US2004096947A1PendingUtilityA1
Building blocks for the solution phase synthesis of oligonucleotides
Priority: Mar 30, 2001Filed: Nov 3, 2003Published: May 20, 2004
Est. expiryMar 30, 2021(expired)· nominal 20-yr term from priority
C07H 19/04C07H 21/04C12P 19/30C12P 19/34
42
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Claims
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.
Claims
exact text as granted — not AI-modifiedWhat 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:
wherein:
R 1 is —H, -hydroxyl, a protected hydroxyl, or a 2′-substituent; and
R 2 and R 3 are, independently, —H or an amino protecting group;
G is N or CH; and
Lev is —C(O)—(CH 2 ) 2 —C(O)—CH 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:
wherein:
R 6 is —H, or —OH;
R 2 , R 3 , R 4 , and R 5 are each, independently, —H or an amino protecting group;
G is N or CH; and
Lev is —C(O)—(CH 2 ) 2 —C(O)—CH 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:
wherein:
R 1 is —H, -hydroxyl, a protected hydroxyl, or a 2′-substituent; and
R 2 and R 3 are, independently, —H or an amino protecting group;
G is N or CH; and
Lev is —C(O)—(CH 2 ) 2 —C(O)—CH 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:
wherein:
R 6 is —H or -hydroxyl;
R 2 , R 3 , R 4 , and R 5 are each, independently, —H or an amino protecting group;
G is N or CH; and
Lev is —C(O)—(CH 2 ) 2 —C(O)—CH 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
formulas:
wherein:
R6 is —H or —OH;
R 2 , R 3 , R 4 , and R 5 are each, independently, —H or an amino protecting group;
G is N or CH; and
Lev is —C(O)—(CH 2 ) 2 —C(O)—CH 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:
wherein:
B X is a nucleobase;
T 1 and T 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 1 , T 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:
wherein:
B 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:
wherein 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:
wherein:
B 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:
wherein 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 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 2 Cl 2 , CHCl 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 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 2 Cl 2 , CHCl 3 , hexane, or pyridine.
64 . The method of claim 60 wherein said polymeric support is a polystyrene polymeric support.Join the waitlist — get patent alerts
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