to the present invention. Representative substitutions for
ring 0 include, but are not limited to, NH, NR, S, CH.sub.2, CHF,
and CF.sub.2. See, e.g., Secrist et al., Abstract 21, Program &
Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides
and their Biological Applications, Park City, Utah, Sep. 16-20,
1992.
[0539] Oligonucleotides may also have sugar mimetics, such as
cyclobutyl moieties, hexoses, cyclohexenyl 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;
5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,700,920;
and 5,859,221, all of which are hereby incorporated by
reference.
[0540] Additional modifications may also be made at other positions
on the oligonucleotide, particularly the 3' position of the sugar
on the 3' terminal nucleotide. For example, one modification of
oligonucleotides involves chemically linking to the oligonucleotide
one or more additional moieties or conjugates which enhance the
activity, cellular distribution or cellular uptake of the
oligonucleotide. Such moieties include but are not limited to 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-5-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., Nuc. 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), or 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).
[0541] Representative United States patents that teach the
preparation of such oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928; and 5,688,941, each of which is herein incorporated by
reference.
[0542] Oligonucleotides can be substantially chirally pure with
regard to particular positions within the oligonucleotides.
Examples of substantially chirally pure oligonucleotides include,
but are not limited to, those having phosphorothioate linkages that
are at least 75% Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361)
and those having substantially chirally pure (Sp or Rp)
alkylphosphonate, phosphoramidate or phosphotriester linkages
(Cook, U.S. Pat. Nos. 5,212,295 and 5,521,302).
[0543] Synthetic RNA molecules and derivatives thereof that
catalyze highly specific endoribonuclease activities are known as
ribozymes. (See, generally, U.S. Pat. No. 5,543,508 to Haseloff et
al., issued Aug. 6, 1996, and U.S. Pat. No. 5,545,729 to Goodchild
et al., issued Aug. 13, 1996.) The cleavage reactions are catalyzed
by the RNA molecules themselves. In naturally occurring RNA
molecules, the sites of self-catalyzed cleavage are located within
highly conserved regions of RNA secondary structure (Buzayan et
al., Proc. Natl. Acad. Sci. U.S.A., 1986, 83, 8859; Forster et al.,
Cell, 1987, 50, 9). Naturally occurring autocatalytic RNA molecules
have been modified to generate ribozymes which can be targeted to a
particular cellular or pathogenic RNA molecule with a high degree
of specificity. Thus, ribozymes serve the same general purpose as
antisense oligonucleotides (i.e., modulation of expression of a
specific gene) and, like oligonucleotides, are nucleic acids
possessing significant portions of single-strandedness. That is,
ribozymes have substantial chemical and functional identity with
oligonucleotides and are thus considered to be equivalents for
purposes of the present invention.
[0544] In certain instances, the oligonucleotide may be modified by
a moiety. A number of moieties have been conjugated to
oligonucleotides in order to enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide, and
procedures for performing such conjugations are available in the
scientific literature. Such moieties have included lipid moieties,
such as cholesterol (Letsinger et al., Proc. Natl. A cad. Sci. USA,
1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.
Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-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-glycero-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), or
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). Representative United States
patents that teach the preparation of such oligonucleotide
conjugates have been listed above. Typical conjugation protocols
involve the synthesis of oligonucleotides bearing an aminolinker at
one or more positions of the sequence. The amino group is then
reacted with the molecule being conjugated using appropriate
coupling or activating reagents. The conjugation reaction may be
performed either with the oligonucleotide still bound to the solid
support or following cleavage of the oligonucleotide in solution
phase. Purification of the oligonucleotide conjugate by HPLC
typically affords the pure conjugate.
[0545] One type of double-stranded RNA is short interfering RNA
(siRNA). In certain embodiments, the backbone of the
oligonucleotide can be modified to improve the therapeutic or
diagnostic properties of the siRNA compound. The two strands of the
siRNA compound can be complementary, partially complementary, or
chimeric oligonucleotides. In certain embodiments, at least one of
the bases or at least one of the sugars of the oligonucleotide has
been modified to improve the therapeutic or diagnostic properties
of the siRNA compound.
[0546] The siRNA agent can include a region of sufficient homology
to the target gene, and be of sufficient length in terms of
nucleotides, such that the siRNA agent, or a fragment thereof, can
mediate down regulation of the target gene. It will be understood
that the term "ribonucleotide" or "nucleotide" can, in the case of
a modified RNA or nucleotide surrogate, also refer to a modified
nucleotide, or surrogate replacement moiety at one or more
positions. Thus, the siRNA agent is or includes a region which is
at least partially complementary to the target RNA. In certain
embodiments, the siRNA agent is fully complementary to the target
RNA. It is not necessary that there be perfect complementarity
between the siRNA agent and the target, but the correspondence must
be sufficient to enable the siRNA agent, or a cleavage product
thereof, to direct sequence specific silencing, such as by RNAi
cleavage of the target RNA. Complementarity, or degree of homology
with the target strand, is most critical in the antisense strand.
While perfect complementarity, particularly in the antisense
strand, is often desired some embodiments can include one or more
but preferably 6, 5, 4, 3, 2, or fewer mismatches with respect to
the target RNA. The mismatches are most tolerated in the terminal
regions, and if present are preferably in a terminal region or
regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5' and/or 3'
terminus. The sense strand need only be sufficiently complementary
with the antisense strand to maintain the over all double-strand
character of the molecule.
[0547] In addition, an siRNA agent will often be modified or
include nucleoside surrogates. Single stranded regions of an siRNA
agent will often be modified or include nucleoside surrogates,
e.g., the unpaired region or regions of a hairpin structure, e.g.,
a region which links two complementary regions, can have
modifications or nucleoside surrogates. Modification to stabilize
one or more 3'- or 5'-terminus of an iRNA agent, e.g., against
exonucleases, or to favor the antisense sRNA agent to enter into
RISC are also favored. Modifications can include C3 (or C6, C7,
C12) amino linkers, thiol linkers, carboxyl linkers,
non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene
glycol, hexaethylene glycol), special biotin or fluorescein
reagents that come as phosphoramidites and that have another
DMT-protected hydroxyl group, allowing multiple couplings during
RNA synthesis.
[0548] siRNA agents include: molecules that are long enough to
trigger the interferon response (which can be cleaved by Dicer
(Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC
(RNAi-induced silencing complex)); and, molecules which are
sufficiently short that they do not trigger the interferon response
(which molecules can also be cleaved by Dicer and/or enter a RISC),
e.g., molecules which are of a size which allows entry into a RISC,
e.g., molecules which resemble Dicer-cleavage products. Molecules
that are short enough that they do not trigger an interferon
response are termed sRNA agents or shorter iRNA agents herein.
"sRNA agent or shorter iRNA agent" as used refers to an iRNA agent
that is sufficiently short that it does not induce a deleterious
interferon response in a human cell, e.g., it has a duplexed region
of less than 60 but preferably less than 50, 40, or 30 nucleotide
pairs. The sRNA agent, or a cleavage product thereof, can down
regulate a target gene, e.g., by inducing RNAi with respect to a
target RNA, preferably an endogenous or pathogen target RNA.
[0549] Each strand of a sRNA agent can be equal to or less than 30,
25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides in
length. The strand is preferably at least 19 nucleotides in length.
For example, each strand can be between 21 and 25 nucleotides in
length. Preferred sRNA agents have a duplex region of 17, 18, 19,
29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more
overhangs, preferably one or two 3' overhangs, of 2-3
nucleotides.
[0550] In addition to homology to target RNA and the ability to
down regulate a target gene, an siRNA agent will preferably have
one or more of the following properties:
[0551] (1) it will, despite modifications, even to a very large
number, or all of the nucleosides, have an antisense strand that
can present bases (or modified bases) in the proper three
dimensional framework so as to be able to form correct base pairing
and form a duplex structure with a homologous target RNA which is
sufficient to allow down regulation of the target, e.g., by
cleavage of the target RNA;
[0552] (2) it will, despite modifications, even to a very large
number, or all of the nucleosides, still have "RNA-like"
properties, i.e., it will possess the overall structural, chemical
and physical properties of an RNA molecule, even though not
exclusively, or even partly, of ribonucleotide-based content. For
example, an siRNA agent can contain, e.g., a sense and/or an
antisense strand in which all of the nucleotide sugars contain
e.g., 2' fluoro in place of 2' hydroxyl. This
deoxyribonucleotide-containing agent can still be expected to
exhibit RNA-like properties. While not wishing to be bound by
theory, the electronegative fluorine prefers an axial orientation
when attached to the C2' position of ribose. This spatial
preference of fluorine can, in turn, force the sugars to adopt a
C.sub.3'-endo pucker. This is the same puckering mode as observed
in RNA molecules and gives rise to the RNA-characteristic
A-family-type helix. Further, since fluorine is a good hydrogen
bond acceptor, it can participate in the same hydrogen bonding
interactions with water molecules that are known to stabilize RNA
structures. Generally, it is preferred that a modified moiety at
the 2' sugar position will be able to enter into H-bonding which is
more characteristic of the OH moiety of a ribonucleotide than the H
moiety of a deoxyribonucleotide. A preferred siRNA agent will:
exhibit a C.sub.3'-endo pucker in all, or at least 50, 75, 80, 85,
90, or 95% of its sugars; exhibit a C.sub.3'-endo pucker in a
sufficient amount of its sugars that it can give rise to a the
RNA-characteristic A-family-type helix; will have no more than 20,
10, 5, 4, 3, 2, or 1 sugar which is not a C.sub.3'-endo pucker
structure.
[0553] A "single strand iRNA agent" as used herein, is an iRNA
agent which is made up of a single molecule. It may include a
duplexed region, formed by intra-strand pairing, e.g., it may be,
or include, a hairpin or pan-handle structure. Single strand iRNA
agents are preferably antisense with regard to the target molecule.
A single strand iRNA agent should be sufficiently long that it can
enter the RISC and participate in RISC mediated cleavage of a
target mRNA. A single strand iRNA agent is at least 14, and more
preferably at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in
length. It is preferably less than 200, 100, or 60 nucleotides in
length.
[0554] Hairpin iRNA agents will have a duplex region equal to or at
least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The
duplex region will preferably be equal to or less than 200, 100, or
50, in length. Preferred ranges for the duplex region are 15-30, 17
to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The
hairpin will preferably have a single strand overhang or terminal
unpaired region, preferably the 3', and preferably of the antisense
side of the hairpin. Preferred overhangs are 2-3 nucleotides in
length.
[0555] Chimeric oligonucleotides, or "chimeras," are
oligonucleotides which contain two or more chemically distinct
regions, each made up of at least one monomer unit, i.e., a
nucleotide in the case of an oligonucleotide compound. These
oligonucleotides typically contain at least one region wherein the
oligonucleotide is modified so as to confer upon the
oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target nucleic acid. Consequently, comparable results can often
be obtained with shorter oligonucleotides when chimeric
oligonucleotides are used, compared to phosphorothioate
oligodeoxynucleotides. Chimeric oligonucleotides of the invention
may be formed as composite structures of two or more
oligonucleotides, modified oligonucleotides, oligonucleosides
and/or oligonucleotide mimetics as described above. Such
oligonucleotides have also been referred to in the art as hybrids
or gapmers. Representative United States patents that teach the
preparation of such hybrid structures include, but are not limited
to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;
5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355;
5,652,356; 5,700,922; and 5,955,589, each of which is herein
incorporated by reference. In certain embodiments, the chimeric
oligonucleotide is RNA-DNA, DNA-RNA, RNA-DNA-RNA, DNA-RNA-DNA, or
RNA-DNA-RNA-DNA, wherein the oligonucleotide is between 5 and 60
nucleotides in length.
[0556] Definitions
[0557] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0558] The term "heteroatom" as used herein means an atom of any
element other than carbon or hydrogen. Preferred heteroatoms are
boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
[0559] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. In preferred embodiments, a straight chain or branched
chain alkyl has 30 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.30 for straight chain, C.sub.3-C.sub.30 for branched
chain), and more preferably 20 or fewer. Likewise, preferred
cycloalkyls have from 3-10 carbon atoms in their ring structure,
and more preferably have 5, 6 or 7 carbons in the ring
structure.
[0560] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six
carbon atoms in its backbone structure. Likewise, "lower alkenyl"
and "lower alkynyl" have similar chain lengths. Preferred alkyl
groups are lower alkyls. In preferred embodiments, a substituent
designated herein as alkyl is a lower alkyl.
[0561] The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl group (e.g., an aromatic or heteroaromatic
group). For example, a benzyl group (PhCH.sub.2--) is an aralkyl
group.
[0562] The terms "alkenyl" and "alkynyl" refer to unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described above, but that contain at least one double or
triple bond respectively.
[0563] The term "aryl" as used herein includes 5-, 6- and
7-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, anthracene, naphthalene,
pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,
triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine,
and the like. Those aryl groups having heteroatoms in the ring
structure may also be referred to as "aryl heterocycles" or
"heteroaromatics." The aromatic ring can be substituted at one or
more ring positions with such substituents as described above, for
example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino,
amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,
alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,
heterocyclyl, aromatic or heteroaromatic moieties, --CF.sub.3,
--CN, or the like. The term "aryl" also includes polycyclic ring
systems having two or more cyclic rings in which two or more
carbons are common to two adjoining rings (the rings are "fused
rings") wherein at least one of the rings is aromatic, e.g., the
other cyclic rings can be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls and/or heterocyclyls.
[0564] The terms ortho, meta and para apply to 1,2-, 1,3- and
1,4-disubstituted benzenes, respectively. For example, the names
1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.
[0565] The terms "heterocyclyl" or "heterocyclic group" refer to 3-
to 10-membered ring structures, more preferably 3- to 7-membered
rings, whose ring structures include one to four heteroatoms.
Heterocycles can also be polycycles. Heterocyclyl groups include,
for example, thiophene, thianthrene, furan, pyran, isobenzofuran,
chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole,
isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine,
indolizine, isoindole, indole, indazole, purine, quinolizine,
isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline,
quinazoline, cinnoline, pteridine, carbazole, carboline,
phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,
phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine,
oxolane, thiolane, oxazole, piperidine, piperazine, morpholine,
lactones, lactams such as azetidinones and pyrrolidinones, sultams,
sultones, and the like. The heterocyclic ring can be substituted at
one or more positions with such substituents as described above, as
for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,
ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, --CF.sub.3, --CN, or the like.
[0566] The terms "polycyclyl" or "polycyclic group" refer to two or
more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls
and/or heterocyclyls) in which two or more carbons are common to
two adjoining rings, e.g., the rings are "fused rings". Rings that
are joined through non-adjacent atoms are termed "bridged" rings.
Each of the rings of the polycycle can be substituted with such
substituents as described above, as for example, halogen, alkyl,
aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,
sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,
carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde,
ester, a heterocyclyl, an aromatic or heteroaromatic moiety,
--CF.sub.3, --CN, or the like.
[0567] As used herein, the term "nitro" means --NO.sub.2; the term
"halogen" designates --F, --Cl, --Br or --I; the term "sulfhydryl"
means --SH; the term "hydroxyl" means --OH; and the term "sulfonyl"
means --SO.sub.2--.
[0568] The terms "amine" and "amino" are art-recognized and refer
to both unsubstituted and substituted amines, e.g., a moiety that
can be represented by the general formula: 36
[0569] wherein R.sub.9, R.sub.10 and R'.sub.10 each independently
represent a group permitted by the rules of valence.
[0570] The term "acylamino" is art-recognized and refers to a
moiety that can be represented by the general formula: 37
[0571] wherein R.sub.9 is as defined above, and R'.sub.11
represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R.sub.8, where m and R.sub.8 are as defined
above.
[0572] The term "amido" is art recognized as an amino-substituted
carbonyl and includes a moiety that can be represented by the
general formula: 38
[0573] wherein R.sub.9, R.sub.10 are as defined above. Preferred
embodiments of the amide will not include imides which may be
unstable.
[0574] The term "alkylthio" refers to an alkyl group, as defined
above, having a sulfur radical attached thereto. In preferred
embodiments, the "alkylthio" moiety is represented by one of
--S-alkyl, --S-alkenyl, --S-alkynyl, and
--S--(CH.sub.2).sub.m--R.sub.8, wherein m and R.sub.8 are defined
above. Representative alkylthio groups include methylthio, ethyl
thio, and the like.
[0575] The term "carbonyl" is art recognized and includes such
moieties as can be represented by the general formula: 39
[0576] wherein X is a bond or represents an oxygen or a sulfur, and
R.sub.11 represents a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R.sub.8 or a pharmaceutically acceptable salt,
R'.sub.11 represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R.sub.8, where m and R.sub.8 are as defined
above. Where X is an oxygen and R.sub.11 or R'.sub.11 is not
hydrogen, the formula represents an "ester". Where X is an oxygen,
and R.sub.11 is as defined above, the moiety is referred to herein
as a carboxyl group, and particularly when R.sub.11 is a hydrogen,
the formula represents a "carboxylic acid". Where X is an oxygen,
and R'.sub.11 is hydrogen, the formula represents a "formate". In
general, where the oxygen atom of the above formula is replaced by
sulfur, the formula represents a "thiolcarbonyl" group. Where X is
a sulfur and R.sub.11 or R'.sub.11 is not hydrogen, the formula
represents a "thiolester." Where X is a sulfur and R.sub.11 is
hydrogen, the formula represents a "thiolcarboxylic acid." Where X
is a sulfur and R.sub.11 is hydrogen, the formula represents a
"thiolformate." On the other hand, where X is a bond, and R.sub.11
is not hydrogen, the above formula represents a "ketone" group.
Where X is a bond, and R.sub.11 is hydrogen, the above formula
represents an "aldehyde" group.
[0577] The terms "alkoxyl" or "alkoxy" as used herein refers to an
alkyl group, as defined above, having an oxygen radical attached
thereto. Representative alkoxyl groups include methoxy, ethoxy,
propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons
covalently linked by an oxygen. Accordingly, the substituent of an
alkyl that renders that alkyl an ether is or resembles an alkoxyl,
such as can be represented by one of --O-alkyl, --O-alkenyl,
--O-alkynyl, --O--(CH.sub.2).sub.m--R.sub.8, where m and R.sub.8
are described above.
[0578] The term "sulfonate" is art recognized and includes a moiety
that can be represented by the general formula: 40
[0579] in which R.sub.41 is an electron pair, hydrogen, alkyl,
cycloalkyl, or aryl.
[0580] The terms triflyl, tosyl, mesyl, and nonaflyl are
art-recognized and refer to trifluoromethanesulfonyl,
p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl
groups, respectively. The terms triflate, tosylate, mesylate, and
nonaflate are art-recognized and refer to trifluoromethanesulfonate
ester, p-toluenesulfonate ester, methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules
that contain said groups, respectively.
[0581] The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent
methyl, ethyl, phenyl, trifluoromethanesulfonyl,
nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl,
respectively. A more comprehensive list of the abbreviations
utilized by organic chemists of ordinary skill in the art appears
in the first issue of each volume of the Journal of Organic
Chemistry; this list is typically presented in a table entitled
Standard List of Abbreviations. The abbreviations contained in said
list, and all abbreviations utilized by organic chemists of
ordinary skill in the art are hereby incorporated by reference.
[0582] The term "sulfate" is art recognized and includes a moiety
that can be represented by the general formula: 41
[0583] in which R.sub.41 is as defined above.
[0584] The term "sulfonylamino" is art recognized and includes a
moiety that can be represented by the general formula: 42
[0585] The term "sulfamoyl" is art-recognized and includes a moiety
that can be represented by the general formula: 43
[0586] The term "sulfonyl", as used herein, refers to a moiety that
can be represented by the general formula: 44
[0587] in which R.sub.44 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl,
or heteroaryl.
[0588] The term "sulfoxido" as used herein, refers to a moiety that
can be represented by the general formula: 45
[0589] in which R.sub.44 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aralkyl, or aryl.
[0590] A "selenoalkyl" refers to an alkyl group having a
substituted seleno group attached thereto. Exemplary "selenoethers"
which may be substituted on the alkyl are selected from one of
--Se-alkyl, --Se-alkenyl, --Se-alkynyl, and
--Se--(CH.sub.2).sub.m--R.sub.7, m and R.sub.7 being defined
above.
[0591] Analogous substitutions can be made to alkenyl and alkynyl
groups to produce, for example, aminoalkenyls, aminoalkynyls,
amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls,
thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or
alkynyls.
[0592] As used herein, the definition of each expression, e.g.
alkyl, m, n, etc., when it occurs more than once in any structure,
is intended to be independent of its definition elsewhere in the
same structure.
[0593] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc.
[0594] As used herein, the term "substituted" is contemplated to
include all permissible substituents of organic compounds. In a
broad aspect, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic,
aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, for example, those described
herein above. In addition, the substituent can be halogen, azide,
alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,
amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,
ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic
moieties, --CF.sub.3, --CN, and the like. The permissible
substituents can be one or more and the same or different for
appropriate organic compounds. For purposes of this invention, the
heteroatoms such as nitrogen may have hydrogen substituents and/or
any permissible substituents of organic compounds described herein
which satisfy the valences of the heteroatoms. This invention is
not intended to be limited in any manner by the permissible
substituents of organic compounds.
[0595] The phrase "protecting group" as used herein means temporary
substituents which protect a potentially reactive functional group
from undesired chemical transformations. Examples of such
protecting groups include esters of carboxylic acids, silyl ethers
of alcohols, and acetals and ketals of aldehydes and ketones,
respectively. The field of protecting group chemistry has been
reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in
Organic Synthesis, 2.sup.nd ed.; Wiley: New York, 1991).
[0596] Certain compounds of the present invention may exist in
particular geometric or stereoisomeric forms. The present invention
contemplates all such compounds, including cis- and trans-isomers,
R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the
racemic mixtures thereof, and other mixtures thereof, as falling
within the scope of the invention. Additional asymmetric carbon
atoms may be present in a substituent such as an alkyl group. All
such isomers, as well as mixtures thereof, are intended to be
included in this invention.
[0597] If, for instance, a particular enantiomer of a compound of
the present invention is desired, it may be prepared by asymmetric
synthesis, or by derivation with a chiral auxiliary, where the
resulting diastereomeric mixture is separated and the auxiliary
group cleaved to provide the pure desired enantiomers.
Alternatively, where the molecule contains a basic functional
group, such as amino, or an acidic functional group, such as
carboxyl, diastereomeric salts are formed with an appropriate
optically-active acid or base, followed by resolution of the
diastereomers thus formed by fractional crystallization or
chromatographic means well known in the art, and subsequent
recovery of the pure enantiomers.
[0598] Contemplated equivalents of the compounds described above
include compounds which otherwise correspond thereto, and which
have the same general properties thereof (e.g., functioning as
analgesics), wherein one or more simple variations of substituents
are made which do not adversely affect the efficacy of the compound
in binding to sigma receptors. In general, the compounds of the
present invention may be prepared by the methods illustrated in the
general reaction schemes as, for example, described below, or by
modifications thereof, using readily available starting materials,
reagents and conventional synthesis procedures. In these reactions,
it is also possible to make use of variants which are in themselves
known, but are not mentioned here.
[0599] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87,
inside cover.
EXEMPLIFICATION
[0600] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention.
Example 1
Oligonucleotide Synthesis Using Phosphoramidite Activators 35-48
(see FIGS. 1-3)
[0601] In certain instances the strength of the activator is
increased by forming the activated salt resulting in decreased
coupling time for RNA Synthesis.
[0602] A decamer RNA molecules (49, 5'-CAUCGCTGAdT-3') was
synthesized on a 394 ABI machine (ALN 0208) using the standard 98
step cycle written by the manufacturer with modifications to a few
wait steps as described below. The solid support was controlled
pore glass (CPG, prepacked, 1 .mu.mole, 500, Proligo Biochemie
GmbH) and the monomers were RNA phosphoramidites with fast
deprotecting groups obtained from Pierce Nucleic Acid Technologies
used at concentrations of 0.15 M in acetonitrile (CH.sub.3CN)
unless otherwise stated. Specifically the RNA phosphoramidites were
5'-O-Dimethoxytrityl-N.sup.6-phenoxyacetyl-2'-O-tbu-
tyldimethylsilyl-adenosine-3'-O-(.beta.-cyanoethyl-N,N'-diisopropyl)phosph-
oramidite,
5'-O-Dimethoxytrityl-N.sup.2-p-isopropylphenoxyacetyl-2'-O-tbut-
yldimethylsilyl-guanosine-3'-O-(.beta.-cyanoethyl-N,N'-diisopropyl)phospho-
ramidite,
5'-O-Dimethoxytrityl-N.sup.4-acetyl-2'-O-tbutyldimethylsilyl-cyt-
idine-3'-O--(.beta.-cyanoethyl-N,N'-diisopropyl)phosphoramidite,
and
5'-O-Dimethoxytrityl-2'-O-tbutyldimethylsilyl-uridine-3'-O-(.beta.-cyanoe-
thyl-N,N'-diisopropyl)-phosphoramidite;
[0603] The coupling times were either 1, 3 or 5 minutes for the
different salt concentrations which themselves were 10, 20 and 40
mol % relative to the 5-(ethylthio)-1H-tetrazole (ETT, 0.25 M, Glen
Research). Diisopropylammonium salt of ETT with required mol % was
obtained by adding calculated amount of anhydrous diisopropylamine
to 0.25 M ETT solution and stored over molecular sieves for 4-6 h.
Details of the other reagents are as follows: Cap A: 5%
Phenoxyacetic anhydride/THF/pyridine, (Glen Research, & Cap
B:10% N-methylimidazole/THF, (Glen Research); Oxidant 0.02 M Iodine
in THF/Water/Pyridine (Glen Research] Detritylation was achieved
with 3% TCA/dichloromethane (Proligo).
[0604] After completion of synthesis the CPG was transferred to a
screw cap RNase free microfuge tube. The oligonucleotide was
cleaved from the CPG with simultaneous deprotection of base and
phosphate groups with 1.0 mL of a mixture of 40% methylamine:
ammonia (1:1)] for 30 minutes at 65.degree. C. The solution was
then lyophilized to dryness.
Example 2
Synthesis of compound 1 (R'=H and R"=C(S)OEt or R',R"=H)
[0605] 46
[0606] A solution of chlorocarbonyl sulfenyl chloride (8.4 mL, 0.1
mol) in dry ether (50 mL) was added dropwise to a cold solution of
thiourea (7.62 g, 0.1 mol) in dry ether (500 mL) and triethylamine
(14 mL, 0.1 mol) cooled with ice-bath in 3 h under an argon
atmosphere. The reaction mixture was stirred at the same
temperature for total of 6 h. The solids were filtered off and the
filtration was concentrated into a crude residue which was further
crystallized with dichlorometrhane-hexanes to give a pure compound
(2.5 g). The mother liquid was then concentrated into a crude
residue which was applied to a column of silica gel eluted with
dichloromethane-metahnol (40:1) to give a pure compound (180 mg).
The total yield is about 30%. .sup.1H-NMR (CDCl.sub.3, 400 MHz):
.delta. 10.46 (br, 1H), 4.38 (q, 2H, J=6.8, 14.4 Hz, CH.sub.2),
1.39 (t, 3H, J=7.2 Hz, CH.sub.3). .sup.3C-NMR (CDCl.sub.3, 100
MHz): 181.01, 177.00, 153.75, 64.68, 14.32.
Example 3
Phosphorothioation of Di- and POLY-Oligothymidine Using Sulfur
Transfer Reagent 1 (R'=H and R"=C(S)OEt or R',R"=H):
[0607] Dinucleotide 2 and hexamer 3 were synthesized on a 394 ABI
machine using the standard 93 step cycle written by the
manufacturer with modifications to a few wait steps as described
below. Activator used was 5-(ethylthio)-1H-tetrazole (0.25 M), and
for PS-oxidation, 0.05 M 1 in anhydrous acetonitrile was used. The
sulfurization time was about 4 min. After completion of the
synthesis, 2 and 3 were deprotected from support by aqueous ammonia
treatment at 55.degree. C. for 1 h. After HPLC purification, the
compound were analysed by LC-MS.
[0608] The results of phosphorothioation of oligothymdine using 1
as the sulfur-transfer agent are shown below.
1 Sequence, Mass Mass Compound all P.dbd.S Calc. Found 2 5' TT 3'
562.46 562.22 3 5' TTTTTT 3' 1843.52 1842.05
Example 4
[0609] Medium/Large Scale Oligonucleotides Synthesis with P.dbd.O,
P.dbd.S and P.dbd.O/P.dbd.S Mixed Backbone
[0610] A. Solid Phase Synthesis of Sequences 23 with P.dbd.O
Backbone and 24 with P.dbd.S Backbone
[0611] 200 .mu.mole syntheses were performed on the .ANG.KTA
OligoPilot 100 in 6.3 mL columns using 500 .ANG. dT-CPG loaded at
97 .mu.mole/g (Prime Synthesis; Aston, Pa.) Detritylation was
performed with 3% dichloroacetic acid (DCA) in dichloromethane
(CH.sub.2Cl.sub.2.) Coupling was accomplished with 2 eq. of DNA
3'-.beta.-cyanoethylphosphoramidites (CEP) or 2.5 eq. RNA
3'-.beta.-cyanoethylphosphoramidites (Pierce Nucleic Acids;
Milwaukee, Wis.) used at 0.2 M in acetonitrile (MeCN). Activator
was 0.6 M 5-Ethylthiotetrazole (American International Chemical;
Natick, Mass.) in MeCN and was used at three-fold excess relative
to RNA CEPs and at 4.5-fold excess to DNA CEPs. Oxidation was via
50 mM I.sub.2 in 90% pyridine 10% H.sub.2O or with 0.05 M
3-ethoxy-1,2,4-dithiazolidine-5-one (EDITH) in MeCN (Q. Xu, et al.
Nucleic Acids Research, Vol. 24, No. 18, pp. 3643-3644). Capping
was with 10% acetic anhydride (Ac.sub.2O) 10% 1-methylimidazole
(1-MeIm) 15% 2,6-lutidine in MeCN.
[0612] After synthesis, support was deblocked in 25 mL 40%
methylamine (MeNH.sub.2) in H.sub.2O for 20 minutes at 60.degree.
C. and 200 rpm, then chilled in dry ice [CO.sub.2(s)] and the
support filtered off in a sintered glass funnel and rinsed with 75
mL dimethylsulfoxide (DMSO) added to the filtrate. To this solution
was added 25 mL triethylammonium trihydrofluoride (TEA.3HF, TREAT)
followed by heating to 60.degree. C. for 20 minutes at 200 rpm.
After chilling in CO.sub.2(s) this solution was diluted with 125 mL
20 mM sodium acetate (NaOAc) and pH 6 confirmed. If necessary, pH
was adjusted with HCl.
[0613] Analysis was performed on an Agilent 1100 series HPLC using
a Dionex 4.times.250 mm DNAPak column. Buffer A was 1 mM EDTA, 25
mM Tris pH 8, 20 mM NaClO.sub.4. Buffer B was 1 mM EDTA, 25 mM Tris
pH 8, 0.4 M NaClO.sub.4. Separation was performed on a 0-40% B
gradient with buffers and column heated to 65.degree. C.
[0614] Materials were purified on an .ANG.KTA Explorer equipped
with a XK26/10 column (Amersham Biosciences; Piscataway, N.J.)
packed to a bed height of 10 cm with Hi Load Q Sepharose. Buffer A
was 1 mM EDTA, 25 mM Tris pH 8. Buffer B was 1 mM EDTA, 25 mM Tris
pH8, 0.4 M NaClO.sub.4. Crude materials were diluted 4-6 fold with
H.sub.2O and loaded. Pooled purified material=8.1 kAU at 96% by ion
exchange (IEX).
[0615] The solutions containing the crude material were diluted 4-6
fold, loaded onto the column in 1-3kAU amounts at 10 mL/min and
eluted with a segmented gradient from 0-60% B. Appropriate
fractions were pooled and this pooled material desalted in 30 mL
amounts over Sephadex G-25 on a BioPilot column (6 cm
dia..times.7.5 cm) against H.sub.2O. The eluate was vacuum
evaporated to less than 25 mL, shell frozen and lyophilized.
[0616] The results from the synthesis of 23 and 24 are presented
below. Note that purification was performed on an .ANG.KTA Explorer
and that "nd" indicates that the value was not determined.
2 Thiolation crude Sequence Agent Quantity % Y % fl IEX
Purification 23 -- -- 43 nd 8.6 kAU @ 94% fl 24 0.05 M 1 CV in 42
71 I 8.1 kAU EDITH 1 min. (= 48% of crude) @ 96% fl 23 =
5'-GCGGAUCAAACCUCACCAAdTdT-3' 24 =
5'-UUGGUGAGGUUUGAUCCGCdTdT-3'
[0617] B. Solid phase synthesis of mixed
phosphorothioate-phosphodiester oligoribonucleotides using phenyl
acetyl disulfide or 3-ethoxy-1,2,4-dithiazoline-5-one
[0618] 200 .mu.mole syntheses were performed on the .ANG.KTA
OligoPilot 100 in 6.3 mL columns using 500 .ANG. dT-CPG loaded at
97 .mu.mole/g (Prime Synthesis; Aston, Pa.) Detritylation was
performed with 3% dichloroacetic acid (DCA) in dichloromethane
(CH.sub.2Cl.sub.2.) Coupling was accomplished with 2 eq. of DNA
CEPs or 2.5 eq. of RNA CEPs (Pierce Nucleic Acids; Milwaukee, Wis.)
used at 0.2 M in acetonitrile (MeCN.) Activator was 0.6 M
5-Ethylthiotetrazole (American International Chemical; Natick,
Mass.) in MeCN and was used at threefold excess relative to RNA
CEPs and at 4.5-fold excess to DNA CEPs. Oxidation was via 50 mM
I.sub.2 in 90% pyridine 10% H.sub.2O. Thiolation was with 0.2 M
phenyl acetyl disulfide (PADS) in 1:1 3-picoline:MeCN or with 0.05
M 3-ethoxy-1,2,4-dithiazolidine-5-one (EDITH) in MeCN (Q. Xu, et
al. Nucleic Acids Research, Vol. 24, No. 18, pp. 3643-3644.)
Capping was with 10% acetic anhydride (Ac.sub.2O) 10%
1-methylimidazole (1-MeIm) 15% 2,6-lutidine in MeCN. When EDITH was
used, capping was performed both before and after the thiolation
reaction (M. Ma, et al. Nucleic Acids Research, 1997, Vol. 25, No.
18, pp. 3590-3593).
[0619] After synthesis, support was deblocked in 25 mL 40%
methylamine (MeNH.sub.2) in H.sub.2O for 20 minutes at 60.degree.
C. and 200 rpm, then chilled in dry ice [CO.sub.2(s)] and the
support filtered off in a sintered glass funnel and rinsed with 75
mL dimethylsulfoxide (DMSO) added to the filtrate. To this solution
was added 25 mL triethylammonium trihydrofluoride (TEA.3HF, TREAT)
followed by heating to 60.degree. C. for 20 minutes at 200 rpm.
After chilling in CO.sub.2(s) this solution was diluted with 125 mL
20 mM sodium acetate (NaAc) and pH 6 confirmed. If necessary, pH
was adjusted with HCl.
[0620] Analysis was performed on an Agilent 1100 series HPLC using
a Dionex 4.times.250 mm DNAPak column. Buffer A was 1 mM EDTA, 25
mM Tris pH 9, 50 mM NaClO.sub.4, 20% MeCN. Buffer B was 1 mM EDTA,
25 mM Tris pH 9, 0.4 M NaClO.sub.4, 20% MeCN. Separation was
performed on a 0-65% B segmented gradient with buffers and column
heated to 65.degree. C.
[0621] Materials were purified on an .ANG.KTA Pilot equipped with a
FineLine70 column packed with TSKgel Q 5PW (Tosoh Biosciences) to a
bed height of 28 cm (=1.08 L) Buffer A was 1 mM EDTA, 25 mM Tris pH
9. Buffer B was 1 mM EDTA, 25 mM Tris pH 9, 0.4 M NaClO.sub.4.
Buffers were heated by a 4 kW buffer heater set at 65.degree. C.,
giving a column outlet temperature of 45.degree. C. The solution
containing the crude material was diluted 4-6 fold and loaded onto
the column at 200 mL/min and eluted with a segmented gradient from
0-60% B. Appropriate fractions were pooled and this pooled material
desalted in 30 mL amounts over Sephadex G-25 on a BioPilot column
(6 cm dia..times.7.5 cm) against H.sub.2O. The eluate was vacuum
evaporated to less than 25 mL, shell frozen and lyophilized.
[0622] The results of the synthesis of 25 and 26 with PADS or EDITH
are shown in FIG. 6. It should be noted that the contact time used
for EDITH is less than that suggested by Q. Xu et al.
[0623] (one vs. two minutes.)
Example 5
Deprotection Conditions
[0624] General
[0625] The following oligonucleotide sequences used for various
deprotection methods.
3 27:5'CUUACGCUGAGUACUUCGAdTdT P = O RNA
28:5'UCGAAGUACUCAGCGUAAGdTdT. P = O/P = S RNA
29:5'GCGGAUCAAACCUCACCAAdTdT. P = O backbone
30:5'GCGGAUCAAACCUCACCAAdTdT. P = O/P= S mixed backbone
31:5'GCGGAUCAAACCUCACCAAdTdT. P = S backbone
32:5'UUGGUGAGGUUUGAUCCGCdTdT. P = O backbone
33:5'UUGGUGAGGUUUGAUCCGCdTdT. P = O/P = S mixed backbone
34:5'UUGGUGAGGUUUGAUCCGCdTdT. P = S backbone
[0626] Method 1
[0627] A volumetric mixture (.about.1:4) of Py.HF and DBU with DMSO
(4.about.5 volume of PYHF) as solvent at 65.degree. C. for 15 mins.
This is a two step reaction condition.
[0628] Control: A .about.1 umole sample of 27 was deprotected by
MeNH.sub.2 at 65.degree. C. for 20 mins and dried. Then it was
treated with a mixture of 0.1 mL TEA.3HF, 0.075 mL TEA and 0.15 mL
DMSO at 65.degree. C. for 1.5 hours. The yield on HPLC was 47/54%
(260 nm and 280 nm) on anion exchange HPLC. A 0.5 .mu.mole OD
sample of dried 27, deprotected by MeNH.sub.2 at 65.degree. C. for
20 mins, was dissolved in premixed 10 .mu.L Py.HF, 50 .mu.L DBU and
50 .mu.L DMSO and heated at 65.degree. C. The yield was 55/53%
after 10 mins, 57/57% after 20 mins, 57/58% after 30 mins and
57/57% after 1 hour. The pH of this 1:5 mixture was found out to be
about 10 by adding in water. Therefore, .about.0.5 .mu.mole of the
MeNH.sub.2 deprotected and dried 27 was deprotected by premixed 6.5
.mu.L Py.HF, 27.4 .mu.L DBU and 26 .mu.L DMSO at 65.degree. C. for
15 mins and 70 mins. The yield was 57/57% after 15 mins and 70
mins. A .about.4 .mu.mole sample of 27 was deprotected by
concentrated ammonia at 65.degree. C. for 1 hour and dried. The
residue was then dissolved in premixed 0.06 mL Py.HF, 0.24 mL DBU,
and 0.3 mL DMSO at 65.degree. C. for 15 mins. The yield was 58/60%.
A .about.4 .mu.mole sample of 27 was deprotected by ethanolic
ammonia at 65.degree. C. for 1 hour and dried. Premixed 0.06 mL
Py.HF, 0.24 mL DBU, and 0.3 mL DMSO were used to treat the RNA at
65.degree. C. for 15 min. The yield was 59/60%.
[0629] Compound 29 was synthesized at 1 .mu.mole scale. It was
deprotected by ethanolic ammonia at 65.degree. C. for 1 hour, then
divided to half (71 OD and 77 OD) and dried. 27 .mu.L Py.HF, 108
.mu.L DBU and 135 .mu.L DMSO were mixed. Half of this mixture was
used to treat the 77 OD sample for 20 mins at 65.degree. C., the
other half was used to treat the 71 OD sample for 30 mins. The
yield was 64/63% after 20 mins and 62/63% after 30 mins. The fully
thioated 31 was deprotected by ethanolic ammonia at 65.degree. C.
for 45 mins. The crude mixture was divided into half and dried, 76
OD in each sample. 20 .mu.L Py.HF, 80 .mu.L DBU and 100 .mu.L DMSO
were premixed, half of it were used to dissolve one sample and the
other half for the other sample. At 65.degree. C., the yield was
64/81% after 20 mins and 63/81% after 30 mins. No PS/PO conversion
was detected on LC-MS.
[0630] Part of 28 was deprotected with MeNH.sub.2 at 65.degree. C.
for 20 mins. The crude mixture was divided into .about.40 OD
samples and dried. The other part was deprotected with ethanolic
ammonia at 65.degree. C. for 40 mins, and also divided into
.about.40 OD samples and dried. One portion of MeNH.sub.2
deprotected sample was desilylated with standard procedures (16
.mu.L TEA.3HF, 12 .mu.L TEA and 24 .mu.L DMSO at 65.degree. C.),
the yield was 37/36% after 30 mins, 41/49% after 1 hour, 38/43%
after 1.5 hours and 42/42% after 2.5 hours. Second portion of
MeNH.sub.2 deprotected sample was desilylated with premixed 9 .mu.L
Py.HF, 36 .mu.L DBU and 36 .mu.L DMSO at 65.degree. C., and the
yield was 44/45% after 15 mins, 46/45% after 30 mins, 45/44% after
1 hour, 45/44% after 1.5 hr and 44/48% after 2.5 hrs. Another
portion of MeNH.sub.2 deprotected sample was desilylated with
premixed 9 .mu.L Py.HF, 31.5 .mu.L DBU and 31.5 .mu.L DMSO at
65.degree. C., and the yield was 42/45% after 15 mins, 45/47% after
30 mins, 45/44% after 1 hour, 45/48% after 1.5 hr and 39/47% after
2.5 hrs. One portion of ethanolic ammonia deprotected sample was
desilylated with standard procedures (16 .mu.L TEA.3HF, 12 .mu.L
TEA and 24 .mu.L DMSO at 65.degree. C.), the yield was 40/39% after
30 mins, 49/51% after 1 hour, 49/51% after 1.5 hour and 47/49%
after 2.5 hour. Second portion of ethanolic ammonia deprotected
sample was desilylated with premixed 9 .mu.L Py.HF, 36 .mu.L DBU
and 36 .mu.L DMSO at 65.degree. C., and the yield was 50/50% after
15 mins, 49/49% after 30 mins, 53/54% after 1 hour, 55/58% after
1.5 hour and 54/54% after 2.5 hrs. Another portion of ethanolic
ammonia deprotected sample was desilylated with premixed 9 .mu.L
Py.HF, 31.5 .mu.L DBU and 31.5 .mu.L DMSO at 65.degree. C., and the
yield was 52/52% after 15 mins, 52/51% after 30 mins, 52/52% after
1 hour, 53/55% after 1.5 hour and 52/55% after 2.5 hour.
[0631] Standard deprotection of 29 gave 47/48% yield. Ethanolic
ammonia deprotection of 29 at 65.degree. C. for 1 hour followed by
15 mins treatment with premixed 105 .mu.L Py.HF, 367.5 .mu.L DBU
and 300 .mu.L DMSO at 65.degree. C. gave 47/49% yield. Part of the
support was treated with ethanolic ammonia for 1.5 hr at 65.degree.
C. and then dissolved in premixed 105 .mu.L Py.HF, 367.5 .mu.L DBU
and 300 .mu.L DMSO at 65.degree. C. for 15 mins, which gave 47/47%
yield.
[0632] Deprotection for 1 hr in ethanolic ammonia at 65.degree. C.
followed by 65.degree. C. and 20 mins/15 mins 1:3.5 mixture
desilylation was applied on 32/34 gave 60/61% and 61/61% yields
respectively. For 33 synthesized on 1 .mu.mole scale, both standard
and Pyridine-HF/DBU deprotections were done, and yields were 41/40%
for standard and 45/43% for Pyridine-HF/DBU method.
[0633] Method 2: One Step Process
[0634] Silyl deprotection reagent: 4 volume desilylation mixture (1
mL Py.HF, 3.5 mL DBU, 4 mL DMSO) per 1 volume of ethanolic ammonia
at 60.degree. C. for 20 mins.
[0635] This method was tested with a .about.40 OD sample of 28
after MeNH.sub.2 deprotection. 20 .mu.L of ethanolic ammonia was
used to dissolve the oligo, and then 80 .mu.L of premixed Py.HF
reagent (1 mL Py.HF+3.5 m DBU+4 mL DMSO) were added in to the
sample. The yield was 49/45% when heated at 60.degree. C. for 20
mins, 1 hour and 2 hours. Under this condition the deprotecion was
complete in 20 minute without any degradation of the RNA.
[0636] Method 3: A Two Step Process.
[0637] Silyl deprotection reagent: 5 .mu.L DMSO and 2.5 .mu.L DBU
per 1 mg of poly{4-vinylpyridinium poly(hydrogen fluoride)] (PVPHF)
at 65.degree. C. for 20 min.
[0638] About 40OD of dried sample of ethanolic ammonia deprotected
27 was dissolved in 50 .mu.L DMSO. 25 .mu.L DBU and 10 mg PVPHF
were added in and heated at 65.degree. C. The yield was 52/51%
after 20 mins, 54/57% after 40 mins and 55/62% after 90 mins. When
the sample was treated with 50 .mu.L DMSO, 30 .mu.L DBU and 10 mg
PVPHF at 65.degree. C., the yield was 48/51% after 20 mins, 50/50%
after 40 mins and 48/48% after 1.5 hours.
[0639] Method 4: One Step Deprotection
[0640] One-step deprotection with PVPHF: for every 10 .mu.L
ethanotic ammonia, add .about.30-40 .mu.L DMSO and 3 mg PVPHF. The
deprotection takes up to 1.5 hours.
[0641] About 400 D dried sample of ethanolic ammonia deprotected 28
was redissolved in 30 ethanolic ammonia, and 90 .mu.L DMSO and 9 mg
PVPHF were added into it. The deprotection was not complete after
20 mins. Yield was 49/51% after 40 mins and 51/51% after 1.5 hours.
A second portion of 28 was redissolved in 25 .mu.L ethanolic
ammonia and 100 .mu.L DMSO with 9 mg PVPHF. The reaction was not
complete after 20 min. The yield was 41/50% after 40 min and 50/57%
after 1.5 hour. When a portion of 28 deprotected by MeNH.sub.2 was
redissovled in 20 .mu.L ethanolic ammonia and 80 .mu.L DMSO with 10
mg PVPHF gave 42/42% yield after 50 mins.
[0642] Method 5
[0643] One-step deprotection with PVPHF: for every 10 .mu.L
ethanolic ammonia, add .about.30-40 .mu.L DMSO, 5 .mu.L DBU and
.about.4.5 mg PVPHF. The deprotection takes up to 40 min.
[0644] A .about.40OD dried sample of MeNH.sub.2 deprotected 28 was
redissolved in 20 .mu.L ethanolic ammonia, and then 80 .mu.L DMSO,
10 .mu.L DBU and 9 mg PVPHF were added into solution. This method
gave 45/45% after 40 min and 46/49% yield after 1.5 hour.
[0645] Method 6: Tris(dimethylamino)sulfur diflurotrimethylsilane
(TAS-F) as silyl deprotecting agent for RNA synthesis
[0646] About 1 .mu.mole methylamine deprotected and dried 27 was
treated with a solution of 0.16 g TAS-F in 0.2 mL of DMF at
55.degree. C. for 2 hours. The reaction was not complete and the
reaction mixture was not homogenous with some gel sitting out of
the solution. 20 .mu.L water was added into the reaction mixture.
The reaction mixture became clear after overnight storing at
55.degree. C. HPLC purification gave 51/55% for this reaction. The
reproducibility of this reaction was not very consistent.
.about.0.6 .mu.mole of 27 was treated with 80 mg TAS-F and 0.2 mL
pyridine at 65.degree. C. Only 22/21% yield was observed after 2
hours. .about.0.6 .mu.mole was treated with 80 mg TAS-F and 0.2 mL
N-methylpyrrolidinone at 65.degree. C. A precipitate was formed
during the course of the reaction and the yield was 34/37% after 2
hrs. .about.0.4 .mu.mole of 27 was treated with 27 mg TAS-F, 0.15
mL N-methylpyrrolidinone and 0.5 mL DMSO at 65.degree. C. for 2
hours. The yield was 35/24%. .about.0.4 .mu.mole was treated with
27 mg TAS-F, 0.15 mLl N-methylpyrolidinone and 0.05 mL DMSO at
65.degree. C. for 2 hours. The yield was 25/25%. .about.0.4
.mu.mole of 27 was treated with 27 mg TAS-F, 0.15 mL
N-methylpyrolidinone and 0.05 mL pyridine at 65.degree. C. for 2
hours. The yield was 22/22%. .about.1 .mu.mole of ethanolic ammonia
deprotected and dried 27 was treated with 75 mg TAS-F and 0.2 mL
DMSO at 65.degree. C. The yield was 39/41% after 2 hours. .about.1
.mu.mole of this sample was treated with 75 mg TAS-F and 0.2 mL DMF
at 65.degree. C. Precipitate formed during the course of the
reaction and the yield was 21/21% after 2 hours. .about.1 mmole of
ammonia deprotected and dried 27 was treated with 75 mg TAS-F and
0.2 mL DMSO at 65.degree. C. The yield was 31/30% after 2 hours.
.about.1 .mu.mole of this sample was treated with 75 mg TAS-F and
0.2 mL DMF at 65.degree. C. Precipitate formed and the yield was
21/24% after 2 hours.
[0647] A .about.40 OD sample of MeNH.sub.2 deprotected (65.degree.
C. 20 mins) and dried 28 sample was treated with 41 mg TASF and 90
.mu.L DMF at 65.degree. C. Injections were done after 30 mins, 1
hr, 2 hr, and then at RT overnight. The reaction did not yield
noticeable amount of product. Another .about.40OD sample was
treated with 41 mg TASF, 90 .mu.L DMF and 40 .mu.L water at
65.degree. C. Injections were done after 30 min, 1 hr, 2 hr, and
then at RT overnight. No major peak was detected in the HPLC for
the product. Same deprotection conditions were applied on
.about.40OD samples of 28 deprotected by ethanolic ammonia
(65.degree. C., 40 min.) and same results were observed: no major
peak.
Example 6
Microwave-mediated Deprotection of a 2'-Silyl Group of RNA
[0648] A. Deprotection 1 (Standard)
[0649] The oligonucleotide was cleaved from the support with
simultaneous deprotection of base and phosphate groups with 2.0 mL
of a mixture of ammonia and 8 M ethanolic methylamine [1:1] for 30
min at 65.degree. C. The vial was cooled briefly on ice and then
the ethanolic ammonia mixture was transferred to a new microfuge
tube. The CPG was washed with 2.times.0.1 mL portions of deionized
water, put in dry ice for 10 min, and then dried in speed vac.
[0650] B. Microwave deprotection of 2'-O-TBDMS group of RNA 47
[0651] Instrument: CEM Discover Explorer, Magnetron Frequency 2450
MHz, Power output 300 Watts, Microwave Applicator: Circular Single
mode, Self Tuning
[0652] Reagents: A) 1 M TBAF in THF, B) TEA.3HF, C) Pyr.HF with
DBU.
[0653] About 12 OD of Oligo 50 or 51 was resuspended in 600 .mu.L
of Reagent A to C. The vial containing the oligonucleotides was
then placed in microwave unit. The solution was irradiated for 2
min. and 4 min. in CEM Discover Explorer.
[0654] Work Up
[0655] Condition A: In case of TBAF after Microwave irradiation
quenched the reaction with water followed by desalting.
[0656] Condition B: The reaction was then quenched with 400 .mu.L
of isopropoxytrimethylsilane (iPrOSiMe.sub.3, Aldrich) and further
incubated on the heating block leaving the caps open for 10 min.
(This causes the volatile isopropxytrimethylsilylfluoride adduct to
vaporize). The residual quenching reagent was removed by drying in
a speed vac. Added 1.5 mL of 3% triethylamine in diethyl ether and
pelleted by centrifuging. The supernatant was pipetted out without
disturbing the pellet. Dry the pellet in speed vac. The crude RNA
was obtained as a white fluffy material in the microfuge tube.
Microwave Deprotection RNA and its MS Analysis
[0657]
4 2'-silyl depro- Com- tection cal. found pound Sequence condition
mass mass 50 5' ACGUCGAUAT 3' TBAF 2 min 3142.95 3142.57 50 5'
ACGUCGAUAT 3' Py.HF 2 min 3142.95 nd 50 5' ACGUCGAUAT 3' Py.HF 4
min 3142.95 nd 51 5'CGUCAAGGCGAT3' TBAF 2min 3832.37 3831.34 51
5'CGUCAAGGCGAT3' TEA.3HF 3832.37 3831.34 2 min 51 5'CGUCAAGGCGAT3'
TEA.3HF 3832.37 3831.34 4 min nd: not determined
Example 7
[0658] The Applicants have surprisingly discovered that impurities
in a composition of single stranded RNA can be readily removed by
HPLC purification of a mixture of single-stranded RNA that has been
annealed to generate double-stranded RNA.
[0659] General Procedure
[0660] A diagram illustrating the overall purification procedure is
presented in FIG. 9. The specific procedure used for the
purification of AL-DP-4014 is presented in FIGS. 11 and 12.
[0661] The analytical conditions used for reverse phase HPLC
purification, ion exchange purification, capillary gel
electrophoresis, and LC-MS are presented below.
[0662] Reverse Phase HPLC:
[0663] Luna C-18 column, 150.times.2.0 mm, temp=25.degree. C.,
flow=0.2 mL/min
[0664] Buffer A: 35 mm TEAA PH=7, 100 mm HFIP
[0665] Buffer B: MeOH
[0666] Gradient: 25% B to 35% B in 50 minutes, ramp to 85% B at 55
minutes, re-equilibrate
[0667] Ion Exchange Chromatography:
[0668] Dnapac PA-100 ion exchange column, 250.times.4 mm,
temp=65.degree. C., flow=1 ml/min
[0669] Buffer A: 50 mm NaClO.sub.4, 25 mm tris pH=9.0, 1 MM EDTA,
20% CAN
[0670] Buffer B: 400 mm NaClO.sub.4, 25 mm tris pH=9.0, 1 MM EDTA,
20% CAN
[0671] Gradient: hold at 0% B for 2.00 min, ramp to 40% B at 17
min, ramp to 65% B at 32 min, ramp to 100% B at 32.5 min.
re-equilibrate
[0672] Capillary Gel Electrophoresis:
[0673] DNA 100R Gel, temp=40.degree. C.
[0674] Separate at 12 KV, reverse polarity
[0675] LC-MS Analysis:
[0676] Chromolith speedrod 50.times.4 mm temp=25.degree. C.,
flow=0.8 mL/min
[0677] Buffer A: 20% MeOH, 10 mm TBAA pH=7.0
[0678] Buffer B: 80% MeOH, 10 mm TBAA pH=7.0
[0679] Gradient: 40% B to 80% B in 19.5 min., ramp to 100% B at 23
minutes re-equilibrate Scan MS in negative ion mode from 500 to
3000
[0680] Results
[0681] The specific procedure used for the purification of
AL-DP-4014 is presented in FIGS. 11 and 12. The chromatographic
data presented in FIGS. 14-18 indicate that the purification
procedure produced AL-DP-4014 in substantially pure form. The
purification procedure was performed as described above for
AL-DP-4127, AL-DP-4139, AND AL-DP-414. The results from analytical
analyses are presented in FIGS. 19-39.
Example 8
Procedure for Quenching Acrylonitrile
[0682] The solid support bound oligonucleotide is treated with
exceess of a mixture of triethylamine (or an amine with pKa=9-12),
an organic solvent (e.g. acetonitrile, THF) and a thiol or a
odorless thiol. The alkylamine would generate the acrylonitlile
which would be scavenged by the thiol. This is an improvement over
the process described by Capaldi et al. Org. Process Res. Dev.
2003, 7, 832-838.
Example 9
2'-O-Methyl-modified, 2'-Fluoro-modified, conjugated, thioate
oligonucleotides
[0683] Step 1. Oligonucleotide Synthesis
[0684] All oligonucleotides were synthesized on an AKTAoligopilot
synthesizer. Commercially available controlled pore glass solid
supports (dT-CPG, rC-CPG, rU-CPG, from Prime Synthesis) or the
in-house synthesized solid supports
(phthalimido-hydroxy-prolinol-CPG, hydroxyprolinol-cholesterol-CPG
described in patent applications: provisional 60/600,703 Filed Aug.
10, 2004 and PCT/US04/11829 Filed Apr. 16, 2004) were used for the
synthesis. RNA phosphoramidites and 2'-O-methyl modified RNA
phosphoramidites with standard protecting groups
(5'-O-dimethoxytrityl-N-6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-O--
N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N-4-ace-
tyl-2'-t-butyldimethylsilyl-cytidine-3'-O-N,N'-diisopropyl-2-cyanoethylpho-
sphoramidite,
5'-O-dimethoxytrityl-N-2-isobutryl-2'-t-butyldimethylsilyl-g-
uanosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-O-N,N'-diisopropy-
l-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N-6-benzoyl-2'-O-methy-
ladenosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N4-acetyl-2'-O-methyl-cytidine-3'-O-N,N'-diisopropyl-
-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N-2-isobutryl-2'-O-meth-
yl-guanosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, and
5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-N,N'-diisopropyl-2-cyanoeth-
ylphosphoramidite) were obtained from Pierce Nucleic Acids
Technologies and ChemGenes Research. The 2'-F phosphoramidites
(5'-O-dimethoxytrityl-N-
-4-acetyl-2'-fluro-cytidine-3'-O-N,N'-diisopropyl-2-cyanoethyl-phosphorami-
dite and
5'-O-dimethoxytrityl-2'-fluro-uridine-3'-O-N,N'-diisopropyl-2-cya-
noethyl-phosphoramidite) were obtained from Promega. All
phosphoramidites were used at a concentration of 0.2 M in
CH.sub.3CN except for guanosine and 2'-O-methyl-uridine, which were
used at 0.2 M concentration in 10% THF/CH.sub.3CN (v/v).
Coupling/recycling time of 16 minutes was used for all
phosphoramidite couplings. The activator was 5-ethyl-thio-tetrazole
(0.75 M, American International Chemicals). For the PO-oxidation,
50 mM iodine in water/pyridine (10:90 v/v) was used and for the
PS-oxidation 2% PADS (GL Synthesis) in 2,6-lutidine/CH.sub.3CN (1:1
v/v) was used. The cholesterol and amino-linker phosphoramidites
were synthesized in house, and used at a concentration of 0.1 M in
dichloromethane for cholesterol and 0.2 M in CH.sub.3CN for the
amino-linker. Coupling/recycling time for both the cholesterol and
the amino-linker phosphoramidites was 16 minutes.
[0685] Step 2. Deprotection of oligonucleotides
[0686] (a) Deprotection of RNAs without the 2'-fluoro modification:
After completion of synthesis, the support was transferred to a 100
mL glass bottle (VWR). The oligonucleotide was cleaved from the
support with simultaneous deprotection of base and phosphate groups
with 40 mL of a 40% aq. methyl amine (Aldrich) 90 mins at
45.degree. C. The bottle was cooled briefly on ice and then the
methylamine was filtered into a new 500 mL bottle. The CPG was
washed three times with 40 mL portions of DMSO. The mixture was
then cooled on dry ice.
[0687] In order to remove the tert-butyldimethylsilyl (TBDMS)
groups at the 2' position, 60 mL triethylamine trihydrofluoride
(Et.sub.3N--HF) was added to the above mixture. The mixture was
heated at 40.degree. C. for 60 minutes. The reaction was then
quenched with 220 mL of 50 mM sodium acetate (pH 5.5) and stored in
the freezer until purification.
[0688] (b) Deprotection of 2'-fluoro modified RNAs: After
completion of synthesis, the support was transferred to a 100 mL
glass bottle (VWR). The oligonucleotide was cleaved from the
support with simultaneous deprotection of base and phosphate groups
with 80 mL of a mixture of ethanolic ammonia (ammonia:ethanol, 3:1
v/v) for 6.5 h at 55.degree. C. The bottle was cooled briefly on
ice and then the ethanolic ammonia mixture was filtered into a new
250 mL bottle. The CPG was washed with twice with 40 mL portions of
ethanol/water (1:1 v/v). The volume of the mixture was then reduced
to .about.30 mL by roto-vap. The mixture was then frozen on dry ice
and dried under vacuum on a speed vac.
[0689] The dried residue was resuspended in 26 mL of triethylamine,
triethylamine trihydrofluoride (Et3N.3HF), and DMSO (3:4:6) and
heated at 60.degree. C. for 90 minutes to remove the
tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The
reaction was then quenched with 50 mL of 20 mM sodium acetate and
the pH was adjusted to 6.5, and the solution was stored in freezer
until purification.
[0690] Step 3. Quantitation of Crude Oligonucleotides
[0691] For all samples, a 10 .mu.L aliquot was diluted with 990
.mu.L of deionised nuclease free water (1.0 mL) and the absorbance
reading at 260 nm was obtained.
[0692] Step 4. Purification of Oligonucleotides
[0693] (a) Unconjugated oligonucleotides: The unconjugated crude
oligonucleotides were first analyzed by HPLC (Dionex PA 100). The
buffers were 20 mM phosphate, pH 11 (buffer A); and 20 mM
phosphate, 1.8 M NaBr, pH 11 (buffer B). The flow rate 1.0 m L/min
and monitored wavelength was 260-280 nm. Injections of 5-15 .mu.L
were done for each sample.
[0694] The unconjugated samples were purified by HPLC on an TSK-Gel
SuperQ-5PW (20) column packed in house (17.3.times.5 cm). The
buffers were 20 mM phosphate in 10% CH.sub.3CN, pH 8.5 (buffer A)
and 20 mM phosphate, 1.0 M NaBr in 10% CH.sub.3CN, pH 8.5 (buffer
B). The flow rate was 50.0 mL/min and wavelengths of 260 and 294 nm
were monitored. The fractions containing the full-length
oligonucleotides were pooled together, evaporated, and
reconstituted to about 100 mL with deionised water.
[0695] (b) Cholesterol-conjugated oligonucleotides: The
cholesterol-conjugated crude oligonucleotides were first analyzed
by LC/MS to determine purity. The 5'-cholesterol conjugated
sequences were HPLC purified on an RPC-Source15 reverse-phase
column packed in house. The buffers were 20 mM TEAA in 10%
CH.sub.3CN (buffer A) and 20 mM TEAA in 70% CH.sub.3CN (buffer B).
The fractions containing the full-length oligonucleotides were then
pooled together, evaporated, and reconstituted to 100 mL with
deionised water. The 3'-cholesterol conjugated sequences were HPLC
purified on an RPC-Source15 reverse-phase column packed in house.
The buffers were 20 mM NaOAc in 10% CH.sub.3CN (buffer A) and 20 mM
NaOAc in 70% CH.sub.3CN (buffer B). The fractions containing the
full-length oligonucleotides were pooled, evaporated, and
reconstituted to 100 mL with deionised water.
[0696] Step 5. Desalting of Purified Oligonucleotides
[0697] The purified oligonucleotides were desalted on an AKTA
Explorer system (Amersham Biosciences) using a Sephadex G-25
column. First, the column was washed with water at a flow rate of
25 mL/min for 20-30 min. The sample was then applied in 25 mL
fractions. The eluted salt-free fractions were combined, dried, and
reconstituted in 50 mL of RNase free water.
[0698] Step 6. Purity Analysis by Capillary Gel Electrophoresis
(CGE), Ion-Exchange HPLC, and Electrospray LC/Ms
[0699] Approximately 0.3 OD of each of the desalted
oligonucleotides were diluted in water to 300 .mu.L and were
analyzed by CGE, ion exchange HPLC, and LC/MS.
5 Calc Found Purity AL-SQ # Sequence Target Mass Mass (%) 2936
HP-NH2- Luc 6915 6915.01 97.8* CUUACGCUGAGUACUUCGAdTs dT 2937
CsUUACGCUGAGUACUUCGAdT Luc 6915 6915.06 95.9* dTdT-HP-NH2 5225
GUCAUCACACUGAAUACCAAU ApoB 7344 7344.70 83 s-Chol 3169
U.sub.F.sub..sup.SU.sub.FGGAUC.sub.FAAAU.sub.FAU.sub.FA- AGA ApoB
7325.39 7325.5 92 U.sub.FUCC.sub.F.sub..sup.SC.sub.F.sub.- .sup.SU
2920 GGAC.sub.FU.sub.FAC.sub.FU.sub.FC.sub.FU.sub.-
FAAGU.sub.FU.sub.FC.sub.F Factor VII 6628.93 6628.45 99.6
U.sub.FAC.sub.FdTsdT 2921 GU.sub.FAGAAC.sub.FU.sub.FU.sub-
.FAGAGU.sub.FAGU.sub.FC Factor VII 6726.04 6725.78 96.0
.sub.FC.sub.FdTsdT 4723 GGAU.sub.FC.sub.FAU.sub.FC.sub.FU-
.sub.FC.sub.FAAGU.sub.FC.sub.FU.sub.F Factor VII 6628.93 6628.47
98.9 U.sub.FAC.sub.FdTsdT 4724 GU.sub.FAAGAC.sub.FU.sub.FU.-
sub.FGAGAU.sub.FGAU.sub.FC Factor VII 6726.04 6725.56 96.3
.sub.FC.sub.CdTsdT 3000 CsGUCU.sub.FGUCU.sub.FGUCCCGGAUCd G6P
6610.94 6611.34 92 TsdT 3002 GsAUCCGGGAC.sub.FAGAC.sub.FAGACG G6P
6806.2 6806.06 93 dTsdT 2918 Chol- Factor VII 7332.93 7333.61 99.9
GGAU.sub.FC.sub.FAU.sub.FC.sub.FU.sub.FC.sub.FAAGU.sub.FC.sub.FU.sub.F
U.sub.FAC.sub.FdTsdT 2919 Chol- Factor VII 7332.93 7333.62 99.6
GGAC.sub.FU.sub.FAC.sub.FU.sub.FC.sub.FU.sub.FAAGU.s-
ub.FU.sub.FC.sub.F U.sub.FAC.sub.FdTsdT 3168
GsGAAUCU.sub.FU.sub.FAU.sub.FAU.sub.FU.sub.FU.sub.FGAU ApoB 7393
7393.3 76.4 CC.sub.FAAs-Chol 3001 CsGUCU.sub.FGUCU.sub.FGUCCCGGAUCd
G6P 7330.94 7331.3 79.4 TsdTs-Chol 4968
CGUCCUU.sub.OMeGAAGAAGAU.sub.OMeG GFP 7504.7 7504.20 92
GU.sub.OMeGC.sub.OMe.sub..sup.SG.sub.OMe.sub..s- up.SC 5226
AUUGGUAUUCAGUGUGAUGA ApoB 7409.5 7409.80 91
C.sub.OMe.sub..sup.SA.sub.OMe.sub..sup.SC 5475
U.sub.OMeU.sub.OMeGGAUC.sub.OMeAAAU.sub.OMeAU
.sub.OMeAAGAU.sub.OMeUCC.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SU
ApoB 7421.7 7421.4 89 3196 CSU.sub.OMeAUGAGCCUGAAGCC.sub.OMeU
a-synuclein 6741.2 6741.01 92.6 .sub.OMeA.sub.OMeAdTsdT 3197
U.sub.OMe.sub..sup.SU.sub.OMeAGGCUUCAGGCUCA a-synuclein 6721.12
6720.93 91.9 U.sub.OMeAGdTsdT 3199
CSU.sub.OMeACGAACCUGAAGCC.sub.OMeU a-synuclein 6724.21 6723.94 92.2
.sub.OMeA.sub.OMeAdTsdT 3200 U.sub.OMe.sub..sup.SU.sub.-
OMeAGGCUUGAGGUUCG a-synuclein 6738.11 6737.88 79.6 U.sub.OMeAGdTsdT
3201 CSU.sub.OMeACGAACCUGAAGCC.sub.OMeU a-synuclein 7444.21 7445.08
91.4 .sub.OMeA.sub.OMeAdTsdTs-Chol 3198
CSU.sub.OMeAUGAGGCUGAAGCC.sub.OMeU a-synuclein 7461.2 7462.02 85.7
.sub.OMeA.sub.OMeAdTsdTs-Chol 3131
ASGAAGC.sub.OMeAGGACCUU.sub.OMeAU ApoB 7471.1 7472.17 97.6
CU.sub.OMeAdTsdTs-Chol 5474 GGAAUCU.sub.OMeU.sub.OMeAU.su-
b.OMeAU.sub.OMeU ApoB 7461.1 7461.9 83 .sub.OMeU.sub.OMeGAUCC.sub.-
OMeAAs-Chol 4967 GC.sub.OMeAGG.sub.OMeAUGUUCUUC.sub.OMeA GFP 7394
7394.80 91 AGGACGs-Chol 3037
A.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SA.sub.OMe.sub..sup.SA.sub.OMe.sub-
..sup.SA.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.S miR-122A 8613.43
8614.53 82.7
A.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.S-
A.sub.OMe.sub..sup.SU.sub.OMe.sub..sup.SU.sub.OMe.sub..sup.S
G.sub.OMe.sub..sup.SU.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SA.sub.OMe.sub-
..sup.SC.sub.OMe.sub..sup.SA.sub.OMe.sub..sup.S
C.sub.OMe.sub..sup.SU.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SC.sub.OMe.sub-
..sup.SA.sub.OMe.sub..sup.S-Chol 3038
A.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SA.sub.OMeA.sub.OMeA.sub.OMeC.sub.-
OMeA.sub.OMe miR-122A 8340.09 8341.23 99.2 C.sub.OMeC.sub.OMeA.sub-
.OMeU.sub.OMeU.sub.OMeG.sub.OMeU.sub.OMeC .sub.OMeA.sub.OMeC.sub.O-
MeA.sub.OMeC.sub.OMeU.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SC
.sub.OMe.sub..sup.SA.sub.OMe.sub..sup.S-Chol 3039
A.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SA.sub.OMe.sub..sup.SC.sub.OMe.sub-
..sup.SA.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.S miR-122A 8613.43
8614.75 86.6
A.sub.OMe.sub..sup.SA.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.S-
A.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SU.sub.OMe.sub..sup.S
G.sub.OMe.sub..sup.SU.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SA.sub.OMe.sub-
..sup.SC.sub.OMe.sub..sup.SA.sub.OMe.sub..sup.S
U.sub.OMe.sub..sup.SU.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SC.sub.OMe.sub-
..sup.SA.sub.OMe.sub..sup.S-Chol 3040
A.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SA.sub.OMeA.sub.OMeC.sub.OMeA.sub.-
OMeA.sub.OMe miR-122A 8340.09 8341.15 85.2 C.sub.OMeA.sub.OMeC.sub-
.OMeU.sub.OMeG.sub.OMeU.sub.OMeC.sub.OMeA .sub.OMeC.sub.OMeA.sub.O-
MeU.sub.OMeU.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.SC.sub.OMe.sub..sup.S
A.sub.OMe.sub..sup.S-Chol
[0700] The strands are shown written 5' to 3'. Lower case "s"
indicates a phosphorothioate linkage. The lower case "d" indicates
a deoxy residue. "HP-NH2" or "NH2-HP" indicates a hydroxyprolinol
amine conjugate. "Chol-" indicates a hydroxyprolinol cholesterol
conjugate. Subscript "OMe" indicates a 2'-O-methyl sugar and
subscript "F" indicates a 2'-fluoro modified sugar. Purity was
determined by CGE except where indicated by an asterisk (in these
two cases, purity was determined by ion-exchange
chromatography).
Example 10
Deprotection methods of RNA (with 2'-OMe, PS, or cholesterol
modifications) using Py.HF and polyvinylpyridine polyHF (PVPHF)
[0701] Step 1. Oligonucleotide Synthesis
[0702] All oligonucleotides were synthesized on an AKTA oligopilot
synthesizer. Commercially available controlled pore glass solid
support (dT-CPG, U-CPG 500') or the hydroxy-prolinol-cholesterol
solid support (described in patent applications: provisional
60/600,703 Filed Aug. 10, 2004 and PCT/US04/11829 Filed Apr. 16,
2004) was used. RNA phosphoramidites with standard protecting
groups, 5'-O-dimethoxytrityl-N--
6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-O-N,N'-diisopropyl-2-cyanoe-
thylphosphoramidite,
5'-O-dimethyloxytrityl-N4-acetyl-2'-t-butyldimethylsi-
lyl-cytidine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N-2-isobutryl-2'-t-butyldimethylsilyl-
guanosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-O-N,N'-diisopropy-
l-2-cyanoethylphosphoramidite and
5'-O-dimethoxytrityl-thymidine-3'-O-N,N'-
-diisopropyl-2-cyanoethylphosphoramidite were used for the
oligonucleotide synthesis. All phosphoramidites were used at a
concentration of 0.2 M in acetonitrile (CH.sub.3CN) except for
guanosine and 2'-OMe uridine which was used at 0.2 M concentration
in 10% THF/acetonitrile (v/v). Coupling/recycling time was 14
minutes with linear flow of 500 cm/h on a 12 mL synthesis column.
The activator was 5-ethyl thiotetrazole (0.75M). For the
PO-oxidation 0.5 M iodine in pyridine with 10% water was used and
for the PS-oxidation 0.2 M PADS in 1:1 mixture of CH.sub.3CN and
2,6-lutidine was used. Capping mixture A was 20% N-methyl imidazole
and 80% CH.sub.3CN and capping mixture B was 25% acetic anhydride,
30% 2,6-lutidine and 45% CH.sub.3CN.
[0703] The oligonucleotides synthesized, scale, support type,
amount and loading are listed below:
6 Alnylam Support, Mass Mass of Loading Scale Synthesis SQ No.
(gram) support (g) (.mu.mol/g) (umol) column 5718 dT 4.15 84 349 12
mL 5719 dT 4.15 84 349 12 mL 3216 dT 4.01 87 349 12 mL 3218 dT 4.08
87 355 12 mL 5474 Hydroxy prolinol 4.1 68.6 281 12 mL cholesterol
5475 rU 3.9 83 324 12 mL
[0704] Step 2, Deprotection
[0705] Four methods of deprotection were employed to achieve the
following two steps of cleavage and deprotection: Step 1) cleavage
of oligonucleotide from support with simultaneous removal of base
and phosphate protecting groups from the oligonucleotide, Step 2)
deprotection of 2'-O-TBDMS groups.
[0706] (a) Deprotection with Pyridine HF: The solid support from a
200 .mu.mol synthesis was treated with 30 mL (1 vol) of MeNH.sub.2
(40%, aqueous) at 45.degree. C. for 1.5 hours. The support was
filtered out and rinsed with 60 mL (2 vol) DMSO. Cool it for about
10 minutes in dry ice, a mixture of 7.5 mL pyridine HF (70%) and 30
mL (1 vol) DMSO was added to the filtrate and rinse solution and it
was heated at 40.degree. C. for 1 hour. The reaction was quenched
with 50 mM sodium phosphate (pH 5.5) and diluted with water to an
appropriate volume.
[0707] (b) Deprotection with Pyridine HF with DBU: The solid
support from a 200 .mu.mol synthesis was treated with 20 mL
MeNH.sub.2 (40%, aqueous) at 45.degree. C. for 1.5 hours. The
support was filtered out and rinsed with 60 mL DMSO. 10 mL DBU was
added in the solution. Cool it for about 10 minutes in dry ice, a
mixture of 6 mL pyridine H F (70%) and 20 mL DMSO were added to the
filtrate and rinse solution and it was heated at 40.degree. C. for
1 hour. The reaction was quenched with 50 mM sodium phosphate (pH
5.5) and diluted with water.
[0708] (c) Deprotection with Polyvinylpyridine polyHF (PVPHF): The
solid support from a 200 .mu.mol synthesis was treated with 30 mL
MeNH.sub.2 (40%, aqueous) at 45.degree. C. for 1.5 hours. The
support was filtered out and rinsed with 90 mL DMSO. Cool it for
about 10 minutes in dry ice, PVPHF (12 g) was added to the filtrate
and rinse solution and it was heated at 40.degree. C. for 1 hour.
The reaction was quenched with 50 mM sodium phosphate (pH 5.5). The
reaction mixture was filtered and the solid was rinsed with
water.
[0709] (d) Deprotection with Polyvinylpyridine polyHF (PVPHF) with
DBU: The solid support from a 200 .mu.mol synthesis was treated 20
mL MeNH.sub.2 (40%, aqueous) at 45.degree. C. for 1.5 hours. The
support was filtered out and rinsed with 80 mL DMSO. 8 mL DBU was
added in the solution. Cool it for about 10 minutes in dry ice, 12
g PVPHF were added into the filtrate and rinse solutions and the
reaction was heated at 40.degree. C. for 1 hour. The reaction was
quenched with 50 mM sodium phosphate (pH 5.5). The reaction mixture
was filtered and the solid was rinsed with water.
[0710] Step 3. Purification of Oligonucleotides
[0711] (a) Ion Exchange HPLC Purification: The buffers used for the
ion exchange purification were 20 mM sodium phosphate, 10%
CH.sub.3CN, pH 8.5 (solvent A) and 20 mM sodium phosphate, 1 M
NaBr, 10% CH.sub.3CN, pH 8.5 (solvent B). When the amount of crude
oligonucleotide was less than 10,000 OD, a Waters 2 cm column with
TSK Gel super Q-5PW resin was used. The flow rate was 10 mL/min and
the gradient was 0 to 20% solvent B over 30 minutes, then 20 to 50%
B over 200 minutes.
[0712] When the amount of crude oligonucleotide was more than
10,000 OD or higher resolution was needed due to contamination with
short oligonucleotides, a Waters 5 cm column with TSK-GEL super
Q-5PW resin was used. The flow rate was 50 mL/min and the gradient
was 0 to 20% solvent B over 30 minutes and then 20 to 50% solvent B
over 200 minutes.
[0713] (b) Reverse phase HPLC Purification: For reverse phase
purification, the buffers were 20 mM sodium acetate, 10% CAN, pH
8.5 (solvent A) and 20 mM sodium acetate, 70% CH.sub.3CN, pH 8.5
(solvent B). A 5 cm Waters column with source 15 RPC was used. The
flow rate was 50 mL/min and the gradient was 0 to 15% solvent B
over 30 minutes followed by 15 to 50% solvent B over 160
minutes.
[0714] Step 4. Desalting of Purified Oligomer
[0715] The purified oligonucleotides were desalted on a Waters 5 cm
column with size exclusion resin Sephadex G-25. The flow rate was
25 mL/min. The eluted salt-free fractions were combined together,
dried down and reconstituted in RNase-free water.
[0716] Step 5. Capillary Gel Electrophoresis (CGE) and Electrospray
LC/Ms
[0717] Approximately 0.15 OD of oligonucleotide was diluted in
water to 150 .mu.L. Mass of the product and purity (as shown below)
were determined by LC/MS analysis and anion exchange HPLC or
CGE.
7 AL-SQ Cal. Obs. Purity Deprotect. # Target Sequence Mass Mass %
Method 5718 RSV GGCUCUUAGCAAAGUCAA 6693 6693 95 Pyridine GdTdT HF
5718 RSV GGCUCUUAGCAAAGUCAA 6693 6693 97 Pyridine GdTdT HF with DBU
5719 RSV CUUGACUUUGCUAAGAGC 6607 6606 95 Pyridine CdTdT HF 5719 RSV
CUUGACUUUGCUAAGAGC 6607 6606 96 Pyridine CdTdT HF with DBU 3216 Apo
B GGAAUCU.sub.OMeU.sub.OMeAU.sub.OMeAU 6716 6717 93 PVPHF
.sub.OMeU.sub.OMeU.sub.OMeGAUCC.sub.OMeAdT 3216 Apo B
GGAAUCU.sub.OMeU.sub.OMeAU.sub.OMeAU 6716 6717 94 PVPHF
MeU.sub.OMeU.sub.OMeGAUCC.sub.OMeAdT with DBU 3218 Apo B
GsGAAUCUUAUAUUUGAU 6650 6651 PVPHF CCAsdT 3218 Apo B
GsGAAUCUUAUAUUUGAU 6650 6651 PVPHF CCAsdT with DBU 5474 Apo B
GGAAUCU.sub.OMeU.sub.OMeAU.sub.OMeA- U 7461 7462 90 Pyridine
.sub.OMeU.sub.OMeU.sub.OMeGAUCC.sub.OMeA.- sub.OMe HF with As-Chol
DBU 5475 Apo B U.sub.OMeU.sub.OMeGGAUC.sub.OMeAAAU.sub.O 7421 7421
93 Pyridine .sub.MeAU.sub.OMeAAGAU.sub.OMeUCC.sub.OMe.sub..sup.S HF
with C.sub.OMe.sub..sup.SU DBU
[0718] Oligonucleotides are shown written 5' to 3'. Lower case "s"
indicates a phosphorothioate linkage. The lower case "d" indicates
a deoxy residue. Subscript "OMe" indicates a 2'-O-methyl sugar.
"Chol-" indicates a hydroxyprolinol cholesterol conjugate.
Example 11
Deprotection methods of chimeric RNA with 2'-fluoro modification
using polyvinylpyridine polyHF (PVPHF)
[0719] Step 1. Oligonucleotide Synthesis
[0720] Synthesis, purification and desalting were same as described
in Example 9, Step 1.
[0721] Step 2. Deprotection
[0722] After the synthesis was completed, .about.30 mL of 0.5 M
piperidine in CH.sub.3CN were pumped through the column at a flow
rate of between 5 and 10 mL/min to remove the cyanoethyl protecting
groups from phosphate linkages while the RNA was still attached to
the support. Then, two methods of deprotection were evaluated to
achieve the following two steps of cleavage and deprotection: Step
1) cleavage of oligonucleotide from support with simultaneous
removal of base protecting groups from the oligonucleotide and Step
2) deprotection of 2'-O-TBDMS groups
[0723] (a) Deprotection with Polyvinylpyridine polyHF (PVPHF): The
solid support from a 200 .mu.mol synthesis was treated with 50 mL
solution of NH.sub.3:ethanol (3:1) at 55.degree. C. for 6 hours.
The support was separated from solution by filtering and was rinsed
with 90 mL DMSO. The solid support was removed by filtering. The
filtrate and rinse solution was cooled for about 10 minutes in dry
ice, PVPHF (12 g) was added, and the solution was heated at
40.degree. C. for 2 hours. Deprotection status was checked after 1
hour, 1.5 hours, and 2 hours. The reaction was quenched with 50 mM
sodium phosphate (pH 5.5). The reaction mixture was filtered and
the solid was rinsed with water.
[0724] (b) Deprotection with Polyvinylpyridine polyHF (PVPHF) with
DBU: The solid support from a 200 .mu.mol synthesis was treated 35
mL MeNH.sub.2 (40%, aqueous) at 55.degree. C. for 6 hours. The
support was filtered out and rinsed with 140 mL DMSO. DBU (7 mL)
was added to the filtrate and rinse solution. The solution was
cooled for about 10 minutes in dry ice, 12 g PVPHF was added, and
the reaction was heated at 40.degree. C. for 2 hour. Deprotection
status was checked after 1 hour, 1.5 hours, and 2 hours. The
reaction was quenched with 50 mM sodium phosphate (pH 5.5). The
reaction mixture was filtered and the solid was rinsed with
water.
Example 12
Deprotection Method for RNA Oligonucleotides
[0725] Step 1. Oligonucleotide Synthesis
[0726] Synthesis, purification and desalting were same as described
in Example X, Step 1. The synthesis of oligonucleotides AL-SQ-5548
(5'-AAA GUG CAC AAC AUU AUA CdTdT-3', where all residues were ribo
except for the two 3' terminal nucleotides which were deoxy
thymidine) and AL-SQ-5549 (5'-GUA UAA UGU UGU GCA CUU UdTdT-3') was
done at 400 .mu.mole scale. The calculated mass of AL-SQ-5548 was
6645.03; the observed mass was 6644.94. The calculated mass of
AL-SQ-5549 was 6609.88; the observed mass was 6609.70.
[0727] Step 2. Deprotection Conditions
[0728] The deprotection was done at 94 .mu.mole scale. Dried CPG
(1.5 g) was placed in a 100 mL Schott bottle. Methyl amine (40%
aqueous, 25 mL) was added to the bottle and the mixture was placed
in a shaker oven at 45.degree. C. for 1.5 h. The mixture was cooled
and filtered into a 250 mL Schott bottle. The CPG was washed three
times with 25 mL DMSO in a funnel. The combined filtrates were
cooled for 10 min in dry ice. HF in pyridine (Aldrich, 20 mL) was
added to the bottle. The mixture was shaken well and placed in a
shaker oven at 40.degree. C. for 1 h. The mixture was cooled to
room temperature and the reaction was quenched by adding 150 mL of
50 mM sodium acetate. The final solution was stored at 4.degree.
C.
[0729] Step 3. Quantitation of Crude Oligonucleotides
[0730] In order estimate the crude yield the following procedure
was used. Since the pyridine present in the crude oligonucleotide
solution absorbs at 254 nm, the absorbance was measured at 280 nm.
A small amount of the crude support was subjected to deprotection
using TEA.3HF instead of HF in pyridine. Absorbance was measured
for this sample at 254 nm and 280 nm. Based on the ratio of
A.sub.254 to A.sub.280 of this sample, the absorbance at 254 nm for
the sample containing pyridine was estimated.
[0731] The amount of full-length product was determined by anion
exchange HPLC. For AL-SQ-5548, the full-length product was 73% of
the total strand concentration and for AL-SQ-5549 full-length
product was 67%. The crude yield was 143 OD/.mu.mole.
Example 13
Synthesis and Deprotection Conditions for RNAs at 1.6 mmol
Scale
[0732] Step 1. Oligonucleotide Synthesis
[0733] The oligonucleotides were synthesized on an AKTA oligopilot
synthesizer. Commercially available controlled pore glass solid
supports (from Prime Synthesis) were used. RNA phosphoramidites and
2'-O-methyl modified RNA phosphoramidites with standard protecting
groups
(5'-O-dimethoxytrityl-N-6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-O--
N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N-4-ace-
tyl-2'-t-butyldimethylsilyl-cytidine-3'-O-N,N'-diisopropyl-2-cyanoethylpho-
sphoramidite,
5'-O-dimethoxytrityl-N-2-isobutryl-2'-t-butyldimethylsilyl-g-
uanosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-O-N,N'-diisopropy-
l-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N-6-benzoyl-2'-O-methy-
l-adenosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N-4-acetyl-2'-O-methyl-cytidine-3'-O-N,N'-diisopropy-
l-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N-2-isobutryl-2'-O-met-
hyl-guanosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite,
and
5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-N,N'-diisopropyl-2-cyanoeth-
ylphosphoramidite) were obtained from Pierce Nucleic Acids
Technologies and ChemGenes Research. The 2'-F phosphoramidites
(5'-O-dimethoxytrityl-N-
-4-acetyl-2'-fluro-cytidine-3'-O-N,N'-diisopropyl-2-cyanoethyl-phosphorami-
dite and
5'-O-dimethoxytrityl-2'-fluro-uridine-3'-O-N,N'-diisopropyl-2-cya-
noethyl-phosphoramidite) were obtained from Promega.
[0734] All phosphoramidites were used at a concentration of 0.15 M
in CH.sub.3CN. The RNA amidite coupling/recycling time was 23
minutes and 2 equivalents of amidite were used. DNA coupling cycle
used 60% activator, 7 min recycling, and 2.0 equivalents of
phosphoramidite. A UV watch was introduced in the "push" step
before the "recycle" step to assure consistency in each coupling
step. The activator was 0.6 M ethylthiotetrazole. For the
PO-oxidation, 50 mM iodine in water/pyridine (10:90 v/v) was used;
4.5 equivalents were added in 2.5 min. For PS-oxidation, 0.2 M PADS
in acetonitrile:2,6-lutidine (1:1) was used with 2-5 column volumes
of thiolation reagent used. The Cap A solution was 20%
1-methylimidazole in acetonitrile. Cap B was acetic
anhydride:2,6-lutidine:acetonitrile (25:30:45). For capping, 1.5
column volumes were added in 1.5 min.
[0735] Step 2. Deprotection Conditions
[0736] The CPG was mixed with 180 mL of aqueous methylamine
(Aldrich) in a 250 mL Schott bottle. The mixture was placed in a
shaker oven at 45.degree. C. for 75 min. The mixture was cooled,
filtered into a 1 L Schott bottle and the CPG was washed three
times with 160 mL of DMSO. The filtrates were combined and cooled
for 10 min in dry ice. TEA.3HF (Alfa Aesar, 270 mL) was added to
the mixture. The bottle was placed in a shaker oven at 40.degree.
C. for 65 min. The mixture was cooled to room temperature and the
reaction was quenched with 1 L of 50 mM sodium acetate.
[0737] Step 3. Purification of Oligonucleotides
[0738] The oligonucleotides were purified by reverse phase HPLC
using a matrix of TSK-GEL, SuperQ-5PW (20) in a 5 cm.times.17-18 cm
column. The temperature was maintained at 55.degree. C. to
65.degree. C. The buffers were 20 mM sodium phosphate, 10% ACN v/v,
pH 8.5 (buffer A) and 20 mM sodium phosphate, 1 M NaBr, 10% ACN, pH
8.5 (buffer B). The flow rates was 60 mL/min. The gradient was from
20% B to 40% B in 160 min.
[0739] The solution of crude oligonucleotide was diluted 5-fold
with buffer A and loaded directly onto the purification column
using a flow rate that loaded about 20 mg crude material (based on
A.sub.260 readings) per mL of column volume. Fractions of 50 mL
were collected.
Incorporation by Reference
[0740] All of the patents and publications cited herein are hereby
incorporated by reference.
Equivalents
[0741] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
59 1 21 DNA Artificial Sequence Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide 1 gcggaucaaa ccucaccaat t 21 2
21 DNA Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 2 uuggugaggu uugauccgct t 21 3 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 3 gcggaucaaa ccucaccaat t 21 4 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 4 uuggugaggu uugauccgct t 21 5 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 5 gcggaacaau ccugaccaat t 21 6 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 6 uuggucagga uuguuccgct t 21 7 10 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 7 caucgctgat 10 8 21 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 8 cuuacgcuga guacuucgat t 21 9 21 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 9 ucgaaguacu cagcguaagt t 21 10 21 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 10 gcggaucaaa ccucaccaat t 21 11 21 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 11 gcggaucaaa ccucaccaat t 21 12 21 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 12 gcggaucaaa ccucaccaat t 21 13 21 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 13 uuggugaggu uugauccgct t 21 14 21 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 14 uuggugaggu uugauccgct t 21 15 21 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 15 uuggugaggu uugauccgct t 21 16 10 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Synthetic
oligonucleotide 16 acgucgauat 10 17 12 DNA Artificial Sequence
Description of Combined DNA/RNA Molecule Synthetic oligonucleotide
17 cgucaaggcg at 12 18 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 18 cuuacgcuga
guacuucgat t 21 19 22 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 19 cuuacgcuga
guacuucgat tt 22 20 21 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 20 gucaucacac
ugaauaccaa u 21 21 23 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 21 uuggaucaaa
uauaagauuc ccu 23 22 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 22 ggacuacucu
aaguucuact t 21 23 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 23 guagaacuua
gaguagucct t 21 24 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 24 ggaucaucuc
aagucuuact t 21 25 22 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 25 guaagacuug
agaugauccc tt 22 26 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 26 cgucugucug
ucccggauct t 21 27 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 27 gauccgggac
agacagacgt t 21 28 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 28 ggaucaucuc
aagucuuact t 21 29 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 29 ggacuacucu
aaguucuact t 21 30 21 RNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 30 ggaaucuuau
auuugaucca a 21 31 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 31 cgucugucug
ucccggauct t 21 32 23 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 32 cguccuugaa
gaagauggug cgc 23 33 23 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 33 auugguauuc
agugugauga cac 23 34 23 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 34 uuggaucaaa
uauaagauuc ccu 23 35 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 35 cuaugagccu
gaagccuaat t 21 36 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 36 uuaggcuuca
ggcucauagt t 21 37 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 37 cuacgaaccu
gaagccuaat t 21 38 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 38 uuaggcuuca
gguucguagt t 21 39 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 39 cuacgaaccu
gaagccuaat t 21 40 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 40 cuaugagccu
gaagccuaat t 21 41 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 41 agaagcagga
ccuuaucuat t 21 42 21 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 42 ggaaucuuau
auuugaucca a 21 43 21 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 43 gcaccaucuu
cuucaaggac g 21 44 23 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 44 acaaacacca
uugucacacu cca 23 45 23 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 45 acaaacacca
uugucacacu cca 23 46 23 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 46 acacacaaca
cugucacauu cca 23 47 22 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 47 acaacaacac
ugucacauuc ca 22 48 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 48 ggcucuuagc
aaagucaagt t 21 49 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 49 ggcucuuagc
aaagucaagt t 21 50 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 50 cuugacuuug
cuaagagcct t 21 51 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 51 cuugacuuug
cuaagagcct t 21 52 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 52 ggaaucuuau
auuugaucca t 21 53 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 53 ggaaucuuau
auuugaucca t 21 54 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 54 ggaaucuuau
auuugaucca t 21 55 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 55 ggaaucuuau
auuugaucca t 21 56 21 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 56 ggaaucuuau
auuugaucca a 21 57 23 RNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 57 uuggaucaaa
uauaagauuc ccu 23 58 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 58 aaagugcaca
acauuauact t 21 59 21 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide 59 guauaauguu
gugcacuuut t 21
* * * * *