U.S. patent application number 11/167145 was filed with the patent office on 2005-10-27 for solution phase synthesis of oligonucleotides.
This patent application is currently assigned to Avecia Limited. Invention is credited to Reese, Colin Bernard, Song, Quanlai.
Application Number | 20050240015 11/167145 |
Document ID | / |
Family ID | 10817430 |
Filed Date | 2005-10-27 |
United States Patent
Application |
20050240015 |
Kind Code |
A1 |
Reese, Colin Bernard ; et
al. |
October 27, 2005 |
Solution phase synthesis of oligonucleotides
Abstract
A process for the synthesis in solution phase of a
phosphorothioate triester is provided. The process comprises the
solution phase coupling of an H-phosphonate with an alcohol in the
presence of a coupling agent to form an H-phosphonate diester. The
H-phosphonate diester is oxidised in situ with a sulfur transfer
agent to produce the phosphorothioate triester. Preferably, the
H-phosphonate and alcohol are protected nucleosides or
oligonucleotides. Oligonucleotide H-phosphonates which can be used
in the formation of phosphorothioate triesters are also
provided.
Inventors: |
Reese, Colin Bernard;
(London, GB) ; Song, Quanlai; (San Marcos,
CA) |
Correspondence
Address: |
PILLSBURY WINTHROP SHAW PITTMAN, LLP
P.O. BOX 10500
MCLEAN
VA
22102
US
|
Assignee: |
Avecia Limited
Manchester
GB
|
Family ID: |
10817430 |
Appl. No.: |
11/167145 |
Filed: |
June 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11167145 |
Jun 28, 2005 |
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10294794 |
Nov 15, 2002 |
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10294794 |
Nov 15, 2002 |
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09485606 |
Apr 26, 2000 |
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6506894 |
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09485606 |
Apr 26, 2000 |
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PCT/GB98/02407 |
Aug 10, 1998 |
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Current U.S.
Class: |
536/25.34 |
Current CPC
Class: |
C07F 9/18 20130101; C07H
19/20 20130101; C07H 21/04 20130101; C07D 207/48 20130101; Y02P
20/55 20151101; C07H 19/10 20130101; C07D 265/33 20130101; C07F
9/165 20130101; C07H 21/00 20130101 |
Class at
Publication: |
536/025.34 |
International
Class: |
C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 13, 1997 |
GB |
9717158.1 |
Claims
1-13. (canceled)
14. A process for introducing a group of formula --S-A into an
H-phosphonate diester moiety, comprising: reacting a substrate
comprising an H-phosphonate diester moiety with a sulfur transfer
agent selected from the group consisting of a) compounds of
formula: 10wherein A represents an aryl, methyl, substituted alkyl
or alkenyl group; b) compounds of formula: 11wherein A represents
an aryl, methyl, substituted alkyl or alkenyl group; and c)
compounds of formula: 12wherein A represents a methyl or alkenyl
group.
15. A process according to claim 14, wherein the sulfur transfer
agent is selected from the group consisting of 13
16. A process according to claim 14 or claim 15, wherein the
H-phosphonate diester is an oligonucleotide H-phosphonate
diester.
17. A process according to claim 16, wherein the oligonucleotide
H-phosphonate diester is a protected oligonucleotide H-phosphonate
diester.
18. A process according to claim 17, further comprising one or more
subsequent deprotection steps, thereby producing a deprotected
oligonucleotide.
19. A process for the preparation of an oligonucleotide, an
oligonucleotide phosphorothioate or a mixed
oligonucleotide/oligonucleoti- de phosphorothioate, said process
comprising: introducing a group of formula --S-A into an
H-phosphonate diester moiety by the process according to claim
14.
20. A process according to claim 19, wherein the sulphur transfer
agent is selected from the group consisting of 14
21. A compound of formula: 15wherein A represents an aryl, methyl,
substituted alkyl or alkenyl group.
22. A compound according to claim 21, wherein A represents a phenyl
group or a --CH.sub.2CH.sub.2AN group.
23. A compound according to claim 22, wherein A represents a phenyl
group and the phenyl group is unsubstituted or is substituted by a
halo or alkyl group.
24. A compound of formula: 16
25. A compound of formula: 17wherein A represents an alkenyl group.
Description
[0001] The present invention provides a method of synthesizing
oligonucleotides and oligonucleotide phosphorothioates in solution
based on H-phosphonate coupling and in situ sulfur transfer,
carried out at Jew temperature. The invention further provides a
process for the stepwise synthesis of oligonucleotides and
oligonucleotide phosphorothioates in which one nucleoside residue
is added at a time, and the block synthesis of oligonucleotides and
oligonucleotide phosphorothioates in which two or more nucleotide
residues are added at a time.
[0002] In the past 15 years or so, enormous progress has been made
in the development of the synthesis of oligodeoxyribonucleotides
(DNA sequences), oligoribonucleotides (RNA sequences) and their
analogues `Methods in Molecular Biology, Vol. 20, Protocol for
Oligonucleotides and Analogs`, Agrawal, S. Ed., Humana Press,
Totowa, 1993. Much of the work has been carried out on a micromolar
or even smaller scale, and automated solid phase synthesis
involving monomeric phosphoramidite building blocks Beaucage, S.
L.; Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862 has
proved to be the most convenient approach. Indeed, high molecular
weight DNA and relatively high molecular weight RNA sequences can
now be prepared routinely with commercially available synthesisers.
These synthetic oligonucleotides have met a number of crucial needs
in biology and biotechnology.
[0003] Following Zamecnik and Stephenson's seminal discovery that a
synthetic oligonucleotide could selectively inhibit gene expression
in Rous sarcoma virus, (Zamecnik, P.; Stephenson, M. Proc. Natl.
Acad. Sci. USA 1978, 75, 280-284), the idea that synthetic
oligonucleotides or their analogues might well find application in
chemotherapy has attracted a great deal of attention both in
academic and industrial laboratories. For example, the possible use
of oligonucleotides and their phosphorothioate analogues in
chemotherapy has been highlighted in the report of Gura, T.
Science, 195, 270, 575-577. The so-called antisense and antigene
approaches to chemotherapy (Oligonucleotides. Antisense Inhibitors
of Gene Expression, Cohen. J. S., Ed., Macmillan, Basingstoke 1989
Moser, H. E.; Dervan, P. B. Science 1987, 238, 645-649), have
profoundly affected the requirements for synthetic
oligonucleotides. Whereas milligram quantities have generally
sufficed for molecular biological purposes, gram to greater than
100 gram quantities are required for clinical trials. Several
oligonucleotide analogues that are potential antisense drugs are
now in advanced clinical trials. If, as seems likely in the very
near future, one of these sequences becomes approved, say, for the
treatment of AIDS or a form of cancer, kilogram or more probably
multikilogram quantities of a specific sequence or sequences will
be required.
[0004] In the past few years, a great deal of work has been carried
out on the scaling-up of oligonucleotide synthesis. Virtually all
of this work has involved building larger and larger synthesisers
and the same phosphoramidite chemistry on a solid support. The
applicant is unaware of any recent improvement in the methodology
of the phosphotriester approach to oligonucleotide synthesis in
solution, which makes it more suitable for large- and even
moderate-scale synthetic work than solid phase synthesis.
[0005] The main advantages that solid phase has over solution
synthesis are (i) that it is much faster, (ii) that coupling yields
are generally higher, (iii) that it is easily automated and (iv)
that it is completely flexible with respect to sequence. Thus solid
phase synthesis is particularly useful if relatively small
quantities of a large number of oligonucleotides sequences are
required for, say, combinatorial purposes. However, if a particular
sequence of moderate size has been identified and approved as a
drug and kilogram quantities are required, speed and flexibility
become relatively unimportant, and synthesis in solution is likely
to be highly advantageous. Solution synthesis also has the
advantage over solid phase synthesis in that block coupling (i.e.
the addition of two or more nucleotide residues at a time) is more
feasible and scaling-up to any level is unlikely to present a
problem. It is much easier and certainly much cheaper to increase
the size of a reaction vessel than it is to produce larger and
larger automatic synthesisers.
[0006] In the past, oligonucleotide synthesis in solution has been
carried out mainly by the conventional phosphotriester approach
that was developed in the 1970s (Reese, C. B., Tetrahedron 1978,
34, 3143-3179; Kaplan, B. E.; Itakura, K. in `Synthesis and
Applications of DNA and RNA`, Narang, S. A., Ed., Academic Press,
Orlando, 1987, pp. 9-45). This approach can also be used in solid
phase synthesis but coupling reactions are somewhat faster and
coupling yields are somewhat greater when phosphoramidite monomers
are used. This is why automated solid phase synthesis has been
based largely on the use of phosphoramidite building blocks; it is
perhaps also why workers requiring relatively large quantities of
synthetic oligonucleotides have decided to attempt the scaling-up
of phosphoramidite-based solid phase synthesis.
[0007] Three main methods, namely the phosphotriester (Reese,
Tetrahedron, 1978), phosphoramidite (Beaucage, S. L. in Methods in
Molecular Biology, Vol. 20, Agrawal, S., Ed., Humana Press, Totowa,
1993, pp 3-61) and H-phosphonate (Froehler, B. C. in Methods in
Molecular Biology, Vol. 20, Agrawal, S., Ed., Humana Press, Totowa,
1993, pp 63-80) approaches have proved to be effective for the
chemical synthesis of oligonucleotides. While the phosphotriester
approach has been used most widely for synthesis in solution, the
phosphoramidite and H-phosphonate approaches have been used almost
exclusively in solid phase synthesis.
[0008] Two distinct synthetic strategies have been applied to the
phosphotriester approach in solution.
[0009] Perhaps the most widely used strategy for the synthesis of
oligodeoxyribonucleotides in solution involves a coupling reaction
between a protected nucleoside or oligonucleotide
3'-(2-chlorophenyl) phosphate (Chattopadhyaya, J. B.; Reese, C. B.
Nucleic Acids Res., 1980, 8, 2039-2054) and a protected nucleoside
or oligonucleotide with a free 5'-hydroxy function to give a
phosphotriester. A coupling agent such as
1-(mesitylene-2-sulfonyl)-3nitro-1,2,4-1H-triazole (MSNT) (Reese,
C. B.; Titmas, R. C.; Yau. L. Tetrahedron Lett., 1978, 2727-2730)
is required. This strategy has also been used in the synthesis of
phosphorothioate analogues. Coupling is then effected in the same
way between a protected nucleoside or oligonucleotide
3'-S-(2-cyanoethyl or, for example, 4-nitrobenzyl) phosphorothioate
(Liu, X.; Reese, C. B. J. Chem. Soc., Perkin Trans. 1, 1995,
1685-1695) and a protected nucleoside or oligonucleotide with a
free 5'-hydroxy function. The main disadvantages of this
conventional phosphotriester approach are that some concomitant
5'-sulfonation of the second component occurs (Reese, C. B.; Zhang,
P.-Z. J. Chem. Soc., Perkin Trans. 1, 1995, 2291-2301) and that
coupling reactions generally proceed relatively slowly. The
sulfonation side-reaction both leads to lower yields and impedes
the purification of the desired products.
[0010] The second strategy for the synthesis of
oligodeoxyribonucleotides in solution involves the use of a
bifunctional reagent derived from an aryl (usually 2-chlorophenyl)
phosphorodichloridate and two molecular equivalents of an additive
such as 1-hydroxybenzotriazole (van der Marel, et al, Tetrahedron
Lett., 1981, 22, 3887-3890). A related bifunctional reagent,
derived from 2,5-dichlorophenyl phosphorodichloridothioate (Scheme
1b), has similarly been used (Kemal, O et al, J. Chem. Soc., Chem.
Commun., 1983, 591-593) in the preparation of oligonucleotide
phosphorothioates.
[0011] The main disadvantages of the second strategy result
directly from the involvement of a bifunctional reagent. Thus the
possibility exists of symmetrical coupling products being formed
and the presence of small quantities of moisture can lead to a
significant diminution in coupling yields.
[0012] It is an objective of certain aspects of the present
invention to provide a new coupling procedure for the synthesis of
oligonucleotides in solution that in many embodiments (a) is
extremely efficient and does not lead to side-reactions, (b)
proceeds relatively rapidly, and (c) is equally suitable for the
preparation of oligonucleotides, their phosphorothioate analogues
and chimeric oligonucleotides containing both phosphodiester and
phosphorothioate diester internucleotide linkages.
[0013] According to a first aspect of the present invention, there
is provided a process for the preparation of a phosphorothioate
triester which comprises the solution phase coupling of an
H-phosphonate with an alcohol in the presence of a coupling agent
thereby to form an H-phosphonate diester and, in situ, reacting the
H-phosphonate diester with a sulfur transfer agent to produce a
phosphorothioate triester.
[0014] The H-phosphonate employed in the process of the present
invention is advantageously a protected nucleoside or
oligonucleotide H-phosphonate, preferably comprising a 5' or a 3'
H-phosphonate function, particularly preferably a 3' H-phosphonate
function. Preferred nucleosides are 2'-deoxyribonucleosides and
ribonucleosides; preferred oligonucleotides are
oligodeoxyribonucleotides and oligoribonucleotides.
[0015] When the H-phosphonate building block is a protected
deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or
oligoribonucleotide derivative comprising a 3' H-phosphonate
function, the 5' hydroxy function is advantageously protected by a
suitable protecting group. Examples of such suitable protecting
groups include acid labile protecting groups, particularly trityl
and substituted trityl groups such as dimethoxytrityl and
9-phenylxanthen-9-yl groups; and base labile-protecting groups such
as FMOC.
[0016] When the H-phosphonate building block is a protected
deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or
oligoribonucleotide derivative comprising a 5' H-phosphonate
function, the 3' hydroxy function is advantageously protected by a
suitable protecting group. Suitable protecting groups include those
disclosed above for the protection of the 5' hydroxy functions of
3' H-phosphonate building blocks and acyl, such as levulinoyl and
substituted levulinoyl, groups.
[0017] When the H-phosphonate is a protected ribonucleoside or a
protected oligoribonucleotide, the 2'-hydroxy function is
advantageously protected by a suitable protecting group, for
example an acid-labile acetal protecting group, particularly
1-(2-fluorophenyl)-4-methoxypiperidine-4-y- l (Fpmp); and
trialkylsilyl groups, often tri(C.sub.1-4-alkyl)silyl groups such
as a tertiary butyl dimethylsilyl group. Alternatively, the
ribonuclecside or oligoribonucleotide may be a 2'-O-alkyl,
2'-O-alkoxyalkyl or 2'-O-alkenyl derivative, commonly a C.sub.1-4
alkyl, C.sub.1-4 alkoxyC.sub.1-4 alkyl or alkenyl derivative in
which case, the 2' position does not need further protection.
[0018] Other H-phosphonates that may be employed in the process
according to the present invention are derived from other
polyfunctional alcohols, especially alkyl alcohols, and preferably
diols or triols. Examples of alkyl diols include ethane-1,2-diol,
and low molecular weight poly(ethylene glycols), such as those
having a molecular weight of up to 400. Examples of alkyl triols
include glycerol and butane triols. Commonly, only a single
H-phosphonate function will be present, the remaining hydroxy
groups being protected by suitable protecting groups, such as those
disclosed hereinabove for the protection at the 5' or 2' positions
of ribonucleosides.
[0019] The alcohol employed in the process of the present invention
is commonly a protected nucleoside or oligonucleotide comprising a
tree hydroxy group, preferably a free 3' or 5' hydroxy group, and
particularly preferably a 5' hydroxy group.
[0020] When the alcohol is a protected nucleoside or a protected
oligonucleotide, preferred nucleosides are deoxyribonuclecsides and
ribonucleosides and preferred oligonucleotides are
oligodeoxyribonucleotides and oligoribonucleotides.
[0021] When the alcohol is a deoxyribonucleoside, ribonucleoside
oligodeoxyribonucleotide or oligoribonucleotide derivative
comprising a free 5'-hydroxy group, the 3'-hydroxy function is
advantageously protected by a suitable protecting group. Examples
of such protecting groups include acyl groups, commonly comprising
up to 16 carbon atoms, such as those derived from gamma keto acids,
such as levulinoyl groups and substituted levulinoyl groups.
Substituted levulinoyl groups include particularly
5-halo-levulinoyl, such as 5,5,5-trifluorolevulinoyl and
benzoylpropionyl groups. Other such protecting groups include fatty
alkanoyl groups, including particularly linear or branched
C.sub.6-16 alkanoyl groups, such as lauroyl groups; benzoyl and
substituted benzoyl groups, such as alkyl, commonly C.sub.1-4
alkyl-, and halo, commonly chloro or fluoro, substituted benzoyl
groups; and silyl ethers, such as alkyl, commonly C.sub.1-4 alkyl,
and aryl, commonly phenyl, silyl ethers, particularly tertiary
butyl dimethyl silyl and tertiary butyl diphenyl silyl groups.
[0022] When the alcohol is a protected deoxyribonucleoside,
ribonucleoside, oligodeoxyribonucleotides or oligoribonucleotide
comprising a free 3'-hydroxy group, the 5'-hydroxy function is
advantageously protected by a suitable protecting group. Suitable
protecting groups are those disclosed above for the protection of
the 5' hydroxy group of deoxyribonucleosides, ribonucleosides,
oligodeoxyribonucleotides and oligoribonucleotide 3' H
phosphonates.
[0023] When the alcohol is a ribonuclecside or an
oligoribonucleotide, the 2'-hydroxy function is advantageously
protected by a suitable protecting group, such as an acetal,
particularly 1-(2-fluorophenyl)-4-methoxypiperi- dine-4-yl (Fpmp);
and trialkylsilyl groups, often tri(C.sub.1-4-alkyl)sily- l groups
such as a tertiary butyl dimethyl silyl group. Alternatively, the
ribonucleoside or oligoribonucleotide may be a 2'-O-alkyl,
2'-O-alkoxyalkyl or 2-'O-alkenyl derivative, commonly a C.sub.1-4
alkyl, C.sub.1-4 alkoxyC.sub.1-4 alkyl or alkenyl derivative, in
which case, the 2' position does not need further protection.
[0024] Other alcohols that may be employed in the process according
to the present invention are non-saccharide polyols, especially
alkyl polyols, and preferably diols or triols. Examples of alkyl
diols include ethane-1,2-diol, and low molecular weight
poly(ethylene glycols), such as those having a molecular weight of
up to 400. Examples of alkyl triols include glycerol and butane
triols. Commonly, only a single free hydroxy group will be present,
the remaining hydroxy groups being protected by suitable protecting
groups, such as those disclosed hereinabove for the protection at
the 5' or 2' positions of ribonucleosides. However, more than one
free hydroxy group may be present if it is desired lo perform
identical couplings on more than one hydroxy group.
[0025] When the H-phosphonate and the alcohol are both protected
nucleosides or oligonucleotides, the invention provides an improved
method for the stepwise and block synthesis in solution of
oligodeoxyribonucleotides, oligoribonucleotides and analogues
thereof, based on H-phosphonate coupling reactions. According to
one preferred aspect of the present invention, protected
nucleosides or oligonucleotides with a 3'-terminal H-phosphonate
function and protected nucleosides or oligonucleotides with a
5'-terminal hydroxy function are coupled in the presence of a
suitable coupling agent to form a protected dinucleoside or
oligonucleotide H-phosphonate intermediate, wherein said
intermediates undergo sulfur-transfer in situ in the presence of a
suitable sulfur-transfer agent.
[0026] In addition to the presence of hydroxy protecting groups,
bases present in nucleosides/nucleotides employed in present
invention are also preferably protected where necessary by suitable
protecting groups. Protecting groups employed are those known in
the art for protecting such bases. For example, A and/or C can be
protected by benzoyl, including substituted benzoyl, for example
alkyl- or alkoxy-, often C.sub.1-4 alkyl- or C.sub.1-4 alkoxy-,
benzoyl; pivaloyl; and amidine, particularly dialkylaminomethyiene,
preferably di(C.sub.1-4-alkyl) aminomethylene such as dimethyl or
dibutyl aminomethylene. G may be protected by a phenyl group,
including substituted phenyl, for example 2,5-dichlorophenyl and
also by an isobutyryl group. T and U generally do not require
protection, but in certain embodiments may advantageously be
protected, for example at O4 by a phenyl group, including
substituted phenyl, for example 2,4-dimethylphenyl or at N3 by a
pivaloyloxymethyl, benzoyl, alkyl or alkoxy substituted benzoyl,
such as C.sub.1-4 alkyl- or C.sub.1-4 alkoxybenzoyl.
[0027] When the alcohol and/or H-phosphonate is a protected
nucleoside or oligonucleotide having protected hydroxy groups, one
of these protecting groups may be removed after carrying out the
process of the first invention. Commonly, the protecting group
removed is that on the 3'-hydroxy function. After the protecting
group has been removed, the oligonucleotide thus formed may be
converted into an H-phosphonate and may then proceed through
further stepwise or block coupling and sulfur transfers according
to the process of the present invention in the synthesis of a
desired oligonucleotide sequence. The method may then proceed with
steps to remove the protecting groups from the internucleotide
linkages, the 3' and the 5'-hydroxy groups and from the bases.
Similar methodology may be applied to coupling 5' H-phosphonates,
wherein the protecting group removed is that on the 5' hydroxy
function.
[0028] In a particularly preferred embodiment, the invention
provides a method comprising the coupling of a
5'-O-(4,4'-dimethoxytrityl)-2'-deoxyr- ibonucleoside
3'-H-phosphonate or a protected oligodeoxyribonucleotide
3'-H-phosphonate and a component with a free 5'-hydroxy function in
the presence of a suitable coupling agent and subsequent in situ
sulfur transfer in the presence of a suitable sulfur-transfer
agent.
[0029] In the process of the present invention, any suitable
coupling agents and sulfur-transfer agents available in the prior
art may be used.
[0030] Examples of suitable coupling agents include alkyl and aryl
acid chlorides, alkane and arene sulfonyl chlorides, alkyl and aryl
chloroformates, alkyl and aryl chlorosulfites and alkyl and aryl
phosphorochloridates.
[0031] Examples of suitable alkyl acid chlorides which may be
employed include C.sub.2 to C.sub.7 alkanoyl chlorides,
particularly pivaloyl chloride. Examples of aryl acid chlorides
which may be employed include substituted and unsubstituted benzoyl
chlorides, such as C.sub.1-4 alkoxy, halo, particularly fluoro,
chloro and bromo, and C.sub.1-4 alkyl, substituted benzoyl
chlorides. When substituted, from 1 to 3 substituents are often
present, particularly in the case of alkyl and halo
substituents.
[0032] Examples of suitable alkanesulfonyl chlorides which may be
employed include C.sub.2 to C.sub.7 alkanesulfonyl chlorides.
Examples of arenesulfonyl chlorides which may be employed include
substituted and unsubstituted benzenesulfonyl chlorides, such as
C.sub.1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and
C.sub.1-4 alkyl, substituted benzenesulfonyl chlorides. When
substituted, from 1 to 3 substituents are often present,
particularly in the case of alkyl and halo substituents.
[0033] Examples of suitable alkyl chloroformates which may be
employed include C.sub.2 to C.sub.1 alkyl chloroformates. Examples
of aryl chloroformates which may be employed include substituted
and unsubstituted phenyl chloroformates, such as C.sub.1-4 alkoxy,
halo, particularly fluoro, chloro and bromo, and C.sub.1-4 alkyl,
substituted phenyl chloroformates. When substituted, from 1 to 3
substituents are often present, particularly in the case of alkyl
and halo substituents.
[0034] Examples of suitable alkyl chlorosulfites which may be
employed include C.sub.2 to C.sub.7 alkyl chlorosulfites. Examples
of aryl chlorosulfites which may be employed include substituted
and unsubstituted phenyl chlorosulfites, such as C.sub.1-4 alkoxy,
halo, particularly fluoro, chloro and bromo, and C.sub.1-4 alkyl,
substituted phenyl chlorosulfites. When substituted, from 1 lo 3
substituents are often present, particularly in the case of alkyl
and halo substituents.
[0035] Examples of suitable alkyl phosphorochloridates which may be
employed include di(C.sub.1 to C.sub.6 alkyl) phosphorochloridates.
Examples of aryl phosphorochloridates which may be employed include
substituted and unsubstituted diphenyl phosphorochloridates, such
as C.sub.1-4 alkoxy, halo, particularly fluoro, chloro and bromo,
and C.sub.1-4 alkyl, substituted diphenyl phosphorochloridates.
When substituted, from 1 to 3 substituents are often present,
particularly in the case of alkyl and halo substituents.
[0036] Further coupling agents that may be employed are the
chloro-, bromo- and (benzotriazo-1-yloxy)-phosphonium and carbonium
compounds disclosed by Wada et al, in J.A.C.S. 1997, 119, pp
12710-12721 (incorporated herein by reference).
[0037] Preferred coupling agents are diaryl phosphorochloridates,
particularly those having the formula (ArO).sub.2POCl wherein Ar is
preferably phenyl, 2-chlorophenyl, 2,4,6-trichlorophenyl or
2,4,6-tribromophenyl.
[0038] The nature of the sulfur-transfer agent will depend on
whether an oligonucleotide, a phosphorothioate analogue or a mixed
oligonucleotide/oligonucleotide phosphorothioate is required.
Sulfur transfer agents employed in the process of the present
invention often have the general chemical formula:
L-S-A
[0039] wherein L represents a leaving group, and A represents an
aryl group, a methyl or a substituted alkyl group or an alkenyl
group. Commonly the leaving group is selected so as to comprise a
nitrogen-sulfur bond. Examples of suitable leaving groups include
morpholines such as morpholine-3,5-dione; imides such as
phthalimides, succinimides and maleimides; indazoles, particularly
indazoles with electron-withdrawing substituents such as
4-nitroindazoles; and triazoles.
[0040] Where a standard phosphodiester linkage is required in the
final product, the sulfur transfer agent, the moiety A represents
an aryl group, such as a phenyl or naphthyl group. Examples of
suitable aryl groups include substituted and unsubstituted phenyl
groups, particularly halophenyl and alkylphenyl groups, especially
4-halophenyl and 4-alkylphenyl, commonly 4-(C.sub.1-4 alkyl)phenyl
groups, most preferably 4-chlorophenyl and p-tolyl groups. An
example of a suitable class of standard phosphodiester-directing
sulfur-transfer agent is an N-(arylsulfanyl)phthalimide
(succinimide or other imide may also be used).
[0041] Where a phosphorothioate diester linkage is requited in the
final product, the moiety A represents a methyl, substituted alkyl
or alkenyl group. Examples of suitable substituted alkyl groups
include substituted methyl groups, particularly benzyl and
substituted benzyl groups, such as alkyl-, commonly C.sub.1-4alkyl-
and halo-, commonly chloro-, substituted benzyl groups, and
substituted ethyl groups, especially ethyl groups substituted at
the 2-position with an electron-withdrawing substituent such as
2-(4-nitrophenyl)ethyl and 2-cyanoethyl groups. Examples of
suitable alkenyl groups are allyl and crotyl. Examples of a
suitable class of phosphorothioate-directing sulfur-transfer agents
are, for example, (2-cyanoethyl)sulfanyl derivatives such as
4-[(2-cyanoethyl)-sulfanyl]morpholine-3,5-dione or a corresponding
reagent such as 3-(phthalimidosulfanyl)propanonitrile.
[0042] A suitable temperature for carrying out the coupling
reaction and sulfur transfer is in the range of approximately
-55.degree. C. to room temperature (commonly in the range of from
10 to 30.degree. C., for example approximately 20.degree. C.), and
preferably from -40.degree. C. to 0.degree. C.
[0043] Organic solvents which can be employed in the process of the
present invention include haloalkanes, particularly
dichloromethane, esters, particularly alkyl esters such as ethyl
acetate, and methyl or ethyl propionate, and basic, nucleophilic
solvents such as pyridine. Preferred solvents for the coupling and
sulfur transfer steps are pyridine, dichloromethane and mixtures
thereof.
[0044] The mole ratio of H-phosphonate to alcohol in the process of
the present invention is often selected to be in the range of from
about 0.9:1 to 3:1, commonly from about 1:1 to about 2:1, and
preferably from about 1.1:1 to about 1.5:1, such as about 1.2:1.
However, where couplings on more than one free hydroxyl are taking
place at the same time, the mole ratios will be increased
proportionately. The mole ratio of coupling agent to alcohol is
often selected to be in the range of from about 1:1 to about 10:1,
commonly from about 1.5:1 to about 5:1 and preferably from about
2:1 to about 3:1. The mole ratio of sulfur transfer agent to
alcohol is often selected to be in the range of from about 1:1 to
about 10:1, commonly from about 1.5:1 to about 5:1 and preferably
from about 2:1 to about 3:1.
[0045] In the process of the present invention, the H-phosphonate
and the alcohol can be pre-mixed in solution, and the coupling
agent added to this mixture. Alternatively, the H-phosphonate and
the coupling agent can be pre-mixed, often in solution and then
added to a solution of the alcohol, or the alcohol and the coupling
agent may be mixed, commonly in solution, and then added to a
solution of the H-phosphonate. In certain embodiments, the
H-phosphonate, optionally in the form of a solution, can be added
to a solution comprising a mixture of the alcohol and the coupling
agent. After the coupling reaction is substantially complete, the
sulfur transfer agent is then added to the solution the
H-phosphonate diester produced in the coupling reaction. Reagent
additions commonly take place continuously or incrementally over an
addition period.
[0046] In the process of the present invention, it is possible to
prepare oligonucleotides containing both phosphodiester and
phosphorothioate diester internucleotide linkages in the same
molecule by selection of appropriate sulfur transfer agents,
particularly when the process is carried out in a stepwise
manner.
[0047] As stated previously, the method of the invention can be
used in the synthesis of RNA, 2'-O-alkyl-RNA, 2'-O-alkoxyalkyl-RNA
and 2'-O-alkenyl-RNA sequences. 2'-O-(F pmp)-5'-O-(4,4
-dimethoxytrityl)-ribonucleoside 3'-H-phosphonates 1 and
2'-O-(alkyl, alkoxyalkyl or
alkenyl)-5'-O-(4,4-dimethoxytrityl)-ribonuclecside
3'-H-phosphonates 2a-c may be prepared, for example, from the
corresponding nucleoside building blocks, ammonium p-cresyl
H-phosphonate and pivaloyl chloride. 1
[0048] The same protocols are used as in the synthesis of DNA and
DNA phosphorothioate sequences (Schemes 2-4). Following the
standard unblocking procedure (Scheme 2, steps v and vi), the Fpmp
protecting groups are removed under mild conditions of acidic
hydrolysis that lead to no detectable cleavage or migration of the
internucleotide linkages (Capaldi, D. C.; Reese, C. B. Nucleic
Acids Res. 1994, 22, 2209-2216). For chemotherapeutically useful
ribozyme sequences, relatively large scale RNA synthesis in
solution is a matter of considerable practical importance. The
incorporation of 2'-O-alkyl, 2'-O-substituted alkyl and
2'-O-alkenyl [especially 2'-O-methyl, 2'-O-allyl and
2'-O-(2-methoxyethyl)]-ribonucleosides (Sprcat, B. S. in `Methods
in Molecular Biology, Vol. 20. Protocols for Oligonucleotides and
Analogs`, Agrawal, S., Ed., Humana Press, Totowa, 1993) into
oligonucleotides is currently a matter of much importance as these
modifications confer both resistance to nuclease digestion and good
hybridisation properties on the resulting oligomers.
[0049] The sulfur transfer step is carried out on the product of
the H-phosphonate coupling in situ, ie without separation and
purification of the intermediate produced by the coupling reaction.
Preferably, the sulfur transfer agent is added to the stirred
mixture resulting from the coupling reaction.
[0050] In addition to the fact that it is carried out in homogenous
solution, the present coupling procedure differs from that followed
in the H-phosphonate approach to solid phase synthesis (Froehler et
al., Methods in Molecular Biology, 1993) in at least two other
important respects. First, it may be carried out at a very low
temperature. Side reactions which can accompany H-phosphonate
coupling (Kuyl-Yeheskiely et al, Rec. Trav. Chim., 1986, 105,
505-506) can thereby be avoided even when di-(2-chlorophenyl)
phosphorodichloridate rather than pivaloyl chloride (Froehler, B.
C.; Matteucci, M. D. Tetrahedron Lett., 1986, 27, 469-472) is used
as the coupling reagent. Secondly, sulfur transfer is carried out
after each coupling step rather than just once following the
assembly of the whole oligomer sequence.
[0051] Protecting groups can be removed using methods known in the
art for the particular protecting group and function. For example,
transient protecting groups, particularly gamma keto acids such as
levulinoyl-type protecting groups, can be removed by treatment with
hydrazine, for example, buffered hydrazine, such as the treatment
with hydrazine under very mild conditions disclosed by van Boom. J.
H.; Burgers, P. M. J. Tetrahedron Lett., 1976, 4875-4878. The
resulting partially-protected oligonucleotides with free 3'-hydroxy
functions may then be converted into the corresponding
H-phosphonates which are intermediates which can be employed for
the block synthesis of oligonucleotides and their phosphorothioate
analogues.
[0052] When deprotecting the desired product once this has been
produced, protecting groups on the phosphorus which produce
phosphorothioate triester linkages are commonly removed first. For
example, a cyanoethyl group can be removed by treatment with a
strongly basic amine such as DABCO, 1,5-diazabicylo[4.3.0]non-5-ene
(DBN), 1,8-diazabicyclo[5.4.0]unde- c-7-ene (DBU) or
triethylamine.
[0053] Phenyl and substituted phenyl groups on the phosphorothioate
internucleotide linkages and on the base residues can be removed by
oximate treatment, for example with the conjugate base of an
aldoxime, preferably that of E-2-nitrobenzaldoxime or
pyridine-2-carboxaldoxime (Reese et al. Nucleic Acids Res. 1981).
Kamimura, T. et al in J. Am. Chem. Soc., 1984, 106 4552-4557 and
Sekine. M. Et al, Tetrahedron, 1985, 41, 5279-5288 in an approach
to oligonucleotide synthesis by the phosphotriester approach in
solution, based on S-phenyl phosphorothioate intermediates: and van
Boom and his co-workers in an approach to oligonucleotide
synthesis, based on S-(4-methylphenyl) phosphorothioate
intermediates (Wreesman, C. T. J. Et al, Tetrahedron Lett., 1985,
26, 933-936) have all demonstrated that unblocking
S-phenylphosphorothioates with oximate ions (using the method of
Reese et al., 1978; Reese, C. B,; Zard, L. Nucleic Acids Res.,
1981, 9, 4611-4626) led to natural phosphodiester internucleotide
linkages. In the present invention, the unblocking of
S-(4-chlorophenyl)-protected phosphorothioates with the conjugate
base of E-2-nitrobenzaldoxime proceeds smoothly and with no
detectable internucleotide cleavage.
[0054] Other base protecting groups, for example benzoyl, pivaloyl
and amidine groups can be removed by treatment with concentrated
aqueous ammonia.
[0055] Trityl groups present can be removed by treatment with acid.
With regard to the overall unblocking strategy in
oligodeoxyribonucleotide synthesis, another important consideration
of the present invention, is that the removal of trityl, often a
5'-terminal DMTr, protecting group (`detritylation`) should proceed
without concomitant depurination, especially of any
6-N-acyl-2'-deoxyadenosine residues. According to an embodiment of
the invention, the present inventors have found that such
depurination, which perhaps is difficult completely to avoid in
solid phase synthesis, can be totally suppressed by effecting
`detritylation` with a dilute solution of hydrogen chloride at low
temperature, particularly ca. 0.45 M hydrogen chloride in
dioxane-dichloromethane (1:8 v/v) solution at -50.degree. C. Under
these reaction conditions, `detritylation` can be completed
rapidly, and in certain cases after 5 minutes or less. For example,
when 6-N-benzoyl-5'-O-(4,4'-dimethoxytrityl- )-2'-deoxyadenosine
was treated with hydrogen chloride in dioxane-dichloromethane under
such conditions, `detritylation` was complete after 2 min, but no
depurination was detected even after 4 hours.
[0056] Silyl protecting groups may be removed by fluoride
treatment, for example with a solution of a tetraalkyl ammonium
fluoride salt such as tetrabutyl ammonium fluoride.
[0057] Fpmp protecting groups may be removed by acidic hydrolysis
under mild conditions.
[0058] This new approach to the synthesis of oligonucleotides in
solution is suitable for the preparation of sequences with (a)
solely phosphodiester, (b) solely phosphorothioate diester and (c)
a combination of both phosphodiester and phosphorothioate diester
internucleotide linkages.
[0059] The invention also relates to the development of block
coupling (as illustrated for example in Scheme 4b). In this
respect, the examples provide an illustration of the synthesis of
d[Tp(s)Tp(s)Gp(s)Gp(s)Gp(s)Gp- (s)Tp(s)T] (ISIS 5320 Ravikuma, V.
T., Cherovallath, Z. S. Nucleosides & Nucleosides 1996, 15,
1149-1155), an octadeoxyribonucleoside heptaphosphorothioate, from
tetramer blocks. This oligonucleotide analogue has properties as an
anti-HIV agent. Other proposed block synthesis targets include
sequences with therapeutic effects, for example, inhibitors of
human thrombin and anti-HIV agents. The method of the invention
furthermore can be used in the synthesis of larger sequences.
[0060] It will be apparent that when the process of the present
invention is applied to block synthesis, a number of alternative
strategies are available in terms of the route to the desired
product. These will depend on the nature of the desired product.
For example, an octamer may be prepared by the preparation of
dimers, coupled to produce tetramers, which are then coupled to
produce the desired octamer. Alternatively, a dimer and a trimer
may be coupled to produce a pentamer, which can be coupled with a
further trimer to produce the desired octamer. The choice of
strategy is at the discretion of the user. However, the common
feature of such block coupling is that an oligomer H-phosphonate
comprising two or more units is coupled with an oligomer alcohol
also comprising two or more units. Most commonly oligonucleotide
3'-H-phosphonates are coupled with oligonucleotides having free
5'-hydroxy functions.
[0061] The process of the present invention can also be employed to
prepare cyclic oligonucleotides, especially cyclic
olicodeoxyribonucleotides and cyclic ribonucleotides. In the
preparation of cyclic oligonucleotides, an oligonucleotides
comprising an H-phosphonate function, often a 3' or 5'
H-phosphonate is prepared, and a free hydroxy function is
introduced by appropriate deprotection. The position of the free
hydroxy function is usually selected to correspond to the
H-phosphonate, for example a 5' hydroxy function would be coupled
with a 3' H-phosphonate, and a 3' hydroxy function would be coupled
with a 5' H-phosphonate. The hydroxy and the H-phosphonate
functions can then be coupled intramolecularly in solution in the
presence of a coupling agent and this reaction is followed by in
situ sulfur transfer.
[0062] According to a further aspect of the present invention,
there is provided novel oligomer H-phosphonates having the general
chemical formula: 2
[0063] wherein
[0064] each B independently is a base selected from A, G, T C or
U;
[0065] each Q independently is H or OR' wherein R' is alkyl,
substituted alkyl, alkenyl or a protecting group;
[0066] each R independently is an aryl, methyl, substituted alkyl
or alkenyl group;
[0067] W is H, a protecting group or an H-phosphonate group of
formula 3
[0068] in which M.sup.+ is a monovalent cation;
[0069] each X independently represent O or S;
[0070] each Y independently represents O or S;
[0071] Z is H, a protecting group or an H-phosphonate group of
formula 4
[0072] in which M.sup.+ is a monovalent cation; and
[0073] n is a positive integer;
[0074] provided that when W is H or a protecting group, that Z is
an H-phosphonate group, and
[0075] that when Z is H or a protecting group, that W is an
H-phosphonate group.
[0076] Preferably, only one of W or Z is an H-phosphonate group,
commonly only Z being an H-phosphonate group.
[0077] When W or Z represents a protecting group, the protecting
group may be one of those disclosed above for protecting the 3' or
5' positions respectively. When W is a protecting group, the
protecting group is a trityl group, particularly a dimethoxytrityl
group. When Z is a protecting group, the protecting group is a
trityl group, particularly a dimethoxytrityl group, or an acyl,
preferably a levulinoyl group.
[0078] The bases A, G and C represented by B are preferably
protected, and bases T and U may be protected. Suitable protecting
groups include those described hereinabove for the protection of
bases in the process according to the first aspect of the present
invention.
[0079] When Q represents a group of OR', and R' is alkenyl, the
alkenyl group is often a C.sub.1-4 alkenyl group, especially allyl
or crotyl group. When R' represents alkyl, the alkyl is preferably
a C.sub.1-4 alkyl group. When R' represents substituted alkyl, the
substituted alkyl group includes alkoxyalkyl groups, especially
C.sub.1-4 alkyoxyC.sub.1-4 alkyl groups such as methoxyethyl
groups. When R' represents a protecting group, the protecting group
is commonly an acid-labile acetal protecting group, particularly
1-(2-fluorophenyl)-4-methoxypiperidine-4-yl (Fpmp) or a
trialkylsilyl groups, often a tri(C.sub.1-4-alkyl)silyl group such
as a tertiary butyl dimethylsilyl group.
[0080] Preferably, X represents O.
[0081] In many embodiments, Y represents S and each R represents
the methyl, substituted alkyl, alkenyl or aryl group remaining from
the sulfur transfer agent(s) employed in the process of the present
invention. Preferably, each R independently represents a methyl
group; a substituted methyl group, particularly a benzyl or
substituted benzyl group, such as an alkyl-, commonly
C.sub.1-4alkyl- or halo-, commonly chloro-, substituted benzyl
group; a substituted ethyl group, especially an ethyl group
substituted at the 2-position with an electron-withdrawing
substituent such as a 2-(4-nitrophenyl)ethyl or a 2-cyanoethyl
group: a C.sub.1-4 alkenyl croup, preferably an allyl and crotyl
group; or a substituted or unsubstituted phenyl group, particularly
a halophenyl or alkylphenyl group, especially 4-halophenyl group or
a 4-alkylphenyl, commonly a 4-(C.sub.1-4 alkyl)phenyl group, and
most preferably a 4-chlorophenyl or a p-tolyl group.
[0082] M+ preferably represents a trialkyl ammonium ion, such as a
tri(C.sub.1-4-alkylammonium) ion, and preferably a triethylammonium
ion.
[0083] n may be 1 up to any number depending on the oligonucleotide
which is intended to be synthesised, particularly up to about 20.
Preferably n is 1 to 16, and especially 1 to 9. H-phosphonate
wherein n represents 1, 2 or 3 can be employed when it is desired
to add small blocks of nucleotide, with correspondingly larger
values of n, for example 5, 6 or 7 or mote being employed if larger
blocks of oligonucleotide are desired to be coupled.
[0084] The H-phosphonates according to the present invention are
commonly in the form of solutions, preferably these employed in the
process of the first aspect of the present invention.
[0085] These H-phosphonates are also useful intermediates in the
block synthesis of oligonucleotides arid oligonucleotide
phosphorothioates. As indicated above, block coupling is much more
feasible in solution phase than in solid phase synthesis.
[0086] The oligonucleotide H-phosphonates can be prepared using
general methods known in the art for the synthesis of nucleoside
H-phosphonates. Accordingly, in a further aspect of the present
invention, there is provided a process for the production of an
oligonucleotide H-phosphonate wherein an oligonucleotide comprising
a free hydroxy function, preferably a 3' or 5' hydroxy function, is
reacted with an alkyl or aryl H-phosphonate salt in the presence of
an activator.
[0087] Preferably, the oligonucleotide is a protected
oligonucleotide, and most preferably a protected
oligodecoxynucleotide or a protected oligoribonucleotide. The
H-phosphonate salt is often an ammonium salt, including alkyl, aryl
and mixed alkyl and aryl ammonium salts. Preferably, the ammonium
salt is an (NH.sub.4).sup.+ or a tri(C.sub.1-4alkyl) ammonium salt.
Examples of alkyl groups which may be present in the H-phosphonate
are C.sub.1-4 alkyl, especially C.sub.2-4 alkyl, groups substituted
with strongly electron withdrawing groups, particularly halo, and
preferably fluoro groups, such as 2,2,2-trifluoroethyl and
1,1,1,3,3,3-hexafluoropropan-2-yl groups. Examples of aryl groups
which may be present include phenyl and substituted phenyl,
particularly alkylphenyl, commonly C.sub.1-4 alkylphenyl and
halophenyl, commonly chlorophenyl groups. Preferably, a substituted
phenyl group is a 4-substituted phenyl group. Particularly
preferred H-phosphonates are ammonium and triethylammonium p-cresyl
H-phosphonates. Activators which may be employed include those
compounds disclosed herein for use as coupling agents, and
particularly diaryl phosphorochloridates and alkyl and cycloalkyl
acid chlorides, such as 1-adamantanecarbonyl chloride, and
preferably pivaloyl chloride. The production of H-phosphonates
preferably takes place in the presence of a solvent often those
solvents disclosed for use in the process of the first aspect of
the present invention, preferably pyridine, dichloromethane and
mixtures thereof.
[0088] One advantage of the present invention for the synthesis of
solely phosphorothioate diesters is that, provided care is taken to
avoid desulfurisation during the unblocking steps [particularly
during heating with aqueous ammonia (for example Scheme 3. step
viii(a))], the synthesis of oligonucleotide phosphorothioates
should not lead to products that are contaminated with standard
phosphodiester internucleotide linkages. In the case of solid phase
oligonucleotide phosphorothioate synthesis, incomplete sulfur
transfer in each synthetic cycle usually leads to a residual
phosphodiester contamination (Zon, G.; Stec, W. J. in
`Oligonucleotides and Analogs. A Practical Approach`, Eckstein, F.,
Ed., IRL Press. Oxford, 1991, pp. 87-108).
[0089] The solution synthesis as proposed by the present invention
has another enormous advantage over solid-phase synthesis in that
the possibility exists of controlling the selectivity of reactions
by working at low or even at very low temperatures. This advantage
extends to the detritylation step (Scheme 3, step i) which can
proceed rapidly and quantitatively below 0.degree. C. without
detectable depurination. After the detritylation step, a relatively
quick and efficient purification can be effected by what has
previously been described as the `filtration` approach (Chaudhuri,
B,; Reese, C. B.; Weclawek, K. Tetrahedron Lett. 1984, 25,
4037-4040). This depends on the fact that phosphotriester (and
phosphorothioate triester) intermediates, but not any remaining
detritylated charged monomers, are very rapidly eluted from short
columns of silica gel by THF-pyridine mixtures.
[0090] The method according to the invention will now be
illustrated with reference to the following examples which are not
intended to be limiting:
[0091] In the Examples, it should be noted, that where nucleoside
residues and internucleotide linkages are italicised, this
indicates that they are protected in some way. In the present
context, A, C, G, and T represent 2'-deoxyadenosine protected on
N-6 with a benzoyl group, 2'-deoxycytidine protected on N-4 with a
benzoyl group, 2'-deoxyguanosine protected on N-2 and on O-6 with
isobutyryl and 2,5-dichlorophenyl groups and unprotected thymine.
For example, as indicated in scheme 3, p(s) and p(s') represent
S-(2-cyanoethyl) and S-(4-chlorophenyl) phosphorothioates,
respectively, and p(H), which is not protected and therefore not
italicised, represents an H-phosphonate monoester if it is placed
at the end of a sequence or attached to a monomer but otherwise it
represents an H-phosphonate diester.
EXAMPLES
Reaction Scheme for Preparation of Dinucleoside Phosphates
[0092] With particular reference to the preparation of dinucleoside
phosphates, Scheme 2 describes in more detail the method of the
invention for the preparation of olicodeoxyribonucleotides and the
phosphorothioate analogues thereof. 56
Reagents and Conditions
[0093] (i) 18, C.sub.5H.sub.5N, CH.sub.2Cl.sub.2, -40.degree. C.,
5-10 min;
[0094] (ii) 19, C.sub.5H.sub.5N, CH.sub.2Cl.sub.2, -40.degree. C.,
15 min, b, C.sub.5H.sub.5N--H.sub.2O (1:1 v/v), -40.degree. C. to
room temp.
[0095] (iii) 4 M HCl/dioxane, CH.sub.2Cl.sub.2, -50.degree. C., 5
min;
[0096] (iv) Ac.sub.2O, C.sub.5H.sub.5N, room temp., 15 h;
[0097] (v) 20, TMG, MeCN, room temp., 12 h;
[0098] (vi) a, conc. aq. NH.sub.3 (d 0.88), 50.degree. C., 15 h, b,
Amberlite IR-120 (plus), Na+ form, H.sub.2O;
[0099] (vii) a, 21, C.sub.5H.sub.5N, CH.sub.2Cl.sub.2, -40.degree.
C., 15 min, b, C.sub.5H.sub.5N--H.sub.2O (1:1 v/v), -40.degree. C.
to room temp.;
[0100] (viii) DBU, Me.sub.3SiCl, CH.sub.2Cl.sub.2, room temp., 30
min;
[0101] (ix) 20, DBU, MeCN, room temp., 12 h.
[0102] From Scheme 2, the synthesis of oligonucleotides proceeds
through intermediates 8, 9, 10 and 11 and the preparation of the
phosphorothioate analogues proceeds through intermediates 8, 9, 12
and 13. Eases 14, 16 and 16 correspond to protected adenine,
protected cytosine and protected guanine. Base 17 corresponds to
thymine which does not require protection. Any conventionally used
protecting group can be used. In the synthesis of RNA, thymine will
be replaced by uracil. Compound 18 is a suitable coupling agent,
and compounds 19 and 21 are suitable sulfur transfer agents. These
compounds are referred to more fully hereinbelow.
[0103] The monomeric building blocks required in the coupling
procedure according to the invention illustrated in Scheme 2 are
triethylammonium 5'-O-(4-4'-dimethoxytrityl)-2'-deoxyribonucleoside
3'-H-phosphonates 8 (Eases B and B'=14-17) which can readily be
prepared in almost quantitative yields from the corresponding
protected nucleoside derivatives by a recently reported procedure
(Ozola. V., Reese. C. B., Song Q. Tetrahedron Lett., 1996, 37,
8621-8624). By way of illustration, triethylammonium
5'-O-(dimethoxytrityl)-2'-deoxyribonucleoside 3'-H-phosphonates 8
were prepared as follows: Ammonium 4-methylphenyl H-phosphonate 30
(2.84 g, 15.0 mmol), 5'-O-(dimethyoxytrityl)-2'-deoxyrib-
onucleoside derivative (5.0 mmol), triethylamine (4.2 ml, 30 mmol)
and dry pyridine (20 ml) were evaporated together under reduced
pressure. The residue was coevaporated again with dry pyridine (20
ml). The residue was dissolved in dry pyridine (40 ml) and the
solution was cooled to -35.degree. C. (industrial methylated
spirits/dry ice bath). Pivaloyl chloride (1.85 ml, 15.0 mmol) was
added dropwise to the stirred solution over a period of 1 min, and
the reactants were maintained at -35.degree. C. After 30 min, water
(5 ml) was added, and the stirred mixture was allowed to warm up to
room temperature. Potassium phosphate buffer (1.0 mol dm.sup.-3, pH
7.0, 250 ml) was added to the products, and the resulting mixture
was concentrated under reduced pressure until all of the pyridine
had been removed. The residual mixture was partitioned between
dichloromethane (250 ml) and water (200 ml). The organic layer was
washed with triethylammonium phosphate buffer (0.5 mol dm.sup.-3,
pH 7.0, 3.times.50 ml), dried (MgSO.sub.4) and then evaporated
under reduced pressure. The reside was fractionated by short column
chromatography on silica gel (25 g). Appropriate fractions, eluted
with dichloromethane-methanol (95:5 to 90:10 v/v), were evaporated
to give (5'-O-(dimethoxytrityl)-2'-deoxyribonucleoside
3'-H-phosphonate 8.
[0104] When triethylammonium
6-N-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-de- ooxyadenosine
3'-H-phosphonate (DMTr-Ap(H))(Ozo/a et al., Tetrahedron, 1996), 8
(B-14), 4-N-benzoyl-3'-O-levulinoyl-2'-deoxycytidine (HO-C-Lev) 9
(B'=15) and di-(2-chlorophenyl) phosphorochloridate 18 were allowed
to react together in pyridine-dichloromethane solution at
-40.degree. C., the corresponding fully-protected dinucleoside
H-phosphonate (DMTr-Ap(H)C-Lev) was obtained apparently in
quantitative yield within 5-10 minutes. The protocol used in this
particular example was the dropwise addition of a solution of
di-(2-chlorophenyl) phosphorochloridate (2.03 g, 6.0 mmol) in
dichloromethane (4 ml) over 5 min to a stirred, dry solution of the
triethylammonium salt of DMTr-Ap(H) 8 (B=14) (3.95 g, ca. 4.8 mmol)
and 4-N-benzoyl-3'-O-levulinoyl-2'-deoxyc- ytidine 9 (B'=15) (1.72
g, 4.0 mmol) in pyridine (36 ml), maintained at -40.degree. C.
(industrial methylated spirits+dry ice bath). After a further
period of 5 min. only one nucleotide product assumed to be
DMTr-Ap(H)C-Lev. and some remaining H-phosphonate monomer 8 (B=14)
could be detected by reverse phase HPLC). However, it should be
noted that these reaction conditions can be varied
appropriately.
[0105] It is particularly noteworthy that such a high coupling
efficiency was achieved with only ca. 20% excess of H-phosphonate
monomer. No attempt was made to isolate the intermediate
dinucleoside H-phosphonate (DMTr-Ap(H)C-Lev).
[0106] N-[(4-Chlorophenyl)sulfanyl]phthalimide 19 (2.32 g, 8.0
mmol) (Behforouz, M.; Kerwood, J. E. J. Org. Chem, 1969, 34, 51-55)
was added to the stirred reactants which were maintained at
-40.degree. C. After 15 min. the products were worked up and
chromatographed on silica gel and the corresponding
S-(4-chlorophenyl) dinucleoside phosphorothioate DMr-Ap(s)C-Lev 10
(E=14, B'=15) was obtained in ca. 99% isolated yield. Thus both
coupling and the sulfur-transfer steps proceeded relatively quickly
and virtually quantitatively at -40.degree. C.
[0107] The four step procedure (Scheme 2, steps iii-vi) for the
unblocking of DMTr-Ap(s')C-Lev 10 (E=14. B'=15) preferably involves
`detritylation`, acetylation of the 5'-terminal hydroxy function,
oximate treatment, and finally treatment with concentrated aqueous
ammonia to remove acyl protecting groups from the base residues and
from the 3'- and 5'-terminal hydroxy functions. In this way,
extremely pure d[ApC] 11 (B=adenin-9-yl, B'=cytosine-1-yl) was
obtained without further purification and isolated as its sodium
salt. The monomeric building blocks 8 (B=17) and 9 (B'=16) were
coupled together in the same way and on the same scale. After
sulfur transfer with N-[(4-chlorophenyl)sulfanyl]phthalimide 19,
the fully protected dinucleoside phosphorothioate DMTr-Tp(s)G-Lev
10 (B=17, B'16) was isolated in ca. 98% yield. Again, very pure
d[TpG] 11 (B=thymin-1-yl, B'=guanin-9-yl) was obtained when this
material was unblocked by the above procedure (Scheme 2, steps
iii-vi).
[0108] The protocol for the preparation of fully-protected
oligonucleotide phosphorothioates differs from that used for
oligonucleotide synthesis only in that sulfur-transfer is effected
with 4-[(2-cyanoethyl)sulfanyl]m- orpholine-3,5-dione 21 or
3-(phthalimidosulfanyl)propanonitrile. However,
4-[(2-cyanoethyl)sulfanylgmorpholine-3,5-dione has the advantage
that the morpholine-3,5-dione produced in the course of
sulfur-transfer is more water-soluble than phthalimide.
Triethylammonium 6-O-(2,5-dichlorophenyl)-
-5'-O-(4,4'-dimethoxytrityl)-2-N-isobutyryl-2'-deoxyguanosine
3'-H-phosphonate (DMTr-Gp(H)) 8 (B=16) [ca. 4.8 mmol],
6-N-benzoyl-3'-O-levulinoyl-2'-deoxyadenosine (HO-A-Lev) 9 (B=14)
14.0 mmol) and di-(2-chlorophenyl) phosphorochloridate 18 [6.0
mmol] were allowed to react together in pyridine-dichloromethane
solution at -40.degree. C. for 5-10 minutes.
4-[(2-Cyanoethyl)sulfanyl]morpholine-3,5- -dione 21 [8.0 mmol]
(Scheme 2, step vii) was then added while the reactants were
maintained at -40.degree. C. After 15 minutes, the products were
worked up and fractionated by chromatography on silica gel to give
the fully-protected dinucleoside phosphorothioate (DMTr-Gp(s)A-Lev)
12 (B=14. B'=16) in 99% isolated yield. This material was unblocked
by a five-step procedure (Scheme 2, steps iii, iv, viii, ix and
vi). Following the `detritylation` and acetylation steps, the
product was treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
under strictly anhydrous conditions to remove the S-(2-cyanoethyl)
protecting group. The 6-O-(2,5-dichlorophenyl) protecting group was
then removed from the guanine residue by oximate treatment, and
finally all of the acyl protecting groups were removed by
ammonolysis. The oximate treatment step can be omitted if the
oligonucleotide phosphorothioate does not contain any
2'-deoxyguanosine residues. Extremely pure d[Gp(s)A] 13
(B=guanin-9-yl. B'=adenin-9-yl) was obtained without further
purification, and was isolated as its sodium salt.
Preparation of 4-[(2-cyanoethyl)sulfanyl]morpholine-3,5-dione
[0109] S-(2-cyanoethyl)isothiouronium chloride was prepared as
follows. Thiourea (304 g), was dissolved with heating in
concentrated hydrochloric acid (500 ml). The resulting solution was
evaporated under reduced pressure and the residual colourless solid
was dissolved in boiling absolute ethanol (1300 ml). The solution
was cooled to room temperature and acrylonitrile (400 cm.sup.3) was
added in portions with stirring. The reactants were heated, under
reflux, for 2 hours. The cooled products were filtered and the
residue was washed with cold ethanol and then dried in vacuo over
calcium chloride.
[0110] Di-(2-cyanoethyl) disulphide was then prepared as follows.
Dichloromethane (400 ml) was added to a stirred solution of
S-(2-cyanoethyl)isothiouronium chloride (83.0 g) in water (500 ml)
at 0.degree. C. (ice-water bath). Sodium perborate tetrahydrate
(44.1 g) was added, and then a solution of sodium hydroxide (30.0
g) in water (250 ml) was added dropwise. The reactants were
maintained at 0.degree. C. (ice-water bath). After 5 hours, the
products were separated and the aqueous layer was extracted with
dichloromethane (3.times.50 ml). The combined organic layers were
dried (MgSO.sub.4) and evaporated under reduced pressure to give a
solid which was recrystallised from methanol (30 ml) to give
colourless crystals.
[0111] Di-(2-cyanoethyl)disulphide (4.51 g) and morpholin-2,6-dione
(5.75 g) were suspended in acetonitrile (10 ml), dichloromethane
(20 ml) and 2.6-lutidine (17.4 ml) and cooled to 0.degree. C.
(ice-water Lath). A solution of bromine (4.28 g) in dichloromethane
(20 ml) was added over 30 minutes. The reaction mixture was allowed
to stir at 0.degree. C. For 1.5 hours. The product was then
precipitated by the addition of ice-cold methanol (50 ml) over 30
minutes and filtered to give the title compound (8.28 g, 82%).
Recrystallisation from ethyl acetate cave
4-[(2-cyanoethyl)sulfanyl]morpholine-3,5-dione as colourless
needles, m.p. 121-122.degree. C.
Reaction Scheme for Preparation of Chimeric Oligonucleotides
[0112] The stepwise synthesis of d[pGp(s)ApC] 25 which has one
phosphorothioate diester and two phosphodiester internucleotide
linkages is illustrated in outline by way of example in Scheme 3.
7
Reagents and Conditions
[0113] (i) 4 M HCl/dioxane, CH.sub.2Cl.sub.2, -50.degree. C., 5
min;
[0114] (ii) 18, C.sub.5H.sub.5N, CH.sub.2Cl.sub.2, -40.degree. C.,
5-10 min;
[0115] (iii) a, 21, C.sub.5H.sub.5N, CH.sub.2Cl.sub.2, -40.degree.
C., 15 min, b, C.sub.5H.sub.5N--H.sub.2O (1:1 v/v), -40.degree. C.
to room temp;
[0116] (iv) a, 19, C.sub.5H.sub.5N, CH.sub.2Cl.sub.2, 40.degree.
C., 15 min, b, C.sub.5H.sub.5N--H.sub.2O (1:1 v/v), -40.degree. C.
to room temp;
[0117] (v) Ac.sub.2O, C.sub.5H.sub.5N, room temp., 15 h;
[0118] (vi) DBU, Me.sub.2SiCl, CH.sub.2Cl.sub.2, room temp., 30
min:
[0119] (vii) 20, DBU, MeCN, room temp., 12 h;
[0120] (viii) a, conc. aq. NH.sub.3(d 0.88), 50.degree. C., 15 h,
b, Ambcriltle IR-120 (plus), Na+ form. H.sub.2O.
[0121] No limitation of scale is anticipated. The reactions shown
in Scheme 3 are not intenced to be limiting and the method of the
invention is equally suitable for the synthesis of RNA,
2'-O-alkyl-RNA and other oligonucleotide sequences.
[0122] All of the reactions involved were used above either in the
preparation of d[ApC] 11 (B=adenin-9-yl, B'=cycsin-1-yl or of
d[Gp(s)A] 13 (E=adenin-9-yl. B'=adenin-9-yl) (Scheme 2).
[0123] First, the fully-protected dinucleoside phosphorothioate
DMTr-Ap(s)C-Lev 10 (B=14. B'=15) [ca. 0.75 mmol) was converted in
four steps and in ca. 96% overall isolated yield (Scheme 3a) into
the partially-protected trimer 23. In Each coupling step, a ca. 20%
excess of H-phosphonate monomer 8 was used, but the excess of
coupling agent 18 depended on the scale of the reaction. In
addition a twofold excess of sulfur-transfer agent 19 or 21 was
used in this example. The products were chromatographed on silica
gel after each "detritylation" step.
[0124] This material was then coupled with DMTr-Tp(H) 8 (B=17) and
the product was converted in three steps and in ca. 93% overall
yield (Scheme 3b) into the fully-protected tetramer 24. The latter
material was unblocked to give d[IpGp(s)ApC] 25 which was isolated
without further purification as its relatively pure (Ca. 96.5% by
HPLC) sodium salt.
[0125] The tetranucleoside triphosphate d[TpGpApC] and the
tetranucleoside triphosphorothioate d[Cp(s)Tp(s)Gp(s)A] were also
prepared by stepwise synthesis in very much the same way. The
protocols followed differed from that outlined in Scheme 3 only
stepwise synthesis in very much the same way. The protocols
followed differed from that outlined in Scheme 3 only inasmuch as
the sulfur-transfer agent 19 was used exclusively in the
preparation of d[TpGpApC] and the sulfur-transfer agent 21 was used
exclusively in the preparation of d[Cp(s)Tp(s)Gp(s)A].
Reaction Scheme for Block Coupling
[0126] By way of illustration, Scheme 4 given herein-below
illustrates an example of block coupling which is part of the
invention. 8
Reagents and Conditions
[0127] (i) N.sub.2H.sub.4H.sub.2O, C.sub.5H.sub.5N-AcOH (3:1 v/v),
0.degree. C., 20 min;
[0128] (ii) a, 30, Me.sub.3C--COCl, C.sub.5H.sub.5N, -35.degree.
C., 30 min, b, Et.sub.2N, H.sub.2O;
[0129] (iii) 18 C.sub.5H.sub.5N, CH.sub.2Cl.sub.2, -35.degree.
C.;
[0130] (iv) a, 21, C.sub.5H.sub.5N, CH.sub.2Cl.sub.2, -35.degree.
C., 10 min, b, C.sub.5H.sub.5N--H.sub.2O (1:1 v/v), -35.degree. C.
to room temp.
[0131] The fully protected octadeoxynucleoside
heptaphosphorothioate 29 which was obtained in 91% isolated yield
is a precursor of d[Tp(s)Tp(s)Gp(s)Gp(s)Gp(s)Gp(s)Tp(s)T]. As
indicated above, block coupling is much more feasible in solution
than in solid phase synthesis.
[0132] This approach is of course not in any way limited to
tetramer coupling. Indeed, it is anticipated that this
H-phosphonate approach will be suitable for coupling quite large
oligonucleotide blocks (for example, 10+10) together.
Reaction Scheme for Preparation of Block H-phosphonates
[0133] For example, partially-protected oligonucleotides 32a and
the corresponding phosphorothioates 33b which can be prepared by
the conventional phosphotriester approved in solution
(Chattopadhyaya, J. B.; Reese, C. B. Nucleic Acids Res., 1980, 8,
2039-2054; Kemal, O., Reese. C. B.; Serafinowska, H. T. J. Chem.
Soc., Chem. Commun., 1983, 591-593) can similarly be converted into
their 3'-H-phosphonates (34a and 34b, respectively) as indicated in
Scheme 5. 9
Reagents and Conditions
[0134] (i) a, 30, Me.sub.3C.COCl, C.sub.5H.sub.5N, -35.degree. C.,
b, Et.sub.3N, H.sub.2O.
Example 1
Ac-Tp(s)Tp(s)Gp(s)G-OH
[0135] HO-Tp(s)Tp(s)Gp(s)G-Lev (5.82 g, 3 mmol) was co-evaporated
with anhydrous pyridine (2.times.20 ml) and redissolved in
anhydrous pyridine (30 ml). Acetic anhydride (1.42 ml, 15 mmol) was
added and the reaction solution was allowed to stir at room
temperature for 12 h. Water (1.5 ml) was added to quench the
reaction. After 10 min, the mixture was cooled to 0.degree. C.
(ice-water bath) and hydrazine hydrate (1.50 g, 30 mmol) in
pyridine (15 ml) and glacial acetic acid (15 ml) was added. The
mixture was stirred at 0.degree. C. For 20 min and was then
partitioned between water (100 ml) and CH.sub.2Cl.sub.2 (100 ml).
The two layers were separated and the organic layer was washed with
water (3.times.50 ml). The oroanic layer was dried (MgSO.sub.4) and
evaporated. The residue was purified by silica gel chrorratooraphy.
Impurities were eluted with methanol-dichloromethane (4:96 v/v) the
main product was eluted with acetone. Evaporation of the
appropriate fractions gave the partially protected
tetradeoxynuciecside triphosphorothicate as colourless solid (5.30
g, 93%).
Example 2
Ac-Tp(s)Tp(s)Gp(s)Gp(H)
[0136] The ammonium salt of 4-methylphenyl H-phosphonate (1.42 g,
7.5 mmol) was dissolved in the mixture of methanol (15 ml) and
triethylamine (2.1 ml, 15 mmol). The mixture vans evaporated and
coevaporated with pyridine (2.times.10 ml) under reduced pressure.
Ac-Tp(s)Tp(s)Gp(s)G-OH (4.71 g, 2.5 mmol) was added and
co-evaporated with dry pyridine (20 ml). The residue was dissolved
in dry pyridine (20 ml) and pivaloyl chloride (1.23 ml, 10 mmol)
was added at -35.degree. C. in 1 min. After 30 min at the same
temperature, water (5 ml) was added and the mixture was allowed to
warm to room temperature and stir for 1 hr. The solution was
partitioned between water (100 ml) and dichloromethane (100 ml).
The organic layer was separated and washed with triethylammonium
phosphate buffer (pH 7.0, 0.5M, 3.times.50 ml), dried (MgSO.sub.4),
and then filtered and applied to a silica gel column (ca. 25 g).
The appropriate fractions, which were eluted with
methanol-dichloromethane (20:80, v/v), were evaporated to give
Ac-Tp(s)Tp(s)Gp(s)Gp(H), as a colourless solid (4.85 g, 94%).
Example 3
Ac-Tp(s)Tp(s)Gp(s)Gp(s)Gp(s)Gp(s)Tp(s)T-Bz
[0137] Ac-Tp(s)Tp(s)Gp(s)Gp(H) (1.229 g, 0.6 mmol) and
HO-Gp(s)Gp(s)Tp(s)T-Bz (0.973 g, 0.5 mmol) were coevaporated with
anhydrous pyridine (2.times.10 ml) and the residue was dissolved in
anhydrous pyridine (10 ml). The solution was cooled to -35.degree.
C. (Industrial methylated spirits-dry ice bath) and
di-(2-chlorophenyl)phosp- horochloridate (0.84 g, 2.5 mmol) in dry
dichloromethane (1 ml) was added over 10 min.
4-[(2-Cyanoethyl)sulfanyl]morpholin-3,5-dione (0.20 g, 1.0 mmol)
was added and the mixture was allowed to stir for 10 min at the
same temperature. Then water-pyridine (0.2 ml, 1:1 v/v) was added
and the mixture was stirred for a further 5 min. The reaction
mixture was then evaporated under reduced pressure. The residue was
dissolved in dichloromethane (100 ml) and the solution was washed
with saturated aqueous sodium bicarbonate solution (3.times.50 ml).
The organic layer was dried (MgSO4) and concentrated under reduced
pressure. The residue was purified by silica gel chromatography.
Firstly, the lipophilic impurities were removed with
methanol-dichloromethane (4:96 v/v), and then the main product was
eluted with acetone. Evaporation of the appropriate fractions gave
fully protected octadeoxynucleoside heptaphosphorothioate as
colourless solid (1.81 g, 91%). The fully-protected
octadeoxynuclecside heptaphosphorothioate which was obtained in 91%
isolated yield is a precursor of d[Tp(s)Tp(s)Gp(s)Gp(s)Gp-
(s)Gp(s)Tp(s)T].
* * * * *