U.S. patent application number 12/206672 was filed with the patent office on 2009-09-03 for 2' deoxy-2'-alkylnucleotide containing nucleic acid.
This patent application is currently assigned to SIRNA THERAPEUTICS INC.. Invention is credited to Leonid Beigelman, Alexander Karpeisky, Anil Modak, Nassim Usman.
Application Number | 20090221807 12/206672 |
Document ID | / |
Family ID | 22817084 |
Filed Date | 2009-09-03 |
United States Patent
Application |
20090221807 |
Kind Code |
A1 |
Usman; Nassim ; et
al. |
September 3, 2009 |
2' Deoxy-2'-Alkylnucleotide Containing Nucleic Acid
Abstract
2'-deoxy-2'-alkylnucleotides useful for stabilizing enzymatic
nucleic acid molecules and antisense molecules.
Inventors: |
Usman; Nassim; (Boulder,
CO) ; Karpeisky; Alexander; (Lafayette, CO) ;
Beigelman; Leonid; (Longmont, CO) ; Modak; Anil;
(Boulder, CO) |
Correspondence
Address: |
MCDONNELL, BOEHNEN, HULBERT AND BERGHOFF, LLP
300 SOUTH WACKER DRIVE, SUITE 3100
CHICAGO
IL
60606
US
|
Assignee: |
SIRNA THERAPEUTICS INC.
San Francisco
CA
|
Family ID: |
22817084 |
Appl. No.: |
12/206672 |
Filed: |
September 8, 2008 |
Related U.S. Patent Documents
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12006585 |
Jan 3, 2008 |
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12206672 |
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11362645 |
Feb 27, 2006 |
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12006585 |
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10104956 |
Mar 21, 2002 |
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11362645 |
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09376687 |
Aug 18, 1999 |
6365374 |
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10104956 |
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08710113 |
Sep 12, 1996 |
5985621 |
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09376687 |
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08218934 |
Mar 29, 1994 |
5639647 |
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Current U.S.
Class: |
536/23.1 |
Current CPC
Class: |
C12N 2310/111 20130101;
C07H 19/048 20130101; C07H 19/04 20130101; C12N 2310/32 20130101;
C12N 2310/3513 20130101; C12N 2310/333 20130101; C12N 2310/335
20130101; C12N 2310/334 20130101; C12N 2310/336 20130101; A61K
48/00 20130101; C12N 2310/122 20130101; C12N 2310/126 20130101;
C12N 15/101 20130101; C12N 15/113 20130101; C12N 2310/322 20130101;
A61K 38/00 20130101; C12N 2310/3523 20130101; C12N 2310/3527
20130101; C12N 15/1136 20130101; C12N 15/1138 20130101; C07H 21/02
20130101; C07H 21/04 20130101; C12N 2310/121 20130101; C12N
2310/123 20130101; C12N 2310/127 20130101; C12N 15/1131 20130101;
C12N 2310/3533 20130101; C12N 2310/3535 20130101; C12N 2310/1241
20130101 |
Class at
Publication: |
536/23.1 |
International
Class: |
C07H 21/02 20060101
C07H021/02 |
Claims
1. A 2'-deoxy-2'-C-allyl nucleoside selected from the group
consisting of 2'-deoxy-2'-C-allyl-beta-D-ribofuranosyl uridine,
2'-deoxy-2'-C-allyl-beta-D-ribofuranosyl cytidine,
2'-deoxy-2'-C-allyl-beta-D-ribofuranosyl adenosine, and
2'-deoxy-2'-C-allyl-beta-D-ribofuranosyl guanosine.
2. An oligonucleotide A RNA comprising one or more 2'-C-allyl
nucleotide 2'-deoxy-2'-C-allyl nucleotides selected from the group
consisting of 2'-deoxy-2'-C-allyl-beta-D-ribofuranosyl uridine,
2'-deoxy-2'-C-allyl-beta-D-ribofuranosyl cytidine,
2'-deoxy-2'-C-allyl-beta-D-ribofuranosyl adenosine, and
2'-deoxy-2'-C-allyl-beta-D-ribofuranosyl guanosine.
3. The oligonucleotide RNA of claim 3, wherein said oligonucleotide
RNA is single stranded.
4. The oligonucleotide RNA of claim 3, wherein said oligonucleotide
RNA is double stranded.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to chemically synthesized ribozymes,
or enzymatic nucleic acid molecules, antisense oligonucleotides and
derivatives thereof.
[0002] The following is a brief history of the discovery and
activity of enzymatic RNA molecules or ribozymes. This history is
not meant to be complete but is provided only for understanding of
the invention that follows. This summary is not an admission that
all of the work described below is prior art to the claimed
invention.
[0003] Prior to the 1970s it was thought that all genes were direct
linear representations of the proteins that they encoded. This
simplistic view implied that all genes were like ticker tape
messages, with each triplet of DNA "letters" representing one
protein "word" in the translation. Protein synthesis occurred by
first transcribing a gene from DNA into RNA (letter for letter) and
then translating the RNA into protein (three letters at a time). In
the mid 1970s it was discovered that some genes were not exact,
linear representations of the proteins that they encode. These
genes were found to contain interruptions in the coding sequence
which were removed from, or "spliced out" of, the RNA before it
became translated into protein. These interruptions in the coding
sequence were given the name of intervening sequences (or introns)
and the process of removing them from the RNA was termed splicing.
After the discovery of introns, two questions immediately arose: i)
why are introns present in genes in the first place, and ii) how do
they get removed from the RNA prior to protein synthesis? The first
question is still being debated, with no clear answer yet
available. The second question, how introns get removed from the
RNA, is much better understood after a decade and a half of intense
research on this question. At least three different mechanisms have
been discovered for removing introns from RNA. Two of these
splicing mechanisms involve the binding of multiple protein factors
which then act to correctly cut and join the RNA. A third mechanism
involves cutting and joining of the RNA by the intron itself, in
what was the first discovery of catalytic RNA molecules.
[0004] Cech and colleagues were trying to understand how RNA
splicing was accomplished in a single-celled pond organism called
Tetrahymena thermophila. They had chosen Tetrahymena thermophila as
a matter of convenience, since each individual cell contains over
10,000 copies of one intron-containing gene (the gene for ribosomal
RNA). They reasoned that such a large number of intron-containing
RNA molecules would require a large amount of (protein) splicing
factors to get the introns removed quickly. Their goal was to
purify these hypothesized splicing factors and to demonstrate that
the purified factors could splice the intron-containing RNA in
vitro. Cech rapidly succeeded in getting RNA splicing to work in
vitro, but something unusual was going on. As expected, splicing
occurred when the intron-containing RNA was mixed with
protein-containing extracts from Tetrahymena, but splicing also
occurred when the protein extracts were left out. Cech proved that
the intervening sequence RNA was acting as its own splicing factor
to snip itself out of the surrounding RNA. They published this
startling discovery in 1982. Continuing studies in the early 1980's
served to elucidate the complicated structure of the Tetrahymena
intron and to decipher the mechanism by which self-splicing occurs.
Many research groups helped to demonstrate that the specific
folding of the Tetrahymena intron is critical for bringing together
the parts of the RNA that will be cut and spliced. Even after
splicing is complete, the released intron maintains its catalytic
structure. As a consequence, the released intron is capable of
carrying out additional cleavage and splicing reactions on itself
(to form intron circles). By 1986, Cech was able to show that a
shortened form of the Tetrahymena intron could carry out a variety
of cutting and joining reactions on other pieces of RNA. The
demonstration proved that the Tetrahymena intron can act as a true
enzyme: i) each intron molecule was able to cut many substrate
molecules while the intron molecule remained unchanged, and ii)
reactions were specific for RNA molecules that contained a unique
sequence (CUCU) which allowed the intron to recognize and bind the
RNA. Zaug and Cech coined the term "ribozyme" to describe any
ribonucleic acid molecule that has enzyme-like properties. Also in
1986, Cech showed that the RNA substrate sequence recognized by the
Tetrahymena ribozyme could be changed by altering a sequence within
the ribozyme itself. This property has led to the development of a
number of site-specific ribozymes that have been individually
designed to cleave at other RNA sequences. The Tetrahymena intron
is the most well-studied of what is now recognized as a large class
of introns. Group I introns. The overall folded structure,
including several sequence elements, is conserved among the Group I
introns, as is the general mechanism of splicing. Like the
Tetrahymena intron, some members of this class are catalytic, i.e.
the intron itself is capable of the self-splicing reaction. Other
Group I introns require additional (protein) factors, presumably to
help the intron fold into and/or maintain its active structure.
While the Tetrahymena intron is relatively large, (413 nucleotides)
a shortened form of at least one other catalytic intron (SunY
intron of phage T4, 180 nucleotides) may prove advantageous not
only because of its smaller size but because it undergoes
self-splicing at an even faster rate than the Tetrahymena
intron.
[0005] Ribonuclease P (RNAseP) is an enzyme comprised of both RNA
and protein components which are responsible for converting
precursor tRNA molecules into their final form by trimming extra
RNA off one of their ends. RNAseP activity has been found in all
organisms tested, but the bacterial enzymes have been the most
studied. The function of RNAseP has been studied since the
mid-1970s by many labs. In the late 1970s, Sidney Altman and his
colleagues showed that the RNA component of RNAseP is essential for
its processing activity; however, they also showed that the protein
component also was required for processing under their experimental
conditions. After Cech's discovery of self-splicing by the
Tetrahymena intron, the requirement for both protein and RNA
components in RNAseP was reexamined. In 1983, Altman and Pace
showed that the RNA was the enzymatic component of the RNAseP
complex. This demonstrated that an RNA molecule was capable of
acting as a true enzyme, processing numerous tRNA molecules without
itself undergoing any change. The folded structure of RNAseP RNA
has been determined, and while the sequence is not strictly
conserved between RNAs from different organisms, this higher order
structure is. It is thought that the protein component of the
RNAseP complex may serve to stabilize the folded RNA in vivo At
least one RNA position important both to substrate recognition and
to determination of the cleavage site has been identified, however
little else is known about the active site. Because tRNA sequence
recognition is minimal, it is clear that some aspect(s) of the tRNA
structure must also be involved in substrate recognition and
cleavage activity. The size of RNAseP RNA (>350 nucleotides),
and the complexity of th6 substrate recognition, may limit the
potential for the use of an RNAseP-like RNA in therapeutics.
However, the size of RNAseP is being trimmed down (a molecule of
only 290 nucleotides functions reasonably well). In addition,
substrate recognition has been simplified by the recent discovery
that RNAseP RNA can cleave small RNAs lacking the natural tRNA
secondary structure if an additional RNA (containing a "guide"
sequence and a sequence element naturally present at the end of all
tRNAs) is present as well.
[0006] Symons and colleagues identified two examples of a
self-cleaving RNA that differed from other forms of catalytic RNA
already reported. Symons was studying the propagation of the
avocado sunblotch viroid (ASV), an RNA virus that infects avocado
plants. Symons demonstrated that as little as 55 nucleotides of the
ASV RNA was capable of folding in such a way as to cut itself into
two pieces. It is thought that in vivo self-cleavage of these RNAs
is responsible for cutting the RNA into single genome-length pieces
during viral propagation. Symons discovered that variations on the
minimal catalytic sequence from ASV could be found in a number of
other plant pathogenic RNAs as well. Comparison of these sequences
revealed a common structural design consisting of three stems and
loops connected by central loop containing many conserved
(invariant from one RNA to the next) nucleotides. The predicted
secondary structure for this catalytic RNA reminded the researchers
of the head of a hammer consisting of three double helical domains,
stems I, II and III and a catalytic core (FIGS. 1 and 2a); thus it
was named as such. Uhlenbeck was successful in separating the
catalytic region of the ribozyme from that of the substrate. Thus,
it became possible to assemble a hammerhead ribozyme from 2 (or 3)
small synthetic RNAs. A 19-nucleotide catalytic region and a
24-nucleotide substrate, representing division of the hammerhead
domain along the axes of stems I and II (FIG. 2b) were sufficient
to support specific cleavage. The catalytic domain of numerous
hammerhead ribozymes have now been studied by both the Uhlenbeck
and Symons groups with regard to defining the nucleotides required
for specific assembly and catalytic activity and determining the
rates of cleavage under various conditions.
[0007] Haseloff and Gerlach showed it was possible to divide the
domains of the hammerhead ribozyme in a different manner, division
of the hammerhead domain along the axes of -stems I and III (FIG.
2c). By doing so, they placed most of the required sequences in the
strand that didn't get cut (the ribozyme) and only a required UH
where H=C, A, U in the strand that did get cut (the substrate).
This resulted in a catalytic ribozyme that could be designed to
cleave any UH RNA sequence embedded within a longer "substrate
recognition" sequence. The specific cleavage of a long mRNA, in a
predictable manner using several such hammerhead ribozymes, was
reported in 1988. A further development was the division of the
catalytic hammerhead domain along the axes of stems III and II
(FIG. 2d, Jeffries and Symons, Nucleic Acids Res. 1989, 17,
1371-1377.)
[0008] One plant pathogen RNA (from the negative strand of the
tobacco ringspot virus) undergoes self-cleavage but cannot be
folded into the consensus hammerhead structure described above.
Bruening and colleagues have independently identified a
50-nucleotide catalytic domain for this RNA. In 1990, Hampel and
Tritz succeeded in dividing the catalytic domain into two parts
that could act as substrate and ribozyme in a multiple-turnover,
cutting reaction (FIG. 3). As with the hammerhead ribozyme, the
hairpin catalytic portion contains most of the sequences required
for catalytic activity while only a short sequence (GUC in this
case) is required in the target. Hampel and Tritz described the
folded structure of this RNA as consisting of a single hairpin and
coined the term "hairpin" ribozyme (Bruening and colleagues use the
term "paperclip" for this ribozyme motif, see, FIG. 3). Continuing
experiments suggest an increasing number of similarities between
the hairpin and hammerhead ribozymes in respect to both binding of
target RNA and mechanism of cleavage. At the same time, the minimal
size of the hairpin ribozyme is still 50-60% larger than the
minimal hammerhead ribozyme.
[0009] Hepatitis Delta Virus (HDV) is a virus whose genome consists
of single-stranded RNA. A small region (.about.80 nucleotides, FIG.
4) in both the genomic RNA, and in the complementary anti-genomic
RNA, is sufficient to support self-cleavage. As the most recently
discovered ribozyme, HDV's ability to self-cleave has only been
studied for a few years, but is interesting because of its
connection to a human disease. In 1991, Been and Perrotta proposed
a secondary structure for the HDV RNAs that is conserved between
the genomic and anti-genomic RNAs and is necessary for catalytic
activity. Separation of the HDV RNA into "ribozyme" and "substrate"
portions has recently been achieved by Been, but the rules for
targeting different substrate RNAs have not yet been determined
fully (see, FIG. 4). Been has also succeeded in reducing the size
of the HDV ribozyme to .about.60 nucleotides.
[0010] The table below lists some of the characteristics of the
ribozymes discussed above:
TABLE-US-00001 TABLE 1 Characteristics of Ribozymes Group I Introns
Size: ~300 to >1000 nucleotides. Requires a U in the target
sequence immediately 5' of the cleavage site. Binds 4-6 nucleotides
at 5' side of cleavage site. Over 75 known members of this class.
Found in Tetrahymena thermophila rRNA, fungal mitochondria,
chloroplasts, phage T4, blue-green algae, and others. RNAseP RNA
(M1 RNA) Size: ~290 to 400 nucleotides. RNA portion of a
ribonucleoprotein enzyme. Cleaves tRNA precursors to form mature
tRNA. Roughly 10 known members of this group all are bacterial in
origin. Hammerhead Ribozyme Size: ~13 to 40 nucleotides. Requires
the target sequence UH immediately 5' of the cleavage site. Binds a
variable number nucleotides on both sides of the cleavage site. 14
known members of this class. Found in a number of plant pathogens
(virusoids) that use RNA as the infectious agent (FIG. 1) Hairpin
Ribozyme Size: ~50 nucleotides. Requires the target sequence GUC
immediately 3' of the cleavage site. Binds 4 nucleotides at 5' side
of the cleavage site and a variable number to the 3' side of the
cleavage site. Only 1 known member of this class. Found in one
plant pathogen (satellite RNA of the tobacco ringspot virus) which
uses RNA as the infectious agent (FIG. 3). Hepatitis Delta Virus
(HDV) Ribozyme Size: ~60 nucleotides (at present). Cleavage of
target RNAs recently demonstrated. Sequence requirements not fully
determined. Binding sites and structural requirements not fully
determined, although no sequences 5' of cleavage site are required.
Only 1 known member of this class. Found in human HDV (FIG. 4).
[0011] Enzymatic nucleic acids act by first binding to a target RNA
(or DNA, see Cech U.S. Pat. No. 5,180,818). Such binding occurs
through the target binding portion of an enzymatic nucleic acid
which is held in close proximity to an enzymatic portion of
molecule that acts to cleave the target RNA. Thus, the enzymatic
nucleic acid first recognizes and then binds a target RNA through
complementary base-pairing, and once bound to the correct site,
acts enzymatically to cut the target RNA. Cleavage of such a target
RNA will destroy its ability to direct synthesis of an encoded
protein. After an enzymatic nucleic acid has bound and cleaved its
RNA target it is released from that RNA to search for another
target and can repeatedly bind and cleave new targets. The
enzymatic nature of a ribozyme is generally advantageous over other
technologies, such as antisense technology (where a nucleic acid
molecule simply binds to a nucleic acid target to block its
translation) since the effective concentration of ribozyme
necessary to effect a therapeutic treatment is lower than that of
an antisense oligonucleotide. This advantage reflects the ability
of the ribozyme to act enzymatically. Thus, a single ribozyme
molecule is able to cleave many molecules of target RNA. In
addition, the ribozyme is a highly specific inhibitor, with the
specificity of inhibition depending not only on the base pairing
mechanism of binding, but also on the mechanism by which the
molecule inhibits the expression of the RNA to which it binds. That
is, the inhibition is caused by cleavage of the RNA target and so
specificity is defined as the ratio of the rate of cleavage of the
targeted RNA over the rate of cleavage of non-targeted RNA. This
cleavage mechanism is dependent upon factors additional to those
involved in base pairing. Thus, it is thought that the specificity
of action of a ribozyme is greater than that of antisense
oligonucleotide binding the same RNA site.
[0012] By the phrase "enzymatic nucleic acid" is meant a catalytic
modified-nucleotide containing nucleic acid molecule that has
complementarity in a substrate binding region to a specified gene
target, and also has an enzymatic activity that specifically
cleaves RNA or DNA in that target. That is, the enzymatic nucleic
acid is able to intramolecularly or intermolecularly cleave RNA or
DNA and thereby inactivate a target RNA or DNA molecule. This
complementarity functions to allow sufficient hybridization of the
enzymatic RNA molecule to the target RNA or DNA to allow the
cleavage to occur. 100% complementarity is preferred, but
complementarity as low as 50-75% may also be useful in this
invention.
[0013] In preferred embodiments of this invention, the enzymatic
nucleic molecule is formed in a hammerhead motif, but may also be
formed in the motif of a hairpin, hepatitis delta virus, group I
intron or RNAseP RNA (in association with an RNA guide sequence).
Examples of such hammerhead motifs are described by Rossi et al.
Aids Research and Human Retroviruses 1992, 8, 183, of hairpin
motifs by Hampel et al. "RNA Catalyst for Cleaving Specific RNA
Sequences," filed Sep. 20, 1989, which is a continuation-in-part of
U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz,
Biochemistry 1989, 28, 4929 and Hampel et al. Nucleic Acids
Research 1990, 18, 299, and an example of the hepatitis delta virus
motif is described by Perrctta and Been, Biochemistry 1992, 31, 16,
of the RNAseP motif by Guerrier-Takada et al. Cell 1983, 35, 849,
and of the Group I intron by Cech et al. U.S. Pat. No. 4,987,071.
These specific motifs are not limiting in the invention and those
skilled in the art will recognize that all that is important in an
enzymatic nucleic acid molecule of this invention is that it has a
specific substrate binding site which is complementary to one or
more of the target gene RNA or DNA regions, and that it have
nucleotide sequences within or surrounding that substrate binding
site which impart an RNA or DNA cleaving activity to the
molecule.
[0014] The invention provides a method for producing a class of
enzymatic cleaving agents or antisense molecules which exhibit a
high degree of specificity for the RNA or DNA of a desired target.
The enzymatic nucleic acid or antisense molecule is preferably
targeted to a highly conserved sequence region of a target such
that specific treatment of a disease or condition can be provided
with a single enzymatic nucleic acid. Such nucleic acid molecules
can be delivered exogenously to specific cells as required. In the
preferred hammerhead motif the small size (less than 60
nucleotides, preferably between 30-40 nucleotides in length) of the
molecule allows the cost of treatment to be reduced compared to
other ribozyme motifs.
[0015] Synthesis of nucleic acids greater than 100 nucleotides in
length is difficult using automated methods, and the therapeutic
cost of such molecules is prohibitive. In this invention, small
enzymatic nucleic acid motifs (e.g., of the hammerhead structure)
are used for exogenous delivery. The simple structure of these
molecules increases the ability of the enzymatic nucleic acid to
invade targeted regions of the mRNA structure. Unlike the situation
when the hammerhead structure is included within longer
transcripts, there are no non-enzymatic nucleic acid flanking
sequences to interfere with correct folding of the enzymatic
nucleic acid structure or with complementary regions.
[0016] Eckstein et al. International Publication No. WO 92/07065,
Perrault et al. Nature 1990, 344, 565-568, Pieken et al. Science
1991, 253, 314-317, Usman, N.; Cedergren, R. J. Trends in Biochem.
Sci. 1992, 17, 334-339, Usman et al. U.S. patent application Ser.
No. 07/829,729, and Sproat, B. European Patent Application
92110298.4 describe various chemical modifications that can be made
to the sugar moieties of enzymatic RNA molecules. Usman et al. also
describe various required ribonucleotides in a ribozyme, and
methods by which such nucleotides can be defined. De Mesmaeker et
al. Syn. Lett. 1993, 677-680 (not admitted to be prior art to the
present invention) describes the synthesis of certain 2'-C-alkyl
uridine and thymidine derivatives. They conclude that " . . . their
use in an antisense approach seems to be very limited."
SUMMARY OF THE INVENTION
[0017] This invention relates to the use of
2'-deoxy-2'-alkylnucleotides in oligonucleotides, which are
particularly useful for enzymatic cleavage of RNA or
single-stranded DNA, and also as antisense oligonucleotides. As the
term is used in this application,
2'-deoxy-2'-alkylnucleotide-containing enzymatic nucleic acids are
catalytic nucleic molecules that contain
2'-deoxy-2'-alkylnucleotide components replacing, but not limited
to, double stranded stems, single stranded "catalytic core"
sequences, single-stranded loops or single-stranded recognition
sequences. These molecules are able to cleave (preferably,
repeatedly cleave) separate RNA or DNA molecules in a nucleotide
base sequence specific manner. Such catalytic nucleic acids can
also act to cleave intramolecularly if that is desired. Such
enzymatic molecules can be targeted to virtually any RNA
transcript.
[0018] Also within the invention are 2'-deoxy-2'-alkylnucleotides
which may be present in enzymatic nucleic acid or even in antisense
oligonucleotides. Contrary to the findings of De Mesmaeker et al.
applicant has found that such nucleotides are useful since they
enhance the stability of the antisense or enzymatic molecule, and
can be used in locations which do not affect the desired activity
of the molecule. That is, while the presence of the 2'-alkyl group
may reduce binding affinity of the oligonucleotide containing this
modification, if that moiety is not in an essential base pair
forming region then the enhanced stability that it provides to the
molecule is advantageous. In addition, while the reduced binding
may reduce enzymatic activity, the enhanced stability may make the
loss of activity of less consequence. Thus, for example, if a
2'-deoxy-2'-alkyl-containing molecule has 10% the activity of the
unmodified molecule, but has 10-fold higher stability in viva then
it has utility in the present invention. The same analysis is true
for antisense oligonucleotides containing such modifications. The
invention also relates to novel intermediates useful in the
synthesis of such nucleotides and oligonucleotides (examples of
which are shown in the Figures), and to methods for their
synthesis.
[0019] Thus, in a first aspect, the invention features
2'-deoxy-2'-alkylnucleotides, that is a nucleotide base having at
the 2'-position on the sugar molecule an alkyl moiety and in
preferred embodiments features those where the nucleotide is not
uridine or thymidine. That is, the invention preferably includes
all those nucleotides useful for making enzymatic nucleic acids or
antisense molecules that are not described by the art discussed
above.
[0020] Examples of various alkyl groups useful in this invention
are shown in FIG. 6, where each R group is any alkyl. These
examples are not limiting in the invention. Specifically, an
"alkyl" group refers to a saturated aliphatic hydrocarbon,
including straight-chain, branched-chain, and cyclic alkyl groups.
Preferably, the alkyl group has 1 to 12 carbons. More preferably it
is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4
carbons. The alkyl group may be substituted or unsubstituted. When
substituted the substituted group(s) is preferably, hydroxyl,
cyano, alkoxy, .dbd.O, .dbd.S, NO.sub.2 or N(CH.sub.3).sub.2,
amino, or SH. The term also includes alkenyl groups which are
unsaturated hydrocarbon groups containing at least one
carbon-carbon double bond, including straight-chain,
branched-chain, and cyclic groups. Preferably, the alkenyl group
has 1 to 12 carbons. More preferably it is a lower alkenyl of from
1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group
may be substituted or unsubstituted. When substituted the
substituted group(s) is preferably, hydroxyl, cyano, alkoxy,
.dbd.O, .dbd.S, NO.sub.2, halogen, N(CH.sub.3).sub.2, amino, or SH.
The term "alkyl" also includes alkynyl groups which have an
unsaturated hydrocarbon group containing at least one carbon-carbon
triple bond, including straight-chain, branched-chain, and cyclic
groups. Preferably, the alkynyl group has 1 to 12 carbons. More
preferably it is a lower alkynyl of from 1 to 7 carbons, more
preferably 1 to 4 carbons. The alkynyl group may be substituted or
unsubstituted. When substituted the substituted group(s) is
preferably, hydroxyl, cyano, alkoxy, .dbd.O, .dbd.S, NO.sub.2 or
N(CH.sub.3).sub.2, amino or SH. The term "alkyl" does not include
alkoxy groups which have an "--O-alkyl" group, where "alkyl" is
defined as described above, where the O is adjacent the 2'-position
of the sugar molecule.
[0021] Such alkyl groups may also include aryl, alkylaryl,
carbocyclic aryl, heterocyclic aryl, amide and ester groups. An
"aryl" group refers to an aromatic group which has at least one
ring having a conjugated pi electron system and includes
carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which
may be optionally substituted. The preferred substituent(s) of aryl
groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy,
alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group
refers to an alkyl group (as described above) covalently joined to
an aryl group (as described above. Carbocyclic aryl groups are
groups wherein the ring atoms on the aromatic ring are all carbon
atoms. The carbon atoms are optionally substituted. Heterocyclic
aryl groups are groups having from 1 to 3 heteroatoms as ring atoms
in the aromatic ring and the remainder of the ring atoms are carbon
atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,
and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl
pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all
optionally substituted. An "amide" refers to an --C(O)--NH--R,
where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester"
refers to an --C(O)--OR', where R is either alkyl, aryl, alkylaryl
or hydrogen.
[0022] In other aspects, also related to those discussed above, the
invention features oligonucleotides having one or more
2'-deoxy-2'-alkylnucleotides (preferably not a 2'-alkyl-uridine or
thymidine); e.g. enzymatic nucleic acids having a
2'-deoxy-2'-alkylnucleotide; and a method for producing an
enzymatic nucleic acid molecule having enhanced activity to cleave
an RNA or single-stranded DNA molecule, by forming the enzymatic
molecule with at least one nucleotide having at its 2'-position an
alkyl group. In other related aspects, the invention features
2'-deoxy-2'-alkylnucleotide triphosphates. These triphosphates can
be used in standard protocols to form useful oligonucleotides of
this invention.
[0023] The 2'-alkyl derivatives of this invention provide enhanced
stability to the oligonucleotides containing them. While they may
also reduce absolute activity in an in vitro assay they will
provide enhanced overall activity in vivo. Below are provided
assays to determine which such molecules are useful. Those in the
art will recognize that equivalent assays can be readily
devised.
[0024] In another aspect, the invention features hammerhead motifs
having enzymatic activity having ribonucleotides at locations shown
in FIG. 5 at 5, 6, 8, 12, and 15.1, and having substituted
ribonucleotides at other positions in the core and in the substrate
binding arms if desired. (The term "core" refers to positions
between bases 3 and 14 in FIG. 5, and the binding arms correspond
to the bases from the 3'-end to base 15.1, and from the 5'-end to
base 2). Applicant has found that use of ribonucleotides at these
five locations in the core provide a molecule having sufficient
enzymatic activity even when modified nucleotides are present at
other sites in the motif. Other such combinations of useful
ribonucleotides can be determined as described by Usman et al.
supra.
[0025] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0026] The drawings will first briefly be described.
DRAWINGS
[0027] FIG. 1 is a diagrammatic representation of the hammerhead
ribozyme domain known in the art, numbered according to Hertel et
al. Nucleic Acids Res. 1992, 20, 3252.
[0028] FIG. 2a is a diagrammatic representation of the hammerhead
ribozyme domain known in the art;
[0029] FIG. 2b is a diagrammatic representation of the hammerhead
ribozyme as divided by Uhlenbeck (Nature 1987, 327, 596-600) into a
substrate and enzyme portion;
[0030] FIG. 2c is a similar diagram showing the hammerhead divided
by Haseloff and Gerlach (Nature 1988, 334, 585-591) into two
portions; and
[0031] FIG. 2d is a similar diagram showing the hammerhead divided
by Jeffries and Symons (Nucleic Acids Res. 1989, 17, 1371-1371)
into two portions.
[0032] FIG. 3 is a representation of general the structure of the
hairpin ribozyme domain known in the art.
[0033] FIG. 4 is a representation of the general structure of the
hepatitis delta virus ribozyme domain known in the art.
[0034] FIG. 5 is a diagrammatic representation of a position
numbered hammerhead ribozyme (according to Hertel et al. Nucleic
Acids Res. 1992, 20, 3252) showing specific substitutions.
[0035] FIG. 6 shows the structures of various 2'-alkyl modified
nucleotides which exemplify those of this invention. R groups are
alkyl groups, Z is a protecting group.
[0036] FIG. 7 is a diagrammatic representation of the synthesis of
2'-C-allyl uridine and cytidine.
[0037] FIG. 8 is a diagrammatic representation of the synthesis of
2'-C-methylene and 2'-C-difluoromethylene uridine.
[0038] FIG. 9 is a diagrammatic representation of the synthesis of
2'-C-methylene and 2'-C-difluoromethylene cytidine.
[0039] FIG. 10 is a diagrammatic representation of the synthesis of
2'-C-methylene and 2'-C-difluoromethylene adenosine.
[0040] FIG. 11 is a diagrammatic representation of the synthesis of
2'-C-carboxymethylidine uridine, 2'-C-methoxycarboxymethylidine
uridine and derivatized amidites thereof. X is CH.sub.3 or alkyl as
discussed above, or another substituent.
[0041] Table 2 is a summary of specified catalytic parameters
(t.sub.A and t.sub.S) on short substrates in vitro, and stabilities
of the noted modified catalytic nucleic acids in human serum. U4
and U7 refer to the uracil bases noted in FIG. 1. Modifications at
the 2'-position are shown in the table.
TABLE-US-00002 TABLE 2 t.sub.1/2 (m) t.sub.1/2 (m) Activity
Stability .beta. = t.sub.S/t.sub.A .times. Entry Modification
(t.sub.A) (t.sub.S) 10 1 U4 & U7 = U 1 0.1 1 2 U4 & U7 =
2'-O-Me-U 4 260 650 3 U4 = 2' = CH.sub.2-U 6.5 120 180 4 U7 = 2' =
CH.sub.2-U 8 280 350 5 U4 & U7 = 2' = CH.sub.2-U 9.5 120 130 6
U4 = 2' = CF.sub.2-U 5 320 640 7 U7 = 2' = CF.sub.2-U 4 220 550 8
U4 & U7 = 2' = CF.sub.2-U 20 320 160 9 U4 = 2'-F-U 4 320 800 10
U7 = 2'-F-U 8 400 500 11 U4 & U7 = 2'-F-U 4 300 750 12 U4 =
2'-C-Allyl-U 3 >500 >1700 13 U7 = 2'-C-Allyl-U 3 220 730 14
U4 & U7 = 2'-C-Allyl-U 3 120 400 15 U4 = 2'-araF-U 5 >500
>1000 16 U7 = 2'-araF-U 4 350 875 17 U4 & U7 = 2'-araF-U 15
500 330 18 U4 = 2'-NH.sub.2-U 10 500 500 19 U7 = 2'-NH.sub.2-U 5
500 1000 20 U4 & U7 = 2'-NH.sub.2-U 2 300 1500 21 U4 = dU 6 100
170 22 U4 & U7 = dU 4 240 600
[0042] FIG. 5 shows base numbering of a hammerhead motif in which
the numbering of various nucleotides in a hammerhead ribozyme is
provided. This is not to be taken as an indication that the Figure
is prior art to the pending claims, or that the art discussed is
prior art to those claims. Referring to FIG. 5, the preferred
sequence of a hammerhead ribozyme in a 5'- to 3'-direction of the
catalytic core is CUGANGAG[base paired with]CGAAA. In this
invention, the use of 2'-C-alkyl substituted nucleotides that
maintain or enhance the catalytic activity and or nuclease
resistance of the hammerhead ribozyme is described. Although
substitutions of any nucleotide with any of the modified
nucleotides shown in FIG. 6 are possible, and were indeed
synthesized, the basic structure composed of primarily 2'-O-Me
nucleotides with selected substitutions was chosen to maintain
maximal catalytic activity (Yang et al. Biochemistry 1992, 31,
5005-5009 and Paolella et al. EMBO J. 1992, 11, 1913-1919) and ease
of synthesis, but is not limiting to this invention.
[0043] Ribozymes from FIG. 5 and Table 2 were synthesized and
assayed for catalytic activity and nuclease resistance. With the
exception of entries 8 and 17, all of the modified ribozymes
retained at least 1/10 of the wild-type catalytic activity. From
Table 2, all 2'-modified ribozymes showed very large and
significant increases in stability in human serum (shown) and in
the other fluids described below (Example 3, data not shown). The
order of most aggressive nuclease activity was fetal bovine
serum>human serum>human plasma>human synovial fluid. As an
overall measure of the effect of these 2'-substitutions on
stability and activity, a ratio .beta. was calculated (Table 2).
This .beta. value indicated that all modified ribozymes tested had
significant, >100->1700 fold, increases in overall stability
and activity. These increases in .beta. indicate that the lifetime
of these modified ribozymes in vivo are significantly increased
which should lead to a more pronounced biological effect.
[0044] More general substitutions of the 2'-modified nucleotides
from FIG. 6 also increased the t.sub.1/2 of the resulting modified
ribozymes. However the catalytic activity of these ribozymes was
decreased >10-old.
[0045] In FIG. 11 compound 37 may be used as a general intermediate
to prepare derivatized 2'-C-alkyl phosphoramidites, where X is
CH.sub.3, or an alkyl, or other group described above.
EXAMPLES
[0046] The following are non-limiting examples showing the
synthesis of nucleic acids using 2'-C-alkyl substituted
phosphoramidites, the syntheses of the amidites, their testing for
enzymatic activity and nuclease resistance.
Example 1
Synthesis of Hammerhead Ribozymes Containing
2'-Deoxy-2'-Alkylnucleotides & Other 2'-Modified
Nucleotides
[0047] The method of synthesis used generally follows the procedure
for normal RNA synthesis as described in Usman, N.; Ogilvie, K. K.;
Jiang, M. -Y.; Cedergren, R. J. J. Am. Chem. Soc. 1987, 109,
7845-7854 and in Scaringe, S. A.; Franklyn, C.; Usman, N. Nucleic
Acids Res. 1990, 18, 5433-5441 and makes use of common nucleic acid
protecting and coupling groups, such as dimethoxytrityl at the
5'-end, and phosphoramidites at the 3'-end (compounds 10, 12, 17,
22, 31, 18, 26, 32, 36 and 38). Other 2'-modified phosphoramidites
were prepared according to: 3 & 4, Eckstein et al.
International Publication No. WO 92/07065; and 5 Kois et al.
Nucleosides & Nucleotides 1993, 12, 1093-1109. The average
stepwise coupling yields were .about.98%. The 2'-substituted
phosphoramidites were incorporated into hammerhead ribozymes as
shown in FIG. 5. However, these 2'-alkyl substituted
phosphoramidites may be incorporated not only into hammerhead
ribozymes, but also into hairpin, hepatitis delta virus, Group 1 or
Group 2 intron catalytic nucleic acids, or into antisense
oligonucleotides. They are, therefore, of general use in any
nucleic acid structure.
Example 2
Ribozyme Activity Assay
[0048] Purified 5'-end labeled RNA substrates (15-25-mers) and
purified 5'-end labeled ribozymes (.about.36-mers) were both heated
to 95.degree. C., quenched on ice and equilibrated at 37.degree.
C., separately. Ribozyme stock solutions were 1 mM, 200 nM, 40 nM
or 8 nM and the final substrate RNA concentrations were .about.1
nM. Total reaction volumes were 50 mL. The assay buffer was 50 mM
Tris-Cl, pH 7.5 and 10 mM MgCl.sub.2. Reactions were initiated by
mixing substrate and ribozyme solutions at t=0. Aliquots of 5 mL
were removed at time points of 1, 5, 15, 30, 60 and 120 m. Each
time point was quenched in formamide loading buffer and loaded onto
a 15% denaturing polyacrylamide gel for analysis. Quantitative
analyses were performed using a phosphorimager (Molecular
Dynamics).
Example 3
Stability Assay
[0049] 500 pmol of gel-purified 5'-end-labeled ribozymes were
precipitated in ethanol and pelleted by centrifugation. Each pellet
was resuspended in 20 mL of appropriate fluid (human serum, human
plasma, human synovial fluid or fetal bovine serum) by vortexing
for 20 s at room temperature. The samples were placed into a
37.degree. C. incubator and 2 mL aliquots were withdrawn after
incubation for 0, 15, 30, 45, 60, 120, 240 and 480 m. Aliquots were
added to 20 mL of a solution containing 95% formamide and 0.5X TBE
(50 mM Tris, 50 mM borate, 1 mM EDTA) to quench further nuclease
activity and the samples were frozen until loading onto gels.
Ribozymes were size-fractionated by electrophoresis in 20%
acrylamide/8M urea gels. The amount of intact ribozyme at each time
point was quantified by scanning the bands with a phosphorimager
(Molecular Dynamics) and the half-life of each ribozyme in the
fluids was determined by plotting the percent intact ribozyme vs
the time of incubation and extrapolation from the graph.
Example 4
[0050]
3',5'-O-(Tetraisopropyl-disiloxane-1,3-diyl)-2'-O-Phenoxythio-carbo-
nyl-Uridine (7)
[0051] To a stirred solution of
3',5'-O-(tetraisopropyl-disiloxane-1,3-diyl)-uridine, 6, (15.1 g,
31 mmol, synthesized according to Nucleic Acid Chemistry, ed. Leroy
Townsend, 1986 pp. 229-231) and dimethylamino-pyridine (7.57 g, 62
mmol) a solution of phenylchlorothionoformate (5.15 mL, 37.2 mmol)
in 50 mL of acetonitrile was added dropwise and the reaction
stirred for 8 h. TLC (EtOAc:hexanes/1:1) showed disappearance of
the starting material. The reaction mixture was evaporated, the
residue dissolved in chloroform, washed with water and brine, the
organic layer was dried over sodium sulfate, filtered and
evaporated to dryness. The residue was purified by flash
chromatography on silica gel with EtOAc:hexanes/2:1 as eluent to
give 16.44 g (85%) of 7.
Example 5
[0052]
3',5'-O-(Tetraisopropyl-disiloxane-1,3-diyl)-2'-C-Allyl-Uridine
(8)
[0053] To a refluxing, under argon, solution of
3',5'-O-(tetraisopropyl-disiloxane-1,3-diyl)-2'-O-phenoxythiocarbonyl-uri-
dine, 7, (5 g, 8.03 mmol) and allyltributyltin (12.3 mL, 40.15
mmol) in dry toluene, benzoyl peroxide (0.5 g) was added
portionwise during 1 h. The resulting mixture was allowed to reflux
under argon for an additional 7-8 h. The reaction was then
evaporated and the product 8 purified by flash chromatography on
silica gel with EtOAc:hexanes/1:3 as eluent. Yield 2.82 g
(68.7%).
Example 6
5'-O-Dimethoxytrityl-2'-C-Allyl-Uridine (9)
[0054] A solution of 8 (1.25 g, 2.45 mmol) in 10 mL of dry
tetrahydrofuran (THF) was treated with a 1 M solution of
tetrabutylammoniumfluoride in THF (3.7 mL) for 10 m at room
temperature. The resulting mixture was evaporated, the residue was
loaded onto a silica gel column, washed with 1 L of chloroform, and
the desired deprotected compound was eluted with
chloroform:methanol/9:1. Appropriate fractions were combined,
solvents removed by evaporation, and the residue was dried by
coevaporation with dry pyridine. The oily residue was redissolved
in dry pyridine, dimethoxytritylchloride (1.2 eq) was added and the
reaction mixture was left under anhydrous conditions overnight. The
reaction was quenched with methanol (20 mL), evaporated, dissolved
in chloroform, washed with 5% aq. sodium bicarbonate and brine. The
organic layer was dried over sodium sulfate and evaporated. The
residue was purified by flash chromatography on silica gel,
EtOAc:hexanes/1:1 as eluent, to give 0.85 g (57%) of 9 as a white
foam.
Example 7
5'-O-Dimethoxytrityl-2'-C-Allyl-Uridine 3'-(2-Cyanoethyl
N,N-diisopropylphosphoramidite) (10)
[0055] 5'-O-Dimethoxytrityl-2'-C-allyl-uridine (0.64 g, 1.12 mmol)
was dissolved in dry dichloromethane under dry argon.
N,N-Diisopropylethylamine (0.39 mL, 2.24 mmol) was added and the
solution was ice-cooled. 2-Cyanoethyl
N,N-diisopropylchlorophosphoramidite (0.35 mL, 1.57 mmol) was added
dropwise to the stirred reaction solution and stirring was
continued for 2 h at RT. The reaction mixture was then ice-cooled
and quenched with 12 mL of dry methanol. After stirring for 5 m,
the mixture was concentrated in vacuo (40.degree. C.) and purified
by flash chromatography on silica gel using a gradient of 10-60%
EtOAc in hexanes containing 1% triethylamine mixture as eluent.
Yield: 0.78 g (90%), white foam.
Example 8
[0056]
3',5'-O(Tetraisopropyl-disiloxane-1,3-diyl)-2'-Allyl-N.sup.4-Acetyl-
-Cytidine (11)
[0057] Triethylamine (6.35 mL, 45.55 mmol) was added dropwise to a
stirred ice-cooled mixture of 1,2,4-triazole (5.66 g, 81.99 mmol)
and phosphorous oxychloride (0.86 mL, 9.11 mmol) in 50 mL of
anhydrous acetonitrile. To the resulting suspension a solution of
3',5'-O-(tetraisopropyl-disiloxane-1,3-diyl)-2'-C-allyl uridine
(2.32 g, 4.55 mmol) in 30 mL of acetonitrile was added dropwise and
the reaction mixture was stirred for 4 h at room temperature. The
reaction was concentrated in vacuo to a minimal volume (not to
dryness). The residue was dissolved in chloroform and washed with
water, saturated aq. sodium bicarbonate and brine. The organic
layer was dried over sodium sulfate and the solvent was removed in
vacuo. The resulting foam was dissolved in 50 mL of 1,4-dioxane and
treated with 29% aq. NH.sub.4OH overnight at room temperature. TLC
(chloroform:methanol/9:1) showed complete conversion of the
starting material. The solution was evaporated, dried by
coevaporation with anhydrous pyridine and acetylated with acetic
anhydride (0.52 mL, 5.46 mmol) in pyridine overnight. The reaction
mixture was quenched with methanol, evaporated, the residue was
dissolved in chloroform, washed with sodium bicarbonate and brine.
The organic layer was dried over sodium sulfate, evaporated to
dryness and purified by flash chromatography on silica gel (3% MeOH
in chloroform). Yield 2.3 g (90%) as a white foam.
Example 9
5'-O-Dimethoxytrityl-2'-C-Allyl-N.sup.4-Acetyl-Cytidine
[0058] This compound was obtained analogously to the uridine
derivative 9 in 55% yield.
Example 10
5'-O-Dimethoxytrityl-2'-C-allyl-N.sup.4-Acetyl-Cytidine
3'-(2-Cyanoethyl N,N-diisopropylphosphoramidite) (12)
[0059] 2'-O-Dimethoxytrityl-2'-C-allyl-N.sup.4-acetyl cytidine (0.8
g, 1.31 mmol) was dissolved in dry dichloromethane under argon.
N,N-Diisopropylethylamine (0.46 mL, 2.62 mmol) was added and the
solution was ice-cooled. 2-Cyanoethyl
N,N-diisopropylchlorophosphoramidite (0.38 mL, 1.7 mmol) was added
dropwise to a stirred reaction solution and stirring was continued
for 2 h at room temperature. The reaction mixture was then
ice-cooled and quenched with 12 mL of dry methanol. After stirring
for 5 m, the mixture was concentrated in vacuo (40.degree. C.) and
purified by flash chromatography on silica gel using
chloroform:ethanol/98:2 with 2% triethylamine mixture as eluent.
Yield: 0.91 g (85%), white foam.
Example 11
2'-Deoxy-2'-Methylene-Uridine
[0060]
2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-u-
ridine 14 (Hansske, F.; Madej, D.; Robins, M. J. Tetrahedron 1984,
40, 125 and Matsuda, A.; Takenuki, K.; Tanaka, S.; Sasaki, T.;
Ueda, T. J. Med. Chem. 1991, 34, 812) (2.2 g, 4.55 mmol ) dissolved
in THF (20 mL) was treated with 1 M TBAF in THF (10 mL) for 20 m
and concentrated in vacuo. The residue was triturated with
petroleum ether and chromatographed on a silica gel column.
2'-Deoxy-2'-methylene-uridine (1.0 g, 3.3 mmol, 72.5%) was eluted
with 20% MeOH in CH.sub.2Cl.sub.2.
Example 12
5'-O-DMT-2'-Deoxy-2'-Methylene-Uridine (15)
[0061] 2'-Deoxy-2'-methylene-uridine (0.91 g, 3.79 mmol) was
dissolved in pyridine (10 mL) and a solution of DMT-CI in pyridine
(10 mL) was added dropwise over 15 m. The resulting mixture was
stirred at RT for 12 h and MeOH (2 mL) was added to quench the
reaction. The mixture was concentrated in vacuo and the residue
taken up in CH.sub.2Cl.sub.2 (100 mL) and washed with sat.
NaHCO.sub.3, water and brine. The organic extracts were dried over
MgSO.sub.4, concentrated in vacuo and purified over a silica gel
column using EtOAc:hexanes as eluant to yield 15 (0.43 g, 0.79
mmol, 22%).
Example 13
5'-O-DMT-2'-Deoxy-2'-Methylene-Uridine 3'-(2-Cyanoethyl
N,N-diisopropylphosphoramidite) (17)
[0062]
1-(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl-.beta.-D-ribofuranosy-
l)-uracil (0.43 g, 0.8 mmol) dissolved in dry CH.sub.2Cl.sub.2 (15
mL) was placed in a round-bottom flask under Ar.
Diisopropylethylamine (0.28 mL, 1.6 mmol) was added, followed by
the dropwise addition of 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (0.25 mL, 1.12 mmol). The
reaction mixture was stirred 2 h at RT and quenched with ethanol (1
mL). After 10 m the mixture evaporated to a syrup in vacuo
(40.degree. C.). The product (0.3 g, 0.4 mmol, 50%) was purified by
flash column chromatography over silica gel using a 25-70% EtOAc
gradient in hexanes, containing 1% triethylamine, as eluant.
R.sub.f 0.42 (CH.sub.2Cl.sub.2:MeOH/15:1)
Example 14
[0063]
2'-Deoxy-2'-Difluoromethylene-3',5'-O-(Tetraisopropyidisiloxane-1,3-
-diyl)-Uridine
[0064] 2'-Keto-3',5'-O-(tetraisopropyidisiloxane-1,3-diyl)uridine
14 (1.92 g, 12.6 mmol) and triphenylphosphine (2.5 g, 9.25 mmol)
were dissolved in diglyme (20 mL), and heated to a bath temperature
of 160.degree. C. A warm (60.degree. C.) solution of sodium
chlorodifluoroacetate in diglyme (50 mL) was added (dropwise from
an equilibrating dropping funnel) over a period of .about.1 h. The
resulting mixture was further stirred for 2 h and concentrated in
vacuo. The residue was dissolved in CH.sub.2Cl.sub.2 and
chromatographed over silica gel.
2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-
-uridine (3.1 g, 5.9 mmol, 70%) eluted with 25% hexanes in
EtOAc.
Example 15
2'-Deoxy-2'-Difluoromethylene-Uridine
[0065]
2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-u-
ridine (3.1 g, 5.9 mmol) dissolved in THF (20 mL) was treated with
1 M TBAF in THF (10 mL) for 20 m and concentrated in vacuo. The
residue was triturated with petroleum ether and chromatographed on
silica gel column. 2'-Deoxy-2'-difluoromethylene-uridine (1.1 g,
4.0 mmol, 68%) was eluted with 20% MeOH in CH.sub.2Cl.sub.2.
Example 16
5'-O-DMT-2'-Deoxy-2'-Difluoromethylene-Uridine (16)
[0066] 2'-Deoxy-2'-difluoromethylene-uridine (1.1 g, 4.0 mmol) was
dissolved in pyridine (10 mL) and a solution of DMT-CI (1.42 g,
4.18 mmol) in pyridine (10 mL) was added dropwise over 15 m. The
resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was
added to quench the reaction. The mixture was concentrated in vacuo
and the residue taken up in CH.sub.2Cl.sub.2 (100 mL) and washed
with sat. NaHCO.sub.3, water and brine. The organic extracts were
dried over MgSO.sub.4, concentrated in vacuo and purified over a
silica gel column using 40% EtOAc:hexanes as eluant to yield
5'-O-DMT-2'-deoxy-2'-difluoromethylene-uridine 16 (1.05 g, 1.8
mmol, 45%).
Example 17
5'-O-DMT-2'-Deoxy-2'-Difluoromethylene-Uridine 3'-(2-Cyanoethyl
N,N-diisopropylphosphoramidite) (18)
[0067]
1-(2'-Deoxy-2'-difluoromethylene-5'-O-dimethoxytrityl-.crclbar.-D-r-
ibofurano-syl)-uracil (0.577 g, 1 mmol) dissolved in dry
CH.sub.2Cl.sub.2 (15 mL) was placed in a round-bottom flask under
Ar. Diisopropylethylamine (0.36 mL, 2 mmol) was added, followed by
the dropwise addition of 2-cyanoethyl
N,N-diiso-propylchlorophosphoramidite (0.44 mL, 1.4 mmol). The
reaction mixture was stirred for 2 h at RT and quenched with
ethanol (1 mL). After 10 m the mixture evaporated to a syrup in
vacuo (40.degree. C.). The product (0.404 g, 0.52 mmol, 52%) was
purified by flash chromatography over silica gel using 20-50% EtOAc
gradient in hexanes, containing 1% triethylamine, as eluant.
R.sub.f 0.48 (CH.sub.2Cl.sub.2:MeOH/15:1).
Example 18
[0068]
2'-Deoxy-2'-Methylene-3',5'-O-(Tetraisopropyldisiloxane-1.3-diyl)-4-
-N-Acetyl-Cytidine 20
[0069] Triethylamine (4.8 mL, 34 mmol) was added to a solution of
POCl.sub.3 (0.65 mL, 6.8 mmol) and 1,2,4-triazole (2.1 g, 30.6
mmol) in acetonitrile (20 mL) at 0.degree. C. A solution of
2'-deoxy-2'-methylene-3',5'-O-(tetraisopropyidi-siloxane-1,3-diyl)
uridine 19 (1.65 g, 3.4 mmol) in acetonitrile (20 mL) was added
dropwise to the above reaction mixture and left to stir at room
temperature for 4 h. The mixture was concentrated in vacuo,
dissolved in CH.sub.2Cl.sub.2 (2.times.100 mL) and washed with 5%
NaHCO.sub.3 (1.times.100 mL). The organic extracts were dried over
Na.sub.2SO.sub.4 concentrated in vacuo, dissolved in dioxane (10
mL) and aq. ammonia (20 mL). The mixture was stirred for 12 h and
concentrated in vacuo. The residue was azeotroped with anhydrous
pyridine (2.times.20 mL). Acetic anhydride (3 mL) was added to the
residue dissolved in pyridine, stirred at RT for 4 h and quenched
with sat. NaHCO.sub.3 (5 mL). The mixture was concentrated in
vacuo, dissolved in CH.sub.2Cl.sub.2 (2.times.100 mL) and washed
with 5% NaHCO.sub.3 (1.times.100 mL). The organic extracts were
dried over Na.sub.2SO.sub.4, concentrated in vacuo and the residue
chromatographed over silica gel.
2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-4-N-ace-
tyl-cytidine 20 (1.3 g, 2.5 mmol, 73%) was eluted with 20% EtOAc in
hexanes.
Example 19
1-(2'-Deoxy-2'-Methylene-5'-O-Dimethoxytrityl-.beta.-D-ribo-furanosyl)-4-N-
-Acetyl-Cytosine 21
[0070]
2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-4-
-N-acetyl-cytidine 20 (1.3 g, 2.5 mmol) dissolved in THF (20 mL)
was treated with 1 M TBAF in THF (3 mL) for 20 m and concentrated
in vacuo. The residue was triturated with petroleum ether and
chromatographed on silica gel column.
2'-Deoxy-2'-methylene-4-N-acetyl-cytidine (0.56 g, 1.99 mmol, 80%)
was eluted with 10% MeOH in CH.sub.2Cl.sub.2.
2'-Deoxy-2'-methylene-4-N-acetyl-cytidine (0.56 9, 1.99 mmol) was
dissolved in pyridine (10 mL) and a solution of DMT-CI (0.81 g, 2.4
mmol) in pyridine (10 mL) was added dropwise over 15 m. The
resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was
added to quench the reaction. The mixture was concentrated in vacuo
and the residue taken up in CH.sub.2Cl.sub.2 (100 mL) and washed
with sat. NaHCO3 (50 mL), water (50 mL) and brine (50 mL). The
organic extracts were dried over MgSO.sub.4, concentrated in vacuo
and purified over a silica gel column using EtOAc:hexanes/60:40 as
eluant to yield 21 (0.88 g, 1.5 mmol, 75%).
Example 20
1-(2'-Deoxy-2'-Methylene-5'-O-Dimethoxytrityl-.beta.-D-ribo-furanosyl)-4-N-
-Acetyl-Cytosine 3'-(2-Cyanoethyl-N,N-diisopropylphosphoramidite)
(22)
[0071]
1-(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl-.beta.-D-ribofuranosy-
l)-4-N-acetyl-cytosine 21 (0.88 g, 1.5 mmol) dissolved in dry
CH.sub.2Cl.sub.2 (10 mL) was placed in a round-bottom flask under
Ar. Diisopropylethylamine (0.8 mL, 4.5 mmol) was added, followed by
the dropwise addition of 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (0.4 mL, 1.8 mmol). The
reaction mixture was stirred 2 h at room temperature and quenched
with ethanol (1 mL). After 10 m the mixture evaporated to a syrup
in vacuo (40.degree. C.). The product 22 (0.82 g, 1.04 mmol, 69%)
was purified by flash chromatography over silica gel using 50-70%
EtOAc gradient in hexanes, containing 1% triethylamine, as eluant.
R.sub.f 0.36 (CH.sub.2Cl.sub.2:MeOH/20:1).
Example 21
[0072] 2'-Deoxy-2'-Difluoromethylene-3',5'-O-(Tetraisopropyl
disiloxane-1,3-diyl)-4-N-Acetyl-Cytidine (24)
[0073] Et.sub.3N (6.9 mL, 50 mmol) was added to a solution of
POCl.sub.3 (0.94 mL, 10 mmol) and 1,2,4-triazole (3.1 g, 45 mmol)
in acetonitrile (20 mL) at 0.degree. C. A solution of
2'-deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-
uridine 23 ([described in example 14] 2.6 g, 5 mmol) in
acetonitrile (20 mL) was added dropwise to the above reaction
mixture and left to stir at RT for 4 h. The mixture was
concentrated in vacuo, dissolved in CH.sub.2Cl.sub.2 (2.times.100
mL) and washed with 5% NaHCO.sub.3 (1.times.100 mL). The organic
extracts were dried over Na.sub.2SO.sub.4 concentrated in vacuo,
dissolved in dioxane (20 mL) and aq. ammonia (30 mL). The mixture
was stirred for 12 h and concentrated in vacuo. The residue was
azeotroped with anhydrous pyridine (2.times.20 mL). Acetic
anhydride (5 mL) was added to the residue dissolved in pyridine,
stirred at RT for 4 h and quenched with sat. NaHCO.sub.3 (5 mL).
The mixture was concentrated in vacuo, dissolved in
CH.sub.2Cl.sub.2 (2.times.100 mL) and washed with 5% NaHCO.sub.3
(1.times.100 mL). The organic extracts were dried over
Na.sub.2SO.sub.4, concentrated in vacuo and the residue
chromatographed over silica gel.
2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-
-4-N-acetyl-cytidine 24 (2.2 g, 3.9 mmol, 78%) was eluted with 20%
EtOAc in hexanes.
Example 22
1-(2'-Deoxy-2'-Difluoromethylene-5'-O-Dimethoxytrityl-.beta.-D-ribofuranos-
yl)-4-N-Acetyl-Cytosine (25)
[0074]
2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyidisiloxane-1,3-
-diyl)-4-N-acetyl-cytidine 24 (2.2 g, 3.9 mmol) dissolved in THF
(20 mL) was treated with 1 M TBAF in THF (3 mL) for 20 m and
concentrated in vacuo. The residue was triturated with petroleum
ether and chromatographed on a silica gel column.
2'-Deoxy-2'-difluoromethylene-4-N-acetyl-cytidine (0.89 g, 2.8
mmol, 72%) was eluted with 10% MeOH in CH.sub.2Cl.sub.2.
2'-Deoxy-2'-difluoromethylene-4-N-acetyl-cytidine (0.89 g, 2.8
mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI
(1.03 g, 3.1 mmol) in pyridine (10 mL) was added dropwise over 15
m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL)
was added to quench the reaction. The mixture was concentrated in
vacuo and the residue taken up in CH.sub.2Cl.sub.2 (100 mL) and
washed with sat. NaHCO.sub.3 (50 mL), water (50 mL) and brine (50
mL). The organic extracts were dried over MgSO.sub.4, concentrated
in vacuo and purified over a silica gel column using
EtOAc:hexanes/60:40 as eluant to yield 25 (1.2 g, 1.9 mmol,
68%).
Example 23
[0075]
1-(2'-Deoxy-2'-Difluoromethylene-5'-O-Dimethoxytrityl-.beta.-D-ribo-
furanosyl)-4-N-Acetylcytosine
3'-(2-cyanoethyl-N,N-diisopropylphosphoramidite) (26)
[0076] 1
-(2'-Deoxy-2'-difluoromethylene-5'-O-dimethoxytrityl-.beta.-D-rib-
ofurano-syl)-4-N-acetylcytosine 25 (0.6 g, 0.97 mmol) dissolved in
dry CH.sub.2Cl.sub.2 (10 mL) was placed in a round-bottom flask
under Ar. Diisopropylethylamine (0.5 mL, 2.9 mmol) was added,
followed by the dropwise addition of 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (0.4 mL, 1.8 mmol). The
reaction mixture was stirred 2 h at RT and quenched with ethanol (1
mL). After 10 m the mixture was evaporated to a syrup in vacuo
(40.degree. C.). The product 26, a white foam (0.52 g, 0.63 mmol,
65%) was purified by flash chromatography over silica gel using
30-70% EtOAc gradient in hexanes, containing 1% triethylamine, as
eluant. R.sub.f 0.48 (CH.sub.2Cl.sub.2:MeOH/20:1).
Example 24
[0077]
2'-Keto-3',5'-O-(Tetraisopropyidisiloxane-1,3-diyl)-6-N-(4-t-Butylb-
enzoyl)-Adenosine (28)
[0078] Acetic anhydride (4.6 mL) was added to a solution of
3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-6-N-(4-t-butylbenzoyl)-adenos-
ine (Brown, J.; Christodolou, C.; Jones, S.; Modak, A.; Reese, C.;
Sibanda, S.; Ubasawa A. J. Chem Soc. Perkin Trans. 11989, 1735)
(6.2 g, 9.2 mmol) in DMSO (37 mL) and the resulting mixture was
stirred at room temperature for 24 h. The mixture was concentrated
in vacuo. The residue was taken up in EtOAc and washed with water.
The organic layer was dried over MgSO.sub.4 and concentrated in
vacuo. The residue was purified on a silica gel column to yield
2'-keto-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-6-N-(4-t-butylben-zoy-
l)-adenosine 28 (4.8 g, 7.2 mmol, 78%).
Example 25
[0079]
2'-Deoxy-2'-methylene-3',5'-O-(Tetraisopropyldisiloxane-1,3-diyl)-6-
-N-(4-t-Butylbenzoyl)-Adenosine (29)
[0080] Under a pressure of argon, sec-butyllithium in hexanes (11.2
mL, 14.6 mmol) was added to a suspension of
triphenylmethylphosphonium iodide (7.07 g,17.5 mmol) in THF (25 mL)
cooled at -78.degree. C. The homogeneous orange solution was
allowed to warm to -30.degree. C. and a solution of
2'-keto-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-6-N-(4-t-butylbenzoyl-
)-adenosine 28 (4.87 g, 7.3 mmol) in THF (25 mL) was transferred to
this mixture under argon pressure. After warming to RT, stirring
was continued for 24 h. THF was evaporated and replaced by
CH.sub.2Cl.sub.2 (250 mL), water was added (20 mL), and the
solution was neutralized with a cooled solution of 2% HCl. The
organic layer was washed with H.sub.2O (20 mL), 5% aqueous
NaHCO.sub.3 (20 mL), H.sub.2O to neutrality, and brine (10 mL).
After drying (Na.sub.2SO.sub.4), the solvent was evaporated in
vacuo to give the crude compound, which was chromatographed on a
silica gel column. Elution with light petroleum ether:EtOAc/7:3
afforded pure
2'-deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-6-N-(4--
t-butylbenzoyl)-adenosine 29 (3.86 g, 5.8 mmol, 79%).
Example 26
[0081] 2'-Deoxy-2'-Methylene-6-N-(4-t-Butylbenzoyl)-Adenosine
[0082]
2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-6-
-N-(4-t-butylbenzoyl)-adenosine (3.86 g, 5.8 mmol) dissolved in THF
(30 mL) was treated with 1 M TBAF in THF (15 mL) for 20 m and
concentrated in vacuo. The residue was triturated with petroleum
ether and chromatographed on a silica gel column.
2'-Deoxy-2'-methylene-6-N-(4-t-butylbenzoyl)-adenosine (1.8 g, 4.3
mmol, 74%) was eluted with 10% MeOH in CH.sub.2Cl.sub.2.
Example 27
[0083]
5'-O-DMT-2'-Deoxy-2'-Methylene-6-N-(4-t-Butylbenzoyl-Adenosine
(29)
[0084] 2'-Deoxy-2'-methylene-6-N-(4-t-butylbenzoyl)-adenosine (0.75
g, 1.77 mmol) was dissolved in pyridine (10 mL) and a solution of
DMT-CI (0.66 g, 1.98 mmol) in pyridine (10 mL) was added dropwise
over 15 m. The resulting mixture was stirred at RT for 12 h and
MeOH (2 mL) was added to quench the reaction. The mixture was
concentrated in vacuo and the residue taken up in CH.sub.2Cl.sub.2
(100 mL) and washed with sat. NaHCO.sub.3, water and brine. The
organic extracts were dried over MgSO.sub.4, concentrated in vacuo
and purified over a silica gel column using 50% EtOAc:hexanes as an
eluant to yield 29 (0.81 g, 1.1 mmol, 62%).
Example 28
[0085]
5'-O-DMT-2'-Deoxy-2'-Methylene-6-N-(4-t-Butylbenzoyl)-Adenosine
3'-(2-Cyanoethyl N,N-diisopropylphosphoramidite) (31)
[0086] 1
-(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl-.beta.-D-ribofuranos-
yl)-6-N-(4-t-butylbenzoyl)-adenine 29 dissolved in dry
CH.sub.2Cl.sub.2 (15 mL) was placed in a round bottom flask under
Ar. Diisopropylethylamine was added, followed by the dropwise
addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. The
reaction mixture was stirred 2 h at RT and quenched with ethanol (1
mL). After 10 m the mixture was evaporated to a syrup in vacuo
(40.degree. C.). The product was purified by flash chromatography
over silica gel using 30-50% EtOAc gradient in hexanes, containing
1% triethylamine, as eluant (0.7 g, 0.76 mmol, 68%). R.sub.f 0.45
(CH.sub.2Cl.sub.2:MeOH/20:1)
Example 29
[0087]
2'-Deoxy-2'-Difluoromethylene-3',5'-O-(Tetraisopropyidisiloxane-1,3-
-diyl)-6-N-(4-t-Butylbenzoyl)-Adenosine
[0088]
2'-Keto-3',5'-O-(tetraisopropyidisiloxane-1,3-diyl)-6-N-(4-t-butyl--
benzoyl)-adenosine 28 (6.7 g, 10 mmol) and triphenylphosphine (2.9
g, 11 mmol) were dissolved in diglyme (20 mL), and heated to a bath
temperature of 160.degree. C. A warm (60.degree. C.) solution of
sodium chlorodifluoroacetate (2.3 g, 15 mmol) in diglyme (50 mL)
was added (dropwise from an equilibrating dropping funnel) over a
period of .about.1 h. The resulting mixture was further stirred for
2 h and concentrated in vacuo. The residue was dissolved in
CH.sub.2Cl.sub.2 and chromatographed over silica gel.
2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-
-6-N-(4-t-butylbenzoyl)-adenosine (4.1 g, 6.4 mmol, 64%) eluted
with 15% hexanes in EtOAc.
Example 30
[0089]
2'-Deoxy-2'-Difluoromethylene-6-N-(4-t-Butylbenzoyl)-Adenosine
[0090]
2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyidisiloxane-1,3-
-diyl)-6-N-(4-t-butylbenzoyl)-adenosine (4.1 g, 6.4 mmol) dissolved
in THF (20 mL) was treated with 1 M TBAF in THF (10 mL) for 20 m
and concentrated in vacuo. The residue was triturated with
petroleum ether and chromatographed on a silica gel column.
2'-Deoxy-2'-difluoromethylene-6-N-(4-t-butylbenzoyl)-adenosine (2.3
g, 4.9 mmol, 77%) was eluted with 20% MeOH in CH.sub.2Cl.sub.2.
Example 31
[0091]
5'-O-DMT-2'-Deoxy-2'-Difluoromethylene-6-N-(4-t-Butyl-benzoyl)-Aden-
osine (30)
[0092]
2'-Deoxy-2'-difluoromethylene-6-N-(4-t-butylbenzoyl)-adenosine (2.3
g, 4.9 mmol) was dissolved in pyridine (10 mL) and a solution of
DMT-CI in pyridine (10 mL) was added dropwise over 15 m. The
resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was
added to quench the reaction. The mixture was concentrated in vacuo
and the residue taken up in CH.sub.2Cl.sub.2 (100 mL) and washed
with sat. NaHCO.sub.3, water and brine. The organic extracts were
dried over MgSO.sub.4, concentrated in vacuo and purified over a
silica gel column using 50% EtOAc:hexanes as eluant to yield 30
(2.6 g, 3.41 mmol, 69%).
Example 32
[0093]
5'-O-DMT-2'-Deoxy-2'-Difluoromethylene-6-N-(4-t-Butyl-benzoyl)-Aden-
osine 3'-(2-Cyanoethyl N,N-diisopropylphosphoramidite) (32)
[0094] 1
-(2'-Deoxy-2'-difluoromethylene-5'-O-dimethoxytrityl-.beta.-D-rib-
ofurano-syl)-6-N-(4-t-butylbenzoyl)-adenine 30 (2.6 g, 3.4 mmol)
dissolved in dry CH.sub.2Cl.sub.2 (25 mL) was placed in a round
bottom flask under Ar. Diisopropylethylamine (1.2 mL, 6.8 mmol) was
added, followed by the dropwise addition of 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (1.06 mL, 4.76 mmol). The
reaction mixture was stirred 2 h at RT and quenched with ethanol (1
mL). After 10 m the mixture evaporated to a syrup in vacuo
(40.degree. C.). 32 (2.3 g, 2.4 mmol, 70%) was purified by flash
column chromatography over silica gel using 20-50% EtOAc gradient
in hexanes, containing 1% triethylamine, as eluant. R.sub.f 0.52
(CH.sub.2Cl.sub.2:MeOH/15:1).
Example 33
[0095]
2'-Deoxy-2'-Methoxycarbonylmethylidine-3',5'-O-(Tetraisopropyldisil-
oxane-1,3-diyl)-Uridine (33)
[0096] Methyl(triphenylphosphoranylidine)acetate (5.4 g, 16 mmol)
was added to a solution of 2'-keto-3',5'-O-(tetraisopropyl
disiloxane-1,3-diyl)-uridine 14 in CH2Cl.sub.2 under argon. The
mixture was left to stir at RT for 30 h. CH.sub.2Cl.sub.2 (100 mL)
and water were added (20 mL), and the solution was neutralized with
a cooled solution of 2% HCl. The organic layer was washed with
H.sub.2O (20 mL), 5% aq. NaHCO.sub.3 (20 mL), H.sub.2O to
neutrality, and brine (10 mL). After drying (Na.sub.2SO.sub.4), the
solvent was evaporated in vacuo to give crude product, that was
chromatographed on a silica gel column. Elution with light
petroleum ether:EtOAc/7:3 afforded pure
2'-deoxy-2'-methoxycarbonylmethylidine-3',5'-O-(tetraisopropyidisiloxane--
1,3-diyl)-uridine 33 (5.8 g, 10.8 mmol, 67.5%).
Example 34
2'-Deoxy-2'-Methoxycarbonylmethylidine-Uridine (34)
[0097] Et.sub.3N.cndot.3 HF (3 mL) was added to a solution of
2'-deoxy-2'-methoxycarboxylmethylidine-3',5'-O-(tetraisopropyldisiloxane--
1,3-diyl)-uridine 33 (5 g, 9.3 mmol) dissolved in CH.sub.2Cl.sub.2
(20 mL) and Et.sub.3N (15 mL). The resulting mixture was evaporated
in vacuo after 1 h and chromatographed on a silica gel column
eluting 2'-deoxy-2'-methoxycarbonylmethylidine-uridine 34 (2.4 g, 8
mmol, 86%) with THF:CH.sub.2Cl.sub.2/4:1.
Example 35
5'-O-DMT-2'-Deoxy-2'-Methoxycarbonylmethylidine-Uridine (35)
[0098] 2'-Deoxy-2'-methoxycarbonylmethylidine-uridine 34 (1.2 g,
4.02 mmol) was dissolved in pyridine (20 mL). A solution of DMT-CI
(1.5 g, 4.42 mmol) in pyridine (10 mL) was added dropwise over 15
m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL)
was added to quench the reaction. The mixture was concentrated in
vacuo and the residue taken up in CH.sub.2Cl.sub.2 (100 mL) and
washed with sat. NaHCO.sub.3, water and brine. The organic extracts
were dried over MgSO.sub.4, concentrated in vacuo and purified over
a silica gel column using 2-5% MeOH in CH.sub.2Cl.sub.2 as an
eluant to yield
5'-O-DMT-2'-deoxy-2'-methoxycarbonylmethylidine-uridine 35 (2.03 g,
3.46 mmol, 86%).
Example 36
[0099] 5'-O-DMT-2'-Deoxy-2'-Methoxycarbonylmethylidine-Uridine
3'-(2-cyanoethyl-N,N-diisopropylphosphoramidite) (36)
[0100] 1
-(2'-Deoxy-2'-2'-methoxycarbonylmethylidine-5'-O-dimethoxytrityl--
.beta.-D-ribofuranosyl)-uridine 35 (2.0 g, 3.4 mmol) dissolved in
dry CH.sub.2Cl.sub.2 (10 mL) was placed in a round-bottom flask
under Ar. Diisopropylethylamine (1.2 mL, 6.8 mmol) was added,
followed by the dropwise addition of 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (0.91 mL, 4.08 mmol). The
reaction mixture was stirred 2 h at RT and quenched with ethanol (1
mL). After 10 m the mixture was evaporated to a syrup in vacuo
(40.degree. C.).
5'-O-DMT-2'-deoxy-2'-methoxycarbonylmethylidine-uridine
3'-(2-cyanoethyl-N,N-diisopropylphosphoramidite) 36 (1.8 g, 2.3
mmol, 67%) was purified by flash column chromatography over silica
gel using a 30-60% EtOAc gradient in hexanes, containing 1%
triethylamine, as eluant. R.sub.f 0.44
(CH.sub.2Cl.sub.2:MeOH/9.5:0.5).
Example 37
[0101]
2'-Deoxy-2'-Carboxymethylidine-3',5'-O-(Tetraisopropyldisiloxane-1,-
3-diyl)-Uridine 37
[0102]
2'-Deoxy-2'-methoxycarbonylmethylidine-3',5'-O-(tetraisopropyldisil-
oxane-1,3-diyl)-uridine 33 (5.0 g, 10.8 mmol) was dissolved in MeOH
(50 mL) and 1 N NaOH solution (50 mL) was added to the stirred
solution at RT. The mixture was stirred for 2 h and MeOH removed in
vacuo. The pH of the aqueous layer was adjusted to 4.5 with 1N HCl
solution, extracted with EtOAc (2.times.100 mL), washed with brine,
dried over MgSO.sub.4 and concentrated in vacuo to yield the crude
acid.
2'-Deoxy-2'-carboxymethylidine-3',5'-O-(tetraisopropyidisiloxane-1,3-diyl-
)-uridine 37 (4.2 g, 7.8 mmol, 73%) was purified on a silica gel
column using a gradient of 10-15% MeOH in CH.sub.2Cl.sub.2.
Uses
[0103] The alkyl substituted nucleotides of this invention can be
used to form stable oligonucleotides as discussed above for use in
enzymatic cleavage or antisense situations. Such oligonucleotides
can be formed enzymatically using triphosphate forms by standard
procedure. Administration of such oligonucleotides is by standard
procedure. See Draper et al. PCT WO 94/ (198/063).
[0104] Other embodiments are within the following claims.
Sequence CWU 1
1
6111RNAArtificial SequenceGeneric target sequence 1nnnnuhnnnn n
11232RNAArtificial SequenceEnzymatic Nucleic Acid 2nnnnncugan
gagnnnnnnn nnncgaaann nn 32314RNAArtificial SequenceGeneric target
sequence 3nnnnngucnn nnnn 14450RNAArtificial SequenceEnzymatic
Nucleic Acid 4nnnnnnagaa nnnnaccaga gaaacacacg uugugguaua
uuaccuggua 50585RNAArtificial SequenceEnzymatic Nucleic Acid
5uggccggcau ggucccagcc uccucgcugg cgccggcugg gcaacauucc gaggggaccg
60uccccucggu aauggcgaau gggac 85636RNAArtificial SequenceEnzymatic
Nucleic Acid 6ucuccaucug augaggccga aaggccgaaa aucccu 36
* * * * *