US2004116685A1PendingUtilityA1
Method for solution phase synthesis of oligonucleotides
Est. expiryOct 19, 2015(expired)· nominal 20-yr term from priority
C07H 19/04B01J 2219/00423C40B 60/14B01J 2219/00527B01J 2219/00722C07C 43/225B01J 2219/00599B01J 2219/00707B01J 2219/00497B01J 2219/00452C07C 57/13B01J 19/0046C07C 43/23C07C 59/60C40B 40/06C07H 19/10C07H 21/00C07B 2200/11C40B 50/08B01J 2219/00686C07F 7/12C40B 40/08
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Claims
Abstract
This invention discloses an improved method for the sequential solution phase synthesis of oligonucleotides. The method lends itself to automation and is ideally suited for large scale manufacture of oligonucleotides with high efficiency.
Claims
exact text as granted — not AI-modified1 . A method for the solution phase synthesis of oligonucleotides comprising:
a) reacting a 5′-protected monomer unit with a starting material to form a reaction mixture containing a product; and b) partitioning the product from the unreacted starting material, unreacted 5′-protected monomer unit, side-products and reagents based on the presence of the 5′-protecting group.
2 . The method of claim 1 wherein the 5′-protected monomer unit has the following formula:
wherein
B is a nucleobase;
A is a 2′-sugar substituent;
A′ is a 2′-sugar substituent;
W is independently selected from the group consisting of a phosphoramidite, a H-phosphonate, a phosphate triester, a methyl phosphonate, a phosphoramidate and a protected oligonucleotide, wherein said protected oligonucleotide has a 3′-terminal group selected from the group consisting of a phosphoramidite, a H-phosphonate, a phosphate triester, a methyl phosphonate, phosphoramidate; and
D-E is an alcohol protecting group(s).
3 . The method of claim 1 further comprising:
c) repeating steps a) and b) in successive cycles a select number of times to yield the desired product.
4 . The method of claim 1 further comprising between steps a) and b)
a1) oxidizing the reaction mixture obtained from step a) to yield a second reaction mixture containing an oxidized 5′-protected monomer unit, an oxidized product and the starting material.
a2) partitioning the oxidized 5′-protected monomer unit, from the remainder of the reaction mixture; and
a3) optionally, isolating the oxidized 5′-protected monomer unit.
5 . The method of claim 2 wherein W is selected from the group consisting of a phosphoramidite, a H-phosphonate and a protected oligonucleotide, wherein said protected oligonucleotide has a 3′-terminal group selected from the group consisting of a phosphoramidite or a H-phosphonate.
6 . The method of claim 2 wherein A and A′ are independently selected from the group consisting of H, 2 H, 3 H, Cl, F, OH, NHOR 1 , NHOR 3 , NHNHR 3 , NHR 3 , ═NH, CHCN, CHCl 2 , SH, SR 3 , CFH 2 , CF 2 H, CR 2 2 Br, —(OCH 2 CH 2 ) n OCH 3 , OR 4 and imidazole; wherein
R 1 is selected from the group consisting of H and an alcohol protecting group;
R 2 is selected from the group consisting of ═O, ═S, H, OH, CCl 3 , CF 3 , halide, optionally substituted C 1 -C 20 alkyl (including cyclic, straight chain, and branched), alkenyl, aryl, C 1 -C 20 acyl, benzoyl, OR 4 and esters;
R 3 is selected from the group consisting of R 2 , R 4 , CN, C(O)NH 2 , C(S)NH 2 , C(O)CF 3 , SO 2 R 4 , amino acid, peptide and mixtures thereof; and
R 4 is selected from the group consisting of an optionally substituted hydrocarbon (C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl), an optionally substituted heterocycle, t-butyldimethylsilyl ether, triisopropylsilyl ether, nucleoside, carbohydrate, fluorescent label and phosphate.
7 . The method of claim 2 wherein A is selected from the group consisting of H, OH, NH 2 , Cl, F, NHOR, —(OCH 2 CH 2 ) n OCH 3 , OR 4 , OSiR 4 3 , wherein
R 4 is selected from the group consisting of an optionally substituted hydrocarbon (C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl), an optionally substituted heterocycle, t-butyldimethylsilyl ether, triisopropylsilyl ether, nucleoside, carbohydrate, fluorescent label and phosphate: and
A′ is H.
8 . The method of claim 2 wherein D is a compound selected such that it has a strong affinity for a solid support or covalently reacts with a derivatized solid support and E is a compound selected such that the oxygen-E bond is easily cleaved.
9 . The method of claim 8 wherein E is selected from the group consisting of a trityl group, a levulinic acid group, or a silyl ether group.
10 . The method of claim 9 wherein the trityl group has the following structure:
wherein D is independently selected from the group consisting of H, OR 4 , an alkyl or substituted alkyl group bearing a diene unit, an alkyl or substituted alkyl group bearing a dienophile unit, an alkoxy or substituted alkoxy group bearing a diene unit, an alkoxy or substituted alkoxy group bearing a dienophile unit. CH 2 ═CHCH═CHCH 2 CH 2 O—, maleimide substituted alkoxy groups, alkoxy groups, an alkylamino or substituted alkylamino group bearing a diene unit, maleimide substituted alkylamino groups or substituted alkylamino groups, an alkylamino group or substituted alkylamino group bearing a dienophile moiety, disulfides, aldehydes, and metal chelators, silyl ethers bearing dienophile or diene units, wherein
R 4 is selected from the group consisting of an optionally substituted hydrocarbon (C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl), an optionally substituted heterocycle, t-butyldimethylsilyl ether, triisopropylsilyl ether, nucleoside, carbohydrate, fluorescent label and phosphate.
11 . The method of claim 10 wherein D has between 1-50 carbon atoms.
12 . The method of claim 10 wherein D has between 1-30 carbon atoms.
13 . The method of claim 10 wherein D is independently selected from the
Y=O, NH, S, NH(CO), (CO)NH, O(CO), (CO)O, NH(CO)NH, NH(CO)O, O(CO)NH, NH(CS)NH, NH(CS)O, O(CS)NH, omitted, SO 2 ,
L=a linking group
X=electron withdrawing group or electron donating group or H
14 . The method of claim 9 wherein the levulinic acid group has the following structure:
wherein D is independently selected from the group consisting of H, OR 4 , an alkyl or substituted alkyl group bearing a diene unit, an alkyl or substituted alkyl group bearing a dienophile unit, an alkoxy or substituted alkoxy group bearing a diene unit, an alkoxy or substituted alkoxy group bearing a dienophile unit, CH 2 ═CHCH═CHCH 2 CH 2 O—, maleimide substituted alkoxy groups, alkoxy groups, an alkylamino or substituted alkylamino group bearing a diene unit, maleimide substituted alkylamino groups or substituted alkylamino groups, an alkylamino group or substituted alkylamino group bearing a dienophile moiety, disulfides, aldehydes, and metal chelators, silyl ethers bearing dienophile or diene units, wherein
R 4 is selected from the group consisting of an optionally substituted hydrocarbon (C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl), an optionally substituted heterocycle, t-butyldimethylsilyl ether, triisopropylsilyl ether, nucleoside, carbohydrate, fluorescent label and phosphate.
15 . The method of claim 14 wherein D has between 1-50 carbon atoms.
16 . The method of claim 14 wherein D has between 1-30 carbon atoms.
17 . The method of claim 14 wherein D is independently selected from the group consisting of the following compounds:
Y=O, NH, S, NH(CO), (CO)NH, O(CO), (CO)O, NH(CO)NH, NH(CO)O, O(O)NH, NH(CS)NH, NH(CS)O, O(CS)NH, omitted, SO 2 ,
L=a linking group
X=electron withdrawing group or electron donating group or H
18 . The method of claim 9 wherein the silyl group is selected from a compound having one of the following structures:
wherein D is independently selected from the group consisting of H, OR 4 , an alkyl or substituted alkyl group bearing a diene unit, an alkyl or substituted alkyl group bearing a dienophile unit, an alkoxy or substituted alkoxy group bearing a diene unit, an alkoxy or substituted alkoxy group bearing a dienophile unit, CH 2 ═CHCH═CHCH 2 CH 2 O—, maleimide substituted alkoxy groups, alkoxy groups, an alkylamino or substituted alkylamino group bearing a diene unit, maleimide substituted alkylamino groups or substituted alkylamino groups, an alkylamino group or substituted alkylamino group bearing a dienophile moiety, disulfides, aldehydes, and metal chelators, silyl ethers bearing dienophile or diene units, wherein
R 4 is selected from the group consisting of an optionally substituted hydrocarbon (C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl), an optionally substituted heterocycle, t-butyldimethylsilyl ether, triisopropylsilyl ether, nucleoside, carbohydrate, fluorescent label and phosphate.
19 . The method of claim 18 wherein D has between 1-50 carbon atoms.
20 . The method of claim 18 wherein D has between 1-30 carbon atoms.
21 . The method of claim 18 wherein D is independently selected from the group consisting of the following compounds:
Y=O, NH, S, NH(CO), (CO)NH, O(CO), (CO)O, NH(CO)NH, NH(CO)O, O(CO)NH, NH(CS)NH, NH(CS)O, O(CS)NH, omitted, SO 2 ,
L=a linking group
X=electron withdrawing group or electron donating group or H
22 . The method of claim 1 wherein the partitioning is performed by eluting the reaction mixture through a solid support.
23 . The method of claim 22 wherein said solid support has an affinity for D.
24 . The method of claim 22 wherein said solid support covalently reacts with D.
25 . The method of claim 24 wherein said covalent reaction is a Diels-Alder reaction.
26 . The method of claim 22 wherein said solid support is selected from the group consisting of a resin, membrane and polymer.
27 . The method of claim 22 wherein said solid support is selected from the group consisting of a hydrophobic reversed phase resin, a thiopropyl sepharose resin, a mercurated resin, an agarose adipic acid hydrazide resin, an avidin resin, an ultrafiltration membrane, Tentagel™, polyethylene glycol and an inorganic oxide, selected from the group consisting of silica gel, alumina, controlled pore glass and zeolite.
28 . The method of claim 27 wherein the hydrophobic reversed phase resin is selected from the group consisting of a C2 to a C18 polystyrene resin.
29 . The method of claim 22 wherein said starting material is a 3′-polyethylene glycol (PEG) derivatized oligonucleotide and the 5′-protected monomer unit is partitioned from the remainder of the reaction mixture using an ultrafiltration membrane.
30 . The method of claim 22 wherein said solid support is derivatized with a group selected from a diene or a dienophile.
31 . The method of claim 30 wherein said diene is selected from the group consisting of 3,5-hexadiene.
32 . The method of claim 30 wherein said dienophile is maleimide.
33 . The method of claim 4 wherein the oxidation is performed in situ.
34 . The method of claim 33 wherein the oxidation is performed with I 2 , pyridine and H 2 O.
35 . The method of claim 33 wherein the oxidation is performed with iodobenzene diacetate.
36 . The method of claim 33 wherein the oxidation is performed with sodium periodate or tetraalkylammonium periodate.
37 . A product formed by the method of claim 1 .
38 . A method for the solution phase synthesis of oligonucleotides comprising:
a) reacting a 5′-protected monomer unit with a starling material to form a reaction mixture containing a product, the 5′-protected monomer unit and the starting material; b) oxidizing the reaction mixture of step a). c) adding the oxidized reaction mixture of step b) to an extraction vessel. d) extracting said reaction mixture with an organic solvent; e) eluting said organic solvent containing the extract from step d) through a chromatography resin chamber containing a solid support, wherein said oxidized 5′-protected monomer unit is retained on the solid support and the oxidized product and the starting material remains in the eluted solvent; f) separating the oxidized product from the starting material by eluting the organic effluent obtained in step e) through a second solid support, wherein the oxidized product is retained on the solid support and the starting material is eluted with the solvent; and g) eluting said oxidized product from said second solid support by washing said second solid support with a dilute acid.
39 . The method of claim 38 further comprising after step g)
g1) neutralizing said effluent obtained in step g) with an organic base; and
g2) concentrating said neutralized effluent and exchanging said oxidized product into acetonitrile by ultrafiltration.
40 . The method of claim 38 further comprising after step g)
g1) eluting said effluent obtained in step g) through a third solid support to neutralize said effluent.
41 . The method of claim 40 wherein said third solid support is Dowex.
42 . The method of claim 38 further comprising between steps e) and f)
e1) washing said solid support to release and isolate the oxidized 5′-protected monomer.
43 . A compound of the following formula:
wherein
R′ is selected from a diene or a dienophile; and
X is selected from the group consisting of a halogen, hydroxyl, OR″ and OAr, wherein R″ is an alkyl or substituted alkyl group and Ar is an aromatic or heteroaromatic group.
44 . The compound of claim 43 wherein R′ is 3,5-hexadiene.
45 . The compound of claim 43 wherein R′ is 2,4-hexadiene.
46 . A compound formed by the Diels-Alder reaction of a compound of claim 43 with a compound selected from the group consisting of a dienophile attached to a solid support, a diene attached to a solid support, a diene and a dienophile.
47 . The compound of claim 46 wherein R′ is selected from 3,5-hexadiene or 2,4-hexadiene.
48 . The compound of claim 46 wherein said dienophile is maleimide.
49 . The compound of claim 46 wherein X is absent, resulting in a positively charged compound.
50 . A compound of the following formula:
wherein
B is a nucleobase;
A is a 2′-sugar substituent;
A′ is a 2′-sugar substituent;
W is independently selected from the group consisting of a phosphoramidite, a H-phosphonate, a phosphate triester, a methyl phosphonate, a phosphoramidate and a protected oligonucleotide, wherein said protected oligonucleotide has a 3′-terminal group selected from the group consisting of a phosphoramidite, a H-phosphonate, a phosphate triester, a methyl phosphonate, a phosphoramidate and a deprotected oligonucleotide; and
R′ is selected from a diene or a dienophile.
51 . The compound of claim 50 wherein W is selected from the group consisting of a phosphoramidite, a H-phosphonate, and a protected oligonucleotide, wherein said protected oligonucleotide has a 3′-terminal group selected from a phosphoramidite or a H-phosphonate.
52 . The compound of claim 50 wherein A and A′ are independently selected from the group consisting of H, 2 H, 3 H, Cl, F, OH, NHOR 1 , NHOR 3 , NHNHR 3 , NHR 3 , ═NH, CHCN, CHCl 2 , SH, SR 3 , CFH 2 , CF 2 H, CR 2 2 Br, —(OCH 2 CH 2 ) n OCH 3 , OR 4 and imidazole; wherein
R′ is selected from the group consisting of H and an alcohol protecting group;
R 2 is selected from the group consisting of ═O, ═S, H, OH, CCl 3 , CF 3 , halide, optionally substituted C 1 -C 20 alkyl (including cyclic, straight chain, and branched), alkenyl, aryl, C 1 -C 20 acyl, benzoyl, OR 4 and esters;
R 3 is selected from the group consisting of R 2 , R 4 , CN, C(O)NH 2 , C(S)NH 2 , C(O)CF 3 , SO 2 R 4 , amino acid, peptide and mixtures thereof;
R 4 is selected from the group consisting of an optionally substituted hydrocarbon (C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 1 -C 20 alkynyl, an optionally substituted heterocycle, t-butyldimethylsilyl ether, triisopropylsilyl ether, nucleoside, carbohydrate, fluorescent label and phosphate.
53 . The compound of claim 50 wherein
A is selected from the group consisting of H, OH, NH 2 , Cl, F, —(OCH 2 CH 2 ) n OCH 3 , NHOR, OR 4 , OSiR 4 3 , wherein R 4 is selected from the group consisting of an optionally substituted hydrocarbon (C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl), an optionally substituted heterocycle, t-butyldimethylsilyl ether, triisopropylsilyl ether, nucleoside, carbohydrate, fluorescent label and phosphate; and A′ is H.
54 . The compound of claim 50 wherein R′ is 3,5-hexadiene.
55 . The compound of claim 50 wherein R′ is 2,4-hexadiene.
56 . A compound formed by the Diels-Alder reaction of a compound of claim 50 with a compound selected from the group consisting of a dienophile attached to a solid support, a diene attached to a solid support, a diene and a dienophile.
57 . The compound of claim 56 wherein R′ is selected from 3,5-hexadiene or 2,4-hexadiene.
58 . The compound of claim 56 wherein said dienophile is maleimide.
59 . A compound of the formula:
wherein
B is a nucleobase;
A is a 2′-sugar substituent;
A′ is a 2′-sugar substituent;
W is independently selected from the group consisting of a phosphoramidite, a H-phosphonate, a phosphate triester, a methyl phosphonate, a phosphoramidate and a protected oligonucleotide, wherein said protected oligonucleotide has a 3′-terminal group selected from the group consisting of a phosphoramidite, a H-phosphonate, a phosphate triester, a methyl phosphonate, a phosphoramidate and a deprotected oligonucleotide; and
Solid support is selected from the group consisting of a crosslinked organic polymer, polystyrene, Tentagel™, polyethylene glycol, an inorganic oxide selected from the group consisting of silica gel, alumina, controlled pore glass and zeolites.
60 . The compound of claim 59 wherein
A and A′ are independently selected from the group consisting of H, 2 H, 3 H, Cl, F, OH, NHOR 1 , NHOR 3 , NHNHR 3 , NHR 3 , ═NH, CHCN, CHCl 2 , SH, SR 3 , CFH 2 , CF 2 H, CR 2 2 Br, —(OCH 2 CH 2 ) n OCH 3 , OR 4 and imidazole; wherein
R 1 is selected from the group consisting of H and an alcohol protecting group;
R 2 is selected from the group consisting of ═O, ═S, H, OH, CCl 3 , CF 3 , halide, optionally substituted C 1 -C 20 alkyl (including cyclic, straight chain, and branched), alkenyl, aryl, C 1 -C 20 acyl, benzoyl, OR 4 and esters;
R 3 is selected from the group consisting of R 2 , R 4 , CN, C(O)NH 2 , C(S)NH 2 , C(O)CF 3 , SO 2 R 4 , amino acid, peptide and mixtures thereof; and
R 4 is selected from the group consisting of an optionally substituted hydrocarbon (C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl), an optionally substituted heterocycle, t-butyldimethylsilyl ether, triisopropylsilyl ether, nucleoside, carbohydrate, fluorescent label and phosphate.
61 . The compound of claim 59 wherein A is selected from the group consisting of H, OH, NH 2 , Cl, F, NHOR, —(OCH 2 CH 2 ) n OCH 3 , OR 4 , OSiR 4 3 , wherein R 4 is selected from the group consisting of an optionally substituted hydrocarbon (C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl), an optionally substituted heterocycle, t-butyldimethylsilyl ether, triisopropylsilyl ether, nucleoside, carbohydrate, fluorescent label and phosphate; and A′ is H.
62 . The compound of claim 59 wherein W is protected oligonucleotide wherein said protected oligonucleotide has a 3′-terminal group selected from the group consisting of polyethylene glycol having a molecular weight between 5,000 and 100,000 and hydroxyl.
63 . A compound of the following formula:
wherein R is selected from the group consisting of a diene.
64 . The compound of claim 63 wherein R is selected from 2,4 pentadiene.
65 . The use of the compounds of claim 63 as capping reagents in oligonucleotide synthesis.
66 . The use of the compounds of claim 63 as a means for removing failure sequences in oligonucleotide synthesis.
67 . A compound of the following formula:
wherein R is selected from the group consisting of a diene.
68 . The compound of claim 67 wherein R is selected from 3,5-hexadiene.
69 . The use of the compounds of claim 67 as capping reagents in oligonucleotide synthesis.
70 . The use of the compounds of claim 67 as a means for removing failure sequences in oligonucleotide synthesis.
71 . A compound of the formula:
wherein R is independently selected from the group consisting of a diene, alkyl group or aryl group, wherein at least two R's are a diene.
72 . The compound of claim 71 wherein R is independently selected from the group consisting of 3,5-hexadiene, phenyl and tert-butyl.
73 . The use of the compounds of claim 71 as capping reagents in oligonucleotide synthesis.
74 . The use of the compounds of claim 71 as a means for removing failure sequences in oligonucleotide synthesis.
75 . A method for the solution phase synthesis of oligonucleotides, comprising:
a) reacting a 5′-protected monomer unit containing a protecting group at the 5′ position which is capable of reacting with a first solid support, with a starting material to form a reaction mixture containing a product, the 5′-protected monomer unit and the starting material; b) circulating said reaction mixture through a chromatography resin chamber containing said first solid support, wherein said 5′-protected monomer unit and said product covalently react with said first solid support and are thereby retained on the solid support; c) washing said first solid support with a first solvent to elute the starting material; d) washing said first solid support containing the retained 5′-protected monomer unit and product with a dilute acid followed by eluting a second organic solvent to release and isolate the product together with the 5′-protected monomer unit; and e) separating the product from the 5′-protected monomer unit by passing the organic effluent obtained in step d) through a second solid support, wherein the 5′-protected monomer unit is retained by the second solid support and the product is eluted with the second solvent.
76 . The method of claim 75 further comprising between steps a) and b):
a1) oxidizing the reaction mixture containing the product, the 5′-protected monomer unit and the starting material to yield a second reaction mixture containing an oxidized 5′-protected monomer unit, an oxidized product and the starting material.
77 . The method of claim 75 wherein said first and second solid supports are independently selected from the group consisting of a resin, polymer and membrane.
78 . The method of claim 75 wherein said first solid support is derivatized with a group selected from a diene or a dienophile.
79 . The method of claim 75 wherein said first solid support is derivatized with maleimide.
80 . The method of claim 75 wherein said second solid support is an ultrafiltration membrane.
81 . The method of claim 75 wherein said solution phase synthesis is automated.
82 . The method of claim 24 wherein said covalent reaction is a reductive amination.Cited by (0)
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