U.S. patent application number 10/700077 was filed with the patent office on 2004-05-20 for building blocks for the solution phase synthesis of oligonucleotides.
Invention is credited to Fernandez, Susana, Ferrero, Miguel, Garcia, Javier, Gotor, Vicente, Sanghvi, Yogesh S..
Application Number | 20040096947 10/700077 |
Document ID | / |
Family ID | 25237291 |
Filed Date | 2004-05-20 |
United States Patent
Application |
20040096947 |
Kind Code |
A1 |
Sanghvi, Yogesh S. ; et
al. |
May 20, 2004 |
Building blocks for the solution phase synthesis of
oligonucleotides
Abstract
The present invention is directed to methods for the preparation
of 3'-O and 5'-O-levulinyl nucleosides from common precursors using
an enzymatic approach.
Inventors: |
Sanghvi, Yogesh S.;
(Encinitas, CA) ; Gotor, Vicente; (Oviedo, ES)
; Ferrero, Miguel; (Oviedo, ES) ; Fernandez,
Susana; (Oviedo, ES) ; Garcia, Javier;
(Colunga, ES) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE - 46TH FLOOR
PHILADELPHIA
PA
19103
US
|
Family ID: |
25237291 |
Appl. No.: |
10/700077 |
Filed: |
November 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10700077 |
Nov 3, 2003 |
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09822903 |
Mar 30, 2001 |
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6677120 |
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Current U.S.
Class: |
435/89 ;
536/27.1 |
Current CPC
Class: |
C12P 19/30 20130101;
C07H 21/04 20130101; C07H 19/04 20130101 |
Class at
Publication: |
435/089 ;
536/027.1 |
International
Class: |
C07H 019/00; C07H
019/22; C12P 019/30 |
Claims
What is claimed:
1. A method for the selective deprotection of a
3',5'-di-O-levulinyl nucleoside comprising selecting a lipase
effective to direct regioselective hydrolysis of one of said
levulinyl positions of the nucleoside; and contacting the
3',5'-di-O-levulinyl nucleoside with said lipase for a time and
under conditions effective to yield the corresponding
3'-O-levulinyl and 5'-O-levulinyl nucleoside.
2. The method of claim 1 wherein said lipase is CAL-A, CAL-B,
PSL-C, porcine pancreatic lipase, Chromobacteriaum viscosum lipase,
Mucor miehei lipase, Humicola lanuginosa lipase, Penicillium
camemberti lipase, or Candida rugosa lipase.
3. The method of claim 2 wherein said lipase is CAL-A.
4. The method of claim 2 wherein said lipase is CAL-B.
5. The method of claim 2 wherein said lipase is PSL-C.
6. A method for the selective deprotection of a
3',5'-di-O-levulinyl nucleoside at the 5'-O-levulinyl position
comprising selecting a lipase effective to direct regioselective
hydrolysis of said 3',5'-di-O-levulinyl nucleoside at the
5'-O-levulinyl position and contacting said 3',5'-di-O-levulinyl
nucleoside with said lipase for a time and under conditions
effective to yield a 3'-O-levulinyl nucleoside.
7. The method of claim 6 wherein said lipase is CAL-B.
8. A method for the selective deprotection of a
3',5'-di-O-levulinyl nucleoside at the 3'-O-levulinyl position
comprising selecting a lipase effective to direct regioselective
hydrolysis of said 3',5'-di-O-levulinyl nucleoside at the
3'-O-levulinyl position and contacting said 3',5'-di-O-levulinyl
nucleoside with said lipase for a time and under conditions
effective to yield a 5'-O-levulinyl nucleoside.
9. The method of claim 8 wherein said lipase is CAL-A.
10. The method of claim 8 wherein said lipase is PSL-C.
11. A method for the selective deprotection of a
3',5'-di-O-levulinyl nucleoside at the 5'-O-levulinyl position
comprising selecting a lipase effective to direct regioselective
hydrolysis of said 3',5'-di-O-levulinyl nucleoside at the
5'-O-levulinyl position and contacting said 3',5'-di-O-levulinyl
nucleoside with said lipase for a time and under conditions
effective to yield a 3'-O-levulinyl nucleoside wherein said
3',5'-di-O-levulinyl nucleoside has one of the following formulas:
10wherein: R.sub.1 is --H, -hydroxyl, a protected hydroxyl, or a
2'-substituent; and R.sub.2 and R.sub.3 are, independently, --H or
an amino protecting group; G is N or CH; and Lev is
--C(O)--(CH.sub.2).sub.2- --C(O)--CH.sub.3.
12. The method of claim 11 wherein said lipase is CAL-B.
13. The method of claim 12 wherein said 3',5'-di-O-levulinyl
nucleoside is an adenosine, cytosine, thymidine, or an N-isobutyl
guanosine.
14. A method for the selective deprotection of a
3',5'-di-O-levulinyl nucleoside at the 3'-O-levulinyl position
comprising selecting a lipase effective to direct regioselective
hydrolysis of said 3',5'-di-O-levulinyl nucleoside at the
3'-O-levulinyl position and contacting said 3',5'-di-O-levulinyl
nucleoside with said lipase for a time and under conditions
effective to yield a 5'-O-levulinyl nucleoside wherein said
3',5'-di-O-levulinyl nucleoside has one of the following formulas:
11wherein: R.sub.6 is --H, or --OH; R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 are each, independently, --H or an amino protecting group;
G is N or CH; and Lev is
--C(O)--(CH.sub.2).sub.2--C(O)--CH.sub.3.
15. The method of claim 14 wherein said lipase is CAL-A.
16. The method of claim 14 wherein said lipase is PSL-C.
17. The method of claim 15 wherein said 3',5'-di-O-levulinyl
nucleoside is 3',5'-di-O-levulinyl thymidine, 3',5'-di-O-levulinyl
cytosine, or 3',5'-di-O-levulinyl N-benzoyl adenosine.
18. The method of claim 16 wherein said 3',5'-di-O-levulinyl
nucleoside is N-isobutylguanosine.
19. A method for the selective deprotection of a
3',5'-di-O-levulinyl nucleoside at the 5'-O levulinyl position
wherein said 3',5'-di-O-levulinyl nucleoside has one of the
following formulas: 12wherein: R.sub.1 is --H, -hydroxyl, a
protected hydroxyl, or a 2'-substituent; and R.sub.2 and R.sub.3
are, independently, --H or an amino protecting group; G is N or CH;
and Lev is --C(O)--(CH.sub.2).sub.2- --C(O)--CH.sub.3; comprising
contacting said 3',5'-di-O-levulinyl nucleoside with CAL-B for a
time and under conditions effective to hydrolyze said
3',5'-di-O-levulinyl nucleoside at the 5'-O-levulinyl position.
20. The method of claim 20 wherein said
3'-,5'-di-O-levulinylnucleoside comprises an adenosine, cytosine,
thymidine, or an N-isobutyl guanosine moiety.
21. A method for the selective deprotection of a
3',5'-di-O-levulinyl nucleoside at the 3'-O-levulinyl position
wherein said 3',5'-di-O-levulinyl nucleoside has one of the
following formulas: 13wherein: R.sub.6 is --H or -hydroxyl;
R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are each, independently, --H
or an amino protecting group; G is N or CH; and Lev is
--C(O)--(CH.sub.2).sub.2--C(O)--CH.sub.3; comprising contacting
said 3',5'-di-O-levulinyl nucleoside with PSL-C for a time and
under conditions effective to hydrolyze said 3',5'-di-O-levulinyl
nucleoside at the 3'-O-levulinyl position.
22. The method of claim 20 wherein said 3'-,5'-di-O-levulinyl
nucleoside comprises an N-isobutylguanosine moiety.
23. A method for the selective deprotection of a
3',5'-di-O-levulinyl nucleoside at the 3'-O-levulinyl position
wherein 3',5'-di-O-levulinyl nucleoside has one of the following
14formulas: 15wherein: R6 is --H or --OH; R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 are each, independently, --H or an amino
protecting group; G is N or CH; and Lev is
--C(O)--(CH.sub.2).sub.2--C(O)--CH.sub.3; comprising contacting
said 3',5'-di-O-levulinyl nucleoside with CAL-A for a time and
under conditions effective to hydrolyze said 3',5'-di-O-levulinyl
nucleoside at the 3'-O-levulinyl position.
24. The method of claim 23 wherein said 3',5'-di-O-levulinyl
nucleoside comprises a thymidine, cytosine, or N-benzoyl adenosine
moiety.
25. A method for protecting a hydroxyl moiety of a nucleic acid
having at least one of a 2'-O, 3'-O, or 5'-O position comprising
reacting said nucleic acid with levulinic acid in the presence of a
coupling agent that is attached to a polymeric support for a time
and under conditions effective to form an ester at said 2'-O, 3'-O
or 5'-O position.
26. The method of claim 25 wherein said nucleic acid is a
nucleoside.
27. The method of claim 25 wherein said coupling agent is a
carbodiimide.
28. The method of claim 25 wherein said carbodiimide is
cyclohexylcarbodiimide.
29. The method of claim 25 wherein said polymeric support is a
polystyrene.
30. The method of claim 25 wherein said polymeric support is a
polyethylene glycol.
31. A method for acylating at least one hydroxyl moiety of a
carbohydrate comprising reacting said carbohydrate with levulinic
acid in the presence of a coupling agent that is attached to a
polymeric support for a time and under conditions effective to form
an ester.
32. The method of claim 31 wherein said coupling agent is a
carbodiimide.
33. The method of claim 32 wherein said carbodiimide is
cyclohexylcarbodiimide.
34. The method of claim 31 wherein said polymeric support is a
polystyrene support.
35. The method of claim 31 wherein said polymeric support is a
polyethylene glycol support.
36. A method for acylating at least one hydroxyl moiety of a
steroid molecule comprising reacting said steroid molecule with
levulinic acid in the presence of a coupling agent that is attached
to a polymeric support for a time and under conditions effective to
form an ester.
37. The method of claim 36 wherein said coupling agent is a
carbodiimide.
38. The method of claim 37 wherein said carbodiimide is
cyclohexylcarbodiimide.
39. The method of claim 36 wherein said polymeric support is a
polystyrene support.
40. The method of claim 36 wherein said polymeric support is a
polyethylene glycol support.
41. A method for protecting a hydroxyl moiety on a compound having
the following formula: 16wherein: B.sub.X is a nucleobase; T.sub.1
and T.sub.2, independently, are OH, a hydroxyl protecting group, an
activated phosphate group, a nucleotide, a nucleoside, or an
oligonucleotide; R is --H, -hydroxyl, a protected hydroxyl or a 2'
substituent group; provided that at least one of T.sub.1, T.sub.2
or R is --OH; comprising reacting said compound with levulinic acid
in the presence of a coupling agent that is attached to a solid
support for a time and under conditions effective to form an ester
between said hydroxyl moiety and the levulinyl group.
42. The method of claim 41 wherein said coupling agent is a
carbodiimide.
43. The method of claim 42 wherein said carbodiimide is a
cyclohexylcarbodiimide.
44. The method of claim 41 wherein said polymeric support is a
polystyrene support.
45. The method of claim 41 wherein said polymeric support is a
polyethyleneglycol support.
46. A method for protecting the 3'-O and 5'-O positions of a
compound having the following formula: 17wherein: B.sub.X is a
nucleobase; and R is --H, or a 2'-substituent; comprising reacting
said compound with levulinic acid in the presence of a coupling
agent that is attached to a solid support for a time and under
conditions effective to form a compound having formula: 18wherein
Lev is a -levulinyl.
47. The method of claim 46 wherein said coupling agent attached to
a polymeric support is cyclohexylcarbodiimide attached to a
polymeric support.
48. The method of claim 47 wherein said polymeric support is a
polystyrene polymeric support.
49. A method for protecting the 3'-O and 5'-O positions of a
compound having the following formula: 19wherein: B.sub.X is a
nucleobase; and R is --H, or a 2'- substituent; comprising reacting
said compound with levulinic acid in the presence of
cyclohexylcarbodiimide that is attached to a polystyrene polymeric
support for a time and under conditions effective to form a
compound having the following formula: 20wherein Lev is
-levulinyl.
50. A method for acylating a hydroxyl moiety comprising reacting
said hydroxyl moiety with levulinic acid in the presence of a
coupling agent that is attached to a polymericic support for a time
and under conditions effective to yield an ester.
51. The method of claim 50 wherein said coupling agent is a
carbodiimide
52. The method of claim 51 wherein said carbodiimide is
cyclohexylcarbodiimide.
53. The method of claim 50 wherein said polymeric support is a
polystyrene.
54. The method of claim 50 wherein said polymeric support is
polyethylene glycol.
55. A method for generating a cyclohexylcarbodiimide derivatized
polymeric support from a cyclohexylurea derivatized polymeric
support comprising reacting said cyclohexylurea derivatized
polymeric support with a dehydrating agent in an organic solvent
for a time and under conditions effective to yield said
cyclohexylcarbodiimide derivatized polymeric support.
56. The method of claim 55 wherein said dehydrating agent is
POCl.sub.3.
57. The method of claim 55 wherein said dehydrating agent is
tosylchloride.
58. The method of claim 55 wherein said organic solvent is
CH.sub.2Cl.sub.2, CHCl.sub.3, hexane, or pyridine.
59. The method of claim 55 wherein said polymeric support is a
polystyrene polymeric support.
60. A method for generating a cyclohexylcarbodiimide derivatized
polymeric support from a cyclohexylurea derivatized polymeric
support comprising the steps of: reacting said cyclohexylurea
derivatized polymer support with a dehydrating agent in an organic
solvent for a time and under conditions effective to form a salt;
contacting said salt with an aqueous solution to form said
cyclohexylcarbodiimide derivatized polymeric support.
61. The method of claim 60 wherein said dehydrating agent is
POCl.sub.3.
62. The method of claim 60 wherein said dehydrating agent is
tosylchloride.
63. The method of claim 60 wherein said organic solvent is
CH.sub.2Cl.sub.2, CHCl.sub.3, hexane, or pyridine.
64. The method of claim 60 wherein said polymeric support is a
polystyrene polymeric support.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for the preparation
of 3'-O and 5'-O-levulinyl nucleosides from common precursors using
an enzymatic approach. These methods are useful for the large-scale
synthesis of oligonucleotides.
BACKGROUND OF THE INVENTION
[0002] It is well known that most of the bodily states in mammals,
including most disease states, are affected by proteins. Such
proteins, either acting directly or through their enzymatic
functions, contribute in major proportion to many diseases in
animals and man. Classical therapeutics has generally focused on
interactions with such proteins in efforts to moderate their
disease causing or disease potentiating functions. Recently,
however, attempts have been made to moderate the actual production
of such proteins by interactions with molecules that direct their
synthesis, such as intracellular RNA. By interfering with the
production of proteins, it has been hoped to affect therapeutic
results with maximum effect and minimal side effects. It is the
general object of such therapeutic approaches to interfere with or
otherwise modulate gene expression leading to undesired protein
formation.
[0003] One method for inhibiting specific gene expression is the
use of oligonucleotides and oligonucleotide analogs as "antisense"
agents. The oligonucleotides or oligonucleotide analogs
complimentary to a specific, target, messenger RNA (mRNA) sequence
are used. Antisense methodology is often directed to the
complementary hybridization of relatively short oligonucleotides
and oligonucleotide analogs to single-stranded mRNA or
single-stranded DNA such that the normal, essential functions of
these intracellular nucleic acids are disrupted. Hybridization is
the sequence specific hydrogen bonding of oligonucleotides or
oligonucleotide analogs to Watson-Crick base pairs of RNA or
single-stranded DNA. Such base pairs are said to be complementary
to one another.
[0004] Oligonucleotides and oligonucleotide analogs are now
accepted as therapeutic agents holding great promise for
therapeutics and diagnostics methods. But applications of
oligonucleotides and oligonucleotide analogs as antisense agents
for therapeutic purposes, diagnostic purposes, and research
reagents often require that the oligonucleotides or oligonucleotide
analogs be synthesized in large quantities.
[0005] Three principal methods have been used for the synthesis of
oligonucleotides. The phosphotriester method, as described by
Reese, Tetrahedron 1978, 34, 3143; the phosphoramidite method, as
described by Beauage, in Methods in Molecular Biology: Protocols
for Oligonucleotides and Analogs; Agrawal, ed.; Humana Press:
Totowa, 1993, Vol. 20, 33-61; and the H-phosphonate method, as
described by Froehler in Methods in Molecular Biology: Protocols
for Oligonucleotides and Analogs Agrawal, ed.; Humana Press:
Totowa, 1993, Vol. 20, 63-80.
[0006] The phosphotriester approach has been widely used for
solution phase synthesis, whereas the phosphoramidite and
H-phophonate strategies have found application mainly in solid
phase syntheses. Recently, Reese reported a new approach to the
solution phase synthesis of oligonucleotides on H-phosphonate
coupling. See, Reese et al. Nucleic Acids Research, 1999, 27,
963-971, and Reese et al. Biorg. Med. Chem. Lett. 1997, 7,
2787-2792, which is incorporated herein by reference. Solution
phase synthesis is the method of choice in producing large-scale
quantities of oligonucleotides.
[0007] These solution phase methods require the use of nucleoside
monomer building blocks bearing protecting groups on the 3'-O
and/or the 5'-O positions. The protecting groups should be stable
to coupling conditions and selectively cleaved without affecting
other protecting groups in the molecule. One such protecting group
is the levulinyl group, --C(O)--(CH.sub.2).sub.2--C(O)--CH.sub.3.
However, the preparation of nucleosides bearing these protecting
groups involves several tedious chemical protection/deprotection
and/or purification steps.
[0008] For example, the 3',5'-di-O-levulinyl protection of
nucleosides can be accomplished using a well-established method
wherein nucleosides are selectively acylated at their hydroxyl
sites by reacting the nucleosides with levulinic acid in the
presence of DCC (dicyclohexylcarbodiimide). Despite the utility of
this method, it suffers from at least one significant problem. The
method requires a large excess of DCC to achieve optimal yields.
The excess DCC is converted to DCU (dicyclohexylcarbodiimide) upon
completion of the reaction, which must be separated from the
reaction mixture. Unfortunately, for large-scale syntheses, the
separation step requires considerable time and expense.
[0009] Prior to the present invention, synthesis of 5'-O-levulinyl
nucleosides was accomplished by reacting parent nucleosides with
levulinic acid and 2-chloro-1-methylpyridinium iodide. Iwai et al.,
Nucleic Acids Res. 1988, 16, 9443-9456; Iwai et al. Tetrahedron
1990, 46, 6673-6688. However, because this method does not afford
selective acyaltion of the 5'-hydroxyl function, additional
purification and deprotection steps are necessary because both
3'-acyl and 3',5'-diacyl derivatives are formed in the reaction.
After the 3',5'-diacyl derivatives are separated by chromatography,
the residue must be treated with DMTrCl to remove the 3'-acyl
compound. Finally, an additional purification by chromatography
isolates the 5'-O-levulinyl derivatives in very low yields.
[0010] Before now, the synthesis of 3'-O-levulinyl nucleosides
(2'-deoxy or 2'-protected) was accomplished by the treatment of
parent nucleosides with levulinic acid or levulinic anhydride and
DCC. One of the major drawbacks of this method is that it requires
that the 5'-hydroxyl function be protected as a 5'-O-DMTr group
prior to acylation with levulinic acid. The 5'-O-DMTr group must
then be removed in an acid medium to afford the 3'-O-protected
nucleosides. See, Reese et al., Nucleic Acids Res. 1999, 27,
963-971, and Reese et al., J. Chem. Soc., Perkin Trans. 1 1999,
1477-1486.
[0011] Commercially viable methods for the large-scale synthesis of
oligonucleotides are constantly being explored. It has been found
that the application of biocatalysts in organic synthesis has
become an attractive alternative to conventional chemical methods.
See, Carrea, et al. Angew. Chem. Int. Ed. 2000, 39, 2226-2254;
Bornscheuer, et al. Hydrolases in Organic Synthesis. Regio- and
Stereoselective Biotransformations; Wiley-VCH: Weinheim, 1999.
Enzymes catalyze reactions with high chemo-, regio-, and
stereoselectivity. See, Ferrero et al. Chem. Rev. 2000, 100,
4319-4347; Ferrero et al., Monatsh. Chem. 2000, 131, 585-616. It
has previously been reported that Candida antarctica lipase B
(CAL-B) catalyzes acylation at the 5'-hydroxyl group of nucleosides
with high selectivity. Pseudomonas cepacia lipase (PSL) shows
unusual regioselectivity towards the secondary alcohol at the
3'-position of 2'-deoxynucleosides. Moris et al., J. Org. Chem.
1993, 58, 653-660; Gotor et al. Synthesis 1992, 626-628.
[0012] In the last few years the use of antisense oligonucleotides
has emerged as an exciting new therapeutic paradigm. As a result,
very large quantities of therapeutically useful oligonucleotides
are required in the near future. In view of the considerable
expense and time required for synthesis of oligonucleotide building
blocks, there has been a longstanding effort to develop successful
methodologies for the preparation of oligonucleotides with
increased efficiency and product purity.
SUMMARY OF THE INVENTION
[0013] Applicants have discovered methods that are useful in, for
example, the large-scale synthesis of oligonucleotides. The methods
of the present invention help to minimize the number of steps
required to yield desired results using an enzymatic approach.
Applicants have found that both 3'-O-levulinyl nucleosides and
5'-O-levulinyl nucleosides can be prepared from a common precursor
the regioselective deprotection of a 3',5'-di-O-levulinyl
nucleoside to yield the desired 3'-O levulinyl nucleoside or
5'-O-levulinyl nucleoside. Surprisingly, it has been found that the
presence of selected lipases in deprotection reaction protocols
gives rise to regioselectivity of deprotection
[0014] According to one embodiment, a method is provided for
regioselectively deprotecting a 3',5'-di-O-levulinylnucleoside
comprising selecting a lipase that is effective to direct a
regioselective hydrolysis of one of the levulinyl positions,
without causing an undesired level of hydrolysis on the other of
the levulinyl positions, and contacting the 3',5'-di-O-levulinyl
nucleoside with the lipase for a time and under conditions
effective to yield either a 3'-O-levulinyl or a 5'-O-levulinyl
nucleoside. Examples of lipases that are amenable to the present
invention include Candida antarctica lipase B (CAL-B), Candida
antarctica lipase A (CAL-A), Pseudomonas cepacia lipase (PSL),
porcine pancreatic lipase, Chromobacteriaum viscosum lipase, Mucor
miehei lipase, Humicola lanuginosa lipase, Penicillium camemberti
lipase, Candida rugosa lipase, and others.
[0015] According to an embodiment of the present invention, a
3',5'-di-O-levulinyl nucleoside is deprotected at the
5'-O-levulinyl position by contacting the diprotected nucleoside
with CAL-B for a time and under conditions effective to
regioselectively hydrolyze the 5'-O-levulinyl position without
affecting the 3'-O-levulinyl position.
[0016] In another embodiment, a 3'-, 5'-di-O-levulinyl nucleoside
is deprotected at the 3'-O levulinyl position by contacting the
diprotected nucleoside with CAL-A or PSL-C for a time and under
conditions effective to regioselectively hydrolyze the
3'-O-levulinyl position without affecting the 5'-O-levulinyl
position.
[0017] In some embodiments of the present invention, methods are
disclosed for regioselectively deprotecting a 3'-,
5'-di-O-levulinyl nucleoside at the 5'-O-levulinyl position wherein
the nucleoside has one of the following formulas: 1
[0018] wherein:
[0019] R.sub.1 is --H, -hydroxyl, a protected hydroxyl, a
2'-substituent or a 2'-protected substituent; and
[0020] R.sub.2 and R.sub.3 are, independently, --H or an amino
protecting group;
[0021] G is N or CH; and
[0022] Lev is --C(O)--(CH.sub.2).sub.2--C(O)--CH.sub.3, the
levulinyl group;
[0023] comprising selecting a lipase that is effective to direct a
regioselective hydrolysis of the 5'-O-levulinyl position, without
causing hydrolysis on the 3'-O-levulinyl position, and contacting
the 3',5'-di-O-levulinyl nucleoside with the lipase for a time and
under conditions effective to yield a 3'-O-levulinyl nucleoside. A
preferred lipase for 5'-O-levulinyl hydrolysis is CAL-B.
[0024] In still further embodiments, methods are provided for
regioselectively deprotecting a nucleoside at the 3'-O-levulinyl
position wherein the nucleoside has one of the following formulas:
2
[0025] wherein:
[0026] R.sub.6 is --H, -hydroxyl;
[0027] R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are each,
independently, --H or an amino protecting group;
[0028] G is N or CH; and
[0029] Lev is --C(O)--(CH.sub.2).sub.2--C(O)--CH.sub.3;
[0030] comprising selecting a lipase that is effective to direct a
regioselective hydrolysis of the 3'-O-levulinyl position, without
causing hydrolysis of the 5'-O-levulinyl position, and contacting
the 3',5'-di-O-levulinyl nucleoside with the lipase for a time and
under conditions effective to yield a 5'-O-levulinyl nucleoside.
Lipases that are preferable for hydrolysis at the 3'-O-levulinyl
positions are, for example, CAL-A or PSL-C.
[0031] In some embodiments of the present invention, methods for
acylating a hydroxyl moiety of a nucleic acid, such as a nucleoside
or a nucleotide, at at least one of a 2'-O, 3'-O, or 5'-O position
are provided comprising reacting the nucleic acid with levulinic
acid in the presence of a coupling agent, such as a carbodiimide,
that is attached to a polymeric support for a time and under
conditions effective to form an ester at the 2'-O, 3'-O or 5'-O
position. Preferred polymeric supports comprise polystyrene or
polyethylene glycol polymeric supports that are attached to
cyclohexylcarbodiimide.
[0032] The present invention includes the esterification or
acylation of any hydroxyl moiety, such as those found in
carbohydrates or steroid molecules, by reacting the compounds
containing the hydroxyl moiety with levulinic acid in the presence
of a coupling agent that is attached to a polymeric support for a
time and under conditions effective to form an ester between the
hydroxyl moieties and the levulinyl group of the levulinic acid. In
some embodiments of the present invention, methods are provided for
acylating at least one hydroxyl moiety on a compound having the
following formula: 3
[0033] wherein:
[0034] B.sub.X is a nucleobase;
[0035] T.sub.1 and T.sub.2 are, independently, -hydroxyl, a
hydroxyl protecting group, an activated phosphate group, a
nucleotide, a nucleoside, or an oligonucleotide;
[0036] R is --H, -hydroxyl, a protected hydroxyl or a 2'
substituent group;
[0037] provided that at least one of T.sub.1, T.sub.2 or R is
-hydroxyl;
[0038] comprising reacting the compound with levulinic acid in the
presence of a coupling agent that is attached to a solid support,
such as PS-cyclohexylcarbodiimide, for a time and under conditions
effective to form an ester between the hydroxyl moiety and the
levulinyl group. In a preferred embodiment, T.sub.1 and T.sub.2 are
--OH and R is --H or a 2'-substituent.
[0039] In one preferred embodiment, methods are provided for
acylating the 3'-O and 5'-O positions of a compound having the
following formula: 4
[0040] wherein:
[0041] B.sub.X is a nucleobase; and
[0042] R is hydroxyl or an optionally protected 2'-substituent
comprising reacting the compound with levulinic acid in the
presence of a coupling agent that is attached to a solid support
for a time and under conditions effective to form a compound having
formula: 5
[0043] wherein Lev is -levulinyl.
[0044] According to one embodiment of the present invention,
methods are provided for generating a cyclohexylcarbodiimide
derivatized polymeric support from a cyclohexylurea derivatized
polymeric support comprising reacting the cyclohexylurea
derivatized polymeric support with a dehydrating agent, such as
tosyl chloride or POCl.sub.3, in an organic solvent for a time and
under conditions effective to yield the cyclohexylcarbodiimide
derivatized polymeric support. In some embodiments, the organic
solvent employed is CH.sub.2Cl.sub.2, CHCl.sub.3, hexane, or
pyridine.
[0045] In a further embodiment of the present invention, a method
is provided for generating a cyclohexylcarbodiimide derivatized
polymeric support from a cyclohexylurea derivatized polymeric
support comprising the steps of reacting the cyclohexylurea
derivatized polymer support with a dehydrating agent for a time and
under conditions effective to form a salt and subsequently
contacting the salt with an aqueous solution, such as aqueous NaOH,
to form the cyclohexylcarbodiimide derivatized polymeric
support.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] The numerous objects and advantages of the present invention
may be better understood by those skilled in the art by reference
to the accompanying detailed description and the following
drawings, in which:
[0047] FIG. 1 shows 3',5'-di-O-acylation of a 2'-deoxynucleoside
using levulinic acid and DCC or levulinic acid and
PS-carbodiimide.
[0048] FIG. 2 shows the enzymatic regioselective hydrolysis of a
3',5'-di-O-levulinyl 2'-deoxynucleoside.
[0049] FIG. 3 shows the enzymatic regioselective hydrolyis of a
3',5'-di-O-levulinyl 2'-substituted nucleoside.
[0050] FIG. 4 is a table depicting the results of regioselective
hydrolysis of nucleosides 2a-2g.
[0051] The present invention is directed to the preparation of
nucleoside building blocks such as 3',5'-di-O-levulinylnucleosides,
3'-O-levulinylnucleosides, and 5'-O-levulinylnucleosides that are
especially useful in the large-scale synthesis of
oligonucleotides.
[0052] According to one embodiment of the present invention, a
method is provided for protecting a hydroxyl moiety of a nucleic
acid at at least one of a 2'-O, 3'-O, or 5'-O position comprising
reacting the nucleic acid with levulinic acid in the presence of a
coupling agent that is attached to a polymeric support for a time
and under conditions effective to form an ester at the 2'-O, 3'-O
or 5'-O position. The nucleic acids of the present invention
include nucleosides, nucleotides, oligonucleosides and
oligonucleotides. In some embodiments, the nucleic acid is a
nucleoside and the polymeric support is a polystyrene support or a
polyethylene glycol support that is coupled to a coupling agent,
such as cyclohexylcarbodiiimide.
[0053] In a preferred embodiment, the nucleic acid has the formula:
6
[0054] wherein:
[0055] B.sub.X is a nucleobase;
[0056] T.sub.1 and T.sub.2 are, independently, hydroxyl, a
protected hydroxyl, an activated phosphate group, a nucleotide, a
nucleoside, or an oligonucleotide; and
[0057] R is --H, -hydroxyl, a protected hydroxyl, or a 2'
substituent group;
[0058] provided that at least one of T.sub.1, T.sub.2 or R is
--OH;
[0059] comprising reacting the compound with levulinic acid in the
presence of a coupling agent that is attached to a solid support
for a time and under conditions effective to form an ester between
the hydroxyl moiety and the levulinyl group. In a preferred
embodiment, T.sub.1 and T.sub.2 are --OH and R is H.
[0060] The protection methods of the present invention are not
limited to acylation of the hydroxyl groups of nucleosides. Any
hydroxyl functionality may be acylated using the methods of the
present invention, including those found in carbohydrate or steroid
molecules.
[0061] According to one method of the present invention, referring
to FIG. 1, 3',5'-di-O-levulinyl nucleosides (2) were prepared from
their corresponding natural nucleosides by treatment with levulinic
acid and PS-carbodiimide in 1,4-dioxane in the presence of DMAP as
a catalyst. Filtering off of the polystyrene beads removed the urea
and the N-levulinylurea derivatives, which were polymer bound.
3',5'-di-O-levulinylthymidine (2a) and
3',5'-di-O-levulinyl-2'-deoxyadeno- sine (2d) were isolated with
91% and 95% yield, respectively. The PS-dicarbodiimide, an
expensive reagent, is recovered by reacting the cyclohexylurea
derivatized polymer support with a dehydrating agent in an organic
solvent. Preferred dehydrating agents include POCl.sub.3 and
tosylchloride. Preferred organic solvents include CH.sub.2Cl.sub.2,
CHCl.sub.3, hexane, and pyridine.
[0062] Referring again to FIG. 1, 3',5'-di-O-levulinyl nucleosides
can alternatively be prepared from the corresponding natural
nucleosides (1) by treatment with 5.2 equivalents of levulinic acid
(LevOH) and dicyclohexylcarbodiimide (DCC) in 1,4-dioxane in the
presence of DMAP as catalyst. The reaction takes place through
activation of the levulinic acid with DCC to obtain the O-acylurea
intermediate. The excess of adduct evolves into the stable
N-acylurea which was isolated like DCU as byproducts in the
process. 3',5'-Dilevulinyl derivatives (2) were obtained in high
yields (70-95%) after flash chromatography. The crude residue of
the reactions was washed with Et.sub.2O to eliminate the N-acylurea
and subsequently dissolved in EtOAc from which the remaining DCU
was separated by filtration. Almost quantitative yields were
achieved for this acylation reaction. The level of purity was based
on their .sup.1H-NMR which showed just traces of DCU and
N-levulinylurea. Under these conditions no acylation was observed
in the amino group of 2'-deoxyadenosine (1d) and 2'-deoxyaguanosine
(1f). In the case of 2'-deoxycytidine (1b), less amount of LevOH
and DCC (3 equivalents) were used to minimize the formation of the
aminoacyl derivative. As a consequence, longer reaction times were
needed and some amount of the starting material remained unchanged.
In spite of that, 68% isolated yield of
3',5'-di-O-levulinyl-2'-deoxycytidine (2b) were obtained after
flash chromatography.
[0063] Regioselective deprotection of the common precursor,
3',5'-di-O-levulinyl nucleoside, at the 5'-O-levulinyl position is
effected by selecting a lipase effective to direct regioselective
hydrolysis at the 5'-O-levulinyl position, without causing
hydrolysis at the 3'-O-levulinyl position, and contacting the
diprotected nucleoside with the lipase for a time and under
conditions effective to hydrolyze the 3',5'-di-O-levulinyl
nucleoside at the 5'-O-levulinyl position. In some embodiments, the
diprotected nucleosides have one of the following formulas: 7
[0064] wherein:
[0065] R.sub.1 is --H, -hydroxyl, a protected hydroxyl, or a
2'-substituent; and
[0066] R.sub.2 and R.sub.3 are, independently, --H or an amino
protecting group;
[0067] G is N or CH; and
[0068] Lev is --C(O)--(CH.sub.2).sub.2--C(O)--CH.sub.3.
[0069] For example, referring to FIG. 2, a
3',5'-di-O-levulinylthymidine (2a) was treated with CAL-B at
40.degree. C. in 0.15M phoshpate buffer (pH=7) containing 18% of
1,4-dioxane. TLC showed total disappearance of the starting
material after 62 h (entry 1, Table 1). After usual workup, as
described by Myers et al. Trends Pharmacol. Sci. 2000, 21, 19-23;
Cook, Nucleosides Nucleotides 1999, 18, 1141-1162; Crooke, et al.
Annu. Rev. Pharmacol. Toxicol. 1996, 36, 107-129; and Matteucci et
al. 1996, 384, 20-22, the contents of which are all incorporated by
reference herein. .sup.1H-NMR spectra clearly indicated the
selective hydrolysis of the 5'-levulinic ester and the presence of
3'-O-levulinylthymidine (3a) as unique product. Traces of thymidine
formed in the enzymatic reaction (showed by TLC) remained in the
aqueous phase after extraction. Thus, pure compound (3a) was
isolated with 85% yield.
[0070] Table 1, shown in FIG. 4, also indicates that substrates
3',5'-di-O-levulinyl cytosine (2b), 3',5'-di-O-levulinyl adenosine
(2d), and 3',5'-di-O-levulinyl-N-isobutylguanosine (2g) exhibit
excellent selectivity towards the 5'-position, when hydrolyzed in
the presence of CAL-B. The absence of the 5'-O-levulinyl derivative
and the high yields with which the reactions take place are
noteworthy. Also, in these cases, TLC showed traces of completely
hydrolyzed nucleoside, which was easily removed with an aqueous
extraction.
[0071] The hydrolysis reaction catalyzed by CAL-B on
N-benzoyl-di-O-levulinyl-2'-deoxycytidine (2c) and
N-benzoyl-di-O-levulinyl-2'-deoxyadenosine (2e) afforded
N-benzoyl-2'-deoxycytidine (1c) and N-benzoyl-2'-deoxyadenosine
(1e), respectively. Although several reaction conditions were
tried, the process takes place without regioselectivity. It seems
that the active site of CAL-B did not accommodate the N-protected
adenosine and cytosine in the same manner as their unprotected
counterparts. While not wishing to be bound to any particular
theory, it is possible that the phenyl group could have some steric
contact within the binding site, which may lead to unfavorable
results.
[0072] 2'-substituted nucleosides are also successfully selectively
deprotected at the 5'-O-levulinyl position. Referring to FIG. 3,
all four nucleosides, 2'-methoxy-3',5'-di-O-levulinyladenosine
(6a), 2'-methoxyethoxy-3',5'-di-O-levulinyladenosine (6b),
2'-methoxy-3',5'-di-O-levulinyl-2'-deoxycytosine (6c), and
2'-methoxyethoxy-3',5'-di-O-levulinyl-5-methyl cytosine (6d) were
selectively hydrolyzed with CAL-B furnishing 7a-7d in high yields.
The times and conditions effective to hydrolyze the nucleosides are
not limited to those exemplified herein. Various times and
conditions are effective to hydrolyze the esters, which will be
recognized by those of skill in the art.
[0073] In one embodiment of the present invention,
3',5'-di-O-levulinyl nucleosides are regioselectively deprotected
at the 3'-O-levulinyl position by selecting a lipase effective to
direct regioselective hydrolysis at the 3'-O-levulinyl position,
without causing hydrolysis at the 5'-O-levulinyl position, and
contacting the diprotected nucleoside with the lipase for a time
and under conditions effective to hydrolyze the
3',5'-di-O-levulinyl nucleoside at the 3'-O-levulinyl position. In
some embodiments, the diprotected nucleosides have one of the
following formulas: 8
[0074] wherein:
[0075] R.sub.6 is --H, or --OH;
[0076] R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are each,
independently, --H or an amino protecting group;
[0077] G is N or CH; and
[0078] Lev is --C(O)--(CH.sub.2).sub.2--C(O)--CH.sub.3.
[0079] For example, referring to FIG. 2, 3'-O-selective hydrolysis
was accomplished by reaction of 2 with immobilized Pseudomonas
cepacia lipase [PSL-C, ratio of 1:3 w/w (2/PSL-C)] at 60.degree. C.
in 0.15M phosphate buffer giving the 5'-O-levulinyl derivative.
Candida antarctica lipase A (CAL-A) also exhibited excellent
selectivity towards the 3'-O-levulinyl position and has the
advantage of requiring lower reaction temperatures than PSL-C
(40.degree. C. instead of 60.degree. C.), shorter reaction times,
and a lower ratio of enzyme/starting material (see FIG. 4). Thus,
5'-O-levulinylthymidine (4a),
N-benzoyl-5'-O-levulinyl-2'-deoxycytidine (4c), and
N-benzoyl-5'-O-levulinyl-2'-deoxyadenosine (4e) were obtained with
high yields (70-85%). The 3'-levulinyl regioisomer was not detected
by TLC or .sup.1H-NMR of the crude reaction mixture. TLC showed
traces of parent nucleosides 1.
[0080] N-isobutyryl-3',5'-di-O-levulinyl-2'-deoxyguanosine (2g) was
not selectively hydrolyzed with CAL-A. However, treatment with
PSL-C afforded the N-isobutyryl-5'-O-levulinyl-2'-deoxyguanosine
(4g), which was isolated after 28 h at 60.degree. C. with 93% yield
(entry 8, Table 1). N-Benzoyl-di-levulinyl derivatives (2c) and
(2e) were both appropriate substrates for both lipases, PSL-C and
CAL-A.
[0081] Treatment of 2'-OR nucleosides, as shown in FIG. 3 with
PSL-C or CAL-A yielded a mixture of 3'-O-levulinyl and
5'-O-levulinyl nucleosides, without the selectivity that was
demonstrated with unprotected 2'-O and 2'-deoxynucleosides. While
not being bound to any particular theory, this may be the result of
steric hindrance caused by the 2'-O-R group, making the
3'-O-levulinyl group inaccessible for selective hydrolysis by
either of these lipases.
[0082] The nucleic acids of the present invention include naturally
occurring and non-naturally occurring nucleosides and nucleotides.
The nucleosides and nucleotides of the present invention are not
limited to monomer units but may also contain a plurality of linked
monomer units, to form dinucleosides, nucleotides, and
oligonucleotides and comprise naturally and non-naturally occurring
nucleobases, sugars, and backbones.
[0083] Non-naturally occurring nucleosides and nucleotides may be
modified by replacing the sugar moiety with an alternative
structure which has primary and secondary alcohol groups similar to
those of ribose. Non-naturally occurring sugars and nucleosidic
bases are typically structurally distinguishable from, yet
functionally interchangeable with, naturally occurring sugars (e.g.
ribose and deoxyribose) and nucleosidic bases (e.g., adenine,
guanine, cytosine, thymine). Thus, non-naturally occurring
nucleobases and sugars include all such structures which mimic the
structure and/or function of naturally occurring species, and which
aid in the binding of the oligonucleotide to a target, or which
otherwise advantageously contribute to the properties of the
oligonucleotide.
[0084] Backbone modifications include modifications to the
phosphate backbone to increase the resistance to nucleases. These
modifications include use of linkages such as methyl phosphonates,
phosphorothioates and phosphorodithioates as well as those
modifications that dramatically alter the nature of the
internucleotide linkage such as non-phosphorus linkages, peptide
nucleic acids (PNA's) and 2'-5' linkages.
[0085] A heterocyclic base moiety (often referred to in the art
simply as a "base" or a "nucleobase") amenable to the present
invention includes both naturally and non-naturally occurring
nucleobases. The heterocyclic base moiety further may be protected
wherein one or more functionalities of the base bears a protecting
group. As used herein, "unmodified" or "natural" nucleobases
include the purine bases adenine and guanine, and the pyrimidine
bases thymine, cytosine and uracil. Modified nucleobases include
other synthetic and natural nucleobases such as 5-methylcytosine
(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and
guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,
5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo
uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and
other 8-substituted adenines and guanines, 5-halo particularly
5-bromo, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine. Further nucleobases include those disclosed in
U.S. Pat. No. 3,687,808, those disclosed in the Concise
Encyclopedia Of Polymer Science And Engineering, pages 858-859,
Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed
by Englisch et al., Angewandte Chemie, International Edition, 1991,
30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15,
Antisense Research and Applications, pages 289-302, Crooke, S. T.
and Lebleu, B., ed., CRC Press, 1993.
[0086] Certain heterocyclic base moieties are particularly useful
for increasing the binding affinity of the oligomeric compounds of
the invention to complementary targets. 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 selected
2'-sugar modifications such as 2'-methoxyethyl groups.
[0087] Representative United States patents that teach the
preparation of heterocyclic base moieties (modified nucleobases)
include, but are not limited to, U.S. Pat. Nos. 3,687,808;
4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain
of which are commonly owned, and each of which is herein
incorporated by reference, and commonly owned U.S. patent
application Ser. No. 08/762,587, filed on Dec. 10, 1996, also
herein incorporated by reference.
[0088] A representative list of 2'-substituent groups amenable to
the present invention include C.sub.1-C.sub.20 alkyl,
C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl,
C.sub.5-C.sub.20 aryl, O-alkyl, O-alkenyl, O-alkynyl, O-alkylamino,
O-alkylalkoxy, O-alkylaminoalkyl, O-alkyl imidazole, S-alkenyl,
S-alkynyl, NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, O-aryl,
S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido,
halogen (particularly fluoro), keto, carboxyl, nitro, nitroso,
nitrile, trifluoromethyl, trifluoromethoxy, imidazole, azido,
hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,
disulfide, silyl, heterocycle, carbocycle, polyamine, polyamide,
polyalkylene glycol, and polyethers of the formula (O-alkyl).sub.m,
where m is 1 to about 10. Preferred among these polyethers are
linear and cyclic polyethylene glycols (PEGs), and (PEG)-containing
groups, such as crown ethers and those which are disclosed by Ouchi
et al. (Drug Design and Discovery 1992, 9, 93), Ravasio et al. (J.
Org. Chem. 1991, 56, 4329) and Delgardo et. al. (Critical Reviews
in Therapeutic Drug Carrier Systems 1992, 9, 249), each of which is
herein incorporated by reference in its entirety. Further sugar
modifications are disclosed in Cook, P. D., Anti-Cancer Drug
Design, 1991, 6, 585-607. Fluoro, O-alkyl, O-alkylamino, O-alkyl
imidazole, O-alkylaminoalkyl, and alkyl amino substitution is
described in U.S. patent application Ser. No. 08/398,901, filed
Mar. 6, 1995, entitled Oligomeric Compounds having Pyrimidine
Nucleotide(s) with 2' and 5' Substitutions, hereby incorporated by
reference in its entirety.
[0089] Additional substituent groups amenable to the present
invention include --SR and --NR.sub.2 groups, wherein each R is,
independently, hydrogen, a protecting group or substituted or
unsubstituted alkyl, alkenyl, or alkynyl. 2'-SR nucleosides are
disclosed in U.S. Pat. No. 5,670,633, issued Sep. 23, 1997, hereby
incorporated by reference in its entirety. The incorporation of
2'-SR monomer synthons are disclosed by Hamm et al., J. Org. Chem.,
1997, 62, 3415-3420. 2'-NR.sub.2 nucleosides are disclosed by
Goettingen, M., J. Org. Chem., 1996, 61, 73-6281; and Polushin et
al., Tetrahedron Lett., 1996, 37, 3227-3230.
[0090] Further substituent groups have one of formula I or II:
9
[0091] wherein:
[0092] Z.sub.0 is O, S or NH;
[0093] J is a single bond, O or C(.dbd.O);
[0094] E is C.sub.1-C.sub.10 alkyl, N(R.sub.1)(R.sub.2),
N(R.sub.1)(R.sub.5), N.dbd.C(R.sub.1)(R.sub.2),
N.dbd.C(R.sub.1)(R.sub.5) or has one of formula III or IV;
[0095] each R.sub.6, R.sub.7, R.sub.8, R.sub.9 and R.sub.10 is,
independently, hydrogen, C(O)R.sub.11, substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical
functional group or a conjugate group, wherein the substituent
groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,
phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and
alkynyl;
[0096] or optionally, R.sub.7 and R.sub.8, together form a
phthalimido moiety with the nitrogen atom to which they are
attached;
[0097] or optionally, R.sub.9 and R.sub.10, together form a
phthalimido moiety with the nitrogen atom to which they are
attached;
[0098] each R.sub.11 is, independently, substituted or
unsubstituted C.sub.1-C.sub.10 alkyl, trifluoromethyl,
cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy,
9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy,
2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or
aryl;
[0099] R.sub.5 is T--L,
[0100] T is a bond or a linking moiety;
[0101] L is a chemical functional group, a conjugate group or a
solid support material;
[0102] each R.sub.1 and R.sub.2 is, independently, H, a nitrogen
protecting group, substituted or unsubstituted C.sub.1-C.sub.10
alkyl, substituted or unsubstituted C.sub.2-C.sub.10 alkenyl,
substituted or unsubstituted C.sub.2-C.sub.10 alkynyl, wherein said
substitution is OR.sub.3, SR.sub.3, NH.sub.3.sup.+,
N(R.sub.3)(R.sub.4), guanidino or acyl where said acyl is an acid
amide or an ester;
[0103] or R.sub.1 and R.sub.2, together, are a nitrogen protecting
group or are joined in a ring structure that optionally includes an
additional heteroatom selected from N and O;
[0104] or R.sub.1, T and L, together, are a chemical functional
group;
[0105] each R.sub.3 and R.sub.4 is, independently, H,
C.sub.1-C.sub.10 alkyl, a nitrogen protecting group, or R.sub.3 and
R.sub.4, together, are a nitrogen protecting group;
[0106] or R.sub.3 and R.sub.4 are joined in a ring structure that
optionally includes an additional heteroatom selected from N and
O;
[0107] Z.sub.4 is OX, SX, or N(X).sub.2;
[0108] each X is, independently, H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 haloalkyl, C(.dbd.NH)N(H)R.sub.5,
C(.dbd.O)N(H)R.sub.5 or OC(.dbd.O)N(H)R.sub.5;
[0109] R.sub.5 is H or C.sub.1-C.sub.8 alkyl;
[0110] Z.sub.1, Z.sub.2 and Z.sub.3 comprise a ring system having
from about 4 to about 7 carbon atoms or having from about 3 to
about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero
atoms are selected from oxygen, nitrogen and sulfur and wherein
said ring system is aliphatic, unsaturated aliphatic, aromatic, or
saturated or unsaturated heterocyclic;
[0111] Z.sub.5 is alkyl or haloalkyl having 1 to about 10 carbon
atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2
to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms,
N(R.sub.1)(R.sub.2)OR.sub.1, halo, SR.sub.1 or CN;
[0112] each q.sub.1 is, independently, an integer from 1 to 10;
[0113] each q.sub.2 is, independently, 0 or 1;
[0114] q.sub.3 is 0 or an integer from 1 to 10;
[0115] q.sub.4 is an integer from 1 to 10;
[0116] q.sub.5 is from 0, 1 or 2; and
[0117] provided that when q.sub.3 is 0, q.sub.4 is greater than
1.
[0118] Representative substituent groups of Formula I are disclosed
in U.S. patent application Ser. No. 09/130,973, filed Aug. 7, 1998,
entitled "Capped 2'-Oxyethoxy Oligonucleotides," hereby
incorporated by reference in its entirety.
[0119] Representative cyclic substituent groups of Formula II are
disclosed in U.S. patent application Ser. No. 09/123,108, filed
Jul. 27, 1998, entitled "RNA Targeted 2'-Modified Oligonucleotides
that are Conformationally Preorganized," hereby incorporated by
reference in its entirety.
[0120] Particularly preferred substituent groups include
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2,
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)- ].sub.2 (where n and
m are from 1 to about 10), C.sub.1 to C.sub.10 lower alkyl,
substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,
SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino and substituted silyl. Another particularly
preferred modification includes 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3 or 2'-MOE, Martin et al., Helv.
Chim. Acta, 1995, 78, 486). A further preferred substituent group
is 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3)- .sub.2 group, also known as
2'-DMAOE. Representative aminooxy substituent groups are described
in co-owned U.S. patent application Ser. No. 09/344,260, filed Jun.
25, 1999, entitled "Aminooxy-Functionalized Oligomers"; and U.S.
patent application Ser. No. 09/370,541, filed Aug. 9, 1999, also
identified by attorney docket number ISIS-3993, entitled
Aminooxy-Functionalized Oligomers and Methods for Making Same;
hereby incorporated by reference in their entirety.
[0121] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F).
Similar modifications may also be made at other positions on
nucleosides and oligomers, particularly the 3' position of the
sugar on the 3' terminal nucleoside or at a 3'-position of a
nucleoside that has a linkage from the 2'-position such as a 2'-5'
linked oligomer and at the 5'-position at a 5'-terminus. Oligomers
may also have sugar mimetics such as cyclobutyl moieties in place
of the pentofuranosyl sugar. Representative United States patents
that teach the preparation of such modified sugars structures
include, but are not limited to, U.S. Pat. Nos. 4,981,957;
5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;
5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;
5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633;
and 5,700,920, certain of which are commonly owned, and each of
which is herein incorporated by reference, and commonly owned U.S.
Pat. No. 5,859,221, also herein incorporated by reference.
[0122] Representative guanidino substituent groups that are shown
in formula III and IV are disclosed in co-owned U.S. patent
application Ser. No. 09/349,040, entitled "Functionalized
Oligomers", filed Jul. 7, 1999, hereby incorporated by reference in
its entirety.
[0123] Representative acetamido substituent groups are disclosed in
U.S. patent application Ser. No. 09/378,568, entitled
"2'-O-Acetamido Modified Monomers and Oligomers", filed Aug. 19,
1999, also identified by attorney docket number ISIS-4071, hereby
incorporated by reference in its entirety.
[0124] Representative dimethylaminoethyloxyethyl substituent groups
are disclosed in International Patent Application PCT/US99/17895,
entitled "2'-O-Dimethylaminoethyloxyethyl-Modified
Oligonucleotides", filed Aug. 6, 1999, also identified by attorney
docket number ISIS-4045, hereby incorporated by reference in its
entirety.
[0125] The methods of the present invention use labile protecting
groups to protect various functional moieties during synthesis.
Protecting groups are used ubiquitously in standard oligonucleotide
synthetic regimes for protection of several different types of
functionality. In general, protecting groups render chemical
functionality inert to specific reaction conditions and can be
appended to and removed from such functionality in a molecule
without substantially damaging the remainder of the molecule. See,
e.g., Green and Wuts, Protective Groups in Organic Synthesis, 2d
edition, John Wiley & Sons, New York, 1991. Representative
protecting groups useful to protect nucleotides during synthesis
include base labile protecting groups and acid labile protecting
groups. Base labile protecting groups are used to protect the
exocyclic amino groups of the heterocyclic nucleobases. This type
of protection is generally achieved by acylation. Two commonly used
acylating groups for this purpose are benzoylchloride and
iso-butyrylchloride. These protecting groups are stable to the
reaction conditions used during oligonucleotide synthesis and are
cleaved at approximately equal rates during the base treatment at
the end of synthesis.
[0126] Hydroxyl protecting groups typically used in oligonucleotide
synthesis may be represented by the group having the formula:
--C(R.sub.1)(R.sub.2)(R.sub.3) wherein each of R.sub.1, R.sub.2 and
R.sub.3 is an unsubstituted or mono-substituted aryl or heteroaryl
group selected from phenyl, naphthyl, anthracyl, and five or six
membered heterocylic rings with a single heteroatom selected from
N, O and S, or two N heteroatoms, including quinolyl, furyl, and
thienyl; where the substituent is selected from halo (i.e., F, Cl,
Br, and I), nitro, C.sub.1-C.sub.4-alkyl or alkoxy, and aryl,
aralkyl and cycloalkyl containing up to 10 carbon atoms; and
wherein R.sub.2 and R.sub.3 may each also be C.sub.1-C.sub.4-alkyl
or aralkyl or cycloalkyl containing up to 10 carbon atoms.
[0127] As will be recognized, additional objects, advantages, and
novel features of this invention will become apparent to those
skilled in the art upon examination of the following examples
thereof, which are not intended to be limiting.
Experimental
[0128] General. Candida antarctica lipase B (CAL-B) was a gift from
Novo Nordisk Co. Candida antarctica lipase A (CAL-A) and
immobilized Pseudomonas cepacia lipase (PSL-C) were purchased from
Roche Diagnostics S. L. and Amano Pharmaceuticals, respectively.
PS-carbodiimide was purchased from Argonaut Technologies (San
Carlos, Calif., EE.UU.). All other reagents were purchased from
Aldrich or Fluka. Solvents were distilled over an adequate
desiccant under nitrogen.
[0129] 3',5'-di-O-Levulinyl-2'-deoxynucleosides (2).
[0130] Method A: To a stirred mixture of 1 (2 mmol) and Et.sub.3N
(1.7 mL, 12 mmol) in 1,4-dioxane (20 mL) under nitrogen, was added
levulinic acid (1.21 g, 10.4 mmol), DCC (2.14 g, 10.4 mmol), and
DMAP (20 mg, 0.16 mmol). The reaction was stirred at room
temperature for 3 hours. In order to minimize the formation of
triprotected cytidine derivative, 6 mmol both of LevOH and DCC, and
5 mmol of Et.sub.3N were used for 1b. The insoluble material was
collected by filtration and the filtrate was evaporated under
vacuum. The residue was taken up in NaHCO.sub.3 (aq) and extracted
with CH.sub.2Cl.sub.2. The combined organic extracts were dried
over Na.sub.2SO.sub.4 and evaporated. Cold Et.sub.2O was added, and
the slurry was scratched until crystallization occurs. The solid
was filtered and washed with cold Et.sub.2O, and then was poured in
EtOAc (MeOH in case of 2f). The insoluble material was filtered and
the filtrate was concentrated to afford the title compounds. The
resulting materials were pure enough to be carried directly on to
the enzymatic hydrolysis step. Further purification by flash
chromatography (EtOAc) give pure 3',5'-di-O-levulinylnucleosides
2a-g.
[0131] Method B: To a stirred mixture of 1(0.4 mmol) and Et.sub.3N
(0.15 mL, 1 mmol) in 1,4-dioxane (5 mL) under nitrogen, was added
levulinic acid (0.14 g, 1.2 mmol), PS-carbodiimide (1.05 g, 1.2
mmol), DMAP (4 mg, 0.032 mmol), and DMAP.cndot.HCl (3 mg, 0.02
mmol). The reaction was stirred at room temperature for 3 hours.
The insoluble material was collected by filtration and the filtrate
was evaporated under vacuum. The residue was taken up in
NaHCO.sub.3 (aq) and extracted with CH.sub.2Cl.sub.2. The combined
organic extracts were dried over Na.sub.2SO.sub.4 and evaporated.
The solid was washed with cold Et.sub.2O to afford
3',5'-di-O-levulinylnucleosides 2a and 2d.
[0132] 3',5'-di-O-Levulinylthymidine (2a).
[0133] R.sub.f (10% MeOH/CH.sub.2Cl.sub.2): 0.45; Mp: 87-89.degree.
C.; IR (KBr): .upsilon. 3315, 3074, 3006, 2967, 2947, 1743, 1689,
and 1660 cm.sup.-1; .sup.1H-NMR (CDCl.sub.3, 300 MHz): d 1.88 (s,
3H, Me), 2.14 (s, 3H, Me-Lev), 2.15 (s, 3H, Me-Lev), 2.18 (m, 1H,
H.sub.2'), 2.41 (m, 1H, H.sub.2'), 2.54 (m, 4H, 2CH.sub.2-Lev),
2.73 (m, 4H, 2CH.sub.2-Lev), 4.19 (m, 1H, H.sub.4'), 4.31 (m, 2H,
H.sub.5'), 5.18 (m, 1H, H.sub.3), 6.28 (dd, 1H, H.sub.1',
.sup.3J.sub.HH 8.5, .sup.3J.sub.HH 5.4 Hz), 7.32 (s,1H, H.sub.6),
and 9.99 (s,1H,NH); .sup.13C-NMR (CDCl.sub.3, 75.5 MHz): d 12.3
(Me), 27.48 (CH.sub.2-Lev), 27.54 (CH.sub.2-Lev), 29.4 (2 Me-Lev),
36.8 (C.sub.2'), 37.5 (2CH.sub.2-Lev), 63.7 (C.sub.5'), 74.2, 81.8,
84.3 (C.sub.1'+C.sub.3'+C.sub.4'), 111.2 (C.sub.5), 134.6
(C.sub.6), 150.3 (C.sub.2), 163.8 (C.sub.4), 171.97 (C.dbd.O Lev),
172.02 (C.dbd.O Lev), and 206.3 (2C.dbd.O Lev); MS (ESI.sup.+,
m/z): 439 [(M+H).sup.+, 100%], and 461 [(M+Na).sup.+, 50].
[0134] 3',5'-di-O-Levulinyl-2'-deoxycytidine (2b).
[0135] R.sub.f (20% MeOH/CH.sub.2Cl.sub.2): 0.67; IR (KBr):
.upsilon. 3390, 2940, 1737, 1715, and 1649 cm.sup.-1; .sup.1H-NMR
(MeOH-d.sub.4, 200 MHz): d 2.39 (s, 3H, Me-Lev), 2.40 (s, 3H,
Me-Lev), 2.43 (m, 1H, H.sub.2'), 2.75 (m, 1H, H.sub.2'), 2.79 (m,
4H, 2CH.sub.2-Lev), 3.02 (m, 4H, 2CH.sub.2-Lev), 4.51 (m, 3H,
H.sub.4'+2H.sub.5'), 5.46 (m, 1H, H.sub.3'), 6.17 (d, 1H, H.sub.5,
.sup.3J.sub.HH 7.6 Hz), 6.45 (dd, 1H, H.sub.1', .sup.3J.sub.HH 8.6,
.sup.3J.sub.HH 5.7 Hz), and 7.98 (dd, 1H, H.sub.6, .sup.3J.sub.HH
7.3 Hz); .sup.13C-NMR (MeOH-d.sub.4, 75.5 MHz): d 29.1
(CH.sub.2-Lev), 29.2 (CH.sub.2-Lev), 29.9 (Me-Lev), 38.9
(2CH.sub.2-Lev), 39.2 (C.sub.2'), 65.5 (C.sub.5'), 76.5, 84.1, 87.9
(C.sub.1'+C.sub.3'+C.sub.4'), 96.8 (C.sub.5), 142.2 (C.sub.6),
158.4 (C.sub.2), 168.0 (C.sub.4), 174.2 (C.dbd.O), 174.4 (C.dbd.O),
and 209.7 (C.dbd.O); MS (ESI.sup.+, m/z): 446 [(M+Na).sup.+, 70%]
and 462 [(M+K).sup.+, 100].
[0136] N-Benzoyl-3',5'-di-O-levulinyl-2'-deoxycytidine (2c).
[0137] R.sub.f (10% MeOH/CH.sub.2Cl.sub.2): 0.61; Mp: 107-109
.degree. C.; IR (KBr): .upsilon. 3233, 1744,1731, and 1668
cm.sup.-1; .sup.1H-NMR (MeOH-d.sub.4, 200 MHz): d 2.31 (s, 3H,
Me-Lev), 2.38 (s, 3H, Me-Lev), 2.50 (m, 1H, H.sub.2'), 2.75 (m, 4H,
2CH.sub.2-Lev), 2.95 (m, 1H, H.sub.2'), 3.05 (m, 4H,
2CH.sub.2-Lev), 4.59 (m, 3H, H.sub.4'+2H.sub.5'), 5.49 (m, 1H,
H.sub.3'), 6.42 (dd, 1H, H.sub.1', .sup.3J.sub.HH 7.7,
.sup.3J.sub.HH 5.7 Hz), 7.75 (m, 4H, H.sub.5+H.sub.m+H.sub.p), 8.15
(m, 2H, H.sub.o), and 8.45 (d, 1H, H.sub.6, .sup.3J.sub.HH 7.6 Hz);
.sup.13C-NMR (MeOH-d.sub.4, 75.5 MHz): d 29.1 (CH.sub.2-Lev), 29.2
(CH.sub.2-Lev), 29.9 (Me-Lev), 38.91 (CH.sub.2-Lev), 38.94
(CH.sub.2-Lev), 39.8 (C.sub.2'), 65.3 (C.sub.5'), 76.5, 85.0, 89.3
(C.sub.1'+C.sub.3'+C.sub.4'), 99.0 (C.sub.5), 129.5, 130.1
(C.sub.o+C.sub.m), 134.4 (C.sub.p), 135.0 (C.sub.i), 146.2
(C.sub.6), 158.0 (C.sub.2), 165.1 (C.sub.4), 169.2 (PhC.dbd.O),
174.3 (C.dbd.O), 174.4 (C.dbd.O), 209.67 (C.dbd.O), and 209.72
(C.dbd.O); MS (ESI.sup.+, m/z): 528 [(M+H).sup.+, 100%], 550
[(M+Na).sup.+, 30], and 566 [(M+K).sup.+, 40].
[0138] 3',5'-di-O-Levulinyl-2'-deoxyadenosine (2d).
[0139] R.sub.f (10% MeOH/CH.sub.2Cl.sub.2): 0.44; IR (KBr):
.upsilon. 3418, 3165, 2923, 1738, 1715, and 1644 cm.sup.-1;
.sup.1H-NMR (MeOH-d.sub.4, 200 MHz): d 2.33 (s, 3H, Me-Lev), 2.39
(s, 3H, Me-Lev), 2.79 (m, 5H, 2CH.sub.2-Lev+1H.sub.2'), 3.00 (m,
4H, 2CH.sub.2-Lev), 3.25 (m, 1H, H.sub.2'), 4.52 (m, 3H,
H.sub.4'+2H.sub.5'), 5.65 (m, 1H, H.sub.3'), 6.61 (dd, 1H,
H.sub.1', .sup.3J.sub.HH 6.0, .sup.3J.sub.HH 7.9 Hz), 8.41 (s, 1H,
H.sub.2 or H.sub.8), and 8.50 (s, 1H, H.sub.8 or H.sub.2);
.sup.13C-NMR (MeOH-d.sub.4, 75.5 MHz): d 29.1 (CH.sub.2-Lev), 29.2
(CH.sub.2-Lev), 29.9 (2Me-Lev), 38.1 (C.sub.2'), 38.9
(2CH.sub.2-Lev), 65.2 (C.sub.5'), 76.5, 84.2, 86.2
(C.sub.1'+C.sub.3'+C.sub.4'), 120.8 (C.sub.5), 141.2 (C.sub.8),
150.7 (C.sub.4), 154.2 (C.sub.2), 157.6 (C.sub.6), 174.2 (C.dbd.O),
174.4 (C.dbd.O), 209.68 (C.dbd.O), and 209.73 (C.dbd.O); MS
(ESI.sup.+, m/z): 448 [(M+H).sup.+, 20%], 470 [(M+Na).sup.+, 80],
and 486 [(M+K).sup.+, 100].
[0140] N-Benzoyl-3',5'-di-O-levulinyl-2'-deoxyadenosine (2e).
[0141] R.sub.f (10% MeOH/CH.sub.2Cl.sub.2): 0.71; Mp:69-71.degree.
C.; IR(KBr): .upsilon. 3412, 3086, 2958, 1738, 1714, and 1685
cm.sup.-1; .sup.1H-NMR (MeOH-d.sub.4, 300 MHz): d 2.28 (s, 3H,
Me-Lev), 2.35 (s, 3H, Me-Lev), 2.75 (m, 4H, 2CH.sub.2-Lev), 2.87
(m, 1H, H.sub.2'), 2.99 (m, 4H, 2CH.sub.2-Lev), 3.30 (m, 1H,
H.sub.2'), 4.52 (m, 3H, H.sub.4'+2H.sub.5'), 5.65 (m, 1H,
H.sub.3'), 6.70 (apparent t, 1H, H.sub.1', .sup.3J.sub.HH 6.8 Hz),
7.75 (m, 3H, 2H.sub.m+H.sub.p), 8.25 (apparent d, 2H, 2H.sub.o,
.sup.3J.sub.HH 7.4 Hz), 8.75 (s, 1H, H.sub.2 or H.sub.8), and 8.88
(s, 1H, H.sub.8 or H.sub.2); .sup.13C-NMR (MeOH-d.sub.4, 75.5 MHz):
d 29.0 (CH.sub.2-Lev), 29.2 (CH.sub.2-Lev), 30.0 (Me-Lev), 37.9
(C.sub.2'), 38.87 (CH.sub.2-Lev), 38.91 (CH.sub.2-Lev), 65.2
(C.sub.5'), 76.4, 84.3, 86.5 (C.sub.1'+C.sub.3'+C.su- b.4'), 125.5
(C.sub.5), 129.7, 130.0 (C.sub.o+C.sub.m), 134.1 (C.sub.p), 135.1
(C.sub.i), 144.5 (C.sub.8), 151.3 (C.sub.4), 153.3(C.sub.6), 153.5
(C.sub.2), 168.1 (PhC.dbd.O), 174.2 (C.dbd.O), 174.3 (C.dbd.O),
209.6 (C.dbd.O), and 209.7 (C.dbd.O); MS (ESI.sup.+, m/z): 552
[(M+H).sup.+, 100%] and 574 [(M+Na).sup.+, 17].
[0142] 3',5'-di-O-Levulinyl-2'-deoxyguanosine (2f).
[0143] R.sub.f (20% MeOH/CH.sub.2Cl.sub.2): 0.65; Mp:
148-150.degree. C.; IR (KBr): .upsilon. 3397, 3153, 2940, and 1711
cm.sup.-1; .sup.1H-NMR(DMSO-d.sub.6, 200 MHz): d 2.16 (s, 3H,
Me-Lev), 2.22 (s, 3H, Me-Lev), 2.60 (m, 5H,
2CH.sub.2-Lev+1H.sub.2'), 2.83 (m, 4H, 2CH.sub.2-Lev), 3.00 (m, 1H,
H.sub.2'), 4.29 (m, 3H, H.sub.4'+2H.sub.5'), 5.35 (m, 1H,
H.sub.3'), 6.22 (dd, 1H, H.sub.1', .sup.3J.sub.HH 5.8,
.sup.3J.sub.HH 8.8 Hz), 6.69 (br s, 2H, NH), and 8.00 (s, 1H,
H.sub.8); .sup.13C-NMR (DMSO-d.sub.6, 50.3 MHz): d 27.5
(CH.sub.2-Lev), 27.7(CH.sub.2-Lev), 29.55 (Me-Lev), 29.60 (Me-Lev),
35.5, 37.4, 37.50 (2CH.sub.2-Lev+C.sub.2'), 63.8 (C.sub.5'), 74.7,
81.5, 82.6 (C.sub.1'+C.sub.3'+C.sub.4'), 116.8 (C.sub.5), 135.1
(C.sub.8), 151.2 (C.sub.4), 154.0 (C.sub.2), 156.9 (C.sub.6), 172.1
(C.dbd.O), 172.2 (C.dbd.O), 206.9 (C.dbd.O), and 207.1 (C.dbd.O);
MS (ESI.sup.+, m/z): 464 [(M+H).sup.+, 22%], 486 [(M+Na).sup.+,
75], and 502 [(M+K).sup.+, 100].
[0144] N-Isobutyryl-3',5'-di-O-levulinyl-2'-deoxyguanosine
(2g).
[0145] R.sub.f (20% MeOH/CH.sub.2Cl.sub.2): 0.85; Mp: 45-47.degree.
C.; IR (KBr): .upsilon. 3413, 2935, 1740, 1714, 1680, and 1613
cm.sup.-1; .sup.1H-NMR (DMSO-d.sub.6, 200 MHz): d 1.23 (d, 6H,
Me-.sup.iBu, .sup.3J.sub.HH 6.5 Hz), 2.15 (s, 3H, Me-Lev), 2.20 (s,
3H, Me-Lev), 2.55-3.19 (several m, 11H,
4CH.sub.2-Lev+2H.sub.2'+CH-.sup.iBu), 4.32 (m, 3H,
H.sub.4'+2H.sub.5'), 5.35 (m, 1H, H.sub.3'), 6.35 (apparent t, 1H,
H.sub.1', .sup.3J.sub.HH 7.2 Hz), 8.35 (s, 1H, H.sub.8), 11.80 (br
s, 1H, NH), and 12.20 (br s, 1H, NH); .sup.13C-NMR (DMSO-d.sub.6,
50.3 MHz): d 18.86 (Me-.sup.iBu), 18.91 (Me-.sup.iBu), 27.5
(CH.sub.2-Lev), 27.6 (CH.sub.2-Lev), 29.5 (Me-Lev), 29.6 (Me-Lev),
34.8 (CH-.sup.iBu), 35.5 (C.sub.2'), 37.38 (CH.sub.2-Lev), 37.45
(CH.sub.2-Lev), 63.7 (C.sub.5'), 74.6, 81.7, 82.9
(C.sub.1'+C.sub.3'+C.sub.4'), 120.3 (C.sub.5), 137.3 (C.sub.8),
148.3, 148.7 (C.sub.2+C.sub.4), 154.8 (C.sub.6), 172.1 (C.dbd.O),
172.2 (C.dbd.O), 180.2 (.sup.iBu-C.dbd.O), 206.9 (C.dbd.O), and
207.1 (C.dbd.O); MS (ESI.sup.+, m/z): 534 [(M+H).sup.+, 100%], 556
[(M+Na).sup.+, 60], and 572 [(M+K).sup.+, 27].
[0146] General procedure for the enzymatic hydrolysis of
3',5'-di-O-levulinyl-2'-deoxynucleosides.
[0147] To a solution of 2 (0.2 mmol) in 1,4-dioxane (0.35 mL) was
added 0.15M phosphate buffer pH=7 (1.65 mL) and the corresponding
lipase [ratio of 2: enzyme was 1:1 (w/w) for CAL-A or CAL-B, and
1:3 (w/w) for PSL-C]. The mixture was allowed to react at 250 rpm
for the time and at the temperature indicated in Table 1. The
reactions were monitored by TLC (10% MeOH/CH.sub.2Cl.sub.2). The
enzyme was filtered off and washed with CH.sub.2Cl.sub.2, the
solvents were distilled under vacuum, and the residue was taken up
in NaHCO.sub.3 (aq) and extracted with CH.sub.2Cl.sub.2. The
combined organic layers were dried over Na.sub.2SO.sub.4 and
evaporated to give monoacylnucleosides 3 or 4. In case of 3b, the
residue was purified by flash chromatography instead of
extraction.
[0148] 3'-O-Levulinylthymidine (3a).
[0149] R.sub.f (10% MeOH/CH.sub.2Cl.sub.2): 0.32; Mp: 50-52.degree.
C.; IR (KBr): .upsilon. 3449, 3065, 2927, and 1706 cm.sup.-1;
.sup.1H-NMR (MeOH-d.sub.4, 200 MHz): d 2.09 (d, 3H, Me, J.sub.HH
1.3 Hz), 2.39 (s, 3H, Me-Lev,), 2.57 (m, 2H, H.sub.2'), 2.80 (t,
2H, CH.sub.2-Lev, .sup.3J.sub.HH 6.0 Hz), 3.05 (t, 2H,
CH.sub.2-Lev, .sup.3J.sub.HH 6.2 Hz), 4.01 (m, 2H, H.sub.5'), 4.29
(m, 1H, H.sub.4'), 5.02 (m, 1H, H.sub.3'), 6.50 (dd, 1H, H.sub.1',
.sup.3J.sub.HH 8.1, .sup.3J.sub.HH 6.5 Hz), and 8.04 (d, 1H,
H.sub.6, J.sub.HH 1.3 Hz); .sup.13C-NMR (CDCl.sub.3, 75.5 MHz): d
12.4 (Me), 27.8 (CH.sub.2-Lev), 29.6 (Me-Lev), 37.1 (C.sub.2'),
37.7 (CH.sub.2-Lev), 62.2 (C.sub.5'), 74.9, 85.0, 85.7
(C.sub.1'+C.sub.3'+C.sub.4'), 111.1 (C.sub.5), 136.5 (C.sub.6),
150.6 (C.sub.2), 164.3 (C.sub.4), 172.4 (2C.dbd.O Lev), and 206.8
(2C.dbd.O Lev); MS (ESI.sup.+, m/z): 363 [(M+Na).sup.+, 100%] and
379 [(M+K).sup.+, 30].
[0150] 3'-O-Levulinyl-2'-deoxycytidine (3b).
[0151] R.sub.f (20% MeOH/CH.sub.2Cl.sub.2): 0.41; .sup.1H-NMR
(MeOH-d.sub.4, 200 MHz): d 2.39 (s, 3H, Me-Lev), 2.47 (m, 1H,
H.sub.2'), 2.67 (m, 1H, H.sub.2'), 2.75 (m, 2H, CH.sub.2-Lev), 3.02
(m, 2H, CH.sub.2-Lev), 4.00 (m, 2H, 2H.sub.5'), 4.30 (m, 1H,
H.sub.4'), 5.49 (m, 1H, H.sub.3'), 6.15 (d, 1H, H.sub.5,
.sup.3J.sub.HH 6.8 Hz), 6.48 (apparent t, 1H, H.sub.1',
.sup.3J.sub.HH 6.8 Hz), and 8.32 (d, 1H, H.sub.6, .sup.3J.sub.HH
7.3 Hz); .sup.13C-NMR (MeOH-d.sub.4, 75.5 MHz): d 29.2
(CH.sub.2-Lev), 29.9 (Me-Lev), 38.9, 39.5 (C.sub.2'+CH.sub.2-Lev),
63.2 (C.sub.5'), 76.9, 87.1, 87.8 (C.sub.1'+C.sub.3'+C.sub.4'),
96.6 (C.sub.5), 143.0 (C.sub.6), 158.2 (C.sub.2), 167.6 (C.sub.4),
174.2 (C.dbd.O), and 209.8 (C.dbd.O).
[0152] 3'-O-Levulinyl-2'-deoxyadenosine (3d).
[0153] R.sub.f (20% MeOH/CH.sub.2Cl.sub.2): 0.66; IR (KBr):
.upsilon. 3292, 2925, 1730, 1715, 1690, 1644, and 1610 cm.sup.-1;
.sup.1H-NMR (MeOH-d.sub.4, 200 MHz): d 2.40 (s, 3H, Me-Lev), 2.76
(m, 1H, H.sub.2'), 2.80 (t, 2H, CH.sub.2-Lev, .sup.3J.sub.HH 6.2
Hz), 3.05 (t, 2H, CH.sub.2-Lev, .sup.3J.sub.HH 6.2 Hz), 3.14 (m,
1H, H.sub.2'), 4.04 (m, 2H, 2H.sub.5'), 4.40 (m, 1H, H.sub.4'),
5.66 (d, 1H, H.sub.3', .sup.3J.sub.HH 6.0 Hz), 6.61(dd, 1H,
H.sub.1', .sup.3J.sub.HH 5.7, .sup.3J.sub.HH 9.1 Hz), 8.39 (s, 1H,
H.sub.2 or H.sub.8), and 8.50 (s, 1H, H.sub.8 or H.sub.2);
.sup.13C-NMR (MeOH-d.sub.4, 75.5 MHz): d 29.1 (CH.sub.2-Lev), 29.9
(Me-Lev), 38.9, 39.0 (C.sub.2'+CH.sub.2-Lev), 64.0 (C.sub.5'),
77.5, 87.6, 87.9 (C.sub.1'+C.sub.3'+C.sub.4'), 121.2 (C.sub.5),
141.9 (C.sub.8), 150.1 (C.sub.4), 153.8 (C.sub.2), 157.8 (C.sub.6),
174.3 (C.dbd.O), and 209.8 (C.dbd.O); MS (ESI.sup.+, m/z): 350
[(M+H).sup.+, 100%], 372 [(M+Na).sup.+, 100], and 388 [(M+K).sup.+,
60].
[0154] N-Isobutyryl-3'-O-levulinyl-2'-deoxyguanosine (3g).
[0155] R.sub.f (20% MeOH/CH.sub.2Cl.sub.2): 0.75; Mp:
170-172.degree. C.; IR (KBr): .upsilon. 3415, 2961, 2929, 2859,
1725, 1686, and 1614 cm.sup.-1; .sup.1H-NMR (MeOH-d.sub.4, 200
MHz): d 1.41 (d, 6H, Me-.sup.iBu, .sup.3J.sub.HH 6.8 Hz), 2.38 (s,
3H, Me-Lev), 2.70-3.09 (m, 7H,
2CH.sub.2-Lev+2H.sub.2'+CH-.sup.iBu), 3.98 (d, 2H, 2H.sub.5',
.sup.3J.sub.HH 3.4 Hz), 4.45 (m, 1H, H.sub.4'), 5.60 (m, 1H,
H.sub.3'), 6.51 (dd, 1H, H.sub.1', .sup.3J.sub.HH 5.9,
.sup.3J.sub.HH 8.4 Hz), and 8.45 (s, 1H, H.sub.8); .sup.13C-NMR
(MeOH-d.sub.4, 50.3 MHz): d 19.6 (Me-.sup.iBu), 29.2
(CH.sub.2-Lev), 30.0 (Me-Lev), 37.2 (CH-.sup.iBu), 38.9, 39.3
(C.sub.2'+CH.sub.2-Lev), 63.3 (C.sub.5'), 76.8, 85.8, 87.3
(C.sub.1'+C.sub.3'+C.sub.4'), 121.5 (C.sub.5), 139.7 (C.sub.8),
150.0, 150.5 (C.sub.2+C.sub.4), 157.6 (C.sub.6), 174.2 (C.dbd.O),
182.0 (.sup.iBu-C.dbd.O), and 209.7 (C.dbd.O); MS (ESI.sup.+, m/z):
436 [(M+H).sup.+, 15%] and 458 [(M+Na).sup.+, 50].
[0156] 5'-O-Levulinylthymidine (4a).
[0157] R.sub.f (10% MeOH/CH.sub.2Cl.sub.2): 0.22; Mp:
141-143.degree. C.; IR (KBr): .upsilon. 3393, 3215, 2934, 1737,
1724, 1643, and 1629 cm.sup.-1; .sup.1H-NMR (DMSO-d.sub.6, 200
MHz): d 1.91 (s, 3H, Me), 2.27 (s, 3H, Me-Lev,), 2.30 (m, 2H,
H.sub.2'), 2.66 (m, 2H, CH.sub.2-Lev), 2.89 (t, 2H, CH.sub.2-Lev,
.sup.3J.sub.HH 6.2 Hz), 4.07 (m, 1H, H.sub.4'), 4.35 (m, 3H,
H.sub.3'+2H.sub.5'), 5.55 (d, 1H, OH), 6.32 (t, 1H, H.sub.1',
.sup.3J.sub.HH 7.0 Hz), 7.6 (s, 1H, H.sub.6'), and 11.45 (s,1H,NH);
.sup.13C-NMR (DMSO-d.sub.6, 50.3 MHz): d 12.02 (Me), 27.4
(CH.sub.2-Lev), 29.4 (Me-Lev), 37.2 (C.sub.2'), 38.4
(CH.sub.2-Lev), 63.8 (C.sub.5'), 70.1 (C.sub.3'), 83.5, 83.6
(C.sub.1'+C.sub.4'), 109.7 (C.sub.5), 135.7 (C.sub.6), 150.3
(C.sub.4), 163.6 (C.sub.2), 172.1 (C.dbd.O Lev), and 206.7 (C.dbd.O
Lev); MS (ESI, m/z): 341 [(M+H).sup.+, 40%], 379 [(M+Na).sup.+,
100], and 379 [(M+K).sup.+, 80].
[0158] N-Benzoyl-5'-O-levulinyl-2'-deoxycytidine (4c).
[0159] R.sub.f (10% MeOH/CH.sub.2Cl.sub.2): 0.37; mp: 50-52.degree.
C. IR (KBr): .upsilon. 3410, 2919, 1738, 1701, and 1650 cm.sup.-1;
.sup.1H-NMR (CDCl.sub.3, 300 MHz): d 2.20 (s, 3H, Me-Lev), 2.25 (m,
1H, H.sub.2'), 2.58 (m, 2H, CH.sub.2-Lev), 2.75 (m, 1H, H.sub.2'),
2.82 (m, 2H, CH.sub.2-Lev), 3.35 (s, 1H, OH), 4.25 (m, 1H,
H.sub.3'), 4.40 (m, 3H, 2H.sub.5'+H.sub.4'), 6.30 (apparent t, 1H,
H.sub.1', .sup.3J.sub.HH 6.2 Hz), 7.55 (m, 4H,
H.sub.5+2H.sub.m+H.sub.p), 7.90 (apparent d, 2H, H.sub.o,
.sup.3J.sub.HH 7.1 Hz), 8.20 (d, 1H, H.sub.6, .sup.3J.sub.HH 7.4
Hz), and 8.78 (s, 1H, NH); .sup.13C-NMR (CDCl.sub.3, 50.3 MHz): d
27.7 (CH.sub.2-Lev), 29.6 (Me-Lev), 37.7 (CH.sub.2-Lev), 41.3
(C.sub.2'), 63.7 (C.sub.5'), 70.6, 84.8, 87.4
(C.sub.1'+C.sub.3'+C.sub.4'), 96.8 (C.sub.5), 127.6, 128.7
(C.sub.o+C.sub.m), 132.8 (C.sub.i), 133.0 (C.sub.p), 144.2
(C.sub.6), 155.1 (C.sub.2), 162.4 (C.sub.4), 166.7 (PhC.dbd.O),
172.6 (C.dbd.O), and 206.8 (C.dbd.O); MS (ESI.sup.+, m/z): 430
[(M+H).sup.+, 20%], 452 [(M+Na).sup.+, 65], and 468 [(M+K).sup.+,
40].
[0160] N-Benzoyl-5'-O-levulinyl-2'-deoxyadenosine (4e).
[0161] R.sub.f (10% MeOH/CH.sub.2Cl.sub.2): 0.50; Mp: 69-71.degree.
C.; IR (KBr): .upsilon. 3413, 2959, 2928, 1726, 1637, and 1616
cm.sup.-1; .sup.1H-NMR (MeOH-d.sub.4, 300 MHz): d 2.30 (s, 3H,
Me-Lev), 2.70 (m, 3H, CH.sub.2-Lev+1H.sub.2'), 2.92 (m, 2H,
CH.sub.2-Lev), 3.15 (m, 1H, H.sub.2'), 4.40 (m, 1H, H.sub.4'), 4.52
(m, 2H, 2H.sub.5'), 4.85 (m, 1H, H.sub.3'), 6.75 (apparent t, 1H,
H.sub.1', .sup.3J.sub.HH 6.2 Hz), 7.80 (m, 3H, 2H.sub.m+H.sub.p),
8.30 (m, 2H, 2H.sub.o), 8.78 (s, 1H, H.sub.2 or H.sub.8), and 8.92
(s, 1H, H.sub.8 or H.sub.2), .sup.13C-NMR (MeOH-d.sub.4, 75.5 MHz):
d 29.0 (CH.sub.2-Lev), 29.9 (Me-Lev), 38.9, 40.8
(CH.sub.2-Lev+C.sub.2'), 65.3 (C.sub.5'), 72.6, 86.51, 86.54
(C.sub.1'+C.sub.3'+C.sub.4'), 125.7 (C.sub.5), 129.7, 130.0
(C.sub.o+C.sub.m), 134.2(C.sub.p), 135.2 (C.sub.i), 144.6
(C.sub.8), 151.4 (C.sub.4), 153.3(C.sub.6), 153.5 (C.sub.2), 168.4
(PhC.dbd.O), 174.5 (C.dbd.O), and 209.7 (C.dbd.O); MS (ESI.sup.+,
m/z): 476 [(M+Na).sup.+, 100%] and 492 [(M+K).sup.+, 53].
[0162] N-Isobutyryl-3'-O-levulinyl-2'-deoxyguanosine (4g).
[0163] R.sub.f (20% MeOH/CH.sub.2Cl.sub.2): 0.60; Mp: 45-47.degree.
C.; IR (KBr): .upsilon. 3415, 2930, 1720, and 1685 cm.sup.-1;
.sup.1H-NMR (MeOH-d.sub.4, 200 MHz): d 1.41 (d, 6H, Me-.sup.iBu,
.sup.3J.sub.HH 6.8 Hz), 2.33 (s, 3H, Me-Lev), 2.59-3.07 (m, 7H,
2CH.sub.2-Lev+2H.sub.2'+CH-.- sup.iBu), 4.32 (m, 1H, H.sub.4'),
4.50 (m, 2H, H.sub.5'), 4.75 (m, 1H, H.sub.3'), 6.50 (apparent t,
1H, H.sub.1', .sup.3J.sub.HH 6.4 Hz), and 8.32 (s, 1H, H.sub.8);
.sup.13C-NMR (MeOH-d.sub.4, 75.5 MHz): d 19.7 (Me-.sup.iBu), 29.0
(CH.sub.2-Lev), 29.9 (Me-Lev), 37.2 (CH-.sup.iBu), 38.9, 41.1
(C.sub.2'+CH.sub.2-Lev), 65.3 (C.sub.5'), 72.6, 86.1, 86.5
(C.sub.1'+C.sub.3'+C.sub.4'), 121.8 (C.sub.5), 139.8 (C.sub.8),
150.0, 150.5 (C.sub.2+C.sub.4), 157.8 (C.sub.6), 174.5 (C.dbd.O),
182.0 (.sup.iBu-C.dbd.O), and 209.7 (C.dbd.O); MS (ESI.sup.+, m/z):
436 [(M+H).sup.+, 20%], 458 [(M+Na).sup.+, 100], and 474
[(M+K).sup.+, 50].
[0164] General Procedure for the Enzymatic Hydrolysis of Thymidine
Tetramer Bearing Levulinyl Protecting Groups at Each of the 3',-O
and 5'-O Terminal Positions
[0165] To a solution of diprotected tetramer in 1,4-dioxane is
added 0.15M phosphate buffer pH=7 and the corresponding lipase
[ratio of tetramer:enzyme is 1:1 (w/w) for CAL-A or CAL-B, and 1:3
(w/w) for PSL-C]. The mixture is allowed to react at 250 rpm for 62
h at 40.degree.. The reactions are monitored by TLC (10%
MeOH/CH.sub.2Cl.sub.2). The enzyme is filtered off and washed with
CH.sub.2Cl.sub.2, the solvents are distilled under vacuum, and the
residue is taken up in NaHCO.sub.3 (aq) and extracted with
CH.sub.2Cl.sub.2. The combined organic layers are dried over
Na.sub.2SO.sub.4 and evaporated to give
monoacylpolynucleotides.
* * * * *