U.S. patent application number 11/696874 was filed with the patent office on 2007-11-15 for process for the synthesis of oligomeric compounds.
Invention is credited to Daniel C. Capaldi, Douglas L. Cole, Andrei Guzaev, Achim Krotz, Muthiah Manoharan, Vasulinga T. Ravikumar.
Application Number | 20070265435 11/696874 |
Document ID | / |
Family ID | 26816507 |
Filed Date | 2007-11-15 |
United States Patent
Application |
20070265435 |
Kind Code |
A1 |
Ravikumar; Vasulinga T. ; et
al. |
November 15, 2007 |
PROCESS FOR THE SYNTHESIS OF OLIGOMERIC COMPOUNDS
Abstract
Synthetic processes are provided wherein oligomeric compounds
are prepared having phosphodiester, phosphorothioate,
phosphorodithioate, or other covalent linkages. The oligomers have
substantially reduced exocyclic adducts deriving from acrylonitrile
or related contaminants.
Inventors: |
Ravikumar; Vasulinga T.;
(Carlsbad, CA) ; Manoharan; Muthiah; (Weston,
MA) ; Capaldi; Daniel C.; (Carlsbad, CA) ;
Krotz; Achim; (San Diego, CA) ; Cole; Douglas L.;
(Half Moon Bay, CA) ; Guzaev; Andrei; (Vista,
CA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
CIRA CENTRE, 12TH FLOOR
2929 ARCH STREET
PHILADELPHIA
PA
19104-2891
US
|
Family ID: |
26816507 |
Appl. No.: |
11/696874 |
Filed: |
April 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11024958 |
Dec 28, 2004 |
7227016 |
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11696874 |
Apr 5, 2007 |
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|
10940360 |
Sep 14, 2004 |
7199236 |
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11024958 |
Dec 28, 2004 |
|
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|
10760940 |
Jan 20, 2004 |
7041816 |
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10940360 |
Sep 14, 2004 |
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|
10232881 |
Aug 30, 2002 |
6858715 |
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10760940 |
Jan 20, 2004 |
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09288679 |
Apr 9, 1999 |
6465628 |
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10232881 |
Aug 30, 2002 |
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60118564 |
Feb 4, 1999 |
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Current U.S.
Class: |
536/23.1 ;
536/25.31 |
Current CPC
Class: |
C07H 21/00 20130101;
Y02P 20/55 20151101 |
Class at
Publication: |
536/023.1 ;
536/025.31 |
International
Class: |
C07H 21/00 20060101
C07H021/00 |
Claims
1-112. (canceled)
113. A method of preparing a phosphorus-linked oilgonucleotide
comprising: providing a sample containing a plurality of
oligonucleotides, said oligonucleotides linked to a solid support
medium, said oligonucleotides comprising a plurality of nucleosides
and a plurality of protected phosphorus linkages; contacting said
oligonucleotides with a deprotecting agent to remove phosphorus
protecting groups from said oligonucleotides, wherein said
oligonucleotides remain linked to said solid support medium during
said contacting; said deprotecting reagent comprising at least one
secondary amine, the conjugate acid of said tertiary amine having a
pKa of from about 8 to about 11; optionally washing said
oligonucleotides; and reacting said oligonucleotides with a
cleaving reagent, whereby the oligonucleotides are cleaved from the
solid support medium.
114. The method of claim 113, wherein the tertiary amine is an
aliphatic amine.
115. The method of claim 114, wherein the aliphatic amine is
(R).sub.3N, where R is alkyl.
116. The method of claim 115, wherein R is ethyl.
117. The method of claim 113, wherein at least one of the
phosphorus protecting groups is a cyanoethyl protecting group.
118. The method of claim 113, wherein at least one of said
nucleotides further comprising a 2'-substituent group selected from
fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected
O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, or polyethers
of the formula (O-alkyl).sub.m, where m is 1 to about 10.
119. The method of claim 118, wherein the 2'substituent group is
O-alkyl or fluoro.
120. The method of claim 113, wherein at least one of the protected
phosphorus linkage is a protected phosphorothioate linkage.
121. An oligonucleotide produced by the method of claim 120.
122. The oligonucleotide of claim 121, comprising less than 1%
acrylonitrile adduct.
123. The oligonucleotide of claim 121, comprising less than 0.5%
acrylonitrile adduct.
124. The oligonucleotide of claim 121, comprising less than 0.1%
acrylonitrile adduct.
125. A method for deprotecting a phosphorus-linked oligonucleotide
comprising a plurality of nucleosides and a plurality of protected
phosphorus linkages of Formula II: ##STR11## wherein: each X is O
or S; Rt is a phosphorus protecting group of formula:
--C(R.sub.10).sub.2--C(R.sub.10).sub.2--W or
--C(R.sub.10).sub.2--(CH.dbd.CH).sub.p--C(R.sub.10).sub.2--W each
R.sub.10 is independently H or lower alkyl; W is electron
withdrawing group; p is 1 to 3; said oligonucleotides being linked
to a solid support medium, the method comprising: providing a
plurality of said phosphorus-linked oligonucleotides; contacting
said oligonucleotides with a deprotecting reagent for a time and
under conditions sufficient to remove said R.sub.t groups from said
oligonucleotides, wherein said oligonucleotides remain linked to
said solid support medium during said contacting; said deprotecting
reagent comprising at least one secondary amine, the conjugate acid
of said tertiary amine having a pKa of from about 8 to about 11;
optionally washing said oligonucleotides; and reacting said
oligonucleotides with a cleaving reagent, whereby the
oligonucleotides are cleaved from the solid support medium.
126. The method of claim 125, wherein R.sub.t, is
--CH.sub.2CH.sub.2--CN.
127. The method of claim 125, wherein the tertiary amine is an
aliphatic amine.
128. The method of claim 127, wherein the aliphatic amine is
(R).sub.3N, where R is alkyl.
129. The method of claim 125, wherein at least one of the
phosphorus protecting groups is a cyanoethyl protecting group.
130. The method of claim 125, wherein at least one of the
nucleotides further comprising a 2'-substituent group selected from
fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected
O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, or polyethers
of the formula (O-alkyl).sub.m, where m is 1 to about 10.
131. The method of claim 130, wherein the 2'-substituent group is
O-alkyl or fluoro.
132. The method of claim 125, wherein at least one of the protected
phosphorus linkage is a protected phosphorothioate linkage.
133. An oligonucleotide produced by the method of claim 132.
134. An oligonucleotide of claim 133, comprising less than 1%
acrylonitrile adduct.
135. An oligonucleotide of claim 133, comprising less than 0.5%
acrylonitrile adduct.
136. An oligonucleotide of claim 133, comprising less than 0.1%
acrylonitrile adduct.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
11/024,958 filed Dec. 28, 2004 published as U.S. Patent Application
US 2005-0137390 A1, which is a continuation of U.S. Ser. No.
10/940,360 filed Sep. 14, 2004, now U.S. Pat. No. 7,199,236, which
is a continuation of U.S. Ser. No. 10/760,940 filed Jan. 20, 2004,
now U.S. Pat. No. 7,041,816, which is a continuation of U.S. Ser.
No. 10/232,881 filed Aug. 30, 2002, now U.S. Pat. No. 6,858,715,
which is a continuation of U.S. Ser. No. 09/288,679 filed Apr. 9,
1999, now U.S. Pat. No. 6,465,628, which claims benefit of U.S.
provisional Ser. No. 60/118,564, filed Feb. 4, 1999 entitled
"Improved Process for the Synthesis of Oligomeric Compounds". The
entire contents of each of the foregoing patents and patent
applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] 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
[0003] 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.
[0004] 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.
[0005] Eritja et al. (Tetrahedron, 48, 4171-4182 (1992)) report the
prevention of acrylonitrile adduct formation of nucleobase moieties
during deprotection of .beta.-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.
[0006] 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
[0007] 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:
[0008] a) providing a sample containing a plurality of oligomers of
the Formula I: ##STR1##
[0009] wherein: [0010] R.sub.1 is H or a hydroxyl protecting group;
[0011] B is a naturally occurring or non-naturally occurring
nucleobase that is optionally protected at one or more exocyclic
hydroxyl or amino groups;
[0012] R.sub.2 has the Formula III or IV: ##STR2##
[0013] wherein [0014] E is C.sub.1-C.sub.10 alkyl,
N(Q.sub.1)(Q.sub.2) or N.dbd.C(Q.sub.1)(Q.sub.2); [0015] each
Q.sub.1 and Q.sub.2 is, independently, H, C.sub.1-C.sub.10 alkyl,
dialkylaminoalkyl, a nitrogen protecting group, a tethered or
untethered conjugate group, a linker to a solid support, or Q.sub.1
and Q.sub.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;
[0016] R.sub.3 is OX.sub.1, SX.sub.1, or N(X.sub.1).sub.2; [0017]
each X.sub.1 is, independently, H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 haloalkyl, C(.dbd.NH)N(H)Z.sub.8,
C(.dbd.O)N(H)Z.sub.8 or OC(.dbd.O)N(H)Z.sub.8;
[0018] Z.sub.8 is H or C.sub.1-C.sub.8 alkyl; [0019] L.sub.1,
L.sub.2 and L.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; [0020] 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.sub.1)(Q.sub.2), O(Q.sub.1), halo,
S(Q.sub.1), or CN; [0021] each q.sub.1 is, independently, from 2 to
10; [0022] each q.sub.2 is, independently, 0 or 1; [0023] m is 0, 1
or 2; [0024] pp is from 1 to 10; and [0025] q.sub.3 is from 1 to 10
with the proviso that when pp is 0, q.sub.3 is greater than 1;
[0026] R.sub.t is a phosphorus protecting group of formula:
--C(R.sub.10).sub.2--C(R.sub.10).sub.2--W or
--C(R.sub.10).sub.2--(CH.dbd.CH).sub.p--C(R.sub.10).sub.2--W
[0027] each R.sub.10 is independently H or lower alkyl;
[0028] W is an electron withdrawing group;
[0029] p is 0to 3;
[0030] each Y.sub.2 is independently, O, CH.sub.2 or NH;
[0031] each Z is independently O or S;
[0032] each X is independently O or S;
[0033] Q is a linker connected to a solid support, --OH or O--Pr
where Pr is a hydroxyl protecting group; and
[0034] n is 1 to about 100;
[0035] b) contacting said sample with a deprotecting reagent for a
time and under conditions sufficient to remove substantially said
R.sub.t groups from said oligomers; and
[0036] c) reacting said oligomers with a cleaving reagent;
[0037] 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.
[0038] Preferably, the methods further comprises a washing step
before step (c).
[0039] 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.
[0040] In some preferred embodiments, the deprotecting reagent
comprises an aliphatic amine, which is preferably triethylamine or
piperidine.
[0041] In further preferred embodiments, the deprotecting agent
comprises a haloalkyl solvent or a cyanoalkyl solvent, which is
preferably acetonitrile or methylene chloride.
[0042] In particularly preferred embodiments, the phosphorus
protecting group is --CH.sub.2--CH.sub.2--C.ident.N or
--CH.sub.2--(CH.dbd.CH).sub.p--CH.sub.2--C.ident.N, where p is an
integer from 1 to 3, with --CH.sub.2--CH.sub.2--C.ident.N or
--CH.sub.2--CH.dbd.CH--CH.sub.2--C.ident.N being preferred, and
--CH.sub.2--CH.sub.2--C.ident.N being particularly preferred.
[0043] 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.
[0044] 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.TM.
NH.sub.2HL, Agropore.TM. NH.sub.2LL (available from Aldrich Chem.
Co. St. Louis. Mo.), 4-methoxytrityl resin, and thiol
2-chlorotrityl resin.
[0045] In some preferred embodiments, Q is --OH or O--Pr.
[0046] 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.sub.3. In
further preferred embodiments, the cleaving reagent comprises an
aqueous metal hydroxide. In yet further preferred embodiments, the
cleaving reagent comprises a phase transfer catalyst. Preferred
phase transfer catalysts include quaternary ammonium salts,
quaternary phosphonium salts, crown ethers and cryptands (i.e.,
crown ethers which are bicyclic or cycles of higher order). It is
more preferred that the phase transfer catalyst be
t-Bu.sub.4N.sup.+OH, or t-Bu.sub.4N.sup.+F.sup.-.
[0047] In further preferred embodiments, the cleaving reagent
comprises NaNH.sub.2.
[0048] In preferred embodiments, the oligomers produced by the
methods of the invention have from 0.001% to about 1% acrylonitrile
adduct, with from about 0.1% to about 1% acrylonitrile adduct being
more preferred, from about 0.1% to about 0.75% acrylonitrile adduct
being even more preferred, and from about 0.1% to about 0.5%
acrylonitrile adduct being even more preferred. In even more
preferred embodiments, the oligomers are substantially free of
detectable acrylonitrile adduct.
[0049] In some preferred embodiments, steps b) and c) are performed
simultaneously.
[0050] In some particularly preferred embodiments, Q is a linker
connected to a solid support; said aliphatic amine is triethylamine
or piperidine; said solvent is acetonitrile or methylene chloride;
and said phosphorus protecting group is
--CH.sub.2--CH.sub.2--C.ident.N or
--CH.sub.2--CH.dbd.CH--CH.sub.2--C.ident.N, and wherein the
deprotecting reagent, said cleaving reagent, or both preferably
further comprises a scavenger.
[0051] In further preferred embodiments, the deprotecting reagent
comprises a secondary alkyl amine which is preferably piperidine,
and said cleaving reagent comprises an alkali metal carbonate,
which is preferably potassium carbonate.
[0052] Also provided by the present invention are methods for
deprotecting a phosphate-linked oligomer, said oligomer having a
plurality of protected phosphorus linkages of Formula II: ##STR3##
wherein:
[0053] Each X is O or S;
[0054] R.sub.t is a phosphorus protecting group of the formula:
--C(R.sub.10).sub.2--C(R.sub.10).sub.2--W or
--C(R.sub.10).sub.2--(CH.dbd.CH).sub.p--C(R.sub.10).sub.2--W [0055]
each R.sub.10 is independently H or lower alkyl; [0056] W is an
electron withdrawing group; [0057] p is 1 to 3; comprising:
[0058] (a) providing a sample containing a plurality of said
phosphate linked oligomers;
[0059] (b) contacting said oligomers with a deprotecting reagent
for a time and under conditions sufficient to remove substantially
all of said R.sub.t groups from said oligomers, said deprotecting
reagent containing 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; and
[0060] (c) reacting said oligomers with a cleaving reagent.
[0061] Preferably, the methods further comprises a washing step
before step (c).
[0062] In some preferred embodiments, the oligomers are in
solution. In other preferred embodiments, the oligomers are linked
to a solid support.
[0063] In some preferred embodiments, said deprotecting reagent
does not cleave said oligomers from said solid support.
[0064] In some preferred embodiments, the deprotecting reagent
comprises an aliphatic amine, which is preferably triethylamine or
piperidine.
[0065] In further preferred embodiments, the deprotecting agent
comprises a haloalkyl solvent or a cyanoalkyl solvent, which is
preferably acetonitrile or methylene chloride.
[0066] In particularly preferred embodiments, the phosphorus
protecting group is --CH.sub.2--CH.sub.2--C.ident.N or
--CH.sub.2--(CH.dbd.CH).sub.p--CH.sub.2--C.ident.N, where p is an
integer from 1 to 3, with --CH.sub.2--CH.sub.2--C.ident.N or
--CH.sub.2--CH.dbd.CH--CH.sub.2--C.ident.N being preferred, and
with --CH.sub.2--CH.sub.2--C.ident.N being particularly
preferred.
[0067] 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.
[0068] 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.TM.
NH.sub.2HL, Agropore.TM. NH.sub.2LL, 4-methoxytrityl resin, and
thiol 2-chlorotrityl resin.
[0069] In some preferred embodiments, the cleaving reagent
comprises an aqueous methanolic solution of a Group I or Group II
metal carbonate, preferably aqueous methanolic potassium carbonate.
In further preferred embodiments, the cleaving reagent comprises an
aqueous metal hydroxide. In yet further preferred embodiments, the
cleaving reagent comprises a phase transfer catalyst. Preferred
phase transfer catalysts include quaternary ammonium salts,
quaternary phosphonium salts, crown ethers and cryptands (i.e.,
crown ethers which are bicyclic or cycles of higher order). It is
more preferred that the phase transfer catalyst be
t-Bu.sub.4N.sup.+OH, or t-Bu.sub.4N.sup.+F.sup.-.
[0070] In further preferred embodiments, the cleaving reagent
comprises NaNH.sub.2.
[0071] In preferred embodiments, the produced by the methods of the
invention oligomers have from about 0.001% to about 1%
acrylonitrile adduct, with from about 0.001% to about 0.5%
acrylonitrile adduct being more preferred, from about 0.001% to
about 0.1% acrylonitrile adduct being even more preferred, and from
about 0.001% to about 0.05% acrylonitrile adduct being even more
preferred. In even more preferred embodiments, the oligomers are
substantially free of acrylonitrile adduct.
[0072] In some preferred embodiments, steps b) and c) are performed
simultaneously.
[0073] In some particularly preferred embodiments, said aliphatic
amine is triethylamine or piperidine; said solvent is acetonitrile
or methylene chloride; and said phosphorus protecting group is
--CH.sub.2--CH.sub.2--C.ident.N or
--CH.sub.2--CH.dbd.CH--CH.sub.2--C.ident.N, and wherein the
deprotecting reagent, said cleaving reagent, or both preferably
further comprises a scavenger.
[0074] In further preferred embodiments, the deprotecting reagent
comprises a secondary alkyl amine which is preferably piperidine,
and said cleaving reagent comprises an alkali metal carbonate,
which is preferably potassium carbonate.
[0075] Also provided by the present invention are methods for
deprotecting a phosphate-linked oligomer, said oligomer having a
plurality of protected phosphorus linkages of Formula II: ##STR4##
wherein:
[0076] Each X is O or S;
[0077] R.sub.t is a phosphorus protecting group of formula:
--C(R.sub.10).sub.2--C(R.sub.10).sub.2--W or
--C(R.sub.10).sub.2--(CH.dbd.CH).sub.p--C(R.sub.10).sub.2--W [0078]
each R.sub.10 is independently H or lower alkyl; [0079] W is an
electron withdrawing group; [0080] p is 1 to 3; comprising:
[0081] (a) providing a sample containing a plurality of said
phosphate linked oligomers;
[0082] (b) contacting said oligomers with a deprotecting reagent
for a time and under conditions sufficient to remove substantially
all of said R.sub.t groups from said oligomers;
[0083] (c) washing said deprotected oligomers with a washing
reagent comprising 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; and
[0084] (d) reacting said oligomers with a cleaving reagent.
[0085] In some preferred embodiments, the oligomers are in
solution. In other preferred embodiments, the oligomers are linked
to a solid support.
[0086] In some preferred embodiments, the deprotecting reagent does
not cleave said oligomers from said solid support.
[0087] In further preferred embodiments, the deprotecting reagent
comprises an aliphatic amine, which is preferably triethylamine or
piperidine. In still further preferred embodiments, the
deprotecting agent comprises a haloalkyl solvent or a cyanoalkyl
solvent which is preferably acetonitrile or methylene chloride.
[0088] In particularly preferred embodiments, the phosphorus
protecting group is --CH.sub.2--CH.sub.2--C.ident.N or
--CH.sub.2--(CH.dbd.CH).sub.p--CH.sub.2--C .ident.N, where p is an
integer from 1 to 3, with --CH.sub.2--CH.sub.2--C.ident.N or
--CH.sub.2--CH.dbd.CH--CH.sub.2--C.ident.N being preferred, and
with --CH.sub.2--CH.sub.2--C.ident.N being particularly
preferred.
[0089] In some preferred embodiments, the deprotecting reagent, the
cleaving reagent or the washing 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.
[0090] 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.TM.
NH.sub.2HL, Agropore.TM. NH.sub.2LL, 4-methoxytrityl resin, and
thiol 2-chlorotrityl resin.
[0091] In some preferred embodiments, the cleaving reagent
comprises an aqueous methanolic solution of a Group I or Group II
metal carbonate, preferably aqueous methanolic potassium carbonate.
In further preferred embodiments, the cleaving reagent comprises an
aqueous metal hydroxide. In yet further preferred embodiments, the
cleaving reagent comprises a phase transfer catalyst. Preferred
phase transfer catalysts include quaternary ammonium salts,
quaternary phosphonium salts, crown ethers and cryptands (i.e.,
crown ethers which are bicyclic or cycles of higher order). It is
more preferred that the phase transfer catalyst be
t-Bu.sub.4N.sup.+OH, or t-Bu.sub.4N.sup.+F.sup.-.
[0092] In further preferred embodiments, the cleaving reagent
comprises NaNH.sub.2.
[0093] In preferred embodiments, the oligomers produced by the
methods of the invention have from 0.001% to about 1% acrylonitrile
adduct, with from about 0.1% to about 1% acrylonitrile adduct being
more preferred, from about 0.1% to about 0.75% acrylonitrile adduct
being even more preferred, and from about 0.1% to about 0.5%
acrylonitrile adduct being even more preferred. In even more
preferred embodiments, the oligomers are substantially free of
detectable acrylonitrile adduct.
[0094] In some particularly preferred embodiments, said aliphatic
amine is triethylamine or piperidine; said solvent is acetonitrile
or methylene chloride; and said phosphorus protecting group is
--CH.sub.2--CH.sub.2--C.ident.N or
--CH.sub.2--CH.dbd.CH--CH.sub.2--C.ident.N, and wherein the
deprotecting reagent, the washing reagent, the cleaving reagent, or
each preferably further comprise a scavenger.
[0095] In further preferred embodiments, the deprotecting reagent
comprises a secondary alkyl amine which is preferably piperidine,
and said cleaving reagent comprises an alkali metal carbonate,
which is preferably potassium carbonate.
[0096] The present invention also provides methods for deprotecting
a phosphate-linked oligomer, said oligomer having a plurality of
phosphorus linkages of Formula II: ##STR5## wherein:
[0097] Each X is O or S;
[0098] R.sub.t is a phosphorus protecting group of the formula:
--C(R.sub.10).sub.2--C(R.sub.10).sub.2--W or
--C(R.sub.10).sub.2--(CH.dbd.CH).sub.p--C(R.sub.10).sub.2--W [0099]
each R.sub.10 is independently H or lower alkyl; [0100] W is an
electron withdrawing group; [0101] p is 1 to 3; comprising:
[0102] (a) providing a sample containing a plurality of said
phosphate linked oligomers; and
[0103] (b) contacting said oligomers with a deprotecting reagent
for a time and under conditions sufficient to remove substantially
all of said R.sub.t groups from said oligomers, said deprotecting
reagent comprising gaseous ammonia.
[0104] Also provided in accordance with the present invention are
compositions comprising phosphodiester, phosphorothioate, or
phosphorodithioate oligonucleotides produced by the methods of the
invention.
[0105] The present invention also provides compositions comprising
phosphodiester, phosphorothioate, or phosphorodithioate
oligonucleotides, said oligonucleotides having from about 0.001% to
about 1% acrylonitrile adduct, with from about 0.1% to about 1%
acrylonitrile adduct being more preferred, from about 0.1% to about
0.75% acrylonitrile adduct being even more preferred, and from
about 0.1% to about 0.5% acrylonitrile adduct being even more
preferred. In particularly preferred embodiments, compositions
comprising phosphodiester, phosphorothioate, or phosphorodithioate
oligonucleotides, said oligonucleotides are provided that are
substantially free of detectable acrylonitrile adduct.
[0106] The present invention also provides composition comprising
oligonucleotides that are substantially free of acrylonitrile
adduct prepared by the methods of the invention.
[0107] Further provided in accordance with the present invention
are methods of preparing a sample of a phosphate linked
oligonucleotide having a substantially reduced content of
acrylonitrile adduct comprising:
[0108] (a) providing a sample containing a plurality of oligomers,
said oligomers having a plurality of phosphorus protecting
groups;
[0109] (b) contacting said oligomers with a deprotecting agent to
remove substantially all of said phosphorus protecting groups from
said oligomers; [0110] said deprotecting reagent comprising 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;
[0111] (c) optionally washing said oligomers; and
[0112] (d) reacting said oligomers with a cleaving reagent.
[0113] Further provided in accordance with the present invention
are methods of preparing a sample of a phosphate linked
oligonucleotide having a substantially reduced content of
acrylonitrile adduct comprising:
[0114] (a) providing a sample containing a plurality of oligomers,
said oligomers having a plurality of phosphorus protecting
groups;
[0115] (b) contacting said oligomers with a deprotecting agent to
remove substantially all of said phosphorus protecting groups from
said oligomers;
[0116] (c) washing said oligomers with a washing reagent, said
washing reagent comprising 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; and
[0117] (d) reacting said oligomers with a cleaving reagent.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0118] The present invention provides methods for the preparation
of oligomeric compounds having phosphodiester, phosphorothioate,
phosphorodithioate, or other internucleoside linkages, and to
composition produced by the methods.
[0119] The methods of the invention are applicable to both solution
phase and solid phase chemistries. Representative solution phase
techniques are described in U.S. Pat. No. 5,210,264, which is
assigned to the assignee of the present invention. In some
preferred embodiments, the methods of the present invention are
employed for use in iterative solid phase oligonucleotide synthetic
regimes. Representative solid phase techniques are those typically
employed for DNA and RNA synthesis utilizing standard
phosphoramidite chemistry, (see, e.g., Protocols For
Oligonucleotides And Analogs, Agrawal, S., ed., Humana Press,
Totowa, N.J., 1993, hereby incorporated by reference in its
entirety). A preferred synthetic solid phase synthesis utilizes
phosphoramidites as activated phosphate compounds. In this
technique, a 5'-protected phosphoramidite monomer is reacted with a
free hydroxyl on the growing oligomer chain to produce an
intermediate phosphite compound, which is subsequently oxidized to
the P.sup.V state using standard methods. This technique is
commonly used for the synthesis of several types of linkages
including phosphodiester, phosphorothioate, and phosphorodithioate
linkages.
[0120] Typically, the first step in such a process is attachment of
a first monomer or higher order subunit containing a protected
5'-hydroxyl to a solid support, usually through a linker, using
standard methods and procedures known in the art. See for example,
Oligonucleotides And Analogues A Practical Approach, Eckstein, F.
Ed., IRL Press, N.Y, 1991, hereby incorporated by reference in its
entirety. The support-bound monomer or higher order first synthon
is then treated to remove the 5'-protecting group. The solid
support bound monomer is then reacted with an activated phosphorous
monomer or higher order synthon which is typically a nucleoside
phosphoramidite, which is suitably protected at the phosphorus
atom, and at any vulnerable exocyclic amino or hydroxyl groups.
Typically, the coupling of the phosphoramidite to the support bound
chain is accomplished under anhydrous conditions in the presence of
an activating agent such as, for example, 1H-tetrazole,
5-(4-nitrophenyl)-1H-tetrazole, or diisopropylamino
tetrazolide.
[0121] The resulting linkage is a phosphite or thiophosphite, which
is subsequently oxidized prior to the next iterative cycle. Choice
of oxidizing or sulfurizing agent will determine whether the
linkage will be oxidized or sulfurized to a phosphodiester,
thiophosphodiester, or a dithiophosphodiester linkage.
[0122] At the end of the synthetic regime, the support-bound
oligomeric chain is typically treated with strong base (e.g., 30%
aqueous ammonium hydroxide) to cleave the completed oligonucleotide
form the solid support, and to concomitantly remove phosphorus
protecting groups (which are typically .beta.-cyanoethyl protecting
groups) and exocyclic nucleobase protecting groups. Without
intending that the invention be bound by any particular theory, it
is believed that the loss of the cyanoethyl phosphorus protecting
group occurs via a .beta.-elimination mechanism, which produces
acrylonitrile as a product. The acrylonitrile is believed to react
in a Michael addition with nucleobase exocyclic amine and/or
hydroxyl moieties, and in particular the N.sup.3 position of
thymidine residues, to form deleterious adducts. The methods of the
present invention significantly reduce the content of such adducts
formed during the removal of phosphorus protecting groups that are
capable of participating in such adduct-forming addition
reactions.
[0123] Thus, in one aspect, the present invention provides
synthetic methods comprising:
[0124] a) providing a sample containing a plurality of oligomers of
the Formula I: ##STR6##
[0125] wherein: [0126] R.sub.1 is H or a hydroxyl protecting group;
[0127] B is a naturally occurring or non-naturally occurring
nucleobase that is optionally protected at one or more exocyclic
hydroxyl or amino groups;
[0128] R.sub.2 has the Formula III or IV: ##STR7##
[0129] wherein [0130] E is C.sub.1-C.sub.10 alkyl,
N(Q.sub.1)(Q.sub.2) or N.dbd.C(Q.sub.1)(Q.sub.2); [0131] each
Q.sub.1 and Q.sub.2 is, independently, H, C.sub.1-C.sub.10 alkll,
dialkylaminoalkyl, a nitrogen protecting group, a tethered or
untethered conjugate group, a linker to a solid support, or Q.sub.1
and Q.sub.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;
[0132] R.sub.3 is OX.sub.1, SX.sub.1, or N(X.sub.1).sub.2; [0133]
each X.sub.1 is, independently, H, C.sub.1-C.sub.10 alkyl,
C.sub.1-C.sub.8 haloalkyl, C(.dbd.NH)N(H)Z.sub.8,
C(.dbd.O)N(H)Z.sub.8 or OC(.dbd.O)N(H)Z.sub.8;
[0134] Z.sub.8 is H or C.sub.1-C.sub.8 alkyl; [0135] L.sub.1,
L.sub.2 and L.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; [0136] 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.sub.1)(Q.sub.2), O(Q.sub.1), halo,
S(Q.sub.1), or CN; [0137] each q, is, independently, from 2 to 10;
[0138] each q.sub.2 is, independently, 0 or 1; [0139] m is 0, 1 or
2; [0140] pp is from 1 to 10; and [0141] q.sub.3 is from 1 to 10
with the proviso that when pp is 0, q.sub.3 is greater than 1;
R.sub.t is a phosphorus protecting group of formula:
--C(R.sub.10).sub.2--C(R.sub.10).sub.2--W or
--C(R.sub.10).sub.2--(CH.dbd.CH).sub.p--C(R.sub.10).sub.2--W [0142]
each R.sub.10 is independently H or lower alkyl; [0143] W is an
electron withdrawing group; [0144] p is 0 to 3;
[0145] each Y.sub.2 is independently, O, CH.sub.2 or NH;
[0146] each Z is independently O or S;
[0147] Each X is independently O or S;
[0148] Q is a linker connected to a solid support, --OH or O--Pr
where Pr is a hydroxyl protecting group; and
[0149] n is 1 to about 100;
[0150] b) contacting said sample with a deprotecting reagent for a
time and under conditions sufficient to remove substantially said
R.sub.t groups from said oligomers; and
[0151] c) reacting said oligomers with a cleaving reagent;
[0152] 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.
[0153] Also provided by the present invention are methods for
deprotecting a phosphate-linked oligomer, said oligomer having a
plurality of protected phosphorus linkages of Formula II: ##STR8##
wherein X and R.sub.t are as defined above, comprising:
[0154] (a) providing a sample containing a plurality of said
phosphate linked oligomers;
[0155] (b) contacting said oligomers with a deprotecting reagent
for a time and under conditions sufficient to remove substantially
all of said R.sub.t groups from said oligomers, said deprotecting
reagent containing 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; and
[0156] (c) reacting said oligomers with a cleaving reagent.
[0157] In further embodiments, the present invention provides
methods for deprotecting a phosphate-linked oligomer, said oligomer
having a plurality of protected phosphorus linkages of formula II
comprising:
[0158] (a) providing a sample containing a plurality of said
phosphate linked oligomers;
[0159] (b) contacting said oligomers with a deprotecting reagent
for a time and under conditions sufficient to remove substantially
all of said R.sub.t groups from said oligomers;
[0160] (c) washing said deprotected oligomers with a washing
reagent comprising 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; and
[0161] (d) reacting said oligomers with a cleaving reagent.
[0162] The present invention also provides methods for deprotecting
a phosphate-linked oligomer, said oligomer having a plurality of
phosphorus linkages of formula II comprising:
[0163] (a) providing a sample containing a plurality of said
phosphate linked oligomers; and
[0164] (b) contacting said oligomers with a deprotecting reagent
for a time and under conditions sufficient to remove substantially
all of said R.sub.t groups from said oligomers, said deprotecting
reagent comprising gaseous ammonia.
[0165] Further provided in accordance with the present invention
are methods of preparing a sample of a phosphate linked
oligonucleotide having a substantially reduced content of
acrylonitrile adduct comprising:
[0166] (a) providing a sample containing a plurality of oligomers,
said oligomers having a plurality of phosphorus protecting
groups;
[0167] (b) contacting said oligomers with a deprotecting agent to
remove substantially all of said phosphorus protecting groups from
said oligomers; [0168] said deprotecting reagent comprising 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;
[0169] (c) optionally washing said oligomers; and
[0170] (d) reacting said oligomers with a cleaving reagent.
[0171] Further provided in accordance with the present invention
are methods of preparing a sample of a phosphate linked
oligonucleotide having a substantially reduced content of
acrylonitrile adduct comprising:
[0172] (a) providing a sample containing a plurality of oligomers,
said oligomers having a plurality of phosphorus protecting
groups;
[0173] (b) contacting said oligomers with a deprotecting agent to
remove substantially all of said phosphorus protecting groups from
said oligomers;
[0174] (c) washing said oligomers with a washing reagent, said
washing reagent comprising 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; and
[0175] (d) reacting said oligomers with a cleaving reagent.
[0176] Thus, in some preferred methods of the invention, a
support-bound or solution phase oligomer having a plurality of
phosphorus protecting groups that are capable of producing
acrylonitrile, or a structurally similar product that can form an
adduct with nucleobase amino groups, is contacted with a
deprotecting reagent that includes at least one amine. The amine is
selected such that it is a sufficiently strong base to effect the
removal of a substantial majority of the phosphorus protecting
groups, but insufficiently strong to cause deprotonation the
thymidine N.sup.3 position, and hence activation of that position
to adduct formation. It has been found that suitable amines include
those whose conjugate acids have a pKa of from about 8 to about 11,
more preferably from about 9 to about 11, even more preferably from
about 10 to about 11. In general, it is preferred that the amine be
an aliphatic amine of the formula (R).sub.3N, (R).sub.2NH, or
RNH.sub.2 where R is alkyl Two particularly suitable amines are
triethylamine and piperidine.
[0177] As used herein, the term "deprotect" or deprotection" is
intended to mean the removal of the vast majority, and more
preferably substantially all phosphorus protecting groups from the
oligomers of interest.
[0178] In preferred embodiments, the deprotecting reagent can be
either an aliphatic amine, or a solution of one or more amines
aliphatic as described above. In more preferred embodiments, the
deprotecting reagent further comprises one or more solvents
selected from the group consisting of alkyl solvents, haloalkyl
solvents, cyanoalkyl solvents, aryl solvents and aralkyl solvents.
Preferably, the solvent is a haloalkyl solvent or a cyanoalkyl
solvent. Examples of particularly suitable solvents are
acetonitrile and methylene chloride.
[0179] In the practice of the present invention, it is greatly
preferred that the vast majority of the cyanoethyl groups be
removed before the oligomer is treated with the relatively strong
conditions of the cleavage reagent (e.g., 30% aqueous ammonium
hydroxide). The rate of deprotection of .beta.-cyanoethyl groups
from oligonucleotides has been shown to exhibit a marked solvent
effect. For example, the half-life of a dimer containing a single
cyanoethyl group in a 1:1 v/v solution of triethylamine in
acetonitrile or methylene chloride is, very approximately, 10 min.
at 25.degree. C., whereas the half-life of the same compound in
triethylamine-pyridine (1:1, v/v) is about ten times longer. Eritja
et al. (Tetrahedron, 48, 4171-4182 (1992)) recommend a three hour
treatment with a 40% solution of triethylamine in pyridine as
sufficient to avoid formation of acrylonitrile adduct to thymidine
residues in oligonucleotides subsequently treated with DBU.
However, it has been discovered that under the conditions described
by Eritja, many of the cyanoethyl protecting groups would remain
intact. While not wishing to be bound by a specific theory, it is
believed that subsequent treatment with ammonium hydroxide (or any
other strong base such as DBU) would lead to the formation of
unacceptable levels of residues having acrylonitrile adducts. Thus,
in the present invention it is preferred that the solvent which is
contained in the deprotection reagent not include pyridine, or
similar heterocyclic base solvents that could extend the time for
removal of oligomer-bound .beta.-cyanoethyl or other electronically
similar protecting groups.
[0180] At present, the detectable limit of acrylonitrile adduct by
HPLC methodologies is believed to be about 0.1%. However, it is
believed that the present methods provide oligomers having as
little as 0.001% of such adduct. Thus, in preferred embodiments,
the oligomers produced by the methods of the invention have from
0.001% to about 1% acrylonitrile adduct, with from about 0.1% to
about 1% acrylonitrile adduct being more preferred, from about 0.1%
to about 0.75% acrylonitrile adduct being even more preferred, and
from about 0.1% to about 0.5% acrylonitrile adduct being even more
preferred. In even more preferred embodiments, the oligomers are
substantially free of detectable acrylonitrile adduct.
[0181] As used herein, the term "acrylonitrile adduct" refers to
adducts to exocyclic nucleobase adducts that result from the
acrylonitrile formed during removal of .beta.-cyanoethyl phosphorus
protecting groups, or similar adducts formed by removal of
protecting groups that form electronically similar products upon
removal. Representative examples of such protecting groups are
those having the formula: --C(R.sub.10).sub.2--C(R.sub.10).sub.2--W
or --C(R.sub.10).sub.2--(CH.dbd.CH).sub.p--C(R.sub.10).sub.2--W
[0182] wherein each R.sub.10 is independently H or lower alkyl, W
is an electron withdrawing group, and p is 1 to 3. The term
"electron withdrawing group" is intended to have its recognized
meaning in the art as a chemical moiety that attracts electron
density, whether through resonance or inductive effects. Examples
of electron withdrawing groups are cyano, nitro, halogen, phenyl
substituted in the ortho or para position with one or more halogen,
nitro or cyano groups, and trihalomethyl groups. Those of skill in
the art will readily recognize other electron withdrawing groups,
as well as other phosphorus protecting groups that have similar
potential to form adducts with exocyclic amino or hydroxyl
functions.
[0183] After contact with the deprotecting reagent, the oligomers
can be further washed prior to reaction with a cleavage reagent, or
reacted with the cleaving reagent directly. The cleaving reagent is
a solution that includes a single reagent or combination of
reagents that effect the cleavage of the deprotected oligomer from
a solid support, and/or, where the oligomer is in solution, effects
cleavage of exocyclic protecting groups, for example 30% aqueous
ammonium hydroxide.
[0184] In the methods of the invention, it is generally
advantageous to effect removal of substantially all phosphorus
protecting groups from the oligomers, and separating the
acrylonitrile or acrylonitrile-like products from the oligomers
prior to exposing oligomers to the more severe basic conditions
that effect cleavage from the solid support, or removal of
exocyclic and/or hydroxyl protecting groups. Thus, in some
preferred embodiments, a washing step is utilized in between
contact with deprotecting reagent and cleaving reagent. In some
preferred embodiments the washing step is performed using one or
more suitable solvents, for example acetonitrile or methylene
chloride. In other preferred embodiments, washing is performed with
a washing reagent that contains one or more amines as is employed
in the deprotecting reagent.
[0185] In some particularly preferred embodiments, a scavenger can
be included in the deprotection reagent, cleaving reagent, washing
reagent, or combinations thereof. In general, the scavenger is a
molecule that reacts with the acrylonitrile or acrylonitrile-like
products of deprotection, lowering the possibility of nucleobase
adduct formation. Suitable scavengers include purines, pyrimidines,
inosine, pyrroles, imidazoles, triazoles, mercaptans, beta amino
thiols, phosphines, phosphites, dienes, ureas, thioureas, amides,
imides, cyclic imides and ketones. Further useful scavengers
include alkylmercaptans, thiols, ethylene glycol, substituted
ethylene glycols, 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, -methoxyphthalimide, p-sulfobenzenediazonium
chloride, p-sulfamidobenzenediazonium chloride.
[0186] 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.TM.
NH.sub.2HL, Agropore.TM. NH.sub.2LL, 4-methoxytrityl resin, and
thiol 2-chlorotrityl resin.
[0187] The methods of the present invention are useful for the
preparation of oligomeric compounds containing monomeric subunits
that are joined by a variety of linkages, including phosphite,
phosphodiester, phosphorothioate, and/or phosphorodithioate
linkages.
[0188] As used herein, the terms "oligomer" or "oligomeric
compound" are used to refer to compounds containing a plurality of
nucleoside monomer subunits that are joined by internucleoside
linkages, preferably phosphorus-containing linkages, such as
phosphite, phosphodiester, phosphorothioate, and/or
phosphorodithioate linkages. The term "oligomeric compound"
therefore includes naturally occurring oligonucleotides, their
analogs, and synthetic oligonucleotides.
[0189] In some preferred embodiments of the compounds of the
invention, substituent W can be an electron withdrawing group
selected such that it facilitates attack by a nucleophile.
Accordingly, W can be any of a variety of electron withdrawing
substituents, provided that it does not otherwise interfere with
the methods of the invention. Preferred non-silyl electron
withdrawing W groups include cyano, NO.sub.2, alkaryl groups,
sulfoxyl groups, sulfonyl groups, thio groups, substituted sulfoxyl
groups, substituted sulfonyl groups, or substituted thio groups,
wherein the substituents are selected from the group consisting of
alkyl, aryl, or alkaryl. Particularly preferred are alkanoyl groups
having the formula R--C(.dbd.O)-- where R is an alkyl group of from
1 to six carbons, with acetyl groups being especially preferred. W
can also be a trisubstituted silyl moiety, wherein the substituents
are alkyl, aryl or both.
[0190] 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.TM.
NH.sub.2HL, Agropore.TM. NH.sub.2LL (available from Aldrich Chem.
Co. St. Louis. Mo.), 4-methoxytrityl resin, and thiol
2-chlorotrityl resin.
[0191] When used as part of the cleaving reagent, contact with
fluoride ion preferably is effected in a solvent such as
tetrahydrofuran, acetonitrile, dimethoxyethane, or water. Fluoride
ion preferably is provided in the form of one or more salts
selected from tetraalkylammonium fluorides (e.g.,
tetrabutylammonium fluoride (TBAF)), potassium fluoride, cesium
fluoride, or triethylammonium hydrogen fluoride.
[0192] The present invention is applicable to the preparation of
phosphate linked oligomers having a variety of internucleoside
linkages including phosphite, phosphodiester, phosphorothioate, and
phosphorodithioate linkages, and other linkages known in the
art
[0193] In preferred embodiments, the methods of the invention are
used for the preparation of oligomeric compounds including
oligonucleotides and their analogs. As used herein, the term
"oligonucleotide analog" means compounds that can contain both
naturally occurring (i.e. "natural") and non-naturally occurring
("synthetic") moieties, for example, nucleosidic subunits
containing modified sugar and/or nucleobase portions. Such
oligonucleotide analogs are typically structurally distinguishable
from, yet functionally interchangeable with, naturally occurring or
synthetic wild type oligonucleotides. Thus, oligonucleotide analogs
include all such structures which function effectively to mimic the
structure and/or function of a desired RNA or DNA strand, for
example, by hybridizing to a target. The term synthetic nucleoside,
for the purpose of the present invention, refers to a modified
nucleoside. Representative modifications include modification of a
heterocyclic base portion of a nucleoside to give a non-naturally
occurring nucleobase, a sugar portion of a nucleoside, or both
simultaneously.
[0194] Representative nucleobases useful in the compounds and
methods described herein include adenine, guanine, cytosine,
uracil, and thymine, as well as other non-naturally occurring and
natural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine, 5-halo
uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudo uracil), 4-thiouracil, 8-halo, oxa, amino, thiol,
thioalkyl, hydroxyl and other 8-substituted adenines and guanines,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methyl-guanine. Further naturally and non naturally occurring
nucleobases include those disclosed in U.S. Pat. No. 3,687,808
(Merigan, et al), in chapter 15 by Sanghvi, in Antisense Research
and Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993,
in Englisch et al., Angewandte Chemie, International Edition, 1991,
30, 613-722 (see especially pages 622 and 623, and in the Concise
Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz
Ed., John Wiley & Sons, 1990, pages 858-859, Cook, P. D.,
Anti-Cancer Drug Design, 1991, 6, 585-607, each of which are hereby
incorporated by reference in their entirety. The term `nucleosidic
base` is further intended to include heterocyclic compounds that
can serve as like nucleosidic bases including certain `universal
bases` that are not nucleosidic bases in the most classical sense
but serve as nucleosidic bases. Especially mentioned as a universal
base is 3-nitropyrrole.
[0195] As used herein, the term "alkyl" includes but is not limited
to straight chain, branch chain, and alicyclic hydrocarbon groups.
Alkyl groups of the present invention may be substituted.
Representative alkyl substituents are disclosed in U.S. Pat. No.
5,212,295, at column 12, lines 41-50, hereby incorporated by
reference in its entirety.
[0196] As used herein, the term "aralkyl" denotes alkyl groups
which bear aryl groups, for example, benzyl groups. The term
"alkaryl" denotes aryl groups which bear alkyl groups, for example,
methylphenyl groups. "Aryl" groups are aromatic cyclic compounds
including but not limited to phenyl, naphthyl, anthracyl,
phenanthryl, pyrenyl, and xylyl.
[0197] As used herein, the term "alkanoyl" has its accustomed
meaning as a group of formula --C(.dbd.O)-alkyl. A preferred
alkanoyl group is the acetoyl group.
[0198] In general, the term "hetero" denotes an atom other than
carbon, preferably but not exclusively N, O, or S. Accordingly, the
term "heterocycloalkyl" denotes an alkyl ring system having one or
more heteroatoms (i.e., non-carbon atoms). Preferred
heterocycloalkyl groups include, for example, morpholino groups. As
used herein, the term "heterocycloalkenyl" denotes a ring system
having one or more double bonds, and one or more heteroatoms.
Preferred heterocycloalkenyl groups include, for example,
pyrrolidino groups.
[0199] In some preferred embodiments of the invention oligomers can
be linked connected to a solid support. Solid supports are
substrates which are capable of serving as the solid phase in solid
phase synthetic methodologies, such as those described in Caruthers
U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777;
4,973,679; and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and
Re. 34,069. Linkers are known in the art as short molecules which
serve to connect a solid support to functional groups (e.g.,
hydroxyl groups) of initial synthon molecules in solid phase
synthetic techniques. Suitable linkers are disclosed in, for
example, Oligonucleotides And Analogues A Practical Approach,
Eckstein, F. Ed., IRL Press, N.Y, 1991, Chapter 1, pages 1-23.
Other linkers include the "TAMRA" linker described by Mullah et.
al., Tetrahedron Letters, 1997, 38, 5751-5754, and the "Q-linker"
described by Pon et. al., Nucleic Acid Research, 1997, 25,
3629-3635.
[0200] Solid supports according to the invention include those
generally known in the art to be suitable for use in solid phase
methodologies, including, for example, controlled pore glass (CPG),
oxalyl-controlled pore glass (see, e.g., Alul, et al., Nucleic
Acids Research 1991, 19, 1527, hereby incorporated by reference in
its entirety), TentaGel Support--an aminopolyethyleneglycol
derivatized support (see, e.g., Wright, et al., Tetrahedron Letters
1993, 34, 3373, hereby incorporated by reference in its entirety)
and Poros--a copolymer of polystyrene/divinylbenzene.
[0201] In some preferred embodiments of the invention hydroxyl
groups can be protected with a hydroxyl protecting group. A wide
variety of hydroxyl protecting groups can be employed in the
methods of the invention. Preferably, the protecting group is
stable under basic conditions but can be removed under acidic
conditions. In general, protecting groups render chemical
functionalities inert to specific reaction conditions, and can be
appended to and removed from such functionalities in a molecule
without substantially damaging the remainder of the molecule.
Representative hydroxyl protecting groups are disclosed by
Beaucage, et al., Tetrahedron 1992, 48, 2223-2311, and also in
Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2,
2d ed, John Wiley & Sons, New York, 1991, each of which are
hereby incorporated by reference in their entirety. Preferred
protecting groups used for R.sub.2, R.sub.3 and R.sub.3a include
dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl
(Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox). The hydroxyl
protecting group can be removed from oligomeric compounds of the
invention by techniques well known in the art to form the free
hydroxyl. For example, dimethoxytrityl protecting groups can be
removed by protic acids such as formic acid, dichloroacetic acid,
trichloroacetic acid, p-toluene sulphonic acid or with Lewis acids
such as for example zinc bromide. See for example, Greene and Wuts,
supra.
[0202] In some preferred embodiments of the invention amino groups
are appended to alkyl or to other groups such as, for example, to
2'-alkoxy groups. Such amino groups are also commonly present in
naturally occurring and non-naturally occurring nucleobases. It is
generally preferred that these amino groups be in protected form
during the synthesis of oligomeric compounds of the invention.
Representative amino protecting groups suitable for these purposes
are discussed in Greene and Wuts, Protective Groups in Organic
Synthesis, Chapter 7, 2d ed, John Wiley & Sons, New York, 1991.
Generally, as used herein, the term "protected" when used in
connection with a molecular moiety such as "nucleobase" indicates
that the molecular moiety contains one or more functionalities
protected by protecting groups.
[0203] Sulfurizing agents used during oxidation to form
phosphorothioate and phosphorodithioate linkages include Beaucage
reagent (see e.g. Iyer, R. P., et.al., J. Chem. Soc., 1990, 112,
1253-1254, and Iyer, R. P., et.al., J. Org. Chem., 1990, 55,
4693-4699); 3-methyl-1,2,4-thiazolin-5-one (MEDITH; Zong, et al.,
Tetrahedron Lett. 1999, 40, 2095); tetraethylthiuram disulfide (see
e.g., Vu, H., Hirschbein, B. L., Tetrahedron Lett., 1991, 32,
3005-3008); dibenzoyl tetrasulfide (see e.g., Rao, M. V., et.al.,
Tetrahedron Lett., 1992, 33, 4839-4842); di(phenylacetyl)disulfide
(see e.g., Kamer, P. C. J., Tetrahedron Lett., 1989, 30,
6757-6760); Bis(O,O-diisopropoxy phosphinothioyl)disulfides (see
Stec et al., Tetrahedron Lett., 1993, 34, 5317-5320);
3-ethoxy-1,2,4-dithiazoline-5-one (see Nucleic Acids Research, 1996
24, 1602-1607, and Nucleic Acids Research, 1996 24, 3643-3644);
Bis(p-chlorobenzenesulfonyl)disulfide (see Nucleic Acids Research,
1995 23, 4029-4033); sulfur, sulfur in combination with ligands
like triaryl, trialkyl, triaralkyl, or trialkaryl phosphines. The
foregoing references are hereby incorporated by reference in their
entirety.
[0204] Useful oxidizing agents used to form the phosphodiester or
phosphorothioate linkages include
iodine/tetrahydrofuran/water/pyridine or hydrogen peroxide/water or
tert-butyl hydroperoxide or any peracid like m-chloroperbenzoic
acid. In the case of sulfurization the reaction is performed under
anhydrous conditions with the exclusion of air, in particular
oxygen whereas in the case of oxidation the reaction can be
performed under aqueous conditions.
[0205] Oligonucleotides or oligonucleotide analogs according to the
present invention hybridizable to a specific target preferably
comprise from about 5 to about 100 monomer subunits. It is more
preferred that such compounds comprise from about 5 to about 50
monomer subunits, more preferably 10 to about 30 monomer subunits,
with 15 to 25 monomer subunits being particularly preferred. When
used as "building blocks" in assembling larger oligomeric
compounds, smaller oligomeric compounds are preferred. Libraries of
dimeric, trimeric, or higher order compounds can be prepared by the
methods of the invention. The use of small sequences synthesized
via solution phase chemistries in automated synthesis of larger
oligonucleotides enhances the coupling efficiency and the purity of
the final oligonucleotides. See for example: Miura, K., et al.,
Chem. Pharm. Bull., 1987, 35, 833-836; Kumar, G., and Poonian,
M.S., J. Org. Chem., 1984, 49, 4905-4912; Bannwarth, W., Helvetica
Chimica Acta, 1985, 68, 1907-1913; Wolter, A., et al., nucleosides
and nucleotides, 1986, 5, 65-77, each of which are hereby
incorporated by reference in their entirety.
[0206] The present invention is amenable to the preparation of
oligomers that can have a wide variety of 2'-substituent groups. As
used herein the term "2'-substituent group" includes groups
attached to the 2' position of the sugar moiety with or without an
oxygen atom. 2'-Sugar modifications amenable to the present
invention include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy,
protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, 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 are
hereby incorporated by reference in their 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, now U.S. Pat. No.
6,166,197, hereby incorporated by reference in its entirety.
[0207] Representative 2'-O-- sugar substituents of formula XII are
disclosed in U.S. patent application Ser. No. 09/130,973, filed
Aug. 7, 1998, entitled Capped 2'-Oxyethoxy Oligonucleotides, no
U.S. Pat. No. 6,172,209, hereby incorporated by reference in its
entirety.
[0208] Sugars having O-substitutions on the ribosyl ring are also
amenable to the present invention. Representative substitutions for
ring O include S, CH.sub.2, CHF, and CF.sub.2, see, e.g., Secrist,
et al., Abstract 21, Program & Abstracts, Tenth International
Roundtable, Nucleosides, Nucleotides and their Biological
Applications, Park City, Utah, Sep. 16-20, 1992, hereby
incorporated by reference in its entirety.
[0209] Representative cyclic 2'-O-- sugar substituents of formula
XIII 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.
[0210] In one aspect of the invention, the compounds of the
invention are used to modulate RNA or DNA, which code for a protein
whose formation or activity it is desired to modulate. The
targeting portion of the composition to be employed is, thus,
selected to be complementary to the preselected portion of DNA or
RNA, that is to be hybridizable to that portion.
[0211] The oligomeric compounds and compositions of the invention
can be used in diagnostics, therapeutics and as research reagents
and kits. They can be used in pharmaceutical compositions by
including a suitable pharmaceutically acceptable diluent or
carrier. They further can be used for treating organisms having a
disease characterized by the undesired production of a protein. The
organism should be contacted with an oligonucleotide having a
sequence that is capable of specifically hybridizing with a strand
of nucleic acid coding for the undesirable protein. Treatments of
this type can be practiced on a variety of organisms ranging from
unicellular prokaryotic and eukaryotic organisms to multicellular
eukaryotic organisms. Any organism that utilizes DNA-RNA
transcription or RNA-protein translation as a fundamental part of
its hereditary, metabolic or cellular control is susceptible to
therapeutic and/or prophylactic treatment in accordance with the
invention. Seemingly diverse organisms such as bacteria, yeast,
protozoa, algae, all plants and all higher animal forms, including
warm-blooded animals, can be treated. Further, each cell of
multicellular eukaryotes can be treated, as they include both
DNA-RNA transcription and RNA-protein translation as integral parts
of their cellular activity. Furthermore, many of the organelles
(e.g., mitochondria and chloroplasts) of eukaryotic cells also
include transcription and translation mechanisms. Thus, single
cells, cellular populations or organelles can also be included
within the definition of organisms that can be treated with
therapeutic or diagnostic oligonucleotides.
[0212] As will be recognized, the steps of the methods of the
present invention need not be performed any particular number of
times or in any particular sequence. 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 intended to be illustrative
and not intended to be limiting.
EXAMPLE
Example 1
Comparative Example
Present Invention Versus Prior Art Method of Erjita et al
[0213] Treatment of cyanoethyl protected oligonucleotide
phosphorothioates with ammonium hydroxide results in the generation
of one equivalent of acrylonitrile (AN) per phosphorothioate
linkage. In the presence of ammonium hydroxide a small percentage
of thymidine residues react with the liberated AN to form
N.sup.3-cyanoethylthymidine (CN-T) residues.
[0214] Nonadecathymidinyloctadecaphosphorothioate (T-19 P.dbd.S)
was synthesized and deprotected under three sets of conditions:
[0215] (a) Ammonium hydroxide, 60.degree. C., 16 h;
[0216] (b) Triethylamine-pyridine (2:3 v/v), 25.degree. C., 3 h
then ammonium hydroxide, 60.degree. C., 16 h;
[0217] (c) Triethylamine-acetonitrile (1:1, v/v), 25.degree. C., 12
h, then ammonium hydroxide, 60.degree. C., 16 h.
[0218] The second set of conditions are those recommended by
Erijta. The crude oligonucleotides obtained by evaporation of the
ammonium hydroxide lysates were detritylated and inspected by
liquid chromatography-mass spectroscopy (LC-MS) in order to
quantify the amount of CN-T present. It was shown that the levels
of CN-T in T-19 P.dbd.S samples subjected to conditions a), b) and
c) were ca. 15%, 2% and less than 0.1%, respectively.
[0219] The results demonstrate that the conditions proposed by
Erijta lead to the formation of oligonucleotides that still contain
high levels of CN-T residues, where as the methods of the present
invention suppress CN-T formation to a level below the detection
limit of the assay.
Example 2
[0220] TABLE-US-00001 Synthesis of fully-modified [SEQ ID NO: 1]
5'-d(TTT-TTT-TTT-TTT-TTT-TTT-T)-3' phosphorothioate 20-mer
[0221] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 30 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.45 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
1 minute. At the end of synthesis, the support was washed with a
solution of triethylamine in acetonitrile (1:1, v/v) for 12 h,
cleaved, deprotected and purified in the usual manner.
Example 3
[0222] TABLE-US-00002 Synthesis of fully-modified [SEQ ID NO: 2]
5'-d(GCC-CAA-GCT-GGC-ATC-CGT-CA)-3' phosphoro-thioate 20-mer
[0223] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 160 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.45 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was washed with a
solution of triethylamine in acetonitrile (1:1, v/v) for 12 h,
cleaved, deprotected and purified in the usual manner.
Example 4
[0224] TABLE-US-00003 Synthesis of fully-modified [SEQ ID NO: 1]
5'-d(TTT-TTT-TTT-TTT-TTT-TTT-T)-3' phosphorothioate 20-mer
[0225] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 30 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.45 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
1 minute. At the end of synthesis, the support was transferred to a
container, stirred with a solution of triethylamine in acetonitrile
(1:1, v/v) for 12 h, filtered, then treated with 30% aqueous
ammonium hydroxide, cleaved, deprotected and purified in the usual
manner.
Example 5
[0226] TABLE-US-00004 Synthesis of fully-modified [SEQ ID NO: 2]
5'-d(GCC-CAA-GCT-GGC-ATC-CGT-CA)-3' phosphorothioate 20-mer
[0227] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 160 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.45 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was transferred to
a container, stirred with a solution of triethylamine in
acetonitrile (1:1, v/v) for 12 h, filtered, then treated with 30%
aqueous ammonium hydroxide, cleaved, deprotected and purified in
the usual manner.
Example 6
[0228] TABLE-US-00005 Synthesis of fully-modified [SEQ ID NO: 1]
5'-d(TTT-TTT-TTT-TTT-TTT-TTT-T)-3' phosphorothioate 20-mer
[0229] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 30 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.45 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
1 minutes. At the end of synthesis, the support was taken in 30%
aqueous ammonium hydroxide along with thymidine, cleaved,
deprotected and purified in the usual manner.
Example 7
[0230] TABLE-US-00006 Synthesis of fully-modified [SEQ ID NO: 2]
5'-d(GCC-CAA-GCT-GGC-ATC-CGT-CA)-3' phosphorothioate 20-mer
[0231] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 160 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.45 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was taken in 30%
aqueous ammonium hydroxide along with thymidine, cleaved,
deprotected and purified in the usual manner.
Example 8
[0232] TABLE-US-00007 Synthesis of fully-modified [SEQ ID NO: 1]
5'-d(TTT-TTT-TTT-TTT-TTT-TTT-T)-3' phosphorothioate 20-mer
[0233] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 30 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.4 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
1 minute. At the end of synthesis, the support was taken in 30%
aqueous ammonium hydroxide along with uridine, cleaved, deprotected
and purified in the usual manner.
Example 9
[0234] TABLE-US-00008 Synthesis of fully-modified [SEQ ID NO: 2]
5'-d(GCC-CAA-GCT-GGC-ATC-CGT-CA)-3' phosphorothioate 20-mer
[0235] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 160 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.45 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was taken in 30%
aqueous ammonium hydroxide along with uridine, cleaved, deprotected
and purified in the usual manner.
Example 10
[0236] TABLE-US-00009 Synthesis of fully-modified [SEQ ID NO: 1]
5'-d(TTT-TTT-TTT-TTT-TTT-TTT-T)-3' phosphorothioate 20-mer
[0237] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 30 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.45 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
1 minutes. At the end of synthesis, the support was taken in 30%
aqueous ammonium hydroxide along with imidazole, cleaved,
deprotected and purified in the usual manner.
Example 11
[0238] TABLE-US-00010 Synthesis of fully-modified [SEQ ID NO: 2]
5'-d(GCC-CAA-GCT-GGC-ATC-CGT-CA)-3' phosphorothioate 20-mer
[0239] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 160 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.45 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was taken in 30%
aqueous ammonium hydroxide along with imidazole, cleaved,
deprotected and purified in the usual manner.
Example 12
[0240] ISIS 2302 [5'-d(GCC--CAA-GCT-GGC-ATC--CGT-CA)-3'] [SEQ ID
NO: 2] was manufactured under Good Manufacturing Practice (GMP)
conditions on a Pharmacia OligoProcess Synthesizer on a 150 mmole
scale using the cyanoethyl phosphoramidites obtained from Pharmacia
and Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.4 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was washed with a
solution of triethylamine in acetonitrile (1:1, v/v) for 30 minutes
and then let stand at room temperature overnight, filtered, washed
with acetonitrile solvent and then treated with 30% aqueous
ammonium hydroxide, cleaved, deprotected and purified in the usual
manner. The oligonucleotide was analyzed by mass spectroscopy to
confirm the elimination of acrylonitrile adduct.
Example 13
[0241] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 30 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.4 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was incubated with
thymidine nucleoside (20 equivalents), deprotected and purified in
the usual manner.
Example 14
[0242] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 30 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.4 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was incubated with
uridine nucleoside (20 equivalents), deprotected and purified in
the usual manner.
Example 15
[0243] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 30 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.4 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was incubated with
inosine nucleoside (20 equivalents), deprotected and purified in
the usual manner.
Example 16
[0244] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 30 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.4 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was incubated with
thymine (25 equivalents), deprotected and purified in the usual
manner.
Example 17
[0245] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 30 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.4 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was incubated with
uracil (25 equivalents), deprotected and purified in the usual
manner.
Example 18
[0246] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 30 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.4 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was incubated with
imidazole (50 equivalents), deprotected and purified in the usual
manner.
Example 19
[0247] The synthesis of the above sequence was performed on a
Pharmacia OligoPilot I Synthesizer on a 30 micromole scale using
the cyanoethyl phosphoramidites obtained from Pharmacia and
Pharmacia's HL 30 primary support. Detritylation was performed
using 3% dichloroacetic acid in toluene (volume/volume). Activation
of phosphoramidites was done with a 0.4 M solution of 1H-tetrazole
in acetonitrile. Sulfurization was performed using a 0.2 M solution
of phenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for
2 minutes. At the end of synthesis, the support was incubated with
benzyl mercaptan (50 equivalents), deprotected and purified in the
usual manner.
Examples 20-27
Oligonucleotide Synthesis.
[0248] Oligodeoxynucleotides were assembled on an ABI 380B DNA
Synthesizer using 5'-O-(4,4'-dimethoxytrityl)nucleoside
3'-O-(carboxymethyloxy)acetate derivatized CPG 1 (shown in Scheme 1
below) phosphoramidite chemistry, and either commercial oxidizer or
3H-1,2-benzodithiol-3-one 1,1-dioxide (0.05 M in MeCN) as the
sulfur-transfer reagent. ##STR9## ##STR10##
[0249] Deoxynucleoside CE phosphoramidites protected in a standard
manner (A.sup.bz, C.sup.bz, G.sup.ib) were used to synthesize
oligonucleotides presented in Examples 21, 23-27. Those used for
the preparation of oligonucleotides presented in Examples 20 and 22
were uniformly protected with either phenoxyacetyl (PAC) or
4-(t-butyl)phenoxyacetyl (tBPA) groups.
Example 20
Two Step Deprotection of Oligonucleotides with Secondary Amines in
an Organic Solvent Followed by Methanolic K.sub.2CO.sub.3.
[0250] Deprotection procedure is exemplified on Scheme 2 for
dodecathymidylate 5. After completeness of oligonucleotide
synthesis a solid support-bound 2 was decyanoethylated with either
2M diethylamine or 1M piperidine in MeCN, dioxane, THF, or DMF (3
mL) for 2 to 12 h. The column was washed with dioxane (10 mL) to
give 3. Other amines, for instance, morpholine, pyrrolidine, or
dimethylamine can also be used on this step.
[0251] The oligonucleotide 4 was released from the solid support 3
by treatment with 0.01 to 0.05 M K.sub.2CO.sub.3 in MeOH (2'5 mL
and 2'20 mL for 1 and 15 mmol syntheses, respectively). Each
portion was passed forth and back through the column for 45 min,
neutralized by passing through short column with Dowex 50W'8
(PyH.sup.+; ca. 1 mL). The combined eluates were evaporated to
dryness, co-evaporated with MeCN (10 mL), and dissolved in water.
Target oligonucleotide 4 was isolated by RP HPLC on a Delta Pak 15
mm C18 300 .ANG. column (3.9.times.300 mm and 7.8.times.300 mm for
1 and 15 mmol syntheses, respectively), using 0.1 M NH.sub.4OAc as
buffer A, 80% aq MeCN as buffer B, and a linear gradient from 0 to
60% B in 40 min at a flow rate 1.5 and 5 mL min.sup.-1,
respectively. Collected fractions were evaporated and detritylated
with 80% aq AcOH for 30 min at room temperature. The solvent was
evaporated, the product was re-dissolved in water and desalted by
injecting on to the same column, then washing with water (10 min)
and eluting an oligonucleotide 5 as an ammonium salt with 50% aq
MeCN (20 min). Homogeneity of 5 was characterized by RP HPLC and
capillary electrophoresis. ESMS: 3764.2 (found); 3765.1
(calculated)
[0252] The efficiency of the deprotection method was verified in
preparation of oligonucleotide phosphorothioates 6 and 7 (Isis
1939) and phosphodiester oligonucleotide 8 in 1 to 15 mmol scale.
[0253] 6: C.sub.5A.sub.2T.sub.11 thioate. ESMS: 5628.3 (found);
5629.6 (calculated). [0254] 7: C.sub.5AC.sub.2ACT.sub.2C.sub.4TCTC
thioate. ESMS: 6438.6 (found); 6440.2 (calculated). [0255] 8:
C.sub.5A.sub.2T.sub.11. ESMS: 5355.8 (found); 5356.4
(calculated).
Example 21
[0256] A solid support-bound oligonucleotide was decyanoethylated
with either 2M diethylamine or 1M piperidine, morpholine, or
diethylamine in MeCN, dioxane, THF, or DMF as described in Example
20. Other amines, for instance, morpholine, pyrrolidine, or
dimethylamine can also be used on this step.
[0257] The solid support was treated with conc. aq ammonia for 2 h
at room temperature, the solution was collected and kept at
55.degree. C. for 8 h. On removal of solvent, the residue was
re-dissolved in water and purified as described in Example 20.
[0258] 9: ESMS: 5980.9 (found); 5982.8 (calculated). [0259] 10:
TGCATC.sub.5AG.sub.2C.sub.2AC.sub.2AT [SEQ ID NO: 5] thioate. ESMS:
6287.8 (found); 6288.0 (calculated).
Example 22
[0259] Deprotection of Synthetic Oligonucleotides with Aqueous
Amines.
[0260] Deprotection procedure is exemplified for oligonucleotide
10. A solid support-bound material (20 .mu.mol) was treated with 1
M aq piperidine for 2 h at room temperature. Other amines, for
instance, morpholine, pyrrolidine, diethylamine, dimethylamine,
ethylamine, or methylamine can also be used on this step.
[0261] The solid support was washed with another portion of the
deprotecting reagent, and combined solutions were evaporated under
reduced pressure. Crude 5'-DMTr protected oligonucleotide was
dissolved in water (5 mL) and purified by semipreparative HPLC on a
DeltaPak C18 column (Waters, 15 mm; 300 .ANG.; 25 ' 100 mm) using
0.1 M NH.sub.4OAc as buffer A, 80% aq MeCN as buffer B, and a
linear gradient from 0 to 40% B in 50 min at a flow rate 15 mL
min.sup.-1. Collected fractions were evaporated, the residue was
treated with 80% aq AcOH for 30 min and evaporated to dryness. The
obtained material was dissolved in 50% aq DMSO and loaded onto the
same column. The column was washed with 0.05 M aq NaOAc (15 min)
and water (15 min) at a flow rate 15 mL min.sup.-1. Elution with
60% aq MeCN and evaporation to dryness gave 23.0 mg (20%) of
desalted oligonucleotide 10 (Na.sup.+ salt), ESMS: 6286.4 (found);
6288.0 (calculated).
Example 23
Deprotection of Synthetic Oligonucleotides with Aqueous Secondary
Amines.
[0262] On completeness of oligonucleotide synthesis, a solid
support-bound material (20 mmol) was treated with an aq amine as
described in Example 22. On evaporation of the solution of the
deprotecting reagent, the residue was treated with ammonium
hydroxide for 8 h at 55.degree. C., and the solvent was evaporated.
The product, 6, was isolated and characterized as described in
Example 22.
Example 24
Deprotection of Synthetic Oligonucleotides with Ammonia in the
Presence of Aminoalkyl Resins as Acrylonitrile Scavengers. Method
A.
[0263] On completeness of oligonucleotide synthesis, a solid
support-bound material (20 mmol) is mixed with an aminoalkyl resin
[for instance, aminoalkyl CPG or polymer-bound
tris(2-aminoethyl)amine] and treated with conc. aq ammonia for 2 h
at room temperature. The solid phase is filtered off, and the
deprotection is completed by keeping the solution at 55.degree. C.
for 8 h. The solvent was evaporated, and the product is isolated
and characterized as described in Example 22.
Example 25
Deprotection of Synthetic Oligonucleotides with Ammonia in the
Presence of Aminoalkyl Resins as Acrylonitrile Scavengers. Method
B.
[0264] A solid support-bound material (20 mmol) is treated with a
flow of conc. aq ammonia for 2 h at room temperature. On leaving
the reaction vessel, the solution is passed through a second column
that contained an aminoalkyl resin as in Example 24, and collected.
Optionally, the collected solution may be recycled by passing again
through both columns. When the releasing of oligonucleotide from
CPG is complete, the oligonucleotide solution is collected and
treated as in Example 24.
Example 26
Deprotection of Synthetic Oligonucleotides with Ammonia in the
Presence of Mercaptanes as Acrylonitrile Scavengers.
[0265] A solid support-bound oligonucleotide was treated with conc.
aq ammonia and thiocresol (0.1 M) for 2 h at room temperature, the
solution was collected and kept at 55 .degree. C. for 8 h. On
removal of solvent, the residue was re-dissolved in water and
extracted twice with methylene chloride. The aqueous layer was
collected, and the product, 5, was isolated and characterized as
described in Example 20. Other thiols, for instance, thiophenol,
mercaptoethanol, 1,3-ethanedithiol, or ethanethiol can also be used
as acrylonitrile scavengers.
Example 27
Deprotection of Synthetic Oligonucleotides with Ammonia in the
Presence of Mercaptoalkylated Resins as Acrylonitrile
Scavengers.
[0266] A solid support-bound oligonucleotide is treated as in
Example 25, but the second column contains a mercaptoalkylated
resin (for instance, reported previously mercaptoalkylated
resins.sup.1 or NovaSyn TG thiol resin). The product is isolated
and characterized as described in Example 20. .sup.1. Salo, H.;
Guzaev, A.; Lonnberg, H. Bioconjugate Chem., 1998, 9, 365-371.
[0267] It is intended that each of the patents, applications,
printed publications, and other published documents mentioned or
referred to in this specification be herein incorporated by
reference in their entirety.
[0268] Those skilled in the art will appreciate that numerous
changes and modifications may be made to the preferred embodiments
of the invention and that such changes and modifications may be
made without departing from the spirit of the invention. It is
therefore intended that the appended claims cover all such
equivalent variations as fall within the true spirit and scope of
the invention.
Sequence CWU 1
1
5 1 19 DNA Artificial Sequence Oligonucleotide primer 1 tttttttttt
ttttttttt 19 2 20 DNA Artificial Sequence Oligonucleotide primer 2
gcccaagctg gcatccgtca 20 3 18 DNA Artificial Sequence
Oligonucleotide 3 cccccaattt tttttttt 18 4 20 DNA Artificial
Sequence Oligonucleotide 4 cccccaccac ttcccctctc 20 5 20 DNA
Artificial Sequence Oligonucleotide 5 tgcatccccc aggccaccat 20
* * * * *