Process for the synthesis of oligomeric compounds

Details Process for the synthesis of oligomeric compounds Process for the synthesis of oligomeric compounds

1999-04-09

2002-10-15

Richter, Johann (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S024300, C536S024500, C536S025310, C536S025330, C536S025340

Reexamination Certificate

active

06465628

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to improved methods for the preparation of oligomeric compounds having phosphodiester, phosphorothioate, phosphorodithioate or other linkages. In preferred embodiments, the methods of the invention provide oligomers that have reduced amounts of unwanted side-products.
BACKGROUND OF THE INVENTION
Antisense and other oligonucleotide therapies have gone beyond academic publications to the level of approved drug as shown by the recent FDA approval of an antisense oligonucleotide therapeutic for ocular cytomegalovirus infections. More and more oligonucleotides are entering the clinic for the treatment of a variety of diseases such as inflammation, cancer, viral disease and others. There is an urgent need for improved methods for the synthesis of oligonucleotides in high quantity and with high quality. Solid phase chemistry is the present method of choice. Typical synthons now used are O-cyanoethyl protected nucleoside phosphoamidite monomers. At the end of the synthesis, the oligonucleotide product is treated typically with 30% aqueous ammonium hydroxide to deprotect the cyanoethyl groups on the phosphorothioate backbone as well as exocyclic amino groups. During this deprotection step, one molecule of acrylonitrile is produced for every cyanoethyl group present.
It is now known that acrylonitrile is a rodent carcinogen and that, at pH 7, it can react with T, dC, dG, dA and dI, resulting in the formation of a variety of adducts. See, Solomon et al.,
Chem
.-
Biol. Interactions
, 51, 167-190 (1984). It is greatly desired to eliminate these impurities in synthesis of oligonucleotides, especially phosphorothioate oligonucleotides.
Eritja et al. (
Tetrahedron
, 48, 4171-4182 (1992)) report the prevention of acrylonitrile adduct formation of nucleobase moieties during deprotection of &bgr;-cyanoethyl protected oligomers by 40% triethylamine in pyridine for 3 hours followed by treatment with 0.5 M DBU/pyridine. However, as will be seen infra, their conditions failed to eliminate adduct formation to a suitable extent.
Given the demand for oligonucleotides and analogs thereof for clinical use, and the known toxicity of acrylonitrile nucleobase adducts, methods of preparing phosphate linked oligomers having reduced amount of such adducts are greatly desired. The present invention is directed to this, as well a other, important ends.
SUMMARY OF THE INVENTION
The present invention provides an improved method for the preparation of phosphate-linked oligomers that have significantly reduced amounts of exocyclic nucleobase adduct resulting from the products of removal of phosphorus protecting groups. In one aspect of the invention, methods are provided comprising:
a) providing a sample containing a plurality of oligomers of the Formula I:
 wherein:
R
1
is H or a hydroxyl protecting group;
B is a naturally occurring or non-naturally occurring nucleobase that is optionally protected at one or more exocyclic hydroxyl or amino groups;
R
2
has the Formula III or IV:
 wherein
E is C
1
-C
10
alkyl, N(Q
1
)(Q
2
) or N═C(Q
1
)(Q
2
);
each Q
1
and Q
2
is, independently, H, C
1
-C
10
alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support, or Q
1
and Q
2
, together, are joined in a nitrogen protecting group or a ring structure that can include at least one additional heteroatom selected from N and O;
R
3
is OX
1
, SX
1
, or N(X
1
)
2
;
each X
1
is, independently, H, C
1
-C
8
alkyl, C
1
-C
8
haloalkyl, C(═NH)N(H)Z
8
, C(═O)N(H)Z
8
or OC(═O)N(H)Z
8
;
Z
8
is H or C
1
-C
8
alkyl;
L
1
, L
2
and L
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;
Y 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(Q
1
)(Q
2
), O(Q
1
), halo, S(Q
1
), or CN;
each q
1
is, independently, from 2 to 10;
each q
2
is, independently, 0 or 1;
m is 0, 1 or 2;
pp is from 1 to 10; and
q
3
is from 1 to 10 with the proviso that when pp is 0, q
3
is greater than 1;
R
t
is a phosphorus protecting group of formula:
—C(R
10
)
2
—C(R
10
)
2
—W or C(R
10
)
2
—(CH═CH)
p
—C(R
10
)
2
—W
each R
10
is independently H or lower alkyl;
W is an electron withdrawing group;
p is 0 to 3;
each Y
2
is independently, O, CH
2
or NH;
each Z is independently O or S;
each X is independently O or S;
Q is a linker connected to a solid support, —OH or
O—Pr where Pr is a hydroxyl protecting group; and n is 1 to about 100;
b) contacting said sample with a deprotecting reagent for a time and under conditions sufficient to remove substantially said R
t
groups from said oligomers; and
c) reacting said oligomers with a cleaving reagent;
wherein said deprotecting reagent comprises at least one amine, the conjugate acid of said amine having a pKa of from about 8 to about 11; said deprotecting reagent optionally further comprising one or more solvents selected from the group consisting of alkyl solvents, haloalkyl solvents, cyanoalkyl solvents, aryl solvents and aralkyl solvents.
Preferably, the methods further comprises a washing step before step (c).
In some preferred embodiments, Q is a linker connected to a solid support. In further preferred embodiments, said deprotecting reagent does not cleave said oligomers from said solid support.
In some preferred embodiments, the deprotecting reagent comprises an aliphatic amine, which is preferably triethylamine or piperidine.
In further preferred embodiments, the deprotecting agent comprises a haloalkyl solvent or a cyanoalkyl solvent, which is preferably acetonitrile or methylene chloride.
In particularly preferred embodiments, the phosphorus protecting group is —CH
2
—CH
2
—C≡N or —CH
2
—(CH═CH)
p
—CH
2
—C≡N, where p is an integer from 1 to 3, with —CH
2
—CH
2
—C≡N or —CH
2
—CH═CH—CH
2
—C≡N being preferred, and —CH
2
—CH
2
—C≡N being particularly preferred.
In some preferred embodiments, the deprotecting reagent or cleaving reagent further comprises a scavenger, which is preferably a purine, a pyrimidine, inosine, a pyrrole, an imidazole, a triazole, a mercaptan, a beta amino thiol, a phosphine, a phosphite, a diene, a urea, a thiourea, an amide, an imide, a cyclic imide, a ketone, an alkylmercaptan, a thiol, ethylene glycol, a substituted ethylene glycol, 1-butanethiol, S-(2-amino-4-thiazolylmethyl)isothiourea hydrochloride, 2-mercaptoethanol, 3,4-dichlorobenzylamine, benzylamine, benzylamine in the presence of carbon disulfide, hydroxylamine, 2-phenylindole, n-butylamine, diethyl ester of acetaminomalonic acid, ethyl ester of N-acetyl-2-cyanoglycine, 3-phenyl-4-(o-fluorophenyl)-2-butanone, 3,4-diphenyl-2-butanone, desoxybenzoin, N-methoxyphthalimide, p-sulfobenzenediazonium chloride, or p-sulfamidobenzenediazonium chloride.
In some preferred embodiments, the scavenger is a resin containing a suitable scavenging molecule bound thereto. Exemplary scavenger resins include polymers having free thiol groups and polymers having free amino groups, for example a polymer-bound amine resin wherein the amine is selected from benzylamine, ethylenediamine, diethylamine triamine, tris(2-aminoethyl)amine, methylamine, methylguanidine, polylysine, oligolysine, Agropore™ NH
2
HL, Agropore™ NH
2
LL (available from Aldrich Chem. Co. St. Louis. Mo.), 4-methoxytrityl resin, and thiol 2-chlorotrityl resin.
In some preferred embodiments, Q is —OH or O—Pr.
In some preferred embodiments, the cleaving reagent comprises an aqueous methanolic solution of a Group I or Group II metal carbonate, preferably aqueous methanolic CaCO
3
. In further preferred embodiments, the cleaving reagent comprises an aqueous metal hydroxide. In yet further

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