U.S. patent application number 14/847150 was filed with the patent office on 2015-12-31 for 5-position modified pyrimidines and their use.
The applicant listed for this patent is SomaLogic, Inc.. Invention is credited to Jeffrey D. Carter, Catherine Fowler, Nebojsa Janjic, John Rohloff.
Application Number | 20150376223 14/847150 |
Document ID | / |
Family ID | 44798979 |
Filed Date | 2015-12-31 |
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United States Patent
Application |
20150376223 |
Kind Code |
A1 |
Rohloff; John ; et
al. |
December 31, 2015 |
5-POSITION MODIFIED PYRIMIDINES AND THEIR USE
Abstract
The present disclosure relates to the field of nucleic acid
chemistry, specifically to 5-position modified uridines as well as
phosphoramidite and triphosphate derivatives thereof. The present
disclosure also relates to methods of making and using the
same.
Inventors: |
Rohloff; John; (Boulder,
CO) ; Janjic; Nebojsa; (Boulder, CO) ; Carter;
Jeffrey D.; (Longmont, CO) ; Fowler; Catherine;
(Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SomaLogic, Inc. |
Boulder |
CO |
US |
|
|
Family ID: |
44798979 |
Appl. No.: |
14/847150 |
Filed: |
September 8, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14069798 |
Nov 1, 2013 |
9163056 |
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14847150 |
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13085256 |
Apr 12, 2011 |
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14069798 |
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61323145 |
Apr 12, 2010 |
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Current U.S.
Class: |
536/26.26 ;
536/26.8; 536/28.54 |
Current CPC
Class: |
A61P 17/04 20180101;
C12N 2310/335 20130101; A61P 17/00 20180101; C07H 19/10 20130101;
C07H 19/06 20130101; C12N 15/115 20130101; A61P 43/00 20180101;
A61P 37/00 20180101; A61P 37/08 20180101; C07H 21/04 20130101 |
International
Class: |
C07H 19/10 20060101
C07H019/10; C07H 19/06 20060101 C07H019/06 |
Claims
1. A C-5 modified aminocarbonylpyrimidine having the following
structure: ##STR00034## wherein R' is selected from the group
consisting of --H, --Ac, -Bz, --CH.sub.2CH.sub.2OCH.sub.3,
--C(O)CH.sub.2OCH.sub.3 and --SiMe.sub.2tBu; R'' is selected from
the group consisting of H, DMT and triphosphate
(--P(O)(OH)--O--P(O)(OH)--O--P(O)(OH).sub.2) or a salt thereof; X
is selected from the group consisting of --H, --OH, --OMe,
--O-allyl, --F, --OEt, --OPr, --OCH.sub.2CH.sub.2OCH.sub.3 and
-azido; R is selected from the group consisting of
--(CH.sub.2).sub.n--R.sup.X1; R.sup.X1 is selected from the group
consisting of: ##STR00035## R.sup.X4 is selected from the group
consisting of a Cl and a nitrile (CN); and n=0-10.
2. A 3'-phosporamidite of a C-5 modified aminocarbonylpyrimidine
having the following structure: ##STR00036## wherein R'' is
selected from the group consisting of H, DMT and triphosphate
(--P(O)(OH)--O--P(O)(OH)--O--P(O)(OH).sub.2) or a salt thereof; X
is selected from the group consisting of --H, --OH, --OMe,
--O-allyl, --F, --OEt, --OPr, --OCH.sub.2CH.sub.2OCH.sub.3 and
-azido; R is selected from the group consisting of
--(CH.sub.2).sub.n--R.sup.X1; R.sup.X1 is selected from the group
consisting of: ##STR00037## R.sup.X4 selected from the group
consisting of a Cl and a nitrile (CN); and n=0-10.
3. A 5'-triphosphate of a C-5 modified aminocarbonylpyrimidine
having the following structure: ##STR00038## wherein R' is selected
from the group consisting of --H, --Ac, -Bz,
--CH.sub.2CH.sub.2OCH.sub.3, --C(O)CH.sub.2OCH.sub.3 and
--SiMe.sub.2tBu; X is selected from the group consisting of --H,
--OH, --OMe, --O-allyl, --F, --OEt, --OPr,
--OCH.sub.2CH.sub.2OCH.sub.3 and -azido; R is selected from the
group consisting of --(CH.sub.2).sub.n--R.sup.X1; R.sup.X1 is
selected from the group consisting of: ##STR00039## R.sup.X4
selected from the group consisting of a Cl and a nitrile (CN); and
n=0-10.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/069,798, filed Nov. 1, 2013. U.S. application Ser. No.
14/069,798 is a continuation of U.S. application Ser. No.
13/085,256, filed Apr. 12, 2011, now abandoned. U.S. application
Ser. No. 13/085,256 claims the benefit of U.S. Provisional
Application Ser. No. 61/323,145, filed Apr. 12, 2010. Each of these
applications is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present disclosure relates to the field of nucleic acid
chemistry, specifically to 5-position modified uridines as well as
phosphoramidites and triphosphates derivatives thereof. The present
disclosure also relates to methods of making and using the same.
The disclosure includes the use of the modified nucleosides as part
of an oligonucleotide or an aptamer.
BACKGROUND
[0003] The following description provides a summary of information
relevant to the present disclosure and is not an admission that any
of the information provided or publications referenced herein is
prior art to the present disclosure.
[0004] There has been considerable interest in developing modified
nucleosides as therapeutic agents, diagnostic agents, and for
incorporation into oligonucleotides. For example, modified
nucleosides such as AZT, ddI, d4T, and others have been used to
treat AIDS. 5-trifluoromethyl-2'-deoxyuridine is active against
herpetic keratitis and
5-iodo-1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)cytosine has
activity against CMV, VZV, HSV-1, HSV-2 and EBV (A Textbook of Drug
Design and Development, Povl Krogsgaard-Larsen and Hans Bundgaard,
Eds., Harwood Academic Publishers, 1991, Ch. 15).
[0005] Modified nucleosides have shown utility in diagnostic
applications. In these applications, the nucleosides are
incorporated into DNA in determinable locations, and various
diagnostic methods are used to determine the location of the
modified nucleosides. These methods include radiolabeling,
fluorescent labeling, biotinylation, and strand cleavage. An
example of strand cleavage involves reaction of the nucleoside with
hydrazine to yield urea nucleosides, then reaction of the urea
nucleoside with piperidine to cause strand cleavage (the
Maxam-Gilbert method).
[0006] Modified nucleosides have also been incorporated into
oligonucleotides. There are several ways in which oligonucleotides
may be useful as therapeutics. Antisense oligonucleotides can bind
certain genetic coding regions in an organism to prevent the
expression of proteins or to block various cell functions. Further,
a process known as the SELEX process, or systematic Evolution of
Ligands for EXponential Enrichment, allows one to identify and
produce oligonucleotides (referred to as "aptamers") that
selectively bind target molecules. The SELEX process is described
in U.S. Pat. No. 5,270,163, the contents of which are hereby
incorporated by reference.
[0007] The SELEX method involves the selection of oligonucleotides
from a mixture of candidates to achieve virtually any desired
criterion of binding affinity and selectivity. Starting from a
random mixture of oligonucleotides, the method involves contacting
the mixture with a target under conditions favorable for binding
(or interacting), partitioning unbound oligonucleotides from
oligonucleotides which have bound to (or interacted with) target
molecules, dissociating the oligonucleotide-target pairs,
amplifying the oligonucleotides dissociated from the
oligonucleotide-target pairs to yield a ligand-enriched mixture of
oligonucleotides, then reiterating the steps of binding,
partitioning, dissociating and amplifying through as many cycles as
desired.
[0008] Modified nucleosides can be incorporated into antisense
oligonucleotides, ribozymes, and oligonucleotides used in or
identified by the SELEX process. These nucleosides can impart in
vivo and in vitro stability of the oligonucleotides to endo and
exonucleases, alter the charge, hydrophilicity or lipophilicity of
the molecule, and/or provide differences in three dimensional
structure.
[0009] Modifications of nucleosides that have been previously
described include 2'-position sugar modifications, 5-position
pyrimidine modifications, 8-position purine modifications,
modifications at exocyclic amines, substitution of 4-thiouridine,
substitution of 5-bromo or 5-iodo-uracil, backbone modifications,
and methylations. Modifications have also included 3' and 5'
modifications such as capping. PCT WO 91/14696, incorporated herein
by reference, describes a method for chemically modifying antisense
oligonucleotides to enhance entry into a cell.
[0010] U.S. Pat. Nos. 5,428,149, 5,591,843, 5,633,361, 5,719,273,
and 5,945,527 which are incorporated herein by reference in their
entirety, describe modifying pyrimidine nucleosides via palladium
coupling reactions. In some embodiments a nucleophile and carbon
monoxide are coupled to pyrimidine nucleosides containing a leaving
group on the 5-position of the pyrimidine ring, preferably forming
ester and amide derivatives.
[0011] A variety of methods have been used to render
oligonucleotides resistant to degradation by exonucleases. PCT WO
90/15065 describes a method for making exonuclease-resistant
oligonucleotides by incorporating two or more phosphoramidite,
phosphoromonothionate and/or phosphorodithionate linkages at the 5'
and/or 3' ends of the oligonucleotide. PCT WO 91/06629 discloses
oligonucleotides with one or more phosphodiester linkages between
adjacent nucleosides replaced by forming an acetal/ketal type
linkage which is capable of binding RNA or DNA.
[0012] It would be advantageous to provide new nucleosides for
therapeutic and diagnostic applications and for inclusion in
oligonucleotides. When incorporated in oligonucleotides, it would
be advantageous to provide new oligonucleotides that exhibit
different high affinity binding to target molecules, and/or show
increased resistance to exonucleases and endonucleases than
oligonucleotides prepared from naturally occurring nucleosides. It
would also be useful to provide nucleotides with modifications that
impart a biological activity other than, or in addition to,
endonuclease and exonuclease resistance.
SUMMARY
[0013] The present disclosure provides 5-position modified uridines
of the following general formula:
##STR00001##
wherein R is selected from the group consisting of
--(CH.sub.2).sub.n--R.sup.X1; R.sup.X1 is selected from the group
consisting of
##STR00002## ##STR00003##
wherein R.sup.X4 is selected from the group consisting of a
branched or linear lower alkyl (C1-C20); halogen (F, Cl, Br, I);
nitrile (CN); boronic acid (BO.sub.2H.sub.2); carboxylic acid
(COOH); carboxylic acid ester (COOR.sup.X2); primary amide
(CONH.sub.2); secondary amide (CONHR.sup.X2); tertiary amide
(CONR.sup.X2R.sup.X3); sulfonamide (SO.sub.2NH.sub.2);
N-alkylsulfonamide (SONHR.sup.X2); wherein R.sup.X2, R.sup.X3 are
independently selected from the group consisting of a branched or
linear lower alkyl (C1-C20); phenyl (C.sub.6H.sub.5); an R.sup.X4
substituted phenyl ring (R.sup.X4C.sub.6H.sub.4), wherein R.sup.X4
is defined above; a carboxylic acid (COOH); a carboxylic acid ester
(COOR.sup.X5), wherein R.sup.X5 is a branched or linear lower alkyl
(C1-C20); and cycloalkyl, wherein
R.sup.X2.dbd.R.sup.X3.dbd.(CH.sub.2)n; wherein n=0-10; wherein X is
selected from the group including, but not limited to --H, --OH,
--OMe, --O-allyl, --F, --OEt, --OPr, --OCH.sub.2CH.sub.2OCH.sub.3
and -azido; wherein R' is selected from the group including, but
not limited to --Ac; -Bz; and --SiMe.sub.2tBu; wherein R'' is
selected from the group including, but not limited to H, DMT and
triphosphate (--P(O)(OH)--O--P(O)(OH)--O--P(O)(OH).sub.2) or a salt
thereof; and wherein
##STR00004##
can be replaced with carbocyclic sugar analogs, .alpha.-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs such as methyl riboside.
[0014] Included are 3'-phosphoramidite and 5'-triphosphate
derivatives of said compounds having the following general
formulas, respectively or salts thereof:
##STR00005##
wherein all moieties are as defined above.
[0015] The compounds of the present disclosure can be incorporated
into oligonucleotides or aptamers using standard synthetic or
enzymatic methods of preparing such compounds.
[0016] Also provided in the present disclosure are methods for
producing the compounds of the present disclosure and the compounds
produced by said methods.
[0017] In one embodiment, a method is provided for preparing a C-5
modified aminocarbonylpyrimidine said method comprising: reacting a
pyrimidine modified at the 5-position with a
trifluoroethoxycarbonyl with an amine in the presence of a base;
and isolating said C-5 modified aminocarbonylpyrimidine.
[0018] In another embodiment, a method is provided for preparing a
3'-phosporamidite of a C-5 modified aminocarbonylpyrimidine said
method comprising: reacting said C-5 modified
aminocarbonylpyrimidine with
cyanoethyldiisopropylchlorophosphoramidite in the presence of a
base; and isolating said 3'-phosporamidite.
[0019] In yet another embodiment, a method is provided for
preparing a 5'-triphosphate of a C-5 modified
aminocarbonylpyrimidine said method comprising:
[0020] a) reacting a C-5 modified aminocarbonylpyrimidine having
the formula:
##STR00006##
wherein R and X are as defined above, with acetic anhydride in the
presence of a base, followed by cleavage of the 5'-DMTgroup with an
acid to form a 3'-acetate of the following structure:
##STR00007##
[0021] b) performing a Ludwig-Eckstein reaction followed by anion
exchange chromatography on the 3'-acetate of step a); and [0022] c)
isolating a 5'-triphosphate of a C-5 modified
aminocarbonylpyrimidine having the following structure or a salt
thereof:
##STR00008##
[0022] DETAILED DESCRIPTION
[0023] Reference will now be made in detail to representative
embodiments of the invention. While the invention will be described
in conjunction with the enumerated embodiments, it will be
understood that the invention is not intended to be limited to
those embodiments. On the contrary, the invention is intended to
cover all alternatives, modifications, and equivalents that may be
included within the scope of the present invention as defined by
the claims.
[0024] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in and are within the scope of the practice of the
present disclosure. The present disclosure is in no way limited to
the methods and materials described.
[0025] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art(s) to which this invention belongs.
Although any methods, devices, and materials similar or equivalent
to those described herein can be used in the practice or testing of
the invention, the preferred methods, devices and materials are now
described.
[0026] All publications, published patent documents, and patent
applications cited in this disclosure are indicative of the level
of skill in the art(s) to which the disclosure pertains. All
publications, published patent documents, and patent applications
cited herein are hereby incorporated by reference to the same
extent as though each individual publication, published patent
document, or patent application was specifically and individually
indicated as being incorporated by reference.
[0027] As used in this disclosure, including the appended claims,
the singular forms "a," "an," and "the" include plural references,
unless the content clearly dictates otherwise, and are used
interchangeably with "at least one" and "one or more." Thus,
reference to "an aptamer" includes mixtures of aptamers, and the
like.
[0028] As used herein, the term "about" represents an insignificant
modification or variation of the numerical value such that the
basic function of the item to which the numerical value relates is
unchanged.
[0029] The term "each" when used herein to refer to a plurality of
items is intended to refer to at least two of the items. It need
not require that all of the items forming the plurality satisfy an
associated additional limitation.
[0030] As used herein, the terms "comprises," "comprising,"
"includes," "including," "contains," "containing," and any
variations thereof, are intended to cover a non-exclusive
inclusion, such that a process, method, product-by-process, or
composition of matter that comprises, includes, or contains an
element or list of elements does not include only those elements
but may include other elements not expressly listed or inherent to
such process, method, product-by-process, or composition of
matter.
[0031] As used herein, the term "nucleotide" refers to a
ribonucleotide or a deoxyribonucleotide, or a modified form
thereof, as well as an analog thereof. Nucleotides include species
that include purines (e.g., adenine, hypoxanthine, guanine, and
their derivatives and analogs) as well as pyrimidines (e.g.,
cytosine, uracil, thymine, and their derivatives and analogs).
Compounds
[0032] In one embodiment, the present disclosure provides compounds
of the following formula:
##STR00009##
wherein R is selected from the group consisting of
--(CH.sub.2).sub.n--R.sup.X1; R.sup.X1 is selected from the group
consisting of
##STR00010## ##STR00011##
wherein R.sup.X4 is selected from the group including, but not
limited to, a branched or linear lower alkyl (C1-C20); halogen (F,
Cl, Br, I); nitrile (CN); boronic acid (BO.sub.2H.sub.2);
carboxylic acid (COOH); carboxylic acid ester (COOR.sup.X2);
primary amide (CONH.sub.2); secondary amide (CONHR.sup.X2);
tertiary amide (CONR.sup.X2R.sup.X3); sulfonamide
(SO.sub.2NH.sub.2); and N-alkylsulfonamide (SONHR.sup.X2); wherein
R.sup.X2, R.sup.X3 are independently selected from the group
including, but not limited to a branched or linear lower alkyl
(C1-C20); phenyl (C.sub.6H.sub.5); an R.sup.X4 substituted phenyl
ring (R.sup.X4C.sub.6H.sub.4), wherein R.sup.X4 is defined above; a
carboxylic acid (COOH); a carboxylic acid ester (COOR.sup.X5),
wherein R.sup.X5 is a branched or linear lower alkyl (C1-C20); and
cycloalkyl, wherein R.sup.X2.dbd.R.sup.X3.dbd.(CH.sub.2)n; wherein
n=0-10; wherein X is selected from the group including, but not
limited to --H, --OH, --OMe, --O-allyl, --F, --OEt, --OPr,
--OCH.sub.2CH.sub.2OCH.sub.3 and -azido; wherein R' is selected
from the group including, but not limited to --H, --Ac, -Bz,
--C(O)CH.sub.2OCH.sub.3, and --SiMe.sub.2tBu; wherein R'' is
selected from the group including, but not limited to --H,
4,4'-dimethoxytrityl (DMT), and triphosphate
(--P(O)(OH)--O--P(O)(OH)--O--P(O)(OH).sub.2) or a salt thereof;
and; wherein
##STR00012##
can be replaced with carbocyclic sugar analogs, .alpha.-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs such as methyl riboside.
[0033] In another embodiment, the present disclosure provides
compounds of the following formula or salts thereof:
##STR00013##
wherein R, R'' and X are as defined above. Compounds of this
general formula are useful for incorporation of the modified
nucleoside into an oligonucleotide by chemical synthesis.
[0034] In yet other embodiments, the present disclosure provides
compounds of the formula or salts thereof:
##STR00014##
wherein R, R' and X are as defined above. Compounds of this general
formula are useful for incorporation of the modified nucleoside
into an oligonucleotide by enzymatic synthesis.
[0035] As used herein, the term "C-5 modified carboxyamideuridine"
or "C-5 modified aminocarbonyluridine" refers to a uridine with a
carboxyamide (--C(O)NH--) modification at the C-5 position of the
uridine including, but not limited to, those moieties (R)
illustrated above. Examples of a C-5 modified carboxyamideuridines
include those described in U.S. Pat. Nos. 5,719,273 and 5,945,527,
as well as, U.S. Provisional Application Ser. No. 61/422,957 (the
'957 application), filed Dec. 14, 2010, entitled "Nuclease
Resistant Oligonucleotides." Representative C-5 modified
pyrimidines include: 5-(N-benzylcarboxyamide)-2'-deoxyuridine
(BndU), 5-(N-benzylcarboxyamide)-2'-O-methyluridine,
5-(N-benzylcarboxyamide)-2'-fluorouridine,
5-(N-isobutylcarboxyamide)-2'-deoxyuridine (iBudU),
5-(N-isobutylcarboxyamide)-2'-O-methyluridine,
5-(N-isobutylcarboxyamide)-2'-fluorouridine,
5-(N-tryptaminocarboxyamide)-2'-deoxyuridine (TrpdU),
5-(N-tryptaminocarboxyamide)-2'-O-methyluridine,
5-(N-tryptaminocarboxyamide)-2'-fluorouridine,
5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2'-deoxyuridine
chloride, 5-(N-naphthylmethylcarboxyamide)-2'-deoxyuridine (NapdU),
5-(N-naphthylmethylcarboxyamide)-2'-O-methyluridine,
5-(N-naphthylmethylcarboxyamide)-2'-fluorouridine or
5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2'-deoxyuridine).
[0036] Specific examples of C-5 modified aminocarbonyluridines,
described herein for purposes of illustration only, include the
following compounds as well as the 5'-triphosphates and
3'-phosphoramidites and salts thereof of said compounds, the
syntheses of which are described in Examples 1-5.
##STR00015##
[0037] Chemical modifications of the C-5 modified uridines
described herein can also be combined with, singly or in any
combination, 2'-position sugar modifications, modifications at
exocyclic amines, and substitution of 4-thiouridine and the
like.
Salts
[0038] It may be convenient or desirable to prepare, purify, and/or
handle a corresponding salt of the compound, for example, a
pharmaceutically-acceptable salt. Examples of pharmaceutically
acceptable salts are discussed in Berge et al. "Pharmaceutically
Acceptable Salts" (1977) J. Pharm. Sci. 66:1-19.
[0039] For example, if the compound is anionic, or has a functional
group which may be anionic (e.g., --COOH may be --COO.sup.-), then
a salt may be formed with a suitable cation. Examples of suitable
inorganic cations include, but are not limited to, alkali metal
ions such as Na.sup.+ and K.sup.+, alkaline earth cations such as
Ca.sup.2+ and Mg.sup.2+, and other cations such as Al.sup.+3.
Examples of suitable organic cations include, but are not limited
to, ammonium ion (i.e., NH.sub.4.sup.+) and substituted ammonium
ions (e.g., NH.sub.3R.sup.x+, NH.sub.2R.sup.x.sub.2.sup.+,
NHR.sup.x.sub.3.sup.+, NR.sup.x.sub.4.sup.+). Examples of some
suitable substituted ammonium ions are those derived from:
ethylamine, diethylamine, dicyclohexylamine, triethylamine,
butylamine, ethylenediamine, ethanolamine, diethanolamine,
piperizine, benzylamine, phenylbenzylamine, choline, meglumine, and
tromethamine, as well as amino acids, such as lysine and arginine.
An example of a common quaternary ammonium ion is
N(CH.sub.3).sub.4.sup.+.
[0040] If the compound is cationic, or has a functional group which
may be cationic (e.g., --NH.sub.2 may be --NH.sub.3.sup.+), then a
salt may be formed with a suitable anion. Examples of suitable
inorganic anions include, but are not limited to, those derived
from the following inorganic acids: hydrochloric, hydrobromic,
hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and
phosphorous.
[0041] Examples of suitable organic anions include, but are not
limited to, those derived from the following organic acids:
2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic,
camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic,
ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic,
glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic,
lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic,
oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic,
phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic,
sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of
suitable polymeric organic anions include, but are not limited to,
those derived from the following polymeric acids: tannic acid,
carboxymethyl cellulose.
[0042] Unless otherwise specified, a reference to a particular
compound also includes salt forms thereof.
Preparation of Oligonucleotides
[0043] In one aspect, the instant disclosure provides methods for
using the modified nucleosides described herein, either alone or in
combination with other modified nucleosides and/or naturally
occurring nucleosides, to prepare modified oligonucleotides. The
automated synthesis of oligodeoxynucleosides is routine practice in
many laboratories (see e.g., Matteucci, M. D. and Caruthers, M. H.,
(1990) J. Am. Chem. Soc., 103:3185-3191, the contents of which are
hereby incorporated by reference). Synthesis of
oligoribonucleosides is also well known (see e.g. Scaringe, S. A.,
et al., Nucleic Acids Res. 18:5433-5441 (1990), hereby incorporated
by reference). As noted above, the phosphoramidites are useful for
incorporation of the modified nucleoside into an oligonucleotide by
chemical synthesis, and the triphosphates are useful for
incorporation of the modified nucleoside into an oligonucleotide by
enzymatic synthesis. (See e.g., Vaught, J. V., et al. (2010) J. Am.
Chem. Soc., 132, 4141-4151; Gait, M. J. "Oligonucleotide Synthesis
a practical approach" (1984) IRL Press (Oxford, UK); Herdewijn, P.
"Oligonucleotide Synthesis" (2005) (Humana Press, Totowa, N.J.
(each of which is incorporated herein by reference in its
entirety).
[0044] As used herein, the terms "modify," "modified,"
"modification," and any variations thereof, when used in reference
to an oligonucleotide, means that at least one of the four
constituent nucleotide bases (i.e., A, G, T/U, and C) of the
oligonucleotide is an analog or ester of a naturally occurring
nucleotide. In some embodiments, the modified nucleotide confers
nuclease resistance to the oligonucleotide. Additional
modifications can include backbone modifications, methylations,
unusual base-pairing combinations such as the isobases isocytidine
and isoguanidine, and the like. Modifications can also include 3'
and 5' modifications, such as capping. Other modifications can
include substitution of one or more of the naturally occurring
nucleotides with an analog, internucleotide modifications such as,
for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.)
and those with charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those with intercalators (e.g.,
acridine, psoralen, etc.), those containing chelators (e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, and those with modified linkages (e.g.,
alpha anomeric nucleic acids, etc.). Further, any of the hydroxyl
groups ordinarily present on the sugar of a nucleotide may be
replaced by a phosphonate group or a phosphate group; protected by
standard protecting groups; or activated to prepare additional
linkages to additional nucleotides or to a solid support. The 5'
and 3' terminal OH groups can be phosphorylated or substituted with
amines, organic capping group moieties of from about 1 to about 20
carbon atoms, polyethylene glycol (PEG) polymers in one embodiment
ranging from about 10 to about 80 kDa, PEG polymers in another
embodiment ranging from about 20 to about 60 kDa, or other
hydrophilic or hydrophobic biological or synthetic polymers.
[0045] Polynucleotides can also contain analogous forms of ribose
or deoxyribose sugars that are generally known in the art,
including 2'-O-methyl, 2'-O-allyl, 2'-O-ethyl, 2'-O-propyl,
2'-O--CH.sub.2CH.sub.2OCH.sub.3,2'-fluoro- or 2'-azido, carbocyclic
sugar analogs, .alpha.-anomeric sugars, epimeric sugars such as
arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,
sedoheptuloses, acyclic analogs and abasic nucleoside analogs such
as methyl riboside. As noted above, one or more phosphodiester
linkages may be replaced by alternative linking groups. These
alternative linking groups include embodiments wherein phosphate is
replaced by P(O)S ("thioate"), P(S)S ("dithioate"),
(O)NR.sup.x.sub.2 ("amidate"), P(O) R.sup.x, P(O)OR.sup.x', CO or
CH.sub.2 ("formacetal"), in which each R.sup.x or R.sup.x' are
independently H or substituted or unsubstituted alkyl (C1-C20)
optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalky, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. Substitution of analogous forms
of sugars, purines, and pyrimidines can be advantageous in
designing a final product, as can alternative backbone structures
like a polyamide backbone, for example.
[0046] If present, a modification to the nucleotide structure can
be imparted before or after assembly of a polymer. A sequence of
nucleotides can be interrupted by non-nucleotide components. A
polynucleotide can be further modified after polymerization, such
as by conjugation with a labeling component.
[0047] As used herein, "nucleic acid," "oligonucleotide," and
"polynucleotide" are used interchangeably to refer to a polymer of
nucleotides and include DNA, RNA, DNA/RNA hybrids and modifications
of these kinds of nucleic acids, oligonucleotides and
polynucleotides, wherein the attachment of various entities or
moieties to the nucleotide units at any position are included. The
terms "polynucleotide," "oligonucleotide," and "nucleic acid"
include double- or single-stranded molecules as well as
triple-helical molecules. Nucleic acid, oligonucleotide, and
polynucleotide are broader terms than the term aptamer and, thus,
the terms nucleic acid, oligonucleotide, and polynucleotide include
polymers of nucleotides that are aptamers but the terms nucleic
acid, oligonucleotide, and polynucleotide are not limited to
aptamers.
[0048] As used herein, the term "at least one nucleotide" when
referring to modifications of a nucleic acid, refers to one,
several, or all nucleotides in the nucleic acid, indicating that
any or all occurrences of any or all of A, C, T, G or U in a
nucleic acid may be modified or not.
[0049] In other aspects, the instant disclosure methods for using
the modified nucleosides described herein, either alone or in
combination with other modified nucleosides and/or naturally
occurring nucleosides, to prepare aptamers and SOMAmers (described
below). In specific embodiments, the aptamers and SOMAmers are
prepared using the general SELEX or improved SELEX process as
described below.
[0050] As used herein, "nucleic acid ligand," "aptamer," "SOMAmer,"
and "clone" are used interchangeably to refer to a non-naturally
occurring nucleic acid that has a desirable action on a target
molecule. A desirable action includes, but is not limited to,
binding of the target, catalytically changing the target, reacting
with the target in a way that modifies or alters the target or the
functional activity of the target, covalently attaching to the
target (as in a suicide inhibitor), and facilitating the reaction
between the target and another molecule. In one embodiment, the
action is specific binding affinity for a target molecule, such
target molecule being a three dimensional chemical structure other
than a polynucleotide that binds to the nucleic acid ligand through
a mechanism which is independent of Watson/Crick base pairing or
triple helix formation, wherein the aptamer is not a nucleic acid
having the known physiological function of being bound by the
target molecule. Aptamers to a given target include nucleic acids
that are identified from a candidate mixture of nucleic acids,
where the aptamer is a ligand of the target, by a method
comprising: (a) contacting the candidate mixture with the target,
wherein nucleic acids having an increased affinity to the target
relative to other nucleic acids in the candidate mixture can be
partitioned from the remainder of the candidate mixture; (b)
partitioning the increased affinity nucleic acids from the
remainder of the candidate mixture; and (c) amplifying the
increased affinity nucleic acids to yield a ligand-enriched mixture
of nucleic acids, whereby aptamers of the target molecule are
identified. It is recognized that affinity interactions are a
matter of degree; however, in this context, the "specific binding
affinity" of an aptamer for its target means that the aptamer binds
to its target generally with a much higher degree of affinity than
it binds to other, non-target, components in a mixture or sample.
An "aptamer," "SOMAmer," or "nucleic acid ligand" is a set of
copies of one type or species of nucleic acid molecule that has a
particular nucleotide sequence. An aptamer can include any suitable
number of nucleotides. "Aptamers" refer to more than one such set
of molecules. Different aptamers can have either the same or
different numbers of nucleotides. Aptamers may be DNA or RNA and
may be single stranded, double stranded, or contain double stranded
or triple stranded regions.
[0051] As used herein, a "SOMAmer" or Slow Off-Rate Modified
Aptamer refers to an aptamer having improved off-rate
characteristics. SOMAmers can be generated using the improved SELEX
methods described in U.S. Publication No. 20090004667, entitled
"Method for Generating Aptamers with Improved Off-Rates.".
[0052] As used herein, "protein" is used synonymously with
"peptide," "polypeptide," or "peptide fragment." A "purified"
polypeptide, protein, peptide, or peptide fragment is substantially
free of cellular material or other contaminating proteins from the
cell, tissue, or cell-free source from which the amino acid
sequence is obtained, or substantially free from chemical
precursors or other chemicals when chemically synthesized.
The SELEX Method
[0053] The terms "SELEX" and "SELEX process" are used
interchangeably herein to refer generally to a combination of (1)
the selection of nucleic acids that interact with a target molecule
in a desirable manner, for example binding with high affinity to a
protein, with (2) the amplification of those selected nucleic
acids. The SELEX process can be used to identify aptamers with high
affinity to a specific target molecule or biomarker.
[0054] SELEX generally includes preparing a candidate mixture of
nucleic acids, binding of the candidate mixture to the desired
target molecule to form an affinity complex, separating the
affinity complexes from the unbound candidate nucleic acids,
separating and isolating the nucleic acid from the affinity
complex, purifying the nucleic acid, and identifying a specific
aptamer sequence. The process may include multiple rounds to
further refine the affinity of the selected aptamer. The process
can include amplification steps at one or more points in the
process. See, e.g., U.S. Pat. No. 5,475,096, entitled "Nucleic Acid
Ligands." The SELEX process can be used to generate an aptamer that
covalently binds its target as well as an aptamer that
non-covalently binds its target. See, e.g., U.S. Pat. No. 5,705,337
entitled "Systematic Evolution of Nucleic Acid Ligands by
Exponential Enrichment: Chemi-SELEX."
[0055] The SELEX process can be used to identify high-affinity
aptamers containing modified nucleotides that confer improved
characteristics on the aptamer, such as, for example, improved in
vivo stability or improved delivery characteristics. Examples of
such modifications include chemical substitutions at the ribose
and/or phosphate and/or base positions. SELEX process-identified
aptamers containing modified nucleotides are described in U.S. Pat.
No. 5,660,985, entitled "High Affinity Nucleic Acid Ligands
Containing Modified Nucleotides," which describes oligonucleotides
containing nucleotide derivatives chemically modified at the 5'-
and 2'-positions of pyrimidines. U.S. Pat. No. 5,580,737, see
supra, describes highly specific aptamers containing one or more
nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F),
and/or 2'-O-methyl (2'-OMe). See also, U.S. Patent Application
Publication No. 20090098549, entitled "SELEX and PHOTOSELEX," which
describes nucleic acid libraries having expanded physical and
chemical properties and their use in SELEX and photoSELEX.
[0056] SELEX can also be used to identify aptamers that have
desirable off-rate characteristics. See U.S. Patent Publication No.
20090004667, entitled "Method for Generating Aptamers with Improved
Off-Rates," which is incorporated herein by reference in its
entirety, describes improved SELEX methods for generating aptamers
that can bind to target molecules. Methods for producing aptamers
and photoaptamers having slower rates of dissociation from their
respective target molecules are described. The methods involve
contacting the candidate mixture with the target molecule, allowing
the formation of nucleic acid-target complexes to occur, and
performing a slow off-rate enrichment process wherein nucleic
acid-target complexes with fast dissociation rates dissociate and
do not reform, while complexes with slow dissociation rates remain
intact. Additionally, the methods include the use of modified
nucleotides in the production of candidate nucleic acid mixtures to
generate aptamers with improved off-rate performance (see U.S.
Patent Publication No. 20090098549, entitled "SELEX and
PhotoSELEX"). (See also U.S. Pat. No. 7,855,054 and U.S. Patent
Publication No. 20070166740). Each of these applications is
incorporated herein by reference in its entirety.
[0057] "Target" or "target molecule" or "target" refers herein to
any compound upon which a nucleic acid can act in a desirable
manner. A target molecule can be a protein, peptide, nucleic acid,
carbohydrate, lipid, polysaccharide, glycoprotein, hormone,
receptor, antigen, antibody, virus, pathogen, toxic substance,
substrate, metabolite, transition state analog, cofactor,
inhibitor, drug, dye, nutrient, growth factor, cell, tissue, any
portion or fragment of any of the foregoing, etc., without
limitation. Virtually any chemical or biological effector may be a
suitable target. Molecules of any size can serve as targets. A
target can also be modified in certain ways to enhance the
likelihood or strength of an interaction between the target and the
nucleic acid. A target can also include any minor variation of a
particular compound or molecule, such as, in the case of a protein,
for example, minor variations in amino acid sequence, disulfide
bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component, which does not substantially
alter the identity of the molecule. A "target molecule" or "target"
is a set of copies of one type or species of molecule or
multimolecular structure that is capable of binding to an aptamer.
"Target molecules" or "targets" refer to more than one such set of
molecules. Embodiments of the SELEX process in which the target is
a peptide are described in U.S. Pat. No. 6,376,190, entitled
"Modified SELEX Processes Without Purified Protein."
Chemical Synthesis
[0058] Methods for the chemical synthesis of compounds provided in
the present disclosure are described herein. These and/or other
well-known methods may be modified and/or adapted in known ways in
order to facilitate the synthesis of additional compounds provided
in the present disclosure.
[0059] With reference to Scheme 1, in one approach the C-5 position
modified aminocarbonylpyrimidines of the instant disclosure are
prepared by reacting a pyrimidine modified at the 5-position with a
trifluoroethoxycarbonyl with an amine in the presence of a base;
and isolating said C-5 modified aminocarbonylpyrimidine.
[0060] In some embodiments, the trifluoroethoxycarbonylpyrimidine
is selected from the group of compounds including, but not limited
to compounds having the following structure:
##STR00016##
wherein X is selected from the group including, but not limited to
--H, --OH, --OMe, --O-allyl, --F, --OEt, --OPr,
--OCH.sub.2CH.sub.2OCH.sub.3 and -azido, and wherein
##STR00017##
can be replaced with carbocyclic sugar analogs, .alpha.-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs such as methyl riboside.
[0061] In some embodiments, the amine is selected from the group
including, but not limited to compounds of the formula RNH.sub.2,
wherein
R is selected from the group consisting of
--(CH.sub.2).sub.n--R.sup.X1; R.sup.X1 is selected from the group
consisting of:
##STR00018## ##STR00019##
wherein R.sup.X4 is selected from the group consisting of a
branched or linear lower alkyl (C1-C20);
##STR00020##
halogen (F, Cl, Br, I); nitrile (CN); boronic acid
(BO.sub.2H.sub.2); carboxylic acid (COOH); carboxylic acid ester
(COOR.sup.X2); primary amide (CONH.sub.2); secondary amide
(CONHR.sup.X2); tertiary amide (CONR.sup.X2R.sup.X3); sulfonamide
(SO.sub.2NH.sub.2); N-alkylsulfonamide (SONHR.sup.X2); wherein
R.sup.X2 and R.sup.X3 are independently selected from the group
consisting of a branched or linear lower alkyl (C1-C20); phenyl
(C.sub.6H.sub.5); an R.sup.X4 substituted phenyl ring
(R.sup.X4C.sub.6H.sub.4), wherein R.sup.X4 is defined above; a
carboxylic acid (COOH); a carboxylic acid ester (COOR.sup.X5),
wherein R.sup.X5 is a branched or linear lower alkyl (C1-C20); and
cycloalkyl, wherein R.sup.X2.dbd.R.sup.X3.dbd.(CH.sub.2)n; and
wherein n=0-10.
[0062] In specific embodiments, the amine is selected from the
group consisting of:
##STR00021##
[0063] In some embodiments the base is a tertiary amine selected
from the group consisting of triethylamine, diisopropylamine and
the like.
[0064] With reference to Scheme 1, the present disclosure also
provides a method for the synthesis of a 3'-phosporamidite of a C-5
modified aminocarbonylpyrimidine comprising: reacting said C-5
modified aminocarbonylpyrimidine with
cyanoethyldiisopropylchlorophosphoramidite in the presence of a
base; and isolating said 3'-phosporamidite. In some embodiments the
C-5 modified aminocarbonylpyrimidine has the following
structure:
##STR00022##
wherein R and X are as defined above. In some embodiments, the base
is a tertiary amine selected from the group consisting of
consisting of triethylamine, diisopropylamine and the like.
[0065] Again with reference to Scheme 1, the present disclosure
also provides a method for the synthesis of a 5'-triphosphate of a
C-5 modified aminocarbonylpyrimidine comprising:
[0066] a) reacting a C-5 modified aminocarbonylpyrimidine having
the formula:
##STR00023##
wherein R and X are as defined above, with acetic anhydride in the
presence of a base, followed by cleavage of the 5'-DMTgroup with an
acid to form a 3'-acetate of the following structure:
##STR00024##
[0067] b) performing a Ludwig-Eckstein reaction followed by anion
exchange chromatography on the 3'-acetate of step a); and
[0068] c) isolating a 5'-triphosphate of a C-5 modified
aminocarbonylpyrimidine having the following structure or a salt
thereof:
##STR00025##
[0069] The base used is selected from the group including, but not
limited to a tertiary amine. In some embodiments the base is
pyridine. The acid used in step a is selected from the group
including, but not limited to dichloroacetic acid, trichloroacetic
acid and 1,1,1,3,3,3-hexafluoro-2-propanol.
[0070] In an alternate approach, the
trifluoroethoxycarbonylpyrimidine has the following structure:
##STR00026##
With reference to Scheme 2, this compound is formed by the reaction
of compound (7) of Scheme 2 with carbon monoxide and
trifluoroethanol in the presence of a palladium catalyst and a
base. The base is selected from the group including, but not
limited to a tertiary amine selected from triethylamine and the
like.
[0071] The present disclosure includes compounds prepared by each
of the above described methods.
EXAMPLES
[0072] The following examples are provided for illustrative
purposes only and are not intended to limit the scope of the
invention as defined by the appended claims. All examples described
herein were carried out using standard techniques, which are well
known and routine to those of skill in the art. Routine molecular
biology techniques described in the following examples can be
carried out as described in standard laboratory manuals, such as
Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd. ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
(2001).
[0073] The following general procedures were employed to produce
the modified nucleosides described in Examples 1-3 and 5. The
nomenclature used herein is based upon the system described by
Matsuda et al. Nucleic Acids Research 1997, 25:2784-2791.
##STR00027##
Example 1
Synthesis of 5'-O-DMT-dU-5-Carboxamides (3a-e)
5'-O-Dimethoxytrityl-5-(4-fluorobenzylaminocarbonyl)-2'-deoxyuridine
(3a)
[0074] The starting material,
5'-O-dimethoxytrityl-5-trifluoroethoxycarbonyl-2'-deoxyuridine (1)
was prepared by the procedure of Matsuda et al (Nomura, Y.; Ueno,
Y.; Matsuda, A. Nucleic Acids Research 1997, 25:2784-2791; Ito, T.,
Ueno, Y.; Matsuda, A. Nucleic Acids Research 2003, 31:2514-2523). A
solution of (1) (9.85 g, 15 mmol), 4-fluorobenzylamine (2a) (2.25
g, 18 mmol, 1.3 eq), triethylamine (4.2 mL, 30 mmol), and anhydrous
acetonitrile (30 mL) was heated under an inert atmosphere at
60-70.degree. C. for 2-24 hours. Quantitative conversion of (1) to
amide (3a) was confirmed by thin layer chromatography (silica gel
60, 5% methanol/dichloromethane) or HPLC. The reaction mixture was
concentrated in vacuo and the residue purified by silica gel flash
chromatography (Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem.
1978, 43:2923) using an eluent of 0-3% methanol in 1%
triethylamine/99% ethyl acetate. Fractions containing pure product
were combined and evaporated. Traces of residual solvents were
removed by co-evaporation with anhydrous acetonitrile, followed by
drying under high vacuum, to afford (3a) as a white solid (6.57 g,
64% yield). .sup.1H-NMR (300 MHz, CD.sub.3CN) .delta. 2.20-2.40
(2H, m), 3.28 (2H, d, J=4.3 Hz), 3.76 (6H, s), 4.01 (1H, dd, J=3.8,
4.2 Hz), 4.26-4.30 (1H, m), 4.48 (2H, bd, J=6.1 Hz), 6.11 (1H, t,
J=6.5 Hz), 6.85-7.46 (13H, m), 7.03-7.36 (4H, m), 8.58 (1H, s),
9.01 (1H, t, J=6.1 Hz). MS (m/z) calcd for
C.sub.38H.sub.36FN.sub.3O.sub.8, 681.25. found 680.4
[M-H].sup.-.
5'-O-Dimethoxytrityl-5-((R)-2-furfurylmethylaminocarbonyl)-2'-deoxyuridine
(3b)
[0075] The compound (3b) was prepared as described for (3a) using
(R)-2-furfurylmethylamine (2b) and isolated as a white solid (9.3
g, 94% yield). The eluent for chromatography was 1%
triethylamine/4% methanol/95% ethyl acetate. .sup.1H-NMR
(CD.sub.3CN) .delta. 1.51-1.57 (1H, m), 1.84-1.94 (3H, m),
2.18-2.38 (2H, m), 3.25-3.52 (4H, m overlap), 3.66-3.93 (3H, m
overlap), 3.78 (6H, s), 3.97-4.02 (1H, m), 4.24-4.29 (1H, m), 6.12
(1H, t, J=6.5), 6.86-7.47 (13H, m), 8.54 (1H, s), 8.83 (1H, bs). MS
(m/z) calcd for C.sub.36H.sub.39N.sub.3O.sub.9, 657.27. found 656.5
[M-H].sup.-.
5'-O-Dimethoxytrityl-5-((S)-2-furfurylmethylaminocarbonyl)-2'-deoxyuridine
(3c)
[0076] The compound (3c) was prepared as described for (3b) using
(S)-2-furfurylmethylamine (2c) and isolated as a white solid (9.9
g, 99% yield). .sup.1H-NMR (CD.sub.3CN) .delta. 1.50-1.59 (1H, m),
1.84-1.95 (3H, m), 2.18-2.40 (2H, m), 3.24-3.50 (4H, m overlap),
3.69-3.97 (3H, m overlap), 3.78 (6H, s), 3.98-4.02 (1H, m),
4.25-4.30 (1H, m), 6.14 (1H, t, J=6.5), 6.87-7.47 (13H, m), 8.54
(1H, s), 8.84 (1H, bs). MS (m/z) calcd for
C.sub.36H.sub.39N.sub.3O.sub.9, 657.27. found 656.5
[M-H].sup.-.
5'-O-Dimethoxytrityl-5-(2-(4-morpholino)ethylaminocarbonyl)-2'-deoxyuridin-
e (3d)
[0077] The compound (3d) was prepared as described for (3a), using
2-(4-morpholino)-ethylamine (2d), and isolated as a white solid
(8.2 g, 80% yield). The eluent for chromatography was 5%
methanol/2% triethylamine/93% dichloromethane. .sup.1H-NMR
(CD.sub.3CN) .delta. 2.21-2.39 (2H, m), 2.39-2.41 (4H, m), 2.48
(2H, t, J=6.2 Hz), 3.27-3.29 (2H, m), 3.41 (2H, dt, J=5.8, 6.2 Hz),
3.61-3.64 (4H, m), 3.78 (6H, s), 3.98-4.02 (1H, m), 4.25-4.30 (1H,
m), 6.10 (1H, t, J=6.4), 6.86-7.47 (13H, m), 8.55 (1H, s), 8.79
(1H, bt, J.about.6 Hz). MS (m/z) calcd for
C.sub.37H.sub.42N.sub.4O.sub.9, 686.30. found 685.7
[M-H].sup.-.
5'-O-Dimethoxytrityl-5-(2-(N-benzimidazolonyl)ethylaminocarbonyl)-2'-deoxy-
uridine (3e)
[0078] The compound (3e) was prepared as described for (3a) using
N-benzimidazolonyl-2-ethylamine (2e) (CAS RN64928-88-7). The eluent
for chromatography was 2% methanol/1% triethylamine/97%
dichloromethane. The pure product was isolated as a tan solid (8.2
g, 74.5% yield). .sup.1H-NMR (CD.sub.3CN) .delta. 2.20-2.36 (2H,
m), 3.27-3.29 (2H, m), 3.60 (2H, q, J=6.5 Hz), 3.758 (3H, s), 3.762
(3H, s), 3.97 (2H, t, J=6.5 Hz), 3.98-4.02 (1H, m), 4.27-4.30 (1H,
m), 6.09 (1H, t, J=6.5 Hz), 6.86-7.48 (13H, m), 6.91-7.10 (4H, m),
8.52 (1H, s), 8.76 (1H, t, J=6.1 Hz). MS (m/z) calcd for
C.sub.40H.sub.39N.sub.5O.sub.9, 733.27. found 732.0
[M-H].sup.-.
Example 2
Synthesis of 5'-O-DMT-Nucleoside CE-Phosphoramidites (4a-4e)
5'-O-Dimethoxytrityl-5-(4-fluorobenzylaminocarbonyl)-3'-O-[(2-cyanoethyl)(-
N,N-diisopropylamino)phosphinyl]-2'-deoxyuridine (4a)
[0079] A solution of DMT-protected nucleoside (3a) (4.00 g, 5.9
mmol) in anhydrous dichloromethane (40 mL) was cooled to
approximately -10.degree. C. under an atmosphere of dry argon.
Diisopropylethylamine (3.1 mL, 17.6 mmol, 3 eq) was added, followed
by dropwise addition of
2-cyanoethyldiisopropylchlorophosphoramidite (1.7 mL, 7.7 mmol, 1.3
eq). The solution was stirred for one hour and complete reaction
was confirmed by thin layer chromatography (silica ge160, ethyl
acetate/hexane). The reaction mixture was partitioned between
ice-cold 2% sodium bicarbonate solution (200 mL) and ethyl acetate
(200 mL). The organic layer was washed with brine, dried over
anhydrous sodium sulfate, filtered, and concentrated. The residue
was purified by silica gel flash chromography using a mobile phase
of 1% triethylamine/99% ethyl acetate. Fractions containing pure
product were combined and evaporated in vacuo (<30.degree. C.).
Traces of residual chromatography solvent were removed by
co-evaporation with anhydrous acetonitrile and drying at high
vacuum to afford (4a) as a white solid foam (4.10 g, 80% yield).
.sup.1H-NMR (CD.sub.3CN, two isomers) .delta. 1.02-1.16 (12H, m),
2.27-2.57 (2H, m), 2.51/2.62 (2H, 2t, J=6.0/6.0 Hz), 3.25-3.37 (2H,
m), 3.50-3.79 (4H, m overlap), 3.738 (3H, s), 3.742 (3H, s),
4.13/4.16 (1H, 2q, J=3.5/3.7 Hz), 4.37-4.43 (1H, m), 4.44-4.47 (2H,
m), 6.09/6.10 (1H, 2t, J=6.4/7.1 Hz), 6.83-7.44 (13H, m), 7.01-7.30
(4H, m), 8.58/8.60 (1H, 2s), 8.98 (1H, b, J.about.5.5 Hz), 9.24
(1H, bs). .sup.31P-NMR (CD.sub.3CN) .delta. 148.01 (s), 148.06 (s).
.sup.19F-NMR (CD.sub.3CN) .delta. -117.65 (m). MS (m/z) calcd for
C.sub.47H.sub.53FN.sub.5O.sub.9P, 881.36. found 880.3
[M-H].sup.-.
5'-O-Dimethoxytrityl-5-((R)-2-furfurylmethylaminocarbonyl)-3'-O-[(2-cyanoe-
thyl)(N,N-diisopropylamino)phosphinyl]-2'-deoxyuridine (4b)
[0080] The compound (4b) was prepared as described for (4a). A 1:1
mixture of diastereomeric phosphoramidites was isolated as a white
solid foam (3.15 g, 62% yield). The eluent for chromatography was
1% treithylamine/20% hexanes/79% ethyl acetate. .sup.1H-NMR
(CD.sub.3CN, two isomers) .delta. 1.14-1.27 (12H, m), 1.51-1.59
(1H, m), 1.86-1.94 (3H, m), 2.27-2.59 (2H, m), 2.54/2.65 (2H, 2t,
J=6.0/5.7 Hz), 3.27-3.38 (2H, m), 3.44-3.97 (9H, m overlap), 3.782
(3H, s), 3.786 (3H, s), 4.11-4.18 (1H, m), 4.39-4.48 (1H, m),
6.11/6.13 (1H, 2t, J=5.6/6.1 Hz), 6.96-7.47 (13H, m), 8.58/8.60
(1H, 2s), 8.75 (1H, bt, J.about.5.4 Hz), 9.36 (1H, bs).
.sup.31P-NMR (CD.sub.3CN) .delta. 148.09 (s), 148.13 (s). MS (m/z)
calcd for C.sub.45H.sub.56N.sub.5O.sub.10P, 857.38. found 856.6
[M-H].sup.-.
5'-O-Dimethoxytrityl-5-((S)-2-furfurylmethylaminocarbonyl)-3'-O-[(2-cyanoe-
thyl)(N,N-diisopropylamino)phosphinyl]-2'-deoxyuridine (4c)
[0081] The compound (4c) was prepared as described for (4b). A 1:1
mixture of diastereomeric phosphoramidites was isolated as a white
solid foam (3.74 g, 74% yield). .sup.1H-NMR (CD.sub.3CN, two
isomers) .delta. 1.14-1.27 (12H, m), 1.51-1.59 (1H, m), 1.86-1.94
(3H, m), 2.28-2.51 (2H, m), 2.53/2.65 (2H, 2t, J=6.0/6.0 Hz),
3.25-3.41 (2H, m), 3.44-4.14 (9H, m overlap), 3.783 (3H, s), 3.786
(3H, s), 4.12-4.19 (1H, m), 4.40-4.49 (1H, m), 6.11/6.13 (1H, 2t,
J=6.3/6.3 Hz), 6.86-7.48 (13H, m), 8.58/8.60 (1H, 2s), 8.75 (1H,
bt, J.about.5.4 Hz), 9.36 (1H, bs). .sup.31P-NMR (CD.sub.3CN)
.delta. 148.09 (s), 148.13 (s). MS (m/z) calcd for
C.sub.45H.sub.56N.sub.5O.sub.10P, 857.38. found 856.5
[M-H].sup.-.
5'-O-Dimethoxytrityl-5-(2-(4-morpholino)ethylaminocarbonyl)-3'-O-[(2-cyano-
ethyl)(N,N-diisopropylamino)phosphinyl]-2'-deoxyuridine (4d)
[0082] The compound (4d) was prepared as described for (4a) except
that the purification used a chromatography eluent of 1%
triethylamine/5% anhydrous ethanol/94% ethyl acetate. The 1:1
mixture of diastereoisomeric phosphoramidites was isolated as a
white solid foam (3.9 g, 75% yield). .sup.1H-NMR (CD.sub.3CN, two
isomers) .delta. 1.04-1.19 (12H, m), 2.28-2.59 (2H, m), 2.43-2.47
(6H, m overlap), 2.53/2.64 (2H, 2t, J=6.2/6.2 Hz), 3.27-3.76 (8H, m
overlap), 3.61-3.65 (4H, m), 3.781 (3H, s), 3.789 (3H, s),
4.12-4.19 (1H, m), 4.39-4.49 (1H, m), 6.11/6.13 (1H, 2t,
J=5.2//5.2), 6.86-7.48 (13H, m), 8.58/8.60 (1H, 2s), 8.78 (1H, bt,
J.about.5.3 Hz), 9.78 (1H, bs). .sup.31P-NMR (CD.sub.3CN) .delta.
148.08 (s), 148.11 (s). MS (m/z) calcd for
C.sub.46H.sub.59N.sub.6O.sub.10P, 886.4. found 885.7
[M-H].sup.-.
5'-O-Dimethoxytrityl-5-(2-(N-benzimidazolonyl)ethylaminocarbonyl)-3'-O-[(2-
-cyanoethyl)(N,N-diisopropylamino)phosphinyl]-2'-deoxyuridine
(4e)
[0083] The compound (4e) was prepared as described for (4a) except
that the purification used a chromatography eluent of 1%
triethylamine/10% anhydrous methanol/89% ethyl acetate. The 1:1
mixture of diastereomeric phosphoramidites was isolated as a white
solid foam (1.6 g, 31% yield). .sup.1H-NMR (CD.sub.3CN, two
isomers) .delta. 1.03-1.18 (12H, m), 2.27-2.57 (2H, m), 2.52/2.63
(2H, 2t, J=6.0/6.0), 3.27-3.37 (2H, m), 3.49-3.80 (6H, m overlap),
3.732 (3H, s), 3.735/3.738 (3H, 2s), 4.00 (2H, bt, J.about.6.0 Hz),
4.12-4.18 (1H, m), 4.30-4.47 (1H, m), 6.08/6.10 (1H, 2t, J=6.3/6.3
Hz), 6.85-7.48 (13H, m), 6.93-7.09 (4H, m), 8.57/8.60 (1H, 2s),
8.82/8.83 (1H, 2bt, J.about.4.3/4.3 Hz), 9.48 (1H, bs).
.sup.31P-NMR (CD.sub.3CN) .delta. 148.07 (s), 148.10 (s).
Example 3
Synthesis of 3'-O-Acetyl-Nucleosides (5a-5e)
5-(4-Fluorobenzylaminocarbonyl)-3'-O-acetyl-2'-deoxyuridine
(5a)
##STR00028##
[0085] The nucleoside (3a) (3.00 g, 4.4 mmol) was dissolved in a
solution of anhydrous pyridine (30 mL) and acetic anhydride (3 mL).
The solution was stirred overnight and concentrated in vacuo to
yield the 3'-O-acetyl-nucleoside. Residual solvent was removed by
co-evaporation with anhydrous toluene (10 mL). The residue was
dissolved in anhydrous dichloromethane (10 mL) and treated with 3%
trichloroacetic acid in dichloromethane (58 mL). The red solution
was stirred overnight, during which time the product crystallized.
The slurry was cooled to -20.degree. C., filtered, and washed with
diethyl ether. The residue was dried in vacuo to afford (5a) as an
off-white solid (1.10 g, 59% yield). .sup.1H-NMR (CD.sub.3CN)
.delta. 2.07 (3H, s), 2.33-2.38 (1H, m), 2.50-2.52 (1H, m),
3.63-3.64 (2H, m), 4.10 (1H, bdd, J=3.1, 5.1 Hz), 4.46 (2H, d,
J=6.0 Hz), 5.19-5.26 (2H, m overlap), 6.15 (1H, t, J=7.0 Hz), 7.15
(2H, tt, J=2.2, 9.0 Hz), 7.31-7.38 (2H, m), 8.79 (1H, s), 9.14 (1H,
bt, J=6.1 Hz), 11.95 (1H, bs). .sup.19F-NMR (CD.sub.3CN) .delta.
-116.02 (tt, J=5.5, 9.0 Hz)). MS (m/z) calcd for
C.sub.19H.sub.20FN.sub.3O.sub.7, 421.13. found 419.8
[M-H].sup.-.
5-((R)-2-Furfurylmethylaminocarbonyl)-3'-O-acetyl-2'-deoxyuridine
(5b)
##STR00029##
[0087] The compound (5b) was prepared from (4b), by the procedure
described for (5a) and isolated by precipitation from a mixture of
dichloromethane and ethyl acetate as a white solid (1.27 g, 73%
yield). .sup.1H-NMR (CDCl.sub.3) .delta. 1.57-2.02 (4H, m), 2.12
(3H, s), 2.46-2.50 (2H, m), 3.03 (1H, bs), 3.43-3.64 (2H, m),
3.75-3.97 (2H, m), 3.78-4.10 (3H. m), 4.20-4.21 (1H, m), 5.40-5.42
(1H, m), 6.35 (1H, dd, J=6.5, 7.7 Hz), 8.91 (1H, t, J=5.5 Hz), 9.17
(1H, s), 9.44 (1H, bs). MS (m/z) calcd for
C.sub.17H.sub.23N.sub.3O.sub.8, 397.15. found 396.1
[M-H].sup.-.
5-((S)-2-Furfurylmethylaminocarbonyl)-3'-O-acetyl-2'-deoxyuridine
(5c)
##STR00030##
[0089] The compound (5c) was prepared from (4c), by the procedure
described for (5a), and isolated by precipitation from a mixture of
dichloromethane and diethyl ether as a slightly orange solid (1.35
g, 77% yield). .sup.1H-NMR (CDCl.sub.3) .delta. 1.57-2.03 (4H, m),
2.12 (3H, s), 2.47-2.51 (2H, m), 2.98 (1H, bs), 3.40-3.68 (2H, m),
3.78-3.95 (2H, m), 3.90-4.12 (3H. m), 4.20-4.21 (1H, m), 5.39-5.42
(1H, m), 6.33 (1H, dd, J=6.7, 7.4 Hz), 8.90 (1H, t, J=5.5 Hz), 9.15
(1H, s), 9.37 (1H, bs). MS (m/z) calcd for
C.sub.17H.sub.23N.sub.3O.sub.8, 397.15. found 395.9
[M-H].sup.-.
5-(2-(4-Morpholino)ethylaminocarbonyl)-3'-O-acetyl-2'-deoxyuridine
(5d)
##STR00031##
[0091] The nucleoside (3d) (1.00 g, 1.37 mmol) was dissolved in a
solution of anhydrous pyridine (10 mL) and acetic anhydride (1 mL).
The solution was stirred overnight and concentrated in vacuo to
yield the 3'-O-acetyl-nucleoside. Residual solvent was removed by
coevaporation with anhydrous toluene (10 mL). The residue was
dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (20 mL) (Leonard, N.
J. Tetrahedron Letters, 1995, 36:7833) and heated at approximately
50.degree. C. for 3 hours. Complete cleavage of the DMT group was
confirmed by tlc. The red solution mixture was quenched by pouring
into well-stirred methanol (200 mL). The resulting yellow solution
was concentrated in vacuo and the residue was dissolved in hot
ethyl acetate (20 mL). The product crystallized upon cooling and
the resulting slurry was aged at -20.degree. C., followed by
filtration and washing with ethyl acetate. The
3'-O-acetyl-nucleoside (5d) was isolated as a white solid (0.46 g,
79% yield). .sup.1H-NMR (DMSO-d6) .delta. 2.07 (3H, s), 2.32-2.45
(7H, m overlap), 2.49-2.52 (1H, m), 3.33-3.40 (2H, m), 3.57 (4H, t,
J=4.5 Hz), 3.60-3.63 (2H, m), 4.09 (1H, bdd, J=3.2, 5.2 Hz),
5.17-5.25 (2H, m), 6.14 (1H, t, J=7.0 Hz), 8.74 (1H, s), 8.89 (1H,
bt, J=5.4 Hz), 11.90 (1H, bs). MS (m/z) calcd for
C.sub.18H.sub.26N.sub.4O.sub.8, 426.18. found 425.0
[M-H].sup.-.
5-(2-(1-(3-Acetyl-benzimidazolonyl))ethylaminocarbonyl)-3'-O-acetyl-2'-deo-
xyuridine (5e)
##STR00032##
[0093] The compound (5e) was prepared as described for (5d) except
that the product crystallized directly when the DMT-cleavage
reaction was poured into methanol. The diacetyl nucleoside (5e) was
isolated by filtration as a white solid (0.55 g, 78% yield).
.sup.1H-NMR (DMSO-d6) .delta. 2.07 (3H, s), 2.30-2.37 (1H, m),
2.49-2.52 (1H, m), 2.63 (3H, s) 3.33 (1H, bs), 3.55-3.64 (4H, m
overlap), 3.99 (2H, t, J=6.4 Hz), 4.09 (1H, bdd, J=2.3, 5.2 Hz),
5.15-5.25 (2H, m), 6.13 (1H, dd, J=6.3, 7.6 Hz), 7.11 (1H, ddd,
J=1.2, 7.6, 7.9 Hz), 7.22 (1H, ddd, J=1.2, 7.6, 7.9 Hz), 7.33 (1H,
dd, J=0.8, 7.9 Hz), 8.02 (1H, dd, J=0.8, 8.0 Hz), 8.05 (1H, bs),
8.83 (1H, bt), 8.71 (1H, s), 11.87 (1H, bs). MS (m/z) calcd for
C.sub.23H.sub.25N.sub.5O.sub.9, 515.17. found 513.9
[M-H].sup.-.
Example 4
Alternative synthesis of 3'-O-Acetyl-Nucleosides (5a-5d)
[0094] The 3'-O-acetyl-nucleosides (5a-d) were also synthesized by
an alternative route (Scheme 2) from the starting material,
3'-O-acetyl-5'-O-dimethoxytrityl-5-iodo-2'-deoxyuridine (7)
(Vaught, J. D., Bock, C., Carter, J., Fitzwater, T., Otis, M.,
Schneider, D., Rolando, J., Waugh, S., Wilcox, S. K., Eaton, B. E.
J. Am. Chem. Soc. 2010, 132, 4141-4151). Briefly, with reference to
Scheme 2, palladium(II)-catalyzed rifluoroethoxycarbonylation of
the iodide afforded the activated ester intermediate (8).
Condensation of (8) with the amines (2a-d) (1.3 eq., triethylamine
(3 eq), acetonitrile, 60-70.degree. C., 2-24 hours), followed by
cleavage of the 5'-O-DMT-protecting group (3% trichloroacetic
acid/dichloromethane or 1,1,1,3,3,3-hexafluoro-2-propanol, room
temperature), afforded (5a-d), identical to the products produced
via intermediates (3a-d) (Scheme 1).
##STR00033##
3'-O-Acetyl-5'-O-dimethoxytrityl-5-(2,2,2-trifluoroethoxycarbonyl)-2'-deo-
xyuridine (8)
[0095] A 500 mL heavy-walled glass pressure reactor was filled with
argon and charged with
3'-O-acetyl-5'-O-dimethoxytrityl-5-iodo-2'-deoxyuridine (7) (15.9
g, 22.8 mmol), anhydrous acetontirile (200 mL), triethylamine (7.6
mL, 54.7 mmol), and 2,2,2-trifluoroethanol (16.4 mL, 228 mmol). The
resulting solution was vigorously stirred and degassed by
evacuation to <100 mmHg for 2 minutes. The flask was filled with
argon and bis(benzonitrile)dichloropalladium(II) (175 mg, 0.46
mmol) was added. The resulting yellow solutionw as again degassed
and then filled with carbon monoxide (99.9%) (Caution Poison Gas!)
from a gas manifold. A pressure of 1-10 psi CO was maintained while
the reaction mixture was vigorously stirred and heated at 60-65
degC for 12 hours. The cooled reaction mixture was filtered
(Caution Poison Gas) to remove black precipitate and concentrated
in vacuo. The orange residue was partitioned with dichloromethane
(120 mL) and 10% sodium bicarbonate (80 mL). The organic layer was
washed with water (40 mL) and dried over sodium sulfate, filtered,
and concentrated to leave a orange foam (17 g). This crude product
could be used as is or further purified by silica gel flash
chromatography with an eluent of 30% hexane/1% triethylamine/69%
ethyl acetate to afford (8) as a colorless solid foam (12.7 g, 80%
yield). .sup.1H-NMR (CD.sub.3CN)) .delta. 2.03 (3H, s), 2.37-2.56
(2H, m), 3.36-3.38 (2H, m), 3.78 (6H, s), 4.15-4.19 (1H, m),
4.37-4.55 (2H, m), 5.21-5.26 (1H, m), 6.09 (1H, t, J=6.1 Hz),
6.84-7.46 (13H, m), 8.53 (1H, s). .sup.19F-NMR (CD.sub.3CN) .delta.
-74.07 (t, J=8.8 Hz). MS (m/z) calcd for
C.sub.35H.sub.33F.sub.3N.sub.2O.sub.10, 698.21. found 697.4
[M-H].sup.-.
Example 5
Synthesis of Nucleoside 5'-O-Triphosphates
5-(4-Fluorobenzylaminocarbonyl)-2'-deoxyuridine-5'-O-triphosphate
(tris-triethylammonium salt) (6a)
[0096] The triphosphate (6a) was synthesized from the
3'-O-acetyl-nucleoside (5a) by the procedure of Ludwig and Eckstein
(Ludwig, J. and Eckstein, F. J. Org. Chem. 1989, 54:631) at 500
.mu.mol-scale (5.times.). The crude triphosphate product, after
ammonolysis and evaporation, was purified by anion exchange
chromatography, as described in the General Procedure (below).
[0097] General Procedure for Anion Exchange HPLC Purification of
Nucleoside Triphosphates.
[0098] Nucleoside triphosphates were purified via anion exchange
chromatography using an HPLC column packed with Source Q resin (GE
Healthcare), installed on a preparative HPLC system, with detection
at 278 nm. The linear elution gradient employed two buffers,
(buffer A: 10 mM triethylammonium bicarbonate/10% acetonitrile, and
buffer B: 1 M triethylammonium bicarbonate/10% acetonitrile), with
the gradient running at ambient temperature from low buffer B
content to high buffer B over the course of the elution. The
desired product was typically the final material to elute from the
column and was observed as a broad peak spanning approximately ten
to twelve minutes retention time (early eluting products included a
variety of reaction by-products, the most significant being the
nucleoside diphosphate). Several fractions were collected during
product elution. Fraction was analyzed by reversed phase HPLC on a
Waters 2795 HPLC with a Waters Symmetry column (PN: WAT054215).
Pure product-containing fractions (typically >90%) were
evaporated in a Genevac VC 3000D evaporator to afford colorless to
light tan resins. Fractions were reconstituted in deionized water
and pooled for final analysis. Product quantitation was performed
by analysis using a Hewlett Packard 8452A Diode Array
Spectrophotometer at 278 nm. Product yields were calculated via the
equation A=.epsilon.CL, where A is the UV absorbance, .epsilon. is
the estimated extinction coefficient and L is the pathlength (1
cm).
[0099] The crude product (6a) was dissolved in approximately 5 mL
of buffer A (Table 1: prep-HPLC Conditions 1). Each purification
injection consisted of a filtered aliquot of approximately 1 mL of
this solution injected into a Waters 625 HPLC with a 486 detector
fitted with a Resource Q 6 mL column (GE Healthcare product code:
17-1179-01) with a mobile phase gradient of 0%-100% buffer B in a
50 minute elution at 12 mL/minute. For (6a) [.epsilon..sub.est.
13,700 cm.sup.-1 M.sup.-1] the isolated purified product was 130
.mu.mol (26% yield). .sup.1H-NMR (D.sub.2O) .delta. 1.15 (27H, t,
J=7.3 Hz), 2.32-2.37 (2H, m), 3.07 (18H, q, J=7.3 Hz), 4.06-4.17
(3H, m overlap), 4.42 (2H, bd, J.about.0.7 Hz), 4.49-4.53 (1H, m),
4.70 (>7H, bs, HOD), 6.12 (1H, t, J=6.8 Hz), 6.96-7.26 (4H, m),
8.45 (1H, s). .sup.19F-NMR (D.sub.2O) .delta. -116.18 (m).
.sup.31P-NMR (D.sub.2O) .delta. -10.58 (d, J=20 Hz), -11.45 (d,
J=20 Hz), -23.29 (t, J=20 Hz). MS (m/z) calcd for
C.sub.17H.sub.21FN.sub.3O.sub.15P.sub.3, 619.02. found 618.0
[M-H].sup.-.
TABLE-US-00001 TABLE 1 Prep-HPLC Conditions 1 Mobile Phase A: 10 mM
triethylammonium bicarbonate/10% acetonitrile B: 1M
triethylammonium bicarbonate/10% acetonitrile Column Resource Q 6
mL HPLC system Waters 625HPLC/486 detector Gradient (% Buffer B in
0%-100% mobile phase) Run Time/flow rate 50 minutes at 12
mL/minute
5-((R)-2-Furfurylmethylaminocarbonyl)-2'-deoxyuridine-5'-O-triphosphate
(tris-triethylammonium salt) (6b)
[0100] The triphosphate (6b) was synthesized from the
3'-O-acetyl-nucleoside (5b) as described for (6a). The crude
product (6b) was purified in a single injection on a Waters 2767
preparatory system with a Waters 2489 detector using a Waters AP-5
column (Waters PN: WAT023331, 50 mm.times.100 mm) packed with 196
mL of Source 15Q resin (GE Healthcare product code: 17-0947-05).
The same buffers as above were used, but the elution gradient was
modified to 25% to 80% buffer B in a 90 minute elution at 50
mL/minute (Table 2: prep-HPLC Conditions 2). A second purification
was performed on a C18 HPLC column to remove residual impurities
(Table 4: prep-HPLC Conditions 4). For (6b) [.epsilon..sub.est
10,200 cm.sup.-1 M.sup.-1] the isolated purified product was 325
.mu.mol (65% yield). .sup.1H-NMR (D.sub.2O) .delta. 1.17 (27H, t,
J=7.3 Hz), 1.49-1.63 (1H, m), 1.77-2.02 (3H, m), 2.34-2.39 (2H, m),
2.85-3.83 (5H, m overlap), 3.08 (18H, q, J=7.3 Hz), 4.01-4.19 (3H,
m overlap), 4.52-4.56 (1H, m), 4.70 (>7H, bs, HOD), 6.15 (1H, t,
J=6.8 Hz), 8.48 (1H, s). .sup.31P-NMR (D.sub.2O) .delta. -10.50 (d,
J=20 Hz), -11.51 (d, J=20 Hz), -23.25 (t, J=20 Hz). MS (m/z) calcd
for C.sub.15H.sub.24FN.sub.3O.sub.16P.sub.3, 595.04. found 594.1
[M-H].sup.-.
TABLE-US-00002 TABLE 2 Prep-HPLC Conditions 2 Mobile Phase A: 10 mM
triethylammonium bicarbonate/10% acetonitrile B: 1M
triethylammonium bicarbonate/10% acetonitrile Column Resource Q 6
mL HPLC system Waters 625HPLC/486 detector Gradient (% Buffer B in
15%-60% mobile phase) Run Time/flow rate 50 minutes at 12
mL/minute
5-((S)-2-Furfurylmethylaminocarbonyl)-2'-deoxyuridine-5'-O-triphosphate
(tris-triethylammonium salt) (6c)
[0101] The triphosphate (6c) was synthesized from the
3'-O-acetyl-nucleoside (5c) as described for (6a). The crude
product (6c) was purified in a single injection on a Waters 2767
preparatory system with a Waters 2489 detector using a Waters AP-5
column (Waters PN: WAT023331, 50 mm.times.100 mm) packed with 196
mL of Source 15Q resin (GE Healthcare product code: 17-0947-05).
The same buffers as above were used, but the elution gradient was
modified to 25% to 80% buffer B in a 90 minute elution at 50
mL/minute (Table 2: prep-HPLC Conditions 2). A second purification
was performed on a C18 HPLC column to remove residual impurities
(Table 4: prep-HPLC Conditions 4). For (6c) [.epsilon..sub.est
10,200 cm.sup.-1 M.sup.-1] the isolated purified product was 255
.mu.mol (51% yield). .sup.1H-NMR (D.sub.2O) .delta. 1.17 (27H, t,
J=7.3 Hz), 1.49-1.63 (1H, m), 1.78-2.01 (3H, m), 2.34-2.39 (2H, m),
2.85-3.82 (5H, m overlap), 3.09 (18H, q, J=7.3 Hz), 4.01-4.19 (3H,
m overlap), 4.52-4.56 (1H, m), 4.70 (>7H, bs, HOD), 6.15 (1H, t,
J=6.7 Hz), 8.48 (1H, s). .sup.31P-NMR (D.sub.2O) .delta. -10.60 (d,
J=20 Hz), -11.42 (d, J=20 Hz), -23.25 (t, J=20 Hz). MS (m/z) calcd
for C.sub.15H.sub.24FN.sub.3O.sub.16P.sub.3, 595.04. found 594.1
[M-H].sup.-.
5-(2-(4-Morpholino)ethylaminocarbonyl)-2'-deoxyuridine-5'-O-triphosphate
(bis-triethylammonium salt) (6d)
[0102] The triphosphate (6d) was synthesized from the
3'-O-acetyl-nucleoside (5d) as described for (6a). The crude
product (6d) was purified with the same equipment and buffers as
used for (6a), but the gradient was modified to run buffer B from
15% to 60% during the 50 minute elution to improve resolution of
products (Table 3: prep-HPLC Conditions 3). For (6d)
[.epsilon..sub.est 10,200 cm.sup.-1 M.sup.-1] the isolated purified
product was 54 .mu.mol (11% yield). .sup.1H-NMR (D.sub.2O) .delta.
1.17 (18H, t, J=7.3 Hz), 2.37-2.41 (2H, m), 2.91-2.98 (2H, m), 3.09
(12H, q, J=7.3 Hz), 3.20-3.27 (4H, m), 3.87-3.90 (4H, m), 3.63-3.68
(2H, m), 4.10-4.18 (3H, m overlap), 4.56-4.60 (1H, m), 4.70
(>7H, bs, HOD), 6.15 (1H, bt, J=6.3 Hz), 8.48 (1H, s).
.sup.31P-NMR (D.sub.2O) .delta. -9.99 (d, J=21 Hz), -11.90 (d, J=20
Hz), -23.19 (t, J=20 Hz). MS (m/z) calcd for
C.sub.16H.sub.27N.sub.4O.sub.16P.sub.3, 624.06. found 623.1
[M-H].sup.-.
TABLE-US-00003 TABLE 3 Prep-HPLC Conditions 3 Mobile Phase A: 10 mM
triethylammonium bicarbonate/10% acetonitrile B: 1M
triethylammonium bicarbonate/10% acetonitrile Column Waters AP-5
with Source Q 196 mL HPLC system Waters 22767HPLC/2489 detector
Gradient (% Buffer B in 25-80% mobile phase) Run Time/flow rate 90
minutes at 50 mL/minute
TABLE-US-00004 TABLE 4 Prep-HPLC Conditions 4 Mobile Phase A: 100
mM triethylammonium B: acetonitrile Column Waters Novapk C18, 19 mm
.times. 300 mm HPLC system Waters 625HPLC/486 detector Gradient (%
Buffer B in 10-25% mobile phase) Run Time/flow rate 30 minutes at
8.5 mL/minute
5-(2-(N-Benzimidazolonyl)ethylaminocarbonyl)-2'-deoxyuridine-5'-O-triphosp-
hate (bis-triethylammonium salt) (6e)
[0103] The triphosphate (6e) was synthesized from the
3'-O-acetyl-nucleoside (5e) as described for (6a). The crude
product (6e) was purified with the same equipment and buffers as
used for (6a), but the gradient was modified to run buffer B from
15% to 60% during the 50 minute elution to improve resolution of
products (Table 3: prep-HPLC Conditions 3). For (6e)
[.epsilon..sub.est 13,700 cm.sup.-1 M.sup.-1] the isolated purified
product was 101 .mu.mol (20% yield). .sup.1H-NMR (D.sub.2O) .delta.
1.17 (18H, t, J=7.3 Hz), 2.17-2.36 (2H, m), 3.09 (12H, q, J=7.3
Hz), 3.60-3.73 (2H, m), 4.01 (2H, t, J=5.4 Hz), 4.03-4.15 (3H, m),
4.45-4.50 (1H, m), 4.70 (>7H, bs, HOD), 6.04 (1H, t, J=6.6 Hz),
6.95-7.12 (4H, m), 8.02 (1H, s). .sup.31P-NMR (D.sub.2O) .delta.
-10.35 (d, J=20 Hz), -11.40 (d, J=20 Hz), -23.23 (t, J=20 Hz). MS
(m/z) calcd for C.sub.19H.sub.24N.sub.5O.sub.16P.sub.3, 671.04.
found 670.1 [M-H].sup.-.
[0104] The foregoing embodiments and examples are intended only as
examples. No particular embodiment, example, or element of a
particular embodiment or example is to be construed as a critical,
required, or essential element or feature of any of the claims.
Further, no element described herein is required for the practice
of the appended claims unless expressly described as "essential" or
"critical." Various alterations, modifications, substitutions, and
other variations can be made to the disclosed embodiments without
departing from the scope of the present invention, which is defined
by the appended claims. The specification, including the examples,
is to be regarded in an illustrative manner, rather than a
restrictive one, and all such modifications and substitutions are
intended to be included within the scope of the invention.
Accordingly, the scope of the invention should be determined by the
appended claims and their legal equivalents, rather than by the
examples given above. For example, steps recited in any of the
method or process claims may be executed in any feasible order and
are not limited to an order presented in any of the embodiments,
the examples, or the claims.
* * * * *