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AMINO ACID NUCL~IC ACIDS
~ield of In~ention:
The invention is in the field of polynucleotide analogs
lacking furanose rings.
sackground o~ the Invention:
Oligonucleotides that bind sequence specifically to
complementary nucleic acids (i.e. sense strand) by hydrogen
10 boncling so as to inhibit gene expresslon are commonly re~erred
tO as antisense oligonucleotides. These synthetic
oligonucleotides bind to target (mRNA) and thereby inhibit
~ranslation of the messenger R~A. This antisense principle
(Uhlmann, E. et al., Chem. ~eviews, 1990, 90, 543-584; and
Stein, C. A. et al., Cancer ~es., 1988, 48, 2659-2688) is used
in nature to regulate gene expression. This antisense
principle has been used in the laboratory not only to inhibit
but also to activate gene expression. Zamecnik and Stephenson
were the first to propose, in 1978, the use of synthetic
oligonucleotides for therapeutic purposes (Stephenson, M. L.;
20 and Zamecnik, P. C., Proc. Natl. Acad. Sci. USA, 1978, 75, 280
and ~85). The speciric inhibition of antisense polynucleotide
is based on the specific Watson-Crick base pairing between the
heterocyclic bases of the antisense oligonucleotide and of
viral nucleic acid. The process of binding of the
2~ oligonucleotides to a complementary nucleic acid is called
hybridization. An oligomer having a base sequence
complementary to that of an mRNA which encodes protein
necessary for the progress of the disease is of partlcular
interest. By hybridizing specifically to the mRNA, the
30 synthesis of proteins encoded by the mRNA may be disturbed.
The preparation of unmodified oligonucleotides, i.e.,
oligonucleotides having a DNA structure, has been the center
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of interest for many research groups in the past decade. The
synthesis via phosphoramidites according to Caruthers
(McBride, L.J.; and Caruthers, M. H., Tetrahedron Letts.,
1983, 24, 245), originally introduced by Letsinger tLetsinger,
5 R.L.; and Lunsford, W. B. , ~. Amer. Chem. Soc., 1976, ~8,
3655) as the phosphite triester method, is currently the most
efficient method for the preparation phosphodiester
oligonucleotides. When normal, i.e., unmodified,
oligonucleotides are used as antisense oligonucleotides, the
10 problems of instability to nucleases and insufficient membrane
penetration have arisen. For antisense oligonucleotides to be
able to inhibit translation they must reach the interior of
the cell unaltered. The properties useful for oligonucleotides
to be used for antisense inhibition include: (i) stability of
the oligonucleotides towards extra- and intracellular enzymes;
1~ (ii) ability to pen-~:rate through the cell membrane; and (iii)
ability to hybridiz~ ~he target DNA or RNA (Agarwal, K. L. et
al., Nucleic Acids Res., 1979, 6, 3009; Agawal, S. et al.,
Proc Na tl Acad Sci . USA. 1988, 85, 7079). Thus, it is of
interest to provide polynucleotide analogs that have superior
20 properties for use as antisense or for use as primers or
hybridization, probes.
Modified polynucleotides have been synthesized in the
past, these polynucleotide modifications include
25 methylphophonates, phosphorothioates, various amidates and the
sugar moieties of the nucleic acid species. These backbone
substitutions confer enhanced stability to some degree but
suffer from the drawback that they result in a chiral
phosphorous in the linkage, thus leading to the formation of
2n diastereomers where n is the number of modified diester
linkages in the oligomer. The presence of these multiple
diastereomers considerably weakens the capability of the
modified olgonucleotide to hybridize to target sequences. Some
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of these substitutions also retain the ability to support a
negative charge and the presence of charged groups decreases
the ability of the compounds to penetrate cell membranes.
There are numerous other disadvantages associated with these
modified linkages, depending on the precise nature of the
linkage.
Some oligonucleotide analogs containing sugar
modlfications have been synthesized. Previously used sugar
modifications of (deoxy)ribose nucleic acids include ~-DNA,
homo DNA, morpholino and thio nucleosides and Peptide Nucleic
Acids (PNA) to provide what has been perceived to be improved
structures, especially structures which have improved cell
uptake. The general synthetic scheme for arriving at such
analogs has been to involve the primary hydroxyl group of a
1~ nucleoside or its nucleotide, either bound to a polymeric
carrier or to a sequence-specified 3'-nucleotide with
phosphorus atom in either the pentavalent or trivalent
oxidation state. Specific coupling procedures have been
referred to as the phosphite triester (phosphoramidite), the
20 phosphorus diester, and the hydrogen phosphonate procedures.
Commercially available monomers and polymeric carriers-bound
monomers are available for such methods havlng protected bases
(G, A, C, T, U and other heterocycles) along with protected
phosphorus atoms to allow storage and prevent non-specific
reactions during the coupling process.
2~
Nucleic acid species containing modified sugars, non-
ionic backbones or acyclic polyamides (PNA) having, to some
degree, one or more of the following properties useful for
gene modulation: to enhance the duplex stability
30 (hy~ridization efficiency), increased target specificity,
stability against nucleases, improved cellular uptake, and
assistance in the important terminating events of nucleic
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acids (e.g. RNase H activity, catalytic cleavage,
hybridization arrest, and others). It has also been suggested
to use carbonate diesters. However, these compounds are highly
unstable, and the carbonate diester link does not maintain a
5 tetrahedral configuration exhibited by the phosphorous in the
phosphodiester. Similarly, carbamate linkages, while achiral,
confer trigonal symmetry and it has been shown that poly dT
having this linkage does not hybridize strongly with poly dA
(Coull, J. M. et al., Tetrahedron Letts., 1987, 28, 745;
Stirchak, E. P. et al., J. Org. Chem., 1987, 52, 4202).
More recently, reports of acyclic sugar analogs have
appeared in the literature (Augustyns, K. A. et al., Nucl~ic
Acids R~s., 1991, 19, 2587-2593). Incorporation of these
acyclic nucleosides into oligonucleotides caused a drop in Tm,
depending on the number of linkers built in the oligomers.
These oligonucleotides are found to be enzymatically stable
and form base pairing with the complementary sequence. Given
the shortcomings of polynucleotides and known polynucleotide
analogs, it is of interest to provide new polynucleotide
20 analogs for use in antisense inhibition and other techniques
employing oligomers.
Such attempts at modifying both the sugar and the
backbone components have some shortcomings for use as
2~ therapeutics and in other methods. Hence, still greater
improvements in these qualities is required before effective
therapeutics, diagnostics and research tools become available.
Accordlngly, there is a long-felt need for improved oligomer
analogs of oligonucleotides as pharmaceuticals compounds.
The present invention provides novel oligonucleotides,
and structural precursor thereof, which have improved
resistance to nuclease digestion, and which have increased
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.. ..
stability under physiological conditions, and which can be
neutral or positi~ely charged that could enhance cell
permeation. Furthermore, the novel oligonucleotides of the
present invention improved hybridization properties with
respect to nucleic acid hy~ridization targets.
The oligomers of the present invention are generally
characterized as comprising a series of constrained linkers or
monomers that is appropriate for binding of heterocyclic bases
to a target nucleic acid in a sequence specific manner. The
constrained linkers described herein, when incorporated into
oligomers, may have a force greater than a single hydrogen
bond, thereby favoring formation of the binding competent
conformation.
The nucleomonomers of the present invention are generally
characterized as moieties or residues that replace the
furanose ring, that is found in naturally occurring
nucleotides, with an amino acid or a modified amino alcohol
residues. Exemplary monomers and oligomers of the invention
20 are shown in formulae 1 through 41. Incorporation of these
monomers described herein into oligonucleotides permits
synthesis of compounds with improved properties, these
improved properties include (i) increased lipophilicity which
results from eliminating the charge associated with
phosphodiester linkages (Dalge, J. M. et al., Nucleic Acids
~es., 1991, 19, 1805) and (ii) resistance to degradation by
en2~es such as nucleases. Oligomers containing these monomers
may be quite stable for hybridization to target sequences and
superior to unmodified nucleoside residues in one or more
applications.
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S -~y of the Invention:
The present invention provides various novel
oligonucleotide analogs having one or more properties that
make the subject compounds superior to conventional
5 oligonucleotides for use in procedures employing
oligonucleotides. The compounds of the invention are
oligonucleotide analogs in which the furanose ring of a
naturally occurring nucleic acid is replaced with an amino
acid or a modified amino alcohol residue. Some embodiments of
the novel compounds of the invention are particularly useful
for the antisense control of gene expression. The compounds
of the invention may also be used as nucleic acid
hybridization probes or as primers.
Another aspect of the invention is to provide monomeric
15 precursors of the oligonucleotide analogs of the invention.
These monomeric precursors may be used to synthesize the
subject polynucleotide analogs.
Another aspect of the invention is to provide
20 formulations of the subject polynucleotide analogs that are
designed for the treatment or prevention of disease
conditions. Yet another aspect of the invention is to provide
methods for treating or preventing diseases, particularly
viral infections and cell growth disorders. The subject
disease treatment methods comprise the step of administering
an effective amount of the su~ject polynucleotide analogs for
use as antisense inhibitors.
Brief Description of the Figures:
Figures 1 through 25 are depictions of chemical reaction
30 sequences usable for synthesizing monomers and
oligonucleotides of the present invention. More specifically,
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Figure 1 shows the synthesis of L-serinol coupled thymine
monomer phosphoramidite with a -CH2-CO- linkage between
thymine and serinol.
,
Figure 2 shows the synthesis of L-serinol coupled
thymine monomer phosphoramidite with a -CH2-CH~- linkage
between thymine and serinol.
Figure 3 and 4 depicts the synthesis of substituted L-
serinol coupled thymine monomer phosphoramidites with a -CH2-
CO- linkage between thymine and serinol.
Figure S shows the synthesis of T-T dimer with 5 atom
long inter nucleotide linkage having hydroxylamine in the
middle of the internucleotide linkage with a -CH2-CO- linkage
15 between thymine and serinol.
Figure 6 depicts the synthesis of thymine monomer
phosphorami~ite in which thymine is connected to an N-
ethylhydroxylamine through a -CH~-CO- linkage.
Figure 7 shows the synthesis of L-serinol coupled thymine
monomer phosphoramidite in which the NH2 group of L-serine is
connected to 2-hydroxyacetyl group and the hydroxy function is
blocked with DMT group. This building block is used for 2'-5'
connection. This figure also depicts the synthesis of thymine
monomer in which the NH2 group of L-serine is connected to a
2'-hydroxyethyl function.
~,
Figure 8 shows the synthesis T-T dimer having a
hydroxamate backbone with 2'-5' linkage. In this dimer one
30 building block is made from L-aspartic acid and thymine and
the other is from L-serine and thymine. This dimer has two
additional amide bond in the backbone.
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.,
Figure 9 depicts the synthesis T-T dimer having
hydroxamate backbone with 2'-5' linkage. In this dimer one
building block is made from L-aspartic acid and thymine and
the other is from L-serine and thymine. This dimer lacks amide
bond between in the backbone.
Figure 10 shows the synthesis of L-serinol-b-alanine
coupled thymine monomer phosphoramidite in which ~-alanine
links thymine and serinol.
Figure 11 shows the synthesis of L-serinol-akylamine
coupled thymine monomer phosphoramidite with alkyamine links
thymine and serinol.
Figure 12 depicts the synthesis T-T dimer having
15 hydroxamate backbone with 4'-5' linkage. In this, the dimer
is made ~rom two L-aspartic acid units and two thymine units
with an acetyl linker between thymine and aspartic acid.
Figure 13 depicts the synthesis T-T dimer having
20 hydroxamate backbone with 4'-5' linkage. In this, the dimer is
made from two L-aspartic acid units and two thymine units with
an ethyl linker between thymine and aspartic acid.
Figure 14 shows the synthesis of N-hydroxyamino acid
coupled thymine building block.
Figure 15 shows the synthesis of L-aspartic acid coupled
thymine building block with an N-hydroxylamine linker between
thymine and aspartic acid.
Figure 16 depicts the synthesis T-T dimer having a
hydroxamate backbone with 4'-5' linkage. In this, the
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carboxylic acid group is coupled to thymine building block
through an N-hydroxylamine linker.
Figure 17 depicts the synthesis thymidineacetic acid
substituted N-hydroxyamino acid building block 1~0 and its
analogue 149. These monomer building blocks. are useful to
create nucleic acid with hydroxamate backbones.
Figure 18 shows the synthesis of thymidineacetic acid
10 substituted hydroxylamine containing amino acid building
blocks 1~7 and 1~8. These monomers are useful to design
nucleic acid having amide backbone with hydroxylamine
functionality.
Figure 19 shows the synthesis of L-serinol coupled
1~ thymidine building block 166 having a hydroxylamine moiety
between thymine and serinol. This building block is useful to
devise nucleic acid of 4'-5' linkages.
Figure 20 depicts the synthesis of glutamic acid-glycine
20 coupled Thymidine monomer 174. This monomer building block is
useful to generate nucleic acid with amide backbones and 2'-5'
linkages.
Figure 21 shows the synthesis of glycinol-glycine coupled
2~ thymidine building block 181 and 182 having a hydroxylamine
moiety between thymine and glycinol. These building blocks are
useful to prepare nucleic acid of 2'-5' linkages.
Figures 22 through 25 indicate the synthesis of ribose
amino acid coupled thymidine building blocks 191, 199 and 207.
These building blocks are useful to prepare oligonucleotides
having ribose-amide backbone.
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Figure 25 depicts the solid phase synthesis of
oligonucleotide 211 having ribose-amide backbone.
Figure 23 shows the synthesis of 1-O-(4,4'-Dimethoxy-
5 trityl)-2-[amino(thyminylacetyl)]-L-propan-3-0-(N,N-diisopropy
l)-~-cyanoethylphosphoramidite.
Figure 24 shows the synthesis of 1-O-(4,4'-Dimethoxy-
trityl)-2-[amino(thyminylacetyl)]-D-propan-3-0-(N,N-diiso-
propyl)-~-cyanoethylphosphoramidite.
Figure 25 shows the synthesis of 2-[(~-(4,4'-Dimethoxy-
trityl)-0-acetyl)amino]-3-thyminyl-L-propan-1-0-(N,N-diisoprop
yl)-~-cyanoethylphosphoramidite.
lS Figure 26 shows the synthesis of N-(Thyminylacetyl~-N-
[[(2-isobutyryl)oxy]ethyl]-O-benzylhydroxylamine.
Figure 27 shows the synthesis of (2R,4S)-1-(tert-
Butyloxycarbonyl)-2-[N3-benzoyl(thymin-l-yl)]methyl-4-phthalim
20 ido-pyrrolidine~
Detailed Description of the Specific ~mbodiments:
A. Definitions and A~re~iations:
In describing the present invention, the following terms
25 will be employed, and are intended to be defined as indicated
below.
As used herein, "antisense" therapy refers to
administration or n situ generation of DNA or RNA oligomers,
or their analogs thereof, which bind specifically to a
complementary target nucleic acid sequence. The binding may be
by conventional base pair complementarily, or the binding may
through other mechanisms, for example, in the case of binding
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to DNA duplexes, through specific interactions in the major
groove of ~he double helix. In general, "antisense" refers to
a range of techniques generally employed under this
description in the art, and includes any therapy which relies
on specific binding to oligonucleotide sequences. The
techniques of antisense gene regulation are well known to the
person o~ ordinary skill in the art of molecular biology
descriptions of antisense gene regulation can be found, for
example, in U.S. Patent 5,107,065, U.S. Patent 5,166,195, U.S.
Patent 5,087,617, and Crooke, Annual Review Pharmacology
Toxicology 1992 32; 329-376.
The terms "Oligomer" or "Oligonucleotide" are used
interchangeably and include naturally occurring compounds such
as RNA and DNA, as well as synthetic analogs thereof,
1~ including compounds of the invention. Unless indicated
otherwise, the terms "oligomer" and "oligonucleotide" refer to
both DNA/RNA and to synthetic analogs thereof. The term
"oligomer" refers to compounds comprising two or more
nucleomonomers covalently attached to each other by a
20 phosphodiester linkage or any other substitute linkages.
Unless indicated otherwise, a lengthy limitation should not be
read into the term "oligomer". Thus, an oligomer can have as
few as two covalently linked nucleomonomers ( a dimer )or may
be significantly longer. Oligomers can be binding competent
and, thus, can base pair with single-stranded or double-
stranded nucleic acid sequences. Oligomers (e.g. dimers -
hexamers) are also useful as synthons for longer oligomers as
described herein. Oligomers may contain abasic sites and
pseudonucleosides.
The Oligomers includes oligonucleotides,
oligonucleosides, polydeoxyribo-nucleotides (containing 2'-
deoxy-D-ribose or modified forms thereof), i.e., DNA,
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polyribonucleotides ( containing D-ribose or modified forms
thereof), i.e., RNA, and any other type of polynucleotide
which is an N-glycoside or C-glycoside of purine or pyrimidine
base, or modified purine or pyrimidine base. Oligomer as used
herein is also intended to include compounds where adjacent
nucleomonomers are linked via hydroxamate linkages. Elements
ordinarily found in oligomers, such as the furanose ring
and/or the phosphodiester linkage can be replaced with any
suitable functionally equivalent element. The term "Oligomer"
is intended to include any structure that serves as a chassis
or support for the bases wherein the chassis permits binding
to target nucleic acids in a sequence-dependent manner.
Oligomers that are currently known can be defined into four
groups that can be characterized as having (i) phosphodiester
or phosphodiester analog (phosphorothiaoate, methyl-
phosphonate, etc) linkages, (ii) substitute linkages that
contain a non-phosphorous isostere (riboacetal, formacetal,
carbamate, etc), (iii) morpholino residues, carbocyclic
residues or other furanose sugars, such as arabinose, or a
hexose in place of ribose or deoxyribose and (iv)
nucleomonomers linked via amide bonds or acyclic
nucleomonomers linked via any suitable substitute linkage.
The term "Nucleomonomer" as used herein refers to a
moiety comprising (1) a base covalently linked to (2) a second
moiety. Nucleomonomers include nucleosides, nucleotides or
bases connected to an amino alcohol. Nucleomonomers can be
linked to form oligomers that bind to target or complementary
base sequences of nucleic acids in a sequence specific manner.
A "second moiety" as used herein refers to a compound
linked to a Nucleomonomer, and includes an amino acid/amino
alcohol moiety, usually serinol, aspartic acid, glutamic acid,
glycine, and those species which contain modifications of the
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amino acid moiety, for example, wherein one or more of the
hydrogen is replaced with other functionality (see formulae
24-41), or one carboxylic acid is functionalized to an
alcohol, amines, thiols, hydroxylamines, and the like.
Nucleomonomers as defined herein are also intended to include
a base linked to an amino acid or amino alcohol and/or amino
acid/alcohol analog having a free carboxyl/hydroxyl group
and/or a free amino group and/or protected forms thereof.
The term "Nucleoside" as used herein refers to an amino
acid and amino alcohol derivative thereof, as described
further below, carrying a purine, pyrimidine, or analogous
forms thereof, as defined below, but lacking a linking moiety
such as a phosphodiester analog or a modified internucleoside
linkage. By "5'" nucleoside is meant the nucleoside which
15 provides the S' carbon coupling point to the linker. The "5'"
end of the linker couples to the 5' nucleoside. The "3'" end
of the linker joins to the 3' position on the next nucleoside.
If a modified nucleoside is present which does not precisely
include a 3' and/or a 5' carbon, it is understood by the
20 person skilled in the art that this "3'" and "S'" terminology
to describe strand polarity used by analogy to DNA and RNA.
, .
The term "Nucleoside" as used herein refers to a base
covalently attached to an amino alcohol/ amino acid analog and
which contain a linker between base and the amino acid/amino
alcohol. The term nucleoside normally includes
ribonucleosides, deoxyribonucleosides, or any other nucleoside
which is an N-glycoside or C-glycoside of a base.
The term "Nucleotide" as used herein refers to a
30 nuc~eoside having a phosphate group or a phosphate analog
(groups with phosphorous in the same oxidation state as in the
phosphate group e.g. thiophosphate, amidate).
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The term "~ase" as used herein refers to a wide variety
of nucleoside ~ase, including purine and pyrimidine
heterocycles and heterocyclic analogs and tautomers thereof.
Purines include adenine, guanine and xanthine and exemplary
5 purine analogs include 8-oxo-N6-methyladenine and 7-
deazaxanthine. Pyrimidines include uracil and cytosine and
their analogs such as 5-methylcytosine, 5-(1-propynyluracil),
5-(1-propynylcytosine), 5-methyluracil and 4,4-ethanocytosine.
"Bases", when joined to a suitable molecular framework, e.g. a
phophodiester backbone, are capable of entering into a base
pairing relationship that occur in double-stranded DNA or-
other double-stranded nucleic acids of similar structure.
Bases may also be capable of entering into a ~ase pairing
relationship in a triple helix nucleic acid.
1~ The term "Sugar Modification" as used herein refers to
any amino acid or amino alcohol moiety other than 2'-
deoxyribose.
The term "Amino Acids/Alcohol" as used herein refers to
20 any natural amino acids and alcohols of both "R' and "S"
isomers.
The term "Nucleoside Linkages" as used herein refers to
the linkage that exlsts within the monomer.
The term "Linkage" as used herein refers to the moiety
that is used to connect the base with amino acid/amino alcohol
and derivatives thereof.
The term "Internucleotide Linkages" as used herein refers
30 to a phophodiester moiety (-O-P(0)(0)-O-) or any other
functionally equivalent moiety that covalently connects
adjacent nucleomonomers.
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The term "Substitute Linkages" as used herein refers to
any analog of the native group or any suitable moiety that
covalently couples adjacent nucleomonomers. Substitute
linkages include phosphodiester analogs, e.g. such as
phosphorothioate and methylphosphonate, and nonphosphorus
containing linkages, e.g. such as amides, hydroxamates,
hydroxylamine. Substitute linkages include the nonphosphorous
containing linkages (2',5' linkages, 3',5' linkages and 4',5'
linkages) of the invention.
The term "Crosslinking moiety" as used herein refers to a
group or moiety in an oligomer that forms a covalent bond with
a target nucleic acid. Crosslinking moieties include covalent
binding species that covalently link an oligomer to target
nucleic acids either spontaneously (e.g. N4,N4-ethanocytosine)
~5 or via photoactivation (e.g. psoralen) and the like.
The term "Blocking Groups" as used herein refers to a
substituent other than H that is covalently coupled to
oligomers or nucleomonomers, either as a protecting group, a
20 coupling group for synthesis, OP032, or other conventional
conjugate such as a solid support, label, antibody, monoclonal
antibody or fragment thereof and the like. As used herein,
"blocking group" is not intended to be construed solely as a
protecting group, according to slang terminology, but is meant
also to include, for example, coupling groups such as a H-
phosphonate or a phosphoramidite.
The term "protecting group" as used herein refers to any
group capable of protecting the 0-atom, S-atom or N-atom to
which it is attached from participating in a reaction or
30 bonding. Such protecting groups for N-atoms on a base moiety
in a Nucleomonomer and their introduction are conventionally
known in the art. Non-limiting examples of suitable protecting
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groups include: diisobutylformamidine, benzoyl, silyl and the
like. Suitable protecting groups for O-atoms and S-atoms are,
for example, DMT, MMT, FMOC or esters. "Protecting groups" as
used herein includes any group capable of preventing the O-
5 atom, S-atom, or N-atom to which it is attached from
participating in a reaction or binding. Such protecting groups
for O-, S-, and N-atoms in nucleomonomers are described and
the methods for their introduction are conventionally known in
the art. Protecting groups also lnclude any group capable of
preventing reactions and bonding at carboxylic acids, thiols
and the like.
The term "Coupling group" as used herein refers to any
group suitable for generating a linkages or substitute linkage
between nucleomonomers such as a hydrogen phosphonate and a
15 phosphoramidite-
The term "Conjugate" or "conjugate moiety" as used herein
refers to any group attached to the oligomer at a terminal end
or within the oligomer itself. Conjugates include solid
20 supports, such as silica gel, controlled pore glass and
polystyrene; labels, such as fluorescent, chemiluminescent,
radioactive atoms or molecules, enzymatic moieties and
reporter groups; oligomer transport agents, such as
polycations, serum proteins and glycoproteins and polymers and
the like. Other conjugate moities include O-cholesterol,
polyethylene glycol (PEG), amino acids, intercalators,
polynucleotide clearing moieties, crosslinking
functionalities, lipids, hydroxama~es, alkylating agents and
the like.
The term "Synthon" as used herein refers to a structural
unit within an oligonucleotide analog of the invention.
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The term "Transfection" as used herein refers to any
method that is suitable for enhanced delivery of oligomers
into cells.
A 5 The term "Subject" as used herein refers to a plant or
animal, including mammal, particularly a human.
The term "Derivatives" and monomeric constituents thereof
ollgomers include those conventionally recognized in the art.
For instance, the oligonucleotides may be covalently linked to
various moieties such as intercalators, substances which
interact specially with the minor groove of the DNA double
helix and other arbitrarily chosen conjugates, such as labels
(radioactive, fluorescent, enzyme, etc.). These additional
moieties may be (but need not be) derivatized through the
15 modified backbone linkage as part of the linkage itself. For
example, intercalators, such as acridine can be linked through
an R-CH2- attached through any a~ailable -OH or SH, e.g.., at
the terminal 5' position of RNA or DNA, the 2' position of
RNA, or an OH or SH engineered into the 5 position of
20 pyrimidines, e.g., instead of the 5 methyl of cytosine, a
derivatized form which contains -CH2CH2CH2OH or -CH2CH2CH2SH in
the 5 position. A wide variety of substituents can be
attached, including those bound through conventional linkages.
Accordingly the indicated OH moieties in the oligomer of
formula (1) may be replaced by phosphonate groups, protected
2~
by standard protecting groups, or activated to prepare
additional linkages to other nucleotides, or may be bound to
. the conjugated substituent. The 5' terminal OH is
conventionally phosphorylatedi the 2'-OH or OH substituents
r at the 3' terminus may also be phosphorylated. The hydroxyls
30 may also be derivatized to standard protecting groups.
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The term " phosphodiester analog" as used herein refers
to an analog of the conventional phosphodiester linkage as
well as alternative linking groups. These alternative linking
groups include, but are not limited to embodiments wherein the
5 O-P(O) is replaced with P(O)S, P(O)NR2, P(O)R, P(O)OR', where
R is H or alkyl (1-7C) and R' is alkyl (1-7C). Not all
phosphodiester analogs in the same oligomer need to identical,
the only requirement being that at least one of these linkages
is a modified internucleotide linkage as described herein.
"Analogous" forms of purines and pyrimidines are those
generally known in the art, many of which are used as
chemotherapeutic agents. An exemplary but not exhaustive list
includes 4-acetylcytosine, 8-hydroxy-N~-methyladenine,
aziridinylcytosine, pseudoisocytosine, 5-
1~ (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil, 5-
carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-
isopentenyladenine, l-methyladenine, l-methylpseudouracil, 1-
methylguanine, l-methylinosine, 2,2-dimethylguanine, 2-
20 methyladenine, 2-methylguanine, 3-methylcytosine, 5-
methylcytosine, NO-methyladenine, 7-methyladenine, 7-
methylguanine, 5-methylaminomethyluracil, 5-
methoxyaminomethyl-2-thiouracil, beta-D-mannosylguanosine, 5'-
methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N~-
isopentenyladenine, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,
gueosine, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
A particularly preferred analog is 5-methylcytosine q
30 (abbreviated herein as "Cme").
- 18 -
- ~= =
CA 02202274 1997-04-09
W O96/14330 P~lr~35ll4599
The term "Isosteric" as used herein refers to the spatial
and orientation properties of zn internucleoside linkage and
the fact that these properties are so similar to those of the
native phosphodiester linkage that the modified oligonuceotide
containing an isosteric bond will replace, substitute for,
mimic and/ or hybridize with a native oligonuclotide.
The term "Ribose-amide" as used herein refers to the
internucleotide linkage that exists between two nucleobases.
The ri~ose-amide internucleotide linkage has com'oination of
ribosef(2'-deoxy) and amino acid functionalities.
Various abbreviations are used in this application to
refer to functional groups and compounds. These abbreviations
are readily understood by the person skilled in the art of
15 organic chemistry, for example, "Ph" refers to phenyl, "Me"
refers to methyl, "(1-7C)" indicates that a given chain
contains anywhere from 1 to 7 carbons, etc.
Description of the Invention:
The present invention provides novel oligonucleotide
analogs containing modified amino acid/amino alcohol linkages
between the bases and the ~ackbones (phosphodiester,
phosphorothioates and others as shown in table 1) also
referred to as modified nucleotide lin~ages. The modifications
2~ or functional equivalent thereof, replacing the sugar moiety
that lies between the backbone and the bases with an amino
acid derivatives, for example as shown in formulae 24. The
present invention is also provides novel nucleomonomers and
methods for their incorporation into oligomers containing the
f nucleomonomers.
The invention provides various nucleomonomer compounds
having the structures of formulae 1-23.
-- 19 --
.
CA 02202274 1997-04-09
W O96/14330 PCTrUS95/14599
HO>;~ R,X/SN/--I~ R
I ase Bas~ 'Bæe
Z~ R Z~N~R Rl >~--R
Bæe IBase
R3/S N
Rl~_~N--R Rl X
B \~A B>~A
7 ~2 8 R2
Base Base Base
R3J\~0 R3/~
Rl N--OH Rl> <N--OH Rl xN--A
HO R2 1 0 B 11 R2
-- 20 --
CA 02202274 1997-04-09
WO 96/14330 PCTJUS95114599
7ase 7ase IBase R
Z`Y N N
RIXN`OH HO~OH BlA
HO R2 R ~ R2 1 4
12 13
Base Basc I ase
R3 y R,/~Y Z~N,R
B R2 B,N--A B~A
16 R~17 R2
Base
Base R Base R Z~ ,A
R3/~OH R3/~OH Rl X
R~ ,N--R Rl>_<N--R >~
HO O HO R2 2 0
18 19
Base
ase Basc I A
N ~ I
1>_<X B X,N~R B_X
HO R2 2 3
21 22
-- 21 --
SUB~lul~ S~ (R~L~ 26)
CA 02202274 1997-04-09
W O 96/14330 PCTrUS95/14599
The oligomers of the invention are polymers comprising
one or more of the subject monomer compounds joined so as to
provide a structural analog of DNA or RNA. The oligomers of
the invention comprise two or more nucleomonomers and may
5 comprises virtually any number of nucleomonomers, although
oligomers of 200 or less nucleomonomers are generally easier
to synthesize. Compounds of formulae 1-23, may be joined to
one another through 4'-5' linkages, 3'-5' linkages, and 2'-5'
linkages, as can be seen in formulae 24-41.
- 22 -
SUBSTITUTE Sl IEET (RULE 26)
CA 02202274 1997-04-09
W O96/14330 - PCTrUS95/14599
1-' 1 1
~ ~ N ~ ~ N
=P--O O=P--O O=p_o
1N~ ~1N
1 . 24A 24 B
The nucleotide linkages iIl the compounds of the invention are
made from amino acids serine and glycine or derivatives
15 thereof. The oligonucleotides of the invention are stable in
vivo, resistant to endogenous nucleases and are able to
hybridi~e to target nucleotide sequences. Exemplary compounds
of this invention are shown in formulae 24 through 41 and are
conformationally more restricted relative to the
20 phosphodiester linkages found in unmodified DNA or RNA. This
conformational restriction may, in part, contribute to the
enhanced binding properties of the subject compounds to
complementary polynucleotide target sequences; however, the
use of the invention is not dependent upon this theory for
25 enhanced binding properties.
In another embodiment, the present invention is directed
- to a modified oligonucleotide or derivatives thereof, wherein
the furanose moiety of a natural oligonucleotide, e.g., DNA or
RNA is replaced with amino acidJamino alcohol moiety and other
modifications that comprises substitution at the amino acid
positions are shown in the formulae 25 to 41. The
internucleotide linkages between adjacent nucleomonomers is a
~JBST~TUTE SHEET (RULE 26)
CA 02202274 1997-04-09
W O96/14330 PCTnUS95/14599
linkage between the 4' and 5' position of adjacent
nucleomonomers. In other words, the phosphodiester
internucleotide linkage, or functional equivalent thereof,
originates from 5'-position of one nucleomonomer and connects
5 the 4'-position of adjacent monomer as exemplified by the
compounds of formulae 24 - 3~:
1 ,
13 ~ ~ N
O=P-O R
5~ O=p_o
N3 ~ o R
25A
2SB
R2~XN~ R2~XN~
O=P--O ~ H
O R~ O=P--O
f H R2~XN_~
26B
- 24 -
SUBSTITUTE SHEET tRULE 2&)
CA 02202274 1997-04-09
PCTnUS95/1459
W O96114330
O O
~ ~ N ~ ~ H R~
o=P--o o=P--o
~ R~B ~ R~ Se
27A I 27B
~ N ~ $ N
HO-N HO-N
~N~ ~ R~ B9~e
28A 28B
~R~ RL~
~N~ ~ ~r- ~
29A
:~0
-- 25 --
SuBsTlTuTE SHEET (RULE 26)
CA 02202274 1997-04-09
W O96/14330 PCTrUS95114599
OX~1 ~
O X--N~ ~XR~ A X--N
O--B--O ~ V X N~ I ~R
a~RI \~RI R V~B
0 X--N~ X--N~ X~x--N~
30A R 30B R 30C
f;,~, R, R,
R2~N~ o ,o_
R2~N~ N~
31A 31B
R2~ , R2~ ,O_H~e
}~O--N }~O--N
Rz~N~ R2~X N
32A 32B
-- 26 --
CA 02202274 1997-04-09
wa 96114330 . PCTAUS95114599
R2~N~ Rz~N--R ~;~X '
~N~ R2 ~ N ~X-N~
33A 33B 33C
Wherein each "R" is independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH2, ONH(CH3), Ph, ~(CH~)y~F; where "x" is 1-7
arbon and "F" is NH2, SH~ OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2, S(O)NH2, S(O)(O)NH2, CH3, Ph.
1~ .
Wherein each "Base" is independently a nucleoside base.
Wherein each ~L~ is independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH2, ONH(CH3), Ph, -(CH2)X-F; where "x" is 1-7
20 carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2! S(O)NHz, S (O)(O)NH2, CH3, Ph.
Wherein each ''Rz'' is independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH2, ONH(CH3), Ph, -(CH2)~-F; where "x" is 1-7
2~ carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2, S(O)NH2, S(O)(O)NH2, CH3, Ph.
Wherein each "R3" ls independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH2! ONH(CH3), Ph, -(CH2)~-F; where "x" is 1-7
carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNHz, S(O)NH2, S(O)(O)NH2, CH3, Ph.
- 27 -
CA 02202274 1997-04-09
W O96/14330 PCTrUS95/14S99
Wherein each "R4" is independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH2, ONH(CH3), Ph, -(CH2)x-F; where "x" is 1-7
carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2, S(O)NH2, S(O)(O)NH2, CH3, Ph.
- Wherein each "A" is independently (CH2)X, CO, CS, S,
S(O), S(O)(O), NH, NOH, NCH3, NR5 and Se, where "x" is 1- 7
carbon.
Wherein each "B" is independently (CH2)X, CO, CS, S,
S(O), S(O)(O), NH, NOH, NCH3, NR5 and Se, where "x" is 1- 7
carbon.
Wherein each "X" is independently (CH2)~, CO, CS, O, S,
S(O), S(O)(O), NH, NOH, NCH3 and NRs, where "x" is 1- 7
1~ carbon.
Wherein each "Z" is independently (CH2)~, CO, CS, S,
S(O), S(O)(O), NH, NOH, NCHI and NR5, where "x" is 1- 7
carbon.
R5 is a H, OH, OMe, CN, NH, NOH, ONCH3, ONH2, ethyl,
propyl, lower alkyl (1-7C), Me, heteroalkyl (1-7C), aryl(6-
7C), -(CH2)XF; where "x" is 1-7C, and "F" is independently H,
OH, SH, OCH3, CN, SCH3, ONH2, ONH(CH3), SNH2, S(O)N~z,
2~ S(O)(O)NH2, CH3, Ph.
Wherein each "V" is independently a phosphodiester
analog, phosphorothioates, methylphosphonates,
phosphorodithioates, boronphosphonates, selenophosphonates,
phosphoramidates, acetamidate, oxyformamido, oxyacetamido,
diisopropylsilyl, carbamate, dimethylene sulfide, dimethylene
sulfoxide, dimethylene sulfone and/ or two to four atom long
internucleoside linkage is selected from carbon, nitrogen,
- 28 -
CA 02202274 1997-04-09
W O96/14330 PCTrUS95/14S99
oxygen, sulfur and selenium. The length of the oligomer may
vary from a dimer to a 200mer, or longer. Preferred modified
internucleotide linkages include the structures for "V" are
shown in Table I.
Additionally, the compounds of formulae may be conjugated
to one or more conjugate moiety. Suitably, conjugate moieties
include O-cholesterol, polyethylene glycol, amino acids,
intercalators, cleaving moieties (e.g., imdazole),
crosslinking functionalities ~e.g., psoralen), lipids,
1~
peptides, alkylating agents, hydroxamaes, and fluorescent
labels. The conjugate moiety may independently replace one or
more of R, Rl, R2, R3, R4, znd R5.
In yet other embodiment, the subject invention provides
15 oligomer structures as indicated in formulae 34-36 and
derivatives thereof:
X ~ Base X~ ~ BaseX ,Z--Base
N X N-Y
~ O ~ R,
O= I--o o= I--o O="--O
R2 ~ ~ Basc ~2 ~ase R2~ ,Z - B~e
34A ~ 34B ~ 34C
. .
Fonnula34
- 29 -
CA 02202274 1997-04-09
WO96/14330 PCT~S95/14599
S R~ ~ ; ~ B~e R7 y ~ B~e N-Y
R~ ~ B~e R~ ~ B ~e \ N ~ ~ B~e
15 In the compounds of formulae 34-36, the linkages between
adjacent nucleomonomers are 3' to 5 t linkages.
Wherein each "R" is independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH., ONH(CH3), Ph, -(CH2)~-F; where "x" is 1-7
20 carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2, S(O)NH2, S(O)(O)NH2, CH3, Ph.
Wherein each "Base" is lndependently a nucleoside base.
Wherein each "Rl~ is independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH2, ONH(CH3), Ph, -(CH2)~-F; where "x" is 1-7
car~on and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2, S(O)NH2, S(O)(O)NH2, CH3, Ph.
Wherein each "R2~ is independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH2, ONH(CH3), Ph, -(CH2)~-F; where "x" is 1-7
carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2, S(O)NH2, S(O)(O)NH2, CH3, Ph.
- 30 -
CA 02202274 1997-04-09
W O96/14330 PCTrUS95/14599
Wherein each "R3~ is independently H, OH, SH, CN, CH3,
OCH3, SCH3, NH2~ ONH ~CH3), Ph, -(CH2),-F; where "x" is 1-7
carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2, S(O)NH2, S(O) (O)NH2, CH3, Ph.
Wherein each "R4~ is independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH2, ONH(CH3), Ph, -~CH2)~-F; where "x" is 1-7
carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2, S(O)NH2, S(O) (O)NH2, CH3, Ph.
Wherein each "A" is independently (CH2)X, CO, CS, S,
S(O), S(O)(O), NH, NOH, NCH3, NRs and Se. Where "x" is 1- 7.
Wherein each "B" is independently (CH2)~, CO, CS, S,
S(O), S(O)(O), NH, NOH, NCH3, NRs and Se. Where "x" is 1- 7.
Wherein each "X" is independently (CH~)~, CO, CS, O, S,
S(O), S(O)(O), NH, NOH, NCH3 and NR, . Where "x" is 1- 7.
Wherein each "Y" is independently (CH2)X, CO, CS, O, S,
20 S(O), S(O)(O), NH, NOH, NCH3 and NRs . Where "x" is 1- 7.
Wherein each "Z" is independently (CH2)~, CO, CS, S,
S(O), S(O)(O), NH, NOH, NCH3 and NR5. Where "x" is 1- 7.
2~ R5 is a H, OH, OMe, CN, NH, NOH, ONCH3, ONH2, ethyl,
propyl, lower alkyl (1-7C), Me, heteroalkyl (1-7C), aryl(6-
7C), -(CH2)~F; where "x" is 1-7C, and "F" is independently H,
OH, SH, OCH3, CN, SCH3, ONH2, ONH(CH3), SNH~, S(O)NH2,
S~O)(O)NH2, CH3, Ph.
Wherein each "V" is independently a phosphodiester
analog, phosphorothioates, methylphosphonates,
phosphorodithioates, boronphosphonates, selenophosphonates,
.
- 31 -
CA 02202274 1997-04-09
W O96114330 PCTrUS95/14599
phosphoramidates and/ or two to four atom long internucleoside
linkage is selected from carbon, nitrogen, oxygen, sulfur and
selenium. The length of the oligomers may vary rrom a dimer to
a 200mer, or longer. Preferred modified internucleotide
linkages include the structures for "V" are shown in Table I.
In another embodiment of the invention, the subject
invention provides oligomers having formulae 37 to 41, or
variants thereof, oligomers comprising novel internucleotide
10 linkages that are 2',5' linkages. These oligonucleotides are
stable in vivo, have improved resistance to endogenous
nucleases, and are able to hy~ridize to target oligonucleotide
sequences.
R,~ R,~
R R4 R R,,
O~ ,0 O~ ,0
R,~0 O R O' ~O
O ,N ~ R2/~N ~
R ~R,,
37A 37B
2~;
O N <~ R2 N~
R ~R4 R
ROXV~B~ 2XN~
R R< R R~
32
38A 38B
CA 02202274 1997-04-09
W O96114330 PCTrUS95/14599
_l_
X-N Rl~ z_tb#
<A X
O~ ,0 <
O `O
X-N X-N,x,
39A 39B
~3
'X,N~ X-N
~A ~A
B~ B~ z_B.x
,N~, X--N
40A 40B
R3
~,N~ i~X-N
V R3 V
,N~ X--N
41A 41B
-- 33 --
CA 02202274 1997-04-09
W 096/14330 P~ 55114599
Wherein each "R" is independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH., ONH(CH3), Ph, -~CH2)X-F; where "x" is 1-7
carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2, S(O)NH2, S(O) (O)NH2, CH3, Ph.
Wherein each "Base" is independently a nucleoside base.
Wherein each "R1~ is independently H, OH, SH, CN, CH3,
O OCH3, SCH3, ONH7, ONH(CH3), Ph, -(CH2)X-F; where "x" is 1-7
carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2, S(O)NH2, S(O)(O)NH2, CH3, Ph.
Wherein each "R2~ is independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH2, ONH(CH3), Ph, -(CH2)X-F; where "x" is 1-7
15 carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2, S(O)NH2, S(O)(O)NH2, CH3, Ph.
Wherein each "R3~ is independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH" ONElCH3), Ph, -(CH2)X-F; where "x" is 1-7
20 carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH7, S(O)NH2, S(O)(O)NH., CH3, Ph.
Wherein each "R4~ is independently H, OH, SH, CN, CH3,
OCH3, SCH3, ONH2, ONH(CH3), Ph, -(CH2)X-F; where "x" is 1-7
25 carbon and "F" is NH2, SH, OH, COOH, OCH3, SCH3, SPh, NOH,
NOH(CH3), SNH2, S(O)NH2, S(O)(O)NH2, CH3, Ph.
Wherein each "A" is independently (CH2)X, CO, CS, S,
S(O), S(O)(O), NH, NOH, NCH3, NRs and Se; where "x" is 1- 7
carbon.
. - 34 -
CA 02202274 1997-04-09
W O 96/14330 PCT~US95/14599
Wherein each "B" is independently (CH2)X, CO, CS, S,
S(O), S(O)(O), NX, NOX, NCH3, NR5 and Se; where "x" is l- 7
carbon. -
Wherein each "X" is independently (CH2)X, CO, CS, O, S,S(O), S~O)(O), NH, NOH, NCH3 and NR5 ; where "x" is l- 7
carbon.
Wherein each "Z" is independently (CH,)X, CO, CS, S,
S(O), S(O)(O), NH, NOH, NCH3 and NR;; where "x" is l- 7
car~on.
R, is a H, OH, OMe, CN, NH, NOH, ONC~3, ONX7, ethyl,
propyl, lower alkyl (1-7C), Me, heteroalkyl (1-7C), aryl(6-
7C), -(CH2)~F; where "x" is 1-7C, and "F" is independently H,
1~ OH, SH, OCH3, CN, SCH3, ONH, ONH(CH3), SNH2, S (O)NH2,
S(O)(O)NH" CH3, Ph.
Wherein each "v" is independently a phosphodiester
analog, phosphorothioates, methylphosphonates,
20 phosphorodithioates, boronphosphonate~, selenophosphonates,
phosphoramidates and/ or two to four atom long internucleoside
linkage is selected from carbon, nitrogen, oxygen, sulfur and
selenium. The length of the oligomer varies from dimer to
200mer. Preferred modified internucleotide linkages include
2~ the structures for "V" are shown in Table I.
In other embodiments of the invention, the subject
invention is directed to an oligomer of the following formulae
~formula 42) and monomeric constituents thereof
~formulae 85-90).
- 35 -
CA 02202274 l997-04-09
W O96/14330 PCTAUS9S/14599
X~ ,X -R
R ~ R
y N-X
0 ~-B ~ ~ .B
4~A 42B R
42C
~ N~ R ~ X-B R ~ Y
~ X H-N X-B~ ~ X-B
42D~ 42E~ 42F~
25 wherein,
X is selected from the group consisting of (CH2)n, where n=1-
3~ CO(CH2)n, where n= 0-2, and (CH2)nSO2, where n=1-2,
Y is selected from the group consisting of CH2, CO, COOH, CS,
and SO2,
Y'is selected from the group consisting of CH2, CO, COOH, CS,
and SO2,
Z is selected from the group consistlng of 0, S, NH, and CH"
- 36 -
CA 02202274 1997-04-09
W O96/14330 PCTnUS95/14S99
R is selected from the group consisting ofCHzOH, CH7NH
CH2NHCHO, CONH2, and COOH,
B is a nucleoside base.
COOH y ~ H_N,x-B
> z ~ ~
Z ~ X-B ~ ~Iy B y
86 87
~ x-B Y,x-B H-N ,x~B
z ~ H ~ Y R ~ y
H ff 90
88 89
wherein,
X is selected from the group consisting of (CHz) n~ where n=1-
3~ CO(CH2)n~ where n= 0-2, and (CH2)nSO2, where n=1-~,
2~ Y is selected from the group consisting of CH" CO, COOH, CS,
and SO2,
Y'is selected from the group consisting of CHz, CO, COOH, CS,
and SO2,
Z is selected from the group consisting of 0, S, NX, and CH"
R is selected from the group consisting ofCH70H, CH.NH~,
CH7NHCHO, CONHz, and COOH,
B is a nucleoside base.
- 37 -
CA 02202274 1997-04-09
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In other embodiments, the invention provides methods for
treating diseases mediated by the presence of a nucleotide
sequence which comprise administering to a subject in need of
such treatment an amount of the above modified
oligonucleotides capable of specifically binding the
nucleotide sequence effective to inactivate the nucleotide
sequence.
In the oligonucleotides of the invention, at least one
10 f the phosphodiester groups included within the "Vs" of
Formulae 24-41 is substituted by the modified internucleoside
linkages described herein. Desirably, multiple phosphodiester
linkages in the unmodified oligonucleotide are substituted by
the modified internucleoside linkage may be used repeatedly in
this structure, or, if desired, a variety of modified
1~ internucleotide linkages may be used in an individual
oligonucleotide. In a preferred embodiment of the su~ject
oligonucleotides these substituent linkages are non-chiral so
as to enhance the ability of the oligonucleotide to hybridize
to a desired target; however, useful compounds of the
20 invention include those embodiments in which chiral forms are
used.
- 38 -
CA 02202274 1997-04-09
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Perferred modified internucleotide linkages include the
structures for i'V" are shown in the Table 1.
~able I
-O-
-S-
--S (O) --
--S (O) (O)--
-Se-
--si--
--C (O) --
--C (S)--
-NH-
-NOH-
-NCH3-
-NR;-
-CH2-
-O-CH2-
-CH2-O-
-O-CH2-CH2-
-CH2-CH,-O-
-CH2-O-CH2-
-O-CH2-O-
-S-CH2-
-CH2-S -
-S -cH2-cH2
--CH2-CH2--S-
-CH2-S-CH,-
-S-CH2-S -
-O-CH2-S-
-S-CH,-O-
-S(O)-CH~-
-CH2-S (O) -
-S ( O ) -cH2-cH2-
-CH2-CH2-S (O) -
--CH2-S ~ O ) -CH2-
-S (O) -CH2-S (O) -
-O-CH2-S(O)-
-S(O)-CH -O-
-S ( O ) ( O ) -CH2-
-CH2-S (O) (O)-
-S ( O ) ( O ) -CH -CH2-
-CH,-CH2-S (O) (O) -
-CH2-S (O) (O) -CH2-
-S (O) (O)-CH2-S (O) (O) -
-O-CH2-S(O)(O)-
-S ( O ) ( O ) -CH2-O-
--S--S--
--S (O)--S (O)--
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W O96114330 PCTrUS9~/14~99
--S (O) (O)--S (O)--(O)--
-Se-CH2~
-CH2-Se-
-Se-CH2-CH2~
-CH2-CH2-Se-
-CH2-Se-CH2-
-Se-CH2-Se-
-O-CH2-Se-
-Se-CH2-O-
-Se (0) -CH2-
-CH2-Se ( O ) -
-Se (0) -CH2-CH2-
-CH2-CH2-Se (O) -
-CH2-Se (O) -CH2-
-Se (0) -CH2-Se (0) -
-O-CH2-Se (O) -
-Se (0) -CH2-0-
-Se (0) (0) -CH2-
-CH2-Se (O) (O) -
-Se (0) (0) -CH,-CH-,-
-CH2-CH2-Se (O) (O) -
-CH2-Se (O) (O) -CH2-
-Se (0) (0) -CH2-Se (0) (0) -
-Se-Se-
-Se (0) -Se (0) -
-Se (0) (0) -Se (0) - (0) -
-O-CH2-Se (O) (O) -
-Se (0) (0) -CH2-0-
-S-CH2-Se-
-Se-CH,-S-
-S (0) -CH2-Se (0) -
-Se (0) -CH2-S (0) -
-S (O) (O)-CH2-Se(O) (O)-
-Se (0) (0) -CH2-S (0) (0) -
--S--S--
--S (O)--S (O)--
--S ( O ) ( O ) - S ( O ) ( O ) -
-Se-Se-
-Se (0) -Se (0) -
-Se (0) (0) -Se (0) (0) -
-N (Rs) -CH2-
-CH2-N (Rs) -
-N (Rs) -CH2-CH2-
-CH2-CH2-N (Rs) -
-CH2-N (Rs) -CH2-
-N (Rs) -0-
-0-N (R5) -
-N (Rs) -0-CH2-
-CH2-0-N (R5) -
-CH2-N (Rs) -0-
-0-N (R5) -CH2-
,
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W O 96114330 . PC~rnUS95/14599
-O-CH2-N (R;)-
-N(R;)-CH2-
-N(R5) -S-
-S-N (R;)-
-N(R;)-S-cH2-
-CH2-S-N(R;)-
-CH2-N (R5) -S-
- -S-N(Rs)-cH2-
-S-CH2-N (R5)-
-N(Rs) -CH2-S-
-N(R5) -S (O) -
-S (O)-N (R5)-
-N(Rs)-S (O) -CH2-
-CH2-S(O) -N (R5)-
-CH2-N(R5)-S(O)-
_s(o)-N (Rs)-cH2-
-S(O)-CH2-N(Rs) -
-N(Rs)-CH,-S(O)-
-N(Rs)-S (O) (O) -
-S(O) (O)-N(Rs)-
-N(Rs)-S(O) (O) -CH.-
-CH2-S (O) (O) -N (Rs) -
1~ -CH2-N (Rs)-S(O) (O)-
-S(O) (O)-N(R;)-CH2-
` -S(O) (O)-CH2-N (Rs)~
-N (R;)-CH2-S (O) (O)-
-O-N ( Rs)-S-
-S-N ( R5)-O-
-O-N (R5)-S (O)-
-S (O) -N (Rs)~O~
2 0 -O-N ( R;) -S (O) (O)-
-S (O) (O) -N (Rs) -O-
--O--S--O--
--O--S (O)--O--
--O--S (O) (O)--O--
-N(R5)-S-N(R5) -
-N(Rs)-S (O) -N (R5)-
-N (R5)-S(O) (O) -N (Rs)~
-CH2-S -O-
-CH2-S ( O ) -O-
-CH2-S ( O ) ( O ) -O-
-CH2-C ( O ) -O-
-CH2-C ( S ) -O-
-CH2-N (R5)-C (O)-O-
-CH2-N (R5) -C(S)-O-
-N (R5)-C (O) -O-CH2-
-N(R5)-C (S)-O-CH2-
-O-C(O) -N (R5)-O-
-O-C(S)-N (R5)-O-
-O-C (O)-N (R5) -CH2-
-O-C(S)-N (R5) -CH2-
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-O-C (O)-CH2-N (Rs)~
-O-C (S)-CH2-N(R5)-
-O-C(O)-CH2-O-N(Rs)-
-O-C(S)-CH2-O-N(R5)-
-O-C (O)-N (R5) -O-CH2-
- O - C ( S ) - N ( R5)-O-CH2-
-O-N (R5)-C (O)-O-CH2-
-O-N (Rs)-C (S) -O-CH,-.
-CH2-O-C(O) -N(Rg)~O~
-CH2-O-C(S) -N (R5) -O-
-CH2-O-C(O)-N (R5)-CH2-
-CH2-O-C(S)-N (R5)-CH2-
-CH2-O-C(O) -CH2-N (R5)-
-CH2-O-C(S)-CH2-N(R;)-
-CHz-O-C(O)-N (R5)-
-CHz-O-C(S)-N(R5)-
-CH2-O-C ( O ) -N ( R5)-O-
-CH2-O-C (S) -N (R5)-O-
-CH2-O-N(R5)-C (O)-O-
-CH2-O-N (R5) -C (S) -O-
-CH2-N (R;) -C (O)-S-
-CH2-N(R5) -C(S) -S-
1~ -N (R5)-C (O)-S-CHz-
-N (R;)-C (S)-S-CH2-
-S-C (O) -N (R5)-O-
-O-C (S)-N(Rs)-S-
-S-C(O) -N (R5)-CH2-
-S-C(S)-N(Rs)~CH2~
-S-C(O)-CH2-N(Rs)-
-S-C(S)-CH2-N(Rs)-
-S-C (O)-CH2-O-N(Rs)-
-O-C (S)-CH2-S-N (R5)-
-O-C (O)-N (R5)-S-CHz-
-S-C (S)-N (Rs)-o-cH2-
-S-N (Rs)-C (O)-O-CH2-
-O-N ( R5)-C(S)-S-CH2-
-CH2-S-C(O)-N(R5) -O-
-CH2-O-C(S)-N (R5)-S-
2~ -CH2-S-C ( O ) -N ( Rs)-CH,-
-CH2-S -C ( S ) -N ( Rs)-CH2-
-CH2-S-C(O)-CH2-N(R5)-
-CH2-S-C(S) -CH2-N (R5)-
-CH2-S-C (O) -N (R5)-
-CH2-S-C ( S ) -N ( R;)-
-CH2-S-C (O) -N (R5)-O-
-CH2-S-C(S)-N (R5)-O-
-CH2-S-N(R5) -C~O)-O-
-CH2-S-N (R5) -C(S~-O-
-CH2-O-C (O) -N (R5)-S-
-CH2-O-C ~S) -N(R5)-S-
-CH2-O-N(R5)-C(O)-S-
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W O96/14330 PCTrUS95/14599
-CH2-O-N(Rs)-C(S)-S-
--N (R5)--N (R,)--
-N (Rs) -N (Rs) -CE~2-
-CH2-N ( R5 ) -N ( R5 ) -
-N=C ( NH2 ) -N ( R5 ) -
-N ( R5 ) -N=C ( NH2 ) -
-S ( O ) -CH,-O-
-O-CH2-S (O) -
-S-CH ( Rs ) -O-
-O-CH (Rs) -S-
-O-CH2-CH=CH-
-S-CH2-CH=C~I-
-S-CH2-C=C-
-N ( R; ) -CH2-N ( Rs ) -
-N (Rs) -C (O) -N (Rs) -
-N (Rs) -C (S) -N (Rs) -
-N (R;) -C (O) -S-
-N (Rs) -C (S) -S-
-N (R;) -C (S) -O-
-N (R5) -C (O) -O-
-O-C (O) -N (Rs) -
-O-C ( S ) -N (Rs ) -
l~i -S-C (O) -N (Rs) -
-S-C (S) -N (R;) -
Rs is a H, OH, OMe, CN, NH, NOH, ONCH3, ONH,, ethyl,
propyl, lower alkyl (1-7C), Me, heteroalkyl (1-7C), aryl(6-
7C), -(CH2)XFi where "x" is 1-7C, and "F" is independently H,
OH, SH, OCH3, CN, SCH3, ONH2, ONH (CH3), SNH2, S (O) NH2,
S (O) (O) NH2, CH3, Ph. Additionally, conjugate one or more
moieties may be joined to the linkage so as to produce an
oligomer conjugate. Suitable conjugate moieties include, O-
cholesterol, polyethylene glycol, amino acids,
25 intercalulators, cleaving moieties (e.g., imdazole),
crosslinking functionalities (e.g., psoralen), lipids,
peptides, alkylating agents, hydroxamates, and fluorescent
labels~
Particularly preferred 4'-5' linkages include
phosphodiester, phosphorothiates, metylphosphonates,
carboxamide, thiocarboxamide, hydroxamate, sulfonamide,
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WO96/14330 PCT~S95/14599
hydroxylamine and carbama~e. The same modifications are
preferred for 2'-5' and 3'-5' linkages as well.
The oligomers of the invention are not limited to
5 oligomers of homogeneous 1 nkage type, and that alternating or
. randomiy distributed substitute linkages including the 2', 5'
linkages are included. Since the oligomers of tne inven.ion
can be synthesized one nucieomonomer residue at a time, each
individual linkage, and/or substitute linkage, and the nature
or each individual "Base" substituent may be seLected
independently so as to produce oligonucleotides having a
desired sequence.
The oligomers of the invention may contain any desired
number c~ the substitute linkages. These substi~ute linkages
15 may be identical to each other or different by virtue of the
embodiments chosen for "V" including other noninvention
substitute linkages. Since the oligomers are prepared
seauentially, any patte n o- linkage o- substitule linkage
~o.ypes, bases and sugar mod ications may be used.
In prefGrred embodimenrs of ~he invention, the substitu~e
linkages Oc the invention alternate in a regular pattern. For
example, one substitute linkage is followed by two
phosphodiester linkages followed by one invention substitute
linkage, eic. Additional embodiments include, for example,
alternating linkages such as a substitute linkage followed by
a pAosphodieste~ analog (e.g., thioate, etc.), followed by a
substitule linkage of the invention followed by a
phosphodies~er analog, etc., i.e., the oligome- Oc .Ae
invention may comprise a one-by-one alternation o_ the two
30 types o substi.ute linkages. Oligomers of the invention
comprising more than one ty?e of linkage may have any of a
number o~ regular patterns ormed by alternations between the
CA 02202274 1997-04-09
W O96114330 PCTnUS95/14599
. , .
different linkage types present between the subuni~s of the
oligomer.
Sugar modifications may be made to one or more
nuclecmonomer residues in oiigomers or the invention; however,
4'-51, 3'-5~ and 2'-5' nucleotide linkage between amino acid
residues are preferred when such modifications are to be
incorporated. Where this is the case, further ab~reviation
may be used to represent the base sequence of the
oligonucleotid- analog. ~or example, i~ standard DNA (or RNA)
the sequences are generally denoted by the sequence of bases
alone, such as, for exam~l_, .~TG CG_ TGA. 'n general, it is
simply sLated in advance w.~ethsr .his rep~esen.s an RNA o~ DNA
sequence. A corresponding rotation system is used herein so
as to represent oligonucleotide analogs with a given base
1~ sequence
Additional Nucl~c~m~nnmc~?A Modifications:
Oligomers o~ the inVenlion may also comprise of various
modirica~ions n addition .o the substitute linkages of the
20 invention. Additional mod- cations include o lgomers where
(i) one o- more nucleomonome- residues are modified at the 2',
3', 4', and 5' posirions, (ii~ one or more covalent
crosslinking moieties are incorporated, (iii) other
noninvention substi~ute l nkages are included, ~iv) other base
analogs, such as 8-oxo-N -methyladenine, are included and (v)
conjugates such as intercalating agents or polylysine that
respectively enhance binding affinity to target nucleic acid
- sequences or that enhance association of the oligomer with
cells are included.
The sequer.ce-speci_i_ ?olynucleotide binding properties
of the oligomers of the invention for singlD-stranded and
duplex targets is compatib~e with further modifications to the
- 45 -
CA 02202274 1997-04-09
W O96/14330 PCT~US95tl4599
oligomer. These further modifications may also confer other
useful properties such as stability to nuclease cieavage (e.g.
in a domain of an oligomer of the invention having
phosphodiester linkages), or ~nhanc~ their ability to permeate
cell membranes, and the like.
The oligomers of the invention may com?rise one or more
substitute linkages such as sulride or sulfone linkages
(Benner, S.A., International Pubiication No. ~O 89/12060),
sulfamate linkages (Internalional Publication No. WO
91/15500l, carbamate or o~her subs~itute linkages in
morpholino-linked oligomers (Sti_^hak, _.~. et al Nucleic.
Acid3 R~s 1989, 17, 6129-61~'i aummer~n, u., e~ al
International Publication No. 216 860) and related linkages.
1~ Thus, exemplary embodiments of invention oligomers
include oiigomers having (1) at least one substitute linkage
and an amino acid that is linked to an adjacent monomer and
(2) one or more non-invention substitute linkages selected
from the sroup consistlng of phos?horothioate,
20 methylphosphonate and thionomethylpnosphonate and/or (3) one
or more phosphodiester linkages and/or (4) purine or
pyrimidine analogs that enr.ance binding affinity for
complementary target sequences. Other exemplary oligomers
would include (1) an oligomer having invention substitute
25 linkages a~ the 3' and/or S' ends and phosphorothioate
linkages elsewhere in the oligome-; (2) oligomers having
invention substitute linkages and standard purine or
pyrimidine bases (e.g. adenine, guanine, cytosine, thymine, or
uracil); (3) oligomers having invention substitute linkages
and one o_ more bases that enhance binding affinity or
30 ?ermeation com?etence of the oligomer (e.g. 5-methylcytosine,
5'(1-propynyl) uracil, 5-(1-?ropynl) cytosine. Also included
- 46 -
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W O96/14330 PCTrUS95/14599
are oligomers containing nucleomonomer residues linked via
hydroxamates.
Synt~e; ~ of Oliqomers:
The oligomers of the inven~ion may be formed using
nucleomonomers of the invention alone or in ~ombination with
conventional nucleomonomers and synthesized using s.andard
solid phase (or solution phase) oligomer synthesis lechniques,
which are now commercially available. In general, the
invention oligomers may be synthesized by 2 method comprising
~he steps of: synthesizing a nucleomonomer or oligomer
svnthon naving 2 protecting gro~l? and a base and a couDling
group capa~le of coupling ~ a r.~cleomonomer or olisomer;
coupling the nucleomonomer cr oligomer synthon tO an acce~tor
nuc~eomonomer or an accep~or olisomer; removing tAe protecting
15 groupi and repeating the cycle as needed until the desired
oligomer is synthesized.
The oligomers of the present invention may be or any
length including those of greater than 40, 50, 100, 200 or 500
20 r.ucleomonomers. In general, preferred oligomers contain 2-30
nucieomonomers. Lengths or greate- th.an o- eau21 to about 8
to 20 nucleomonomers may be us-ful for therapeutic or
diagnostic applications provided tney have a suitable base
sequence. Short oligomers containing 2, 3, 4 or 5
nucleomonomers are specifically included in the present
invention and may be used as synthons.
- Oligomers having a randomi2ed sequence and containing
about 6, 7 or 8 nucleomonome-s may be used as primers that are
used in cloning or ampli,ication protocols that use random
30 sequence primers, provided that the oligomer contains about 1
or 2 residues at the 3' end that can serve as a primer for
- a7 -
CA 02202274 1997-04-09
W O96/14330 PCTrUS95/14599
polymerases or reverse transc-iptases or that olherwise do not
interfere with polymerase activity.
In addition to the linkages described for the rirst time
herein, the oligomers o' the invenllon may comprise
conventional phosphodiester linkages or can contain other
substitute linkages such as phosphoramidate linkages in
addition to the invention substitute linkages. These
substitu~e linkages include, but are no~ limited to,
embodiments whereln a moie~v of tn- rormula-O-P(O)(S)-O-
("phosphorothioate"), -O-P(O)(NR~ )-X2, -O-P(O) ( Rl;)-O-, -O-
P(S) (R )-O- ("thionoalkylphosphonate"), -P(O) (oR3)-X', -O-
C(O)-X, o- -O-C(O) (NR,--)-X--, wherein R-- is ~ (or a sal.) or
alkyl (1-12C including metnvl and ethyl) and R3 s alkyl
(1=9C) and the linkage is joined to adjacent nucleomonomers
15 through an -O- or -S- bonded to a carbon of the nucleomonomer
and X' is O or S. Phosphorothioate and phosphodlester
linkages are well known. Particularly preferred substitute
linkages for use in the oligomers o' the present invention
include phosphodiester, phosphorothioate, methyl?hosphonate
20 and thionomethylphosphonale substi.ute linkages.
Phosphorothioate and methylpnosphonate substitute linkages
conrer added stability ~o the oligomer need be identical,
particularly preferred oligomers o_ the invention contain one
or more phosphorothioate or methylphosphonate substitute
linkages.
Oligomers of the invention and the segments thereof may
be synthesized using methods that are known to the personof
ordianry skill in the art. The synthetic methods known in the
area and described Aerein can be used to synthesize oligomers
containing substitute linkages of the invention, 2S well as
30 other linkages or substitute linkages known in the art, using
appropriately protected nucleomonomers. Methods for the
synthesis or oligomers ha~ing phosphorous containing linkages
CA 02202274 1997-04-09
W O96/1~330 PCTrUSg5/14599
are found, for example, in Froehie~, ~., et al., Nucleic Acids
~es., 1986, 1~, 5399-5467; Nucleic Aci~s Res , 1988, 1~,
4831-4839; Nucleosides ~ Nucleotid~s, 1987, o, 287-291;
~roehler, B , TetraAedron Letts , 1986, 27, 5575-5578;
Caruthers, M.H. in Oligodeoxyn~cleo.id~s Antisens~ Inhi~i.ions
of Gene Expressio.~, 1989, J.S. Cohen, editor, CRC Press, Boca
Raton, p7-24; Reese, C.B. et al, Tetrahedron Letts, 198~, 26,
2245-2248. Synthesis of the methyl~hosphonate linked
oligomers via methyl phosphonamidite chemistry has also been
10 described (Agrawal, S. et al., Tet~2nedron Letts., 1987, 2~,
3539-3542; Klem, R.E., et al, Inte-national Publication Number
WO 92/07864).
Oligomers containlng linkages o~ the present invention
15 are also convenientLy syn.hesized by preparation of dimer or
trimer compounds by solution phase chemistry followed by
conversion of the synthon IO a derivative that is incorporated
into oligomers by either solid or solution phase chemistry.
Typicai synthons are 5' DMT or MMT blocked 3' Dhosphonate or
Dhosphoramida~e derivatives which are prepared by standard
20 methods (see: Gait, M.J. ed., Oligonucleotid2 Synthesis; A
Prac~ic21 Appro2ch 1984, RT Press, Oxford).
Synthons that are inciuded in the scope of the present
invention include dlmers, t~imers, t tramers, hexamers and
longer oligomer made by solid or solution phàse synthesis.
25 Trime~s and Longer synthons may contain more than one type of
linkage. The synthons may include any base as described above
or Z', 3', 4' and 5' groups such as OH, DMTO, MMTO, O-allyl,
phosDhate, a phosphonate or an amidite as described above.
Ribose-amide o'igonucLeotides could be synthesized by
30 using standard soli~ phase DeDtide synthesis (Fmoc chemistry)
conditions (see figure 26).
49
CA 02202274 1997-04-09
W O 96/14330 PCTAUS95114599
Blockin~ Groups For the Synthesis of the C.l~v~..d o~ t~e
Invention:
1. Couplinc aroUDs.
Suitable coupling groups are, for example, H-phosphonate,
a methylphosphonomidite, or a phosphoramidile. >
Phosphoramidites that can be used include ~-
cyanoethylphosphoramidites (preferred).
Methylphosphonamidites, alkylphosphonamidites (including
ethylphosphonamidites and propylphosphonamidites) can also be
used. Exemplary phosphoramidites are shown in figures 1 to
21.
Suitable "couplir.g groups" 2t the ~', 3', 4' or 5i
position ror oligomer synthesis via phospnoramidite triester
chemistry, referred to herein as "amidite" chemistry, include
1~ N,N-diisopropylamino-~-cyanoethoxyphosphir.e, N-
N,diisopropylamino-methoxyphosphine, N,N-diethylamino--
cyanoethoxyphosphine, and (N-morpholino)-methoxyphosphine
(Moore, M.F. et al, J Org Che~.., 198~, 50, 2019-2025;
Uznanski, A.W., et al, Tetranedron ~etts. ,1987,28, 3401-3404;
20 Bjergarde, K., et 21, Nucl Acids ~es., 1991, la, 5843-5850;
Dahl, O. Sul f ur ~eports, 1991, 11, 167-132). Related
coupling groups such as N,N-diisopropylamino-methyl-phosphine
or N,N-diethylamino-methyl-phosphine can aLso be used to
prepare methylphosphonates. Methylphosphona~e oligomers can
25 be conveniently synthesized using coupling groups such as N,N-
diisopropylamino-methylphosphoramidite. Synthesis of
nucleomonomer amidites of the invention can be accomplisAec`
cor ~ntional methods (for exampl-, Gryaznov, S.M., et 21, 1~.;~..
Ac.us ~es., 1992, ~Q, 1879-1882; Vinayak, R., et al, Nucl
30 Acids ~es ., 1992, 2G, 1265-125g; Sinha, N.D., et al, Nucl
Acids ~es., 1984, 1~, 4539-4557; and othe~ references cited
herein).
-- 50 --
CA 02202274 1997-04-09
W Og6/14330 PCTnUS95/14599
2. DrOteCtinC G-OUDS.
Protecting groups such as diisobutylformamidine, benzoyl,
isobutyryl, FMOC, dialkylformamidine, dialkylacetamidine or
other groups known in the a-t can be used to protect the
5 exocyclic nilrogen of the cytosine, adenine or guanine
heterocycles. Alternatively, cytidine can be directly
incorporated into oligomers without a protec~ing group at the
exocyclic nitrogen using described methods (Gryazno~, S.M. et
al, J Amer Chem Soc., 1991, 113, 5876-5877; Gryaznov, S.M. et
al, Nucl Acids Res., 1992, _0, ;879-1882; Kung, P.-P. et al,
Tetranedro,rl Letts., 1992, 33, 5869-~872).
Suitable protecting groups are DMT (dimethoxy trityl), Bz
(benzoyl), Bu (isobutyryl~, phenoxyacetyl, MMT
(monomethoxytrityl) or FMOC al the 5' terminus and/o- hydrogen
15 phosphonate, methyl phosphoramidite, methyl phosphonamidite,
~-cyanoethylphosphoramidite, TBS (t-butyldimethylsilyl) or
TBDPS (t-butyldiphenylsilyl) at the 3'-terminus.
P-eferred protecling grou?s are Bz (benzoyl), DMT
20 (dlmethoxytrityl), MMT (monomethoxy~rityl) or FMOC a, the 5'
terminus or position and/o- TBS, hydrogen phosphonate,
methylphosphoramidite, methyl-phosphonamidite, ~-
cyanoethylphosphoramidite at the 3'-terminus. However, it is
intended that the position of the blocking groups can be
2~ reversed as needed (e.g., a phosphoramidite at the 5' position
and DMT at the 3'-position). In general, the nucleomonomers
and oligomers of the invention can be derivatized to such
"blocking groups" as indicated ir. the relevant formulas by
methods known in the art.
Con jugates:
The subject invention also provides fo- "conjugates" of
the oligomers of the invention. "Conjugates" of conventional
-- Si --
CA 02202274 1997-04-09
W O96/14330 PCTrUS95114599
oligomers are known to the person of ordinary skill in tAe
art. For example, the oligomers of the in~ention may be
covalently linked to various moieties such as, for example,
intercalators, and compounds which interact specifically with
5 the minor groove of the DNA double helix. Other moieties for
conjugation to the subject oligomers include, labels, (e.g.,
radioactive, fluorescent, enzyme) or moieties which facilitate
cell association using cleavable linkers and the like.
Suitable radiolabels include 32p, 35S, 3H, l3'I and 14C; and
suitable fluorescent labels include fluorescence, resoru~in,
rhodamin~, BODIPY (Molecular Drobes) and Texas red; suitable
enzvmes include alkaline ~hosphatase and horseradish
Deroxidase. Other compounds which can be used as co~aler.tly
linked moieties include biotin, antibodies or antibody
fragments, asialoglycoprotein, transferrin and the HIV Tat
15 protein can also conveniently be linked to the oligomers of
the invention.
These additional moieties can be derivatized through any
convenient moiety. For example, intercalators, such as
20 acridine or psoralen can be linked to the oligomers of the
in~er.tion ~hrough any available - OH o~ -S.:l, e.g., 2. the
terminal 5'-position of the oligomer, the 2'-positions o~ RNA,
or an OH, NH2, COOH or SH incorporated into the 5-position of
pyrimidines. A derivatized form which contains, for example,
- CH2CH2CH2,OH or -CH2CH2CH2SH in the 5-position of pyrimidines
is convenient. Conjugates including polylysine or lysine can
be synthesized as described and can further enhance the
binding affinity of an oligomer to its target nucleic acid
sequence (Lemaitre, M. et al., Proc Natl Acad Sci. USA,1987,
8a, 6a8-652; Lemaitre, M. e~ al., Nucleosides a.~d Nucleotides,
30 1987, 6, 31-~-315).
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W O96/14330 PCT~US95114599
A wide variety of subs~i,uen~s can be attached, includins
those bound through linkages or substitute linkages. The -OH
moieties in the oligomers can be r placed by phosphate groups,
protected by standard protecting groups, or coupling groups to
5 prepare additional linkages to other nucleomonomers, or can be
~ound to the conjugated su~s.ituen_. The 5'-terminal OH can
be phosphorylated; the 2'-OH or OH substituents at the 3'-
terminus can also be phosphorylated. The hydroxyls can also
be derivatized to standard ?rotecting groups.
Oligomers of thë invenlion can be covalently derivatized
to moieties that facilita~e -ell 2ssociatl0n using clea~able
linkers. Suilable conjugates aiso in_lude solid suppor~s for
oligomer synthesis anà to facilita~e detection or nucleic acid
sequences. Solid supports include, but are not limiled to,
15 silica gel, controlled pore glass, polystyrene, and magnetic
glass beads.
Su~ar Modi fications:
Derivatives can be made by substitution on the sugars.
20 Among the preferred derivatives o the oligomers of the
invention are the 2'-0-allyi or 3'-allyl group appears to
enhance permeation ability and stability to nuclease
degradation, but does not appear to diminish the a finity of
the oligomer for single chain or duplex targets. In
particular, in ribose-amide backbone oligonucleotides,
different functionalities could be introduced at the 1', 2',
3', 4' and 5' positions of the ribose moiety to improve the
- pharmacokinetic properties o- the corresponding
oligonucleotides.
30 Su~stitute T-i n~eS:
The oligomers of the invention may also contain one or
more "substitute linkages", in addition to the 2'-5' , 3'-5'
CA 02202274 1997-04-09
WO96/14330 . PCT~S95tl459s
and 4'-5' linkages disclosed herein, which are generally
understood in the art. These "substitute linkages" include
phosphorothioate, methylpnospnonate, thionomethylphosphonate,
phosphorodithioate, alkylphosDhonates, morpholino sulfamide,
boranophosphate (-O-P(OCH3)(BH3)-O-), siloxane (-O-Si (X4) (X4)-
O-; X4 is 1 - 6C alkyl or phenyl) and phosp~oramidate
(methoxyethylamine (-O-P (OCH,CH,OCH3) (O) -O-) and the like),
and are synthesized as described in the generally available
literature including the following references (Sood, A., et al
J . Am .Chem .Soc., 1990, 112, 9000-9001; WO 91/08213; WO
90/15.065; WO 91/15500; Stirchak, E.P. et al Nucleic Acid Res.,
1989, 17, 6129-6141; U.S. 3aten- 5,034,506; U.S. Patent
5,142, oa7; Hewil_, J.M. e~ a:, ~ucleosides & Nucl~otides,
1992, ll, 1661-1666; Summer~on, J. et al International
Publication No. 216 860). Substitute linkages that can be
used in the oligomers disclosed herein also include the
sulfonamide (-O-SO2-NH-), sulfide (-CH,-S-CH2-), sulfonate (-O-
SO,-CH2-). carbamate (O-C(O)-NH-, -NH-C(O)-O-),
dimethylhydra~ino (-CH,-NCH3-), sulfamate (-O-S(O) (O)-N-; -N-
S(O)(O)-N-), 3'-amine (-NH-CH,-, N-methylhydroxylamine (-CH,-
20 NCH3-O-) and 2',5' linkages (such as 2',5' carbamate (2' -
N(H)-C(O)-O- 5'), 5', 2' ca~bamare (2'-O-C(O)-N(H)- 5'), ~',2'
methylcar~amate (2'-O-C(O)-N(CH3)-5') and 5',2' thioformacetal
(2'-O-CH2-S-5'). Additional substitute linkages that are
suitable include amide linkages described by Buchardt, O. et
25 al, (International Publication No. WO 92/20702), and those
described by Cook, P.D. et 21, ( International Publication No.
WO 92/20822), De Mesmaeker, A. et al., (International
Publication No. WO 92/20823) and as described in
PCT/US92/04294.
Except where specifically indicated, the substitute
linkages, such as a formacetal linkage, -O- CH2-O-, are linked
to either the 4', 3', 2' carbon of a nucleomonomer on the
- 54 -
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WO96/14330 PCT~S95/14599
. .
left side and to the 5' carbon of a nucleomonomer on the right
side. The designations of a 4', 3', 2' or 5' carbon can be
modified accordingly when a structure other than ribose,
deoxyribose or arabinose is linked to an adjacent
5 nucleomonomer. Such struclures include xylose, a hexose,
morpholino ring, carbocyclic ring (e.g. cyclopentane) and the
like.
The use of carbamate, carbonate, sulfide, sulfoxide,
sulfone, N-methylhydroxylamine and dimethylhydrazino linkages
in synthons or oligomers hzs been described (Vaseur, J-J. et
al, J Amer Cnem Soc., 1992, 114, ~006-4007; WO 89/12060;
Musicki, B. et al, J Org Cnem., 1990, ~, 4231-4233; Revnolds,
~.C. etal., J .Org .Chem., 1992, - , 2983-298;; Mertes, M.P.,
et al, J ~ed. CAem., 1969, 12, 154-157; Mungall, W.S., et
al, J. Org . Chem., 1977, ~2, 703-706; Stirchak, E.P., et al,
J. Org. Cnem., 1987, ~, 4202-4206; Wang, ~., et al,
Tetr2hedro~ Letts., 1991, 32, 738~-7388; Inte~national
Application No. PCT US91/03680). Substitute linkage(s) can be
utilized in the oligomers for a number of purposes such as to
20 turther facilitate binding with c-mplementary .arget nucleic
acid sequences and/or to increase the stability of the
oligomers toward nucleases.
Bases:
Suitable bases for use 2s nucleoside bases in the
compounds of the invnetion include no- only the naturally
occurring purine and pyrimidine bases, but also analogs of
these heterocyclic bases and tautomers thereo- Such analogs
include alkylated purines or pyrimidines, acylated purines or
30 pyrimidines, or other heterocycles. Such "analogous purines"
and "analogous pyrimidines" or purine or pyrimidine analogs
are those generally known in the art, some of which are used
as cAemotherapeutic agents. An exemplary, bul not exhaustive,
CA 02202274 1997-04-09
W O96/14330 PCTrUS95/14599
list includes N4N4- ethanocytosine, 7-deazaxanthosine, 7-
deazaguanosine, 8-oxo-NG-methyladenine, 4-acetylcytosine, 5-
(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil, 5-
5 carboxymethylaminomethyl uracil, inosine, N-iso~entenyl-
adenine, 1-methyladenine, 2-methylguanine, S-methylcytosine,
N-methyladenine, 7-methylguanine, 5-methylaminomethyl u-acil,
5-methoxy aminomethyl-2-thiouracil, 2-thiouracil, 4-
thiouracil, 5-(1-propynyl)-4-thiouracil, 5-(1-propynyl)-2-
thiouracil, 5-(1-propynyl)-2-thiocytosine, 2-thiocytosine, and
2,6-diaminopurine. In addition to these base analogs,
pyrimidine analogs including 6-azacytosine, 6-azathvmidine and
5-t_ifluoromethyluracll described in Cook, D.P., et al,
_nternational Publication No. WO 92/02258 (incorporated nerein
by reference) can be conveniently incorporated into the
15 invention oligomers.
Incorporation of 4-thiouridine and 2-thiothymidine into
oligomers has been described (Nikiforov, T.T. et al,
Tetranedron Letts., 1992, 33, 2379-2382; Clivio, D., et al
20 T-t~anedron Letts., 1992 33:65-68; Nikiforov, T. , et al,
~-trahedron Letts., 1991 32:2505-2508; Xu, Y.-Z., et al
T~trahedro.n Letts., 1991 32:2817-2820; Clivio, D,, et al
Tetranedron Letts., 1992 33:69-72; Connolly, B.A., et al.,
Nucl. Acids ~es., 1989 17:4957-4974). Preferred bases include
2~ adenine, guanine, thymine, uracil, cytosine, ~-methylcytosine,
5-(1-propynyl) uracil, cytosine, 5-methylcytosine, 5-(1-
propynyl) uracil, 5-(1-propynyl) cytosine, 8-oxo-N~-
methyladenine, 7-deaza-7-methylguanine, 7-deaza-7-
methyladenine and 7-deazaxanthosine.
Co~alent ~on~ n~ ~oie~y:
Included in some of the oligomers of the invention is a
moiety which is capable of effecting at least one covalent
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WO96/14330 PCT~S95/1459s
. . .
bond between the oligomer and the duplex. Multiple covalent
bonds can also be formed by pro~iding a multiplicity of suc~.
crosslinking moieties. The co~alent bond is preferably to a
base residue in the targe. s~rAnd, but can aiso be made with
other portions of the target, ncludins the saccharide or
phosphodiester. The reaction nature of the moiety which
effects crosslinking determines the nature of the target in
the duplex. Preferred crosslinking moieties include acylating
and alkylaling agen~s, and, in particular, those position~d
relative to the seouence sDeci~icity-conferring portion so as
to permit reaction with t.he target location in the strand.
It is clear tha. the he_erocycl~ need not be a purine or
pyrimidine; indeed the pseudo-base to whic,- the reactive
function is attached need not be a he.erocycle at all. Any
l~ means of attaching the rea~tive group is satisfactory so long
as the positioning is correct.
Polarity o~ Oli~omers:
In their most genera rorm, the symbol 3'----5' indica~es
20 a stretch of oligomer in which the linkages are consisten~ly
rormed between the 5'-hydroxyi of the amino acid residu- of
the nucleomonomer to the le^t with the 3'- (or 2'- for
oligomers having 2'-5' linkages, or 4' for oligomers having
4'-5' linkages) hydroxyl o~ the amino acid residue of the
nuc7eomonomer to the right (i.e., a region of uniform
polarity), thus leaving the 5'-hydroxyl of the rightmost
nucleomonomer amino acid resiàue free for additional
- conjugation. Analogously, 5'----3' indicates a stretch of
oligomer in the opposite orientation wherein the linkages are
formed ~etween the 3'-hydroxyl of the amino acid residue of
30 the left nucleomonomer and the 5'-hydroxyl of the amino acid
residue of the nucleomonomer on the right, thus leaving the
3'-hydroxyl of the rishtmost nucleomonomer residue free for
CA 02202274 1997-04-09
W O96/14330 PCTrUS95/14599
additional conjugation. The same Ihing is true ror 5'----4'
stretch of oligomers.
Ph~-m~nti~ally ~t ~ le Salts:
The in~ention also provides for various salts of all
compounds disclosed herein, including pharmaceutically
acceptable salts for adminislration to an animal or human.
Pharmaceutically acceptable salts and such salt forming
materials are well known in the art. Pharmaceu~ically
acceptable salts are preferably metal or ammonium salts of the
oligomers of the invention and include alkali or alka?ine
earth metal salts, e.g., the sodium. potassium, magnesium or
calcium salt; or advantageously easily crystallizing ammonium
salts derived from ammonia or organic amines, such as mono-,
di- or tri-lower (alkyl, cycloalkyl or hydroxyalkyl)-amides,
15 lower alkylenediamines or lower (hydroxyalkyl or arylalkyl)-
alkylammonium bases, e.g. methylamine, diethylamine,
triethylamine, dicyclohexylamine, triethanolamine,
ethylenediamine, tris-(hydroxymethyl)-aminomethane or benzyl-
trimethylammonium hydroxide. The oligomers of the invention
20 may form acia addition salts, p~eferably of therapeutically
acceptable inorganic or organic acids, such as strong mineral
acids, for example hydrophilic, e.g., hydrochloric or
hydrobromic acid; sulfuric, phosphoric; aliphatic or aromatic
carboxylic or sulfonic acids, e.g., formic, acetic, propionic,
25 succinic, glycollic, lactic, malic, tartaric, gluconic,
citric, ascorbic, maleic, fumaric, hydroxymaleic, pyruvic,
phenylacetic, benzoic, 4-aminobenzoic, anthranilic, 4-
hydroxynbenzoic, salicylic, 4-aminosalicylic, methanesulfonic,
ethanesul-onic, hydroxyethanesulfonic, benzenesulfonic,
sulfanilic or cyclohexylsulfamic acid and the like.
Utilit~ and ~ministration:
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W O96/14330 PCTAUS95/14s99
As t:ne oligomers o~ the invention are ca?able of
signiricant single-stranded or double-stranded target n~cleic
acid binding activity to form duplexes, triplexes or other
forms of stable associa_ion, with naturally occurring
polynucleotides and structural analogs thereo , the oligomers
of the invention may be used in most procedures that empioy
conventional oligomers. Thus, the oligomers of the invention
may be used as, for example, polynucleotide hybridization
probes, primers for the polymerase chain reaction and similar
cyclic amDli ication reac.ions, sequencing primers, and the
like. The oligomers o the invention may also be used in the
diagnosis 2nd therapy of diseases. Tnerapeut-c applicalions
of the oligomers of the in~Jention include the sDeciflc
inhibition or the expression of genes (o- inhibit translction
of RNA sequences encoded by those genes) that are associated
1~ with either the establishment or the maintenance of a
pathological condition through the use or antisense oligomers.
The oligomers of the invention may be used to mediate
antisense inhibition OL- numerous genetic targets. Exemplary
genes o- RNAs encoded by those genes that can be targeted
20 through antisense employing the oligomers include those that
encode en~ymes, hormones, seru~ proteins, .ransme.mbrane
proteins, adhesion molecuies (LFA-l, GPII /III3, ELAM-î, VACM-
1, ICAM-l, E-selection, and the like), receptor molecules
including cytokine receptors, cytokines (IL-1, IL-2, IL-3, IL-
4, IL-6 and the like), oncogenes, growth factors, and
interleukins. Target genes or RNAs can be associated wi.h anv
pathological condition such as those associated with
- inflarnmatory conditions, cardiovascular disorders, immune
reactions, cancer, viral infections, bacterial infections,
yeas. infections, parasite infections and the like.
Oligomers of the present invention are suita~le fo- use
in both ~-. vivo and ex vivo therapeutic applications.
_ 59 _
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W O96/14330 PCTrUS95/14S99
Indications for ex vivo uses include treatment of cells such
as bone marrow or peripheral blood in conditions such as
leukemia (chronic myelogenous leukemia, acure lymphocytic
leukemia) or viral infection. Targel genes or RNAs encoded by
those genes that can serve as targets for cancer treatments
include oncogens, such as ras, k-ras, bc1-2, c-myb, bcr, c-
myc, c-abl or overexpressed sequences such as mdm2, oncostatin
M, IL-6 (Kaposi's sarcoma), HER-2 and translocations such as
bcr-abl. Viral gene sequences or RNAs encoded by those genes
such as polymerase or reverse transcriptase genes of
herpesviruses such as CMV, HSV-1, HSV-2, retroviruses sucn as
HTLV-l, HIV-1, HIV-2, or o~her DNA or RNA -~iruses such as HBV,
HPV, VZV, influenza virus, adenoviruses, flaviviruses,
rhinovirus and the like are also suitable targets.
Application of specifically binding oligomers can be used in
1~ conjunction with other therapeutic treatments. Other
therapeutic uses for oligomers of the invention include (1)
modulation of inflammatory responses by modulating expression
of genes such as IL-1 receptor, IL-l, ICAM-1 or E-Selection
that play a role in mediating inflammation and (2) modulation
20 cf cellular proliferation in conditions such as arterial
occlusion (restenosis) after angioplasty by modulating the
expression of ~a) grow-h or mitogenic factors such as non-
muscle myosin, myc, fox, PCNA, PDGF or FGF or their recep.ors,
or (b) cell proliferation ~actors such as c-myb. Other
suitable proliferation factors or signal transduction factors
such as TGFx, IL-6, gINF, protein kinase C, tyrosine kinases
(such as p210, pl90), may be targeted for treatment of
psoriasis or other conditions. In addition, EGF receptor,
TGFa or MHC alleles may be targeted in autoimmune diseas-s.
Delivery of oligomers of the invention into cells can be
enhanced by any suitable method including calcium phosphate,
DMSO, giycerol or dextran ..ansfection, electroporation or by
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WO96/14330 PCT~S95/14599
the use of cationic anionic and/or neu~ral lipid composilions
or liposomes by methods described (International Publications
Nos. WO 90/14074, WO 91/16024, r~o 91/17g2~, U.S. Paten-
4,897,355). The oligomers can be lnt-oduced into cells Dy
~; complexion with caLionic lipids such 25 DOT.M~ (wAich may or
may not form liposomes) which complex is then contacted with
the cells. Suitable cationic lipids i~.clude but are not
limited to N-(2,3-di(9-(Z)-oc.adecenyloxyl))-prop-1-yl-N,N, N-
trimethylammonium (DOTMA) and its salts, 1-O-oleyl-2-O-oleyl-
3-dimethylaminopropyl-~-hydroxyethylammonium and its salts and
2,2-bls (o7 eyloxy)-3-(trimethylammonio) propane and its salts.
Enhanced delive_y of the inventi~.. oligomers can also be
mediated by the us2 of ~ riruses such as Sendai virus
(Bartzatt, R., Bio~2chnol Appl ~iochem., 1989, 11,133-135J or
1~ adeno~irus (Wagner, _. e~ al, Proc Natl Acad Sci. USA, 1992,
8~, 6099-6013J, (i~J polyamine or polycation conjugates using
compounds such as polylysine, protamin or Na, Nl~-bis
(ethylJspermine (Wagner, E. et al, Proc Natl Acad Sci. USA,
1991, 88, 42~5-4259; Zenke, M. et al, Proc. Natl. Acad. Sci.
20 US.', 1990, 87, 36;5-3659; Chan.~, B.K. et al, Biochem B o?nys
~es Commu..., 1988, 157, 264-270; U.S. 3atent 5,138,045); (iii)
lipopolyamine complexes usins compounds such as lipospermine
~Behr, J.-P. et al, Proc Natl Acad Sci. USA, 1989, 86, 6982-
6986; Loeffler, J. 3.. et al, J. Neurochem., 1990, 54, 1812-
25 1815)i (iv) anioni^, neutral or DH sens~tive l pids usingcompounds including anionic ?hospholipids such as phosp,halidyl
glycerol, cardiolipin, phosphatidic acid or
phosphatidylethanoiaminQ (Lee, K.-D. et al, Bioch2m Biophys
ACT~, 1992, llQ3, 185-157; Cheddar, G. et al, Arch Biochem
30 BioDnys, 1992, 294, 188-192; Yoshimura, T., et 21, Biocnem
In_., 1990, 20, 697-706); (v) conjugates with compounds such
as transfer-in or biotin or (vi) conj ugates with proteins
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W O96/14330 PCTrUS95/14599
(including albumin or antibodies), glycoproteins or polymers
(including polyethylene glycol) that enhance pharmacokinetic
properties of oligomers in a subject. As used herein,
transfeclion refers to any method that is suitable for
delivery of oligomers lnto cells. Any reagent such as a lipid
or any agent such as a vlrus that can be used in transfec~ion
protocols is collectively rererred ,o herein as a "permea~ion
enhancing agent". Delivery of the oligomers into cells can be
via cotransfection with other nucleic acids such as (i)
expressable DNA fragments encodins a protein(s) or a prolein
fragment or (ii) translatable RNAs that encode 2 protein(s) o-
a protein fragment.
The oligomers of the invention can thus be incorporated
into any suitable formulation that enhances delivery of the
15 oligomers into cells. Suitable pharmaceutical formulations
also include those commonly used in applications where
compounds are delivered into cells or tissues by topical
a~ministration. Compounds such as polyethylene glycol,
propylene glycol, azone, nonoxonyl-9, oleic acid, DMSO,
20 polyamines or lipopolyamlnes can b- used in topical
preparations that contain the o igomers.
The invention oligomers can be conveniently used as
reagents for research or production purposes where inhibition
of gene expression is desired. There are currently very few
reagents available that efficiently and specifically inhibit
the expression o- a targe. gene by any mechanism. Oligomers
that have been previously reported to inhibit target gene
expression frequently have nonspecific effects and;or do not
reduce target gene expression to very low levels tless than
30 about 40% of uninhibited levels).
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-
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W O 96/14330 PCTrUS95/14599
Thus, the oligomers 2S described herein constitute a
reagent that may be used in melhoas of inhibi~ing exDrGssion
of a selected protein or p-otelns in z su~ject _r in c~lls
wherein the proteins are encoded by DNA sequencGs and the
5 proteins are translated from RNA seauences, comprising the
steps of: introducing an oligomer o~ the lnventi~n ln.o the
cells; and permitting Ihe cligomer to rorm a .rl?lex with Ine
DNA or RNA or a duplex with the DNA or RNA wnere~v e.YprGssion
of the protein or proteins ~s inhibiteà. The me_nods and
compound of the present invention are suitable fo_ modulating
gene expression in both ?rocaryo~ic and eucaryot-c cells such
as bacterial, ,~ungal ~arasite, yeas_ and mammal_an cells.
RNase H "compelent" o~ RNase H "incompe~ent" oligomers
can be easily designed using the substitute linkages of the
1~ in~ention. RN2se H compelent oligomers can comprise one or
more RNase H competent domains com?rised of linked RNase H
competent nucleomonomers. ~llgomers naving modi~ications suc.
as 2'-substitutions (2'-O-allyl and the likG) or cerlain
unch2rged linkages ~methylDhosDhona_G, ?hosDhora.mldate and the
20 like) are usually incompe~en. as _ substrate that is
recogni2ed by and/c- 2cted on ~v RNase ~ Nase .-: comperencG
can acilitate antisense oligome- unction by degrading the
target RNA in an RNA-oligomer duplex tDagle, J.M. et al, Nucl
Acids Res., 1990, 18, 4751-4757; Walder, J.A. et al,
2 International Publication Number W3 83/0535B~. The enzyme
cleaves RNA in RNA-DNA du?lexes.
- In order to retain RNase H com?e.ence, an oligomer
requires a RNase H comDzter.- domain of tAree or more comDeter._
contiguous nucleomonomers located withln it (Qua-lin, R.â., e~
30 al, N~cl Acids ~es.,1989, 17, 7253-7262). Design of
oligomers resistant .o nuclease digestion will have terminal
linkage, sugar and/or ~ase modifications ~o erfec. nuclease
.
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W O96/14330 PCTrUS95/14599
resistance. Thus, the ollgomers c~n be designed to have
modified nucleomonomer residues at either or both the 5'-
and/or 3'- ends, while having an internal RNase H competent
domain. Exemplary oligomers ~hat '-el2' n RNase H competence
~; would generally have unifo-m ?olarity and would comprise about
2 to about 12 nucleomonomers at the 5'- end and at the 3'- end
which stabilize the oligomer to nuclezse degradation and about
three to about 26 nucleomonomers that function as a RNase H
competent domain between the ~Nase H incomDe.ent 3' and 5'-
ends. Varialions on such an oligomer would include (l) a
10shorter RNase H competent domain comp-ising 1 or 2 RNase H
competent linkages o_ subsritute linkages, (2) a longer RNase
H incom~e~ent domain coI;Lprising U? ~o '5, 20 or more
substltute linkages or nucleomonomers, (3) a longer RNase H
competent domain comp-ising up to 30, 40 or more linkages, (4)
15 oligomers with only a single RNase H incompetent domain at the
3' end or a~ the 5' end.
Oligomers containing 2S rew as about 8 nucleomonomers may
be used to effect inhibition cf target ?rotein(s) expression
20 by formation o duDlex or t-iplex struc~ures with target
nucleic 2C'- sequences. However, linear oligomers used to
inhibit target protein expression via duplex or t_iplex
formation will preferably have rrom about 10 ~o about 20
nucleomonomer residues.
01igomers containing substitute linkages of the invention
can be conveniently ci-cularized as described (International
Publication No. WO 92/19732; Kool, E.T. J f~ Chem Soc.,l991,
~, 6265-6266; Prakash, G. et al, J ~m Chem Soc., 1992, 114,
3523-3527). Such oligomers are suitable for binding to
30 single-s~-anded or double stranded nucleic acid targets.
Circular oligomers can be of various sizes. Such oligomers in
a size range o~ about 22-50 nucleomonomers can be conveniently
-- 64 --
CA 02202274 1997-04-09
WO96/14330 PCT~S95/14599
prepared. The circular oligomers can have f_om about three .o
about six nucleomonomer residues in the loop region that
separate binding domains or the oligomer 25 described
~Prakash, G. ibid). Oligomers can ~e enzymatically
circularized through a lerminal ~hosphate by ligase or by
chemical means via linkage through the ~'- and 3'- terminal
sugars and/or bases.
The oligomers can be utilized to modulate targel gene
expression by inhibiting the interaction of nucleic acid
binding proteins resDonsible for modulatins transcription
(Maher, L.J., et al, ~cience, 1989, 24~, 725-730) or
Lranslation. The ol gomers are l.lUS suitable as sequence-
speciric agents that compete with nucleic acid binding
proteins (includins ribosomes, RNA polymerases, DNA
1~ polymerases, translational initiation factors, transcription
~actors that either increase or decrease transcription,
protein-hormone transcription factors and the like).
ADDropriately design-d oligomers can thus be used to increase
.arge~ protein synthesis through mechanisms such as binding ~o
20 or near a regulato_y site tnat transcri?tion ~acto-s us- to
r-~ress expr-ssion or by ir.hibilir.g the exDression or a
sel-c~ed represso~ protein itself.
The invention oligomers, comprising additional
modirications that enhance binding affinity can be designed to
contain secondary or tertiary structures, such as pseudoknoLs
o- pseudo-half-knots (r, cker, O.J. et al, Science, 1992, 257,
958-961). Such structures can have a more stable secondary or
~ertiary structure than corresponding unmodified oligomers.
- The enhanced stability of such s~ructures would rely on the
30 increased binding affinit-y between regions of self
complementary in a single oligomer or regions of complementary
-b~.ween two or more oligomers that form a given structure.
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W O96114330 PCTrUS95/145~9
Such struc~ures can be used lo mimic s~ruc~ures such as tne
HIV TAR structure in order to interfere with binding by the
HIV Tat protein (a protein that binds to TAR). A similar
approach can be utilized with other Iranscri2tion or
5 translation factors that recognize higher nucleic acid
structures such as slems, ioops, hairpins, knots and the like.
Alternatively, the invention oligomers can be used to (1)
disrupt or (2) bind to such structures as a method to (1)
interfere with or (2) enhance the binding of proteins to
nucleic acid structures.
~ n addition to their use in antisense o~ ~_iple helix
therapies, ~he oligomers of the invent~ 5n can also be a?Dlied
as therapeutic or diagnost c agents tAat runclion by direct
displacement of one strand in a duplex nucleic acid.
15 Displacement of a strand in a natural duplex such as
chromosomal DNA or duplex viral DNA, RNA or hybrid DNA/RNA is
possible for oligomers with a high binding affinity ror their
complementary seauence is not great enough to efficiently
displace a DNA or RNA strand in a duplex. Therapeutic
20 e ficacy o, oligome-s that function by D-looping woul~ result
from high a rini~y Dinding to a comDlementary sequence that
resul~s in modulation of the normal bioiogical function
associated with the nucleic acid target. Types o~ target
nucleic acids include bu. are no. limited to (i) gene
sequences including exons, introns, exon/intro,, junctions,
?romo~er/enhancer regions and 5' or 3' untranslated regions,
(ii) regions of nucleic acids that utili~e secondary structure
in order too function (e.g. the HIV TAR stem-loop element or
tRNAs), (iii) nucleic acids that serve structural or other
-unctions such as teLomeres, centromeres or replication
30 o~igins (virus, bacteria and the like) and (iv) any other
duplex region. It is clear that oligomers can be synthesized
Wit~l discrete functional domains wherein one region of an
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wos61l433o PCT~S95/14599
oligomer binds to a target by D-looping while an adjacent
region binds a target molecuis by szy, rormlng a triple helix
or binding as an aptamer to 2 pro,ein. Alternatively, a D-
looping oligomer can bind to each strand in a duplex by
switching the strand to which the oligomer binds (i.e. by
having one region of the oligomer tha~ binds to one strand and
another region that binds to the complementary strand). The
controlling elements that diclate the mode of binding (i.e.
triple helix or DOloop) are the sequence or the oligomer and
the inherent affinity built into the oligomer. Base
recognitiOn rules in Watson-C_ick duplex binding differ from
those in Hoogsteen controlle~ ~riplex binding. Because of
tnis, the oligomer base seau-n.-e c~n be used ~3 di_tate tne
type of bindir.s -ules an oligomer wlll u.i1ize. D-loop
structures are formed in nature by enzyme-medi2ted processes
1~ (Harris, L.D. et al., et al., J Biol CAem., 1987, ~h~, 9285-
9292) or are associated with regions where DNA replication
occurs (Jacobs, H.T. et al., Nucl .~cids ~es, 1989, 17, 8949-
8966). D-loops that aris~ rrom t~2 binding of oligomers can
result from a one or two steD ?rocess. ~irect displacement of
20 a target s~rand will give -ise .o a D-loop by a single binding
even~. However, D-looping can a`so occur by forming a t~iple
helix which facilitates a strand displ2cement envent leading
to the D-loop.
Ribozvmes containing substitu~e linkages of tne inventicn
can be designed in order to desigr. sDecies with altered
characteristics. Ribozymes that cleave single stranded RNA or
- DNA (Robertson, D.L., et al., Nature, 1990, 344, 467-468j
have been desc-ibed. Therapeut c applications for ribozymes
- have been Dostulated (Sarver, ~. et al., Science, l990, 247,
1222-1225; International Publica~ion Number WO 9l/04319).
Secondary or tertiary structure necessary for ribozyme
function can be 2f~ ected by design of aDproDriate oligomer
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sequences. For example, ribozymes having nuclease stable
targeting domains containing substitute linkages of the
invention can have higher af~_nity, while maintaining base
pairing specificity, for larget sequences. Because of t:ne
higher 2ffinity and/or nuclease stability of the inventio..
substitule linkages shorter -ecognition domains in a ribozyme
(an advantage in manufacturing) can be designed which can lead
to more favorable substrate turnover (an advantage in ribozyme
function).
In therapeutic applications, the oligomers o the
inventicn may be utilized in a manner appropriate for
treatmen~ of a variety o~ conditions by inhibiting expression
of appropriate targe. genes. For such therapy, the oiigomers
can b ,~ormulated for a variety of modes o~ administration,
1~ including systemic, topical or localized adminis~ration.
Techniques and formulations generally can be found in
Reminaton's Pharmaceutical Sciences. Merck Publishing Co.,
Easton, PA, latest edition. The oligomer active ingredient is
generally combined with a ca-rier such as a diluent or
20 ex-ipient which can include fillers, extenders, binders,
wettino agents, disintegranls, surface-active agents, or
lubricanls, depending on the nature of the mode of
administration and dosage forms. Typical dosage forms include
tablets, powders, liquid preparations including suspensions,
emulsions and solutions, granules, capsules and suppositories,
2~
as well as liquid prepar2tions for injections, including
liposome preparations.
Fo- systemic administration, injection is preferred,
including intramuscular, int~avenous, intraperitoneal, and
30 subcutaneous. For injection, the oligomers of the invenlion
are formulated in liquid solutions, prererably in
physiologrcally compatible buffe_s such as Hank's solution or
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Ringer's solution. In addition, the oligomers can be
formulated in solid form and redissoived or suspended
immediately prior to use. Lyophilized rorms zre also
included. Dosages that can be used for sys~emic
administration prererably range from a~out 0.01 mg/Kg to 50
mg/Kg admin'stered once or twice per day. However, different
dosing schedules can be utilizsd depending on (i) the potency
or an individual oligomer at innibiting the ac.ivity of its
targe~ DN~ or RNA, (ii) the severity or exten~ of a
palnologiczl disease stat- associated with a given targ~t
gene, or (iii) the pharmacokine~ic behavior of a given
oligomer.
Sys~emic adminis~ration ca.. also be by transmucosal or
transdermal means, or the compounds can be administered
15 orally. For transmucosal o- transdermal administration,
penetrates appropriate to the Darrier to be permeated are used
in the formulation. Such penetrates a~e generally known in
the ar-, and include, for example, bile salts and fusici_ acid
de-iv2tives Lor ~-ansm~cos-,l aaministr2tion. n addition,
20 ds_ergen~s can be used .o r2cilltate permeation. Transmucos2
adm nistration can be throug,i use of nasal sprays, for
example, o- suppositories. For oral administration, the
oligomers are formulated into conventional oral adminis.ration
forms such as capsules, tablets, and tonics.
For topical adminisiration, the oligomers of the
invention are formulated into ointments, salves, gels, or
- c-lams, as is generally known i-. the art. Formulation o~ the
inven.ion oligomers for ocular indications such as viral
inLections would be based on standard compositions known in
30 t,ie art.
..
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In addition to use in therapy, the oligomers of the
invention can be used as diagnostic reagents to detect the
presence or absence o~ ~he ~arget r.ucleic acid sequences to
which they specifically bind. The enhanced binding affinity
of the invention oligomers is an advantage for their use as
primers and probes. Diagnoslic .ests can be conducted by
hybridization through either double or triple helix formation
which is then detected by conventional means. For example,
the oligomers can be labeled using radioactive, fluorescent,
or chromogenic labels and tAe presence of label bound to solid
support detected. Alternatively, the presence of a double or
triple helix can be detec~ed bv 2n~ibodies which specifically
recognize these -orms. Means 5_ conducting assays using such
oligomers as probes are generally known.
1~ The use of oligomers o the invention substitute linkages
25 diagnostic agents by triple helix formation is advantageous
since triple helices Lorm unde_ mild conditions and the assays
can thus be carried out witnou~ subjecring test specimens too
harsh conditions. Diagnos, ~ assays based on detection of RN.
20 for identifica~ion o~ bacte-ia, _~ngi or proto,oa sequences
often re~uired iso'a~ion o ~iA -om samples o~ organisms
grown in the laboratory, which is laborious and time
consuming, as RNA is extremely sensitive to ubiquitous
nucleases.
2~
The oligomer probes can also incorporate additional
modificalions such as modified sugars and/or substitute
linkages that render the oligomer especially nuclease stable,
and would thus be useful ro_ assays conducted in the he
presence or cell o- tissue extracrs wnich normally contain
30 nuclease aclivity. Oligome-s containing terminal
modifications oLten retain their capacity to bind to
complimentary sequences without 1 oss of specificity (Uhlmann
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WO96/14330 PCT~S95/14599
et al., Chemical Reviews, 1990, 9~, 5a3-584). As set rorth
above, the invention probes can also contain linkers that
permit specific binding to alternate DNA strands b~
incorporating a linker that Dermi.S such binding (Froehler,
B.C. et al., Biocnemistry, 1992, 31, i603-1609); rorne et al.,
J Am Chem Soc., l99O, 112, 2435-2437).
Incorporation or base analogs of the present invenlion
into probes thzt also contain covalent crosslinking agen.s has
lO the poten~ial tO increase s-nsitiv ty an~ reduce background in
diagnostic or detec~ion assays. _n addition, the use o
crosslinking agents will permit novel assay modiric~_ions such
as (1) tne use Oc the crosslink to lncrease probe
discrimin2tion, (~) incorpora~ion of a denaturing wash s~ep ~o
red~-e background and ~3) carrying ou~ hybridization and
1~ cro3slinking at o~ near the melting temperature o~ the hybrid
to reduce secondary s~ructure in the target and to increase
probe specificity. Modifications of hybridization conditions
have been previously desc~ibed (Gamper et al., Nuc7eic Acids
~es., 1986, ~, 9943).
Oligomers of th- invenrion are suitabie for use in
diagnostic assays that empioy methods wherein ~itA~; the
oligomer or nucleic acid to be delected ar2 covaiently
attached to a solid support as described (U.S. Patent No.
25 4,775,619). The oligomers are also suitable for use in
diagnostic assays that rely on poiymerase chain reaction
techniques to ampliry target sequences according to desc-ibeq
~ methods (_uropean Patent Publication No. 0 393 744).
Oligomers of the invention containing a 3' terminus that can
~ serve as a primer are comp2.ible with polvmerases used in
polymerase chain reaction methods such as the Taq or Vent-A
(New England Biolabs) poly~erase. Oligome~s of the invention
can thus De u. li-ed 2S primers ir. ~C~ prolocols.
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W O96/14330 PCT~US95/145g9
The oligomers of the invention are useLul as primers tAa
are discrete sequences or as primers with a -andom sequence.
Random sequence primers can be generally about 6, 7, or 8
nucleomonomers in length. Such primers can be used in various
nucleic acid amplification protocols (PCR, ligase chain
reaction, etc.) or in cloning protocols. The substitute
linkages of the inven~ion generally do not interfere with the
capacity of the oligo~er .o function as a primer. Oligomers
of the invention having 2'-modifications at sites other than
the 3' .erminal resiàue, other modifications that render the
oligomer RNase H incompetent or otherwise nuclease stable can
be advantageously used as ~robes o- rime-s for RNA o- DN.~
sequenc~s n cellular extr2c~s or other solu_ions tha. contai~.
nucl~as-s. Thus, the oligomers can be used in protocols for
amplirying nucleic acid in a sample by mixing the oligomer
15 with a sample containing ~arget nucleic acid, followed by
hybridization of the oligomer with the target nucleic acid and
amplifying the target nucleic acid by PCR, LCR or other
suitable methods.
The oligomers deriva.ized tO chelating agents such 25
EDTA, DTPA or analogs of l,2-diaminocy~loAexane acetic ac d
can be u_ilized in various invitro diagnostic assays as
described (U.S. Patent Nos. 4,772,548, 4,7G7,440 and
4,707,352). Alternatively, oligomers of the invention can be
derivati~ed with crosslinking agents such as 5-(3-
iodoacetamidoprop-l-yl) 2'-deoxyu-idine or 5-(3-(4-
bromobu~yramido) prop-l-yl)-2'-deoxyuridine and used ln
varlous ssay methods or kits as described (International
Publication No. W0 90/14353).
In addition to the foregoing uses, the ability or the
oligomers to inhibit gene expression can be verified in i~-
vitro s~stems by measuring the levels of exDression in subject
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cells or in recombinanl systems, Dy an suilab'e method
(Graessmann, M. et al, Nuc12ic Acids R-s., 1991, 19, 53-59).
The invention havir.g been desc-ibed above, the following
examples are orfered to better explain the invention. The
examples are offered to illustrate the invention and should
not be interpreted as limiting the invention.
EXAMPT, ~. S
O~erview of the S~nthesis of the Nucl~mt nt~^r Svnthon and
Oli~omers:
The oligomers o the inven_i^n can be synthesized using
~eactions known i- the ~t o- oligonuc 7 eo,ide ~erivative
synthesis. Se- e.c. Flando-, J. and Yam, S.Y., Tetrah2dron
Letts., 1990, ~1, 597-600; Mattson, R.J. et al., ~ Org Chem.,
1990, 55, 2552-2554; Chung, C.K. et 21., J Org Chem., 1989,
S4, 2767-2769.
As can be seen from the varie.y of substitute linkages
specifically listed in Table 1, the substitute linkages of the
20 invention can vary so as ~o contain one or more nitrogen,
sulrur, and/or oxygen atoms in their s~ruc~ure. TAe posi~ions
o these atoms in the substi_ute linkage can va-y from the
~5 ~ ~r end, to the "middle" to the "2'" or "3'" and ".'" end.
In this section, 2 series of representative synthesis reaction
2~ figures are set fcrth which provide routes to various
locations and combinations of ni~rogen and oxygen atoms within
the substitute linkages.
-
The synthesis iliustrated in figures 1-25 be modified as
~ is known to those practicing in the area of oliyonucleotide
chemistry. For exampl-, although protection o the bases is
no~ always indicated in the synthesis figures, such may be
d-sirable and can be accomplished using reagents and
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W O 96/14330 PCTrUS95/14599
techniques known in the 2rt. See, e.g. Pro~ective Groups in
Organic Synthesis (Theodora W. Greene, John Wiley and Sons,
1981~. Similarly, although th~e use of prote_ti~-e g-oups is
shown in some cases, it is nol always necessary ~o block the
reactants in order to synthesize the exemplified invention
oligomers.
" cxample 1
The first five s~eps sAown in Figure 1 relate to the
preparation of isobutryl protected serinol amino acid alcohol.
The sixth and subsequent steps in Figure l are directed tO the
synthesis of the serinol subs i.uted thymine phopAoramidite
building block.
In step 1 of Figure ~, the amino group of Ihe serine
amino acid is protected by rea~ting 1 with di-te-;-butyl
1~ dicarbonate to yield compound 2. Other equivalent protec~ing
groups may be used. Tn the next step, the ~-hydroxyl group of
Compound 2 is blocked witA dihydropyran to give rully
protected amino acid 3. The amnio acid 3 is then reacted
with diborane-dimethyi sulfide complex to pro~ide alcohol 4,
20 wAich on exposure to isobulryl chloride gave ~. Tris red~c_ion
reaction can also be carried out using isobu~yl cnoloroforma~e
and sodium borohydride (see: K. Ramasamy, R. K. Olsen and T.
~mery, Syntnesis, 1982, 42). Reaction of 5 with
trifluoroacetic acid for 30 m nutes followed by washing with
25 NaHCO3 afforded 6.
Thymine acetic acid 7 w25 prepared as desc~ibed in the
literature (see: L. Kosynkina, W. Wang and T. C.
Liang, Tetranedron Let~s, 1994, ~5, 5173). Coupling of 7 wi.h 6
30 unàer mixed anhydride condition provided 8.
Dim2thoxylritylation of 8 wi_h DMTCl gave compound 9, which on
hydrolysis with base afforded 10. Phophysitylation of 10 under
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PCTAUS95/14599
W 096/14330
standard condition provided tne serinoi coupLed thymine
building block 11. This synthon can then be added into a
growing oligomer using conven~ional chemist-y. Any DNA
synthesis chemistry such as pnosphoramidate or phosphona~e
5 chemistry can be used to link monomers or dimers in a manner
analogous IO that set forth abo~e.
~xample 2
In reaction Figure 2, tnymine acetaldehyde 13 was
10 produced by the Ireatment of thymin~ with bromoacetaldehyde
dimet~ylactal followed by hydrolysis of 12 with aqueous TFA.
.~ldehyde 13 and _mine the 6 ar~ then coupled and the
correspondir.g inlermedia~- W2S _ranstorm~d ~0 the
phosphoroamidite building bloc.~ 17 in a mannsr analogous to
the steps used ir. Figure 1.
1~
Example 3
In reac-ior. Figure 3, th~ starting material is a ~-
substituted amino acid 18. Th~ substituted amino acid could be
transformed inlo the phospno_oamidite building block 27 by
20 following th2 rJ-ocedure o- the steps used in Flgures 1 and 2.
Exampie 4
In Figure 4, the starting amino alcohol 21 is oxidized
with CrO3Jpyridine mixture to give an aldehyde 28. The
25 aldehyde which or. reaction with alkyl halide in the presence
of a base shoul~ yield compour.d 29. The amino alcohol 29 could
then be trar.sformed to the building block 35 in a manner
analogous to the steps used in figure 1 and 2.
30Example 5
Turning to Figure 5, the first four steps are essentially
the same st ?s as used in Figure 1, ir. this aspartic acid is
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PCTnUS95/14599
W O96114330
used instead of serine. Aspartic acld methylester 36 gave
fully protected alcohol 40, which on selective depro~ection
with acelic acid provided 41. Oxidation of 41 with
CrO3/pyridine gave the corresponding aldehyde 42. Reductive
amination or the aldehyde 42 with o-benzylhydroxyl amine in
the presence of sodium triacetoxy~orohydride snould give 43
(see: T. Kolasa and M. ~. Miller, ~. Or~. Chem., 1990, 55,
1711). The alcohol 39 is then converted to an aldehyde 46,
essentially using the same reaction conditions as said above
10 but with an allyl~c pro.ecting group for the hydroxyl function
of 39. Coupling of the aldehyae 4~ and the Ayàroxylamine 43 in
presence of sodium r_iacetoxyborohydriae fcliowed by
depro~ec~ion or the amino pro~ecti.ng groups should afrord the
bisamine 48. The bisamir.e 48 could then be conver~ed to a
1~ dimer ~3 by following the s~eps used in figure l.
Exam?le 6
In Figure 6, coupiing o- alcohol ~4 with O-
ben2ylhy~roxylamine ~ under .~itsunob~ reaction condition
20 (see: O. Mitsunobu, Syn~nesis, 1981, ' ) provides compoun~ ~6.
The in.ermediate ~6 on hydrogenztion followed by acetylation
should give ~7. ~xposure of ~7 to TF.~ deblocks the "TBDMSi"
prQtecting group and gives ~8. Coupling of ~8 with 7 followed
by dimethoxytritylation could provide 60. The final ~uilding
25 block ~2 should be accom?lished from 60 by base hydrolysis
foliowed by phosphitylation.
Example 7
In Figure 7, the se~inol 4 is converted to a haiide ~9
and alkylated with thymine to provide 63. The pro~ecting
groups in Ç3 are removed, coupled wi~h DMT-protected
hydroxyacetic acid and phosphi.ylated to yield 66.
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WO96114330 PCT~S95/14599
Example 8
In Figure 8, the alcohol 64 is coupled with N-
hydroxylaminopropanoic ~cid 69 to give 70. Alkylation o~
thymine with a halide 73 gives 74 whic~ on deprotection,
coupling with 76 followed by hydrolysis could afford 78.
Condensation of 78 with 70 followed by pnosphitylation should
give the hydroxamate dimer 80.
ExamDle 9
In Figure 9, N-hydroxylamino proDano~c aldehyde 81 is
used to couple the alcohol 64. The dimer 88 is DreDared rrom
83 2.-~t 86 by f~llowir.g .he s,e?s used n f_gure 8.
~xample l0
In Figure l0, alkylation ~see: T. Kolasa and M. J.
Mille~, J. Org. C~em., l990, ~., 4246) OL a-bromo~
amino?roDanoic acid methylester 89 with thymin~ would produce
90. The intermediate 90 o~ hy~rolysls with sodium hydroxide
gives an acid 9l wAich iS c~?led with 6 to provide 92. The
20 compound 9~ is then conver~ed in~o tAe pnospAoroamidite
~ul aing olock 9~ using the S_2pS desc~ibed i.. igure i.
Example ll
In Figure ll, thymine is alkylated with an alkylamine
halide 96 (see: R. K. Olsen, ~. Ramasamy and T. Emery, J. Org.
Che.~.., 1984, 4~, 3527 and Islam et al., J. Med. Chem., lg94,
37, 2g3-30~ for tAe preparation or aminoalkyl halide) to give
97. ~xposure of the compound 97 to TFA followed by alkylkation
would _ffo_d l00. The building block l03 is obtained from l00
by d .~e_noxytrityiation, hydrolysis, followed by
phosphityation.
Fxample 12
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Figure 1~ ls an alternative roule ~o a hydroxamate
backbone dimer 111 from N-hydroxylamine 43 and an aldehyde 107
which in turn prepared from asparti- acid.
~xample 13
In Figure 13, the dimer 11~ s prepared from the
intermediate 108 and 13 by following the same steps of
reactions described in figure 2.
ExamDle i4
In Figure 1~, N-hydroxylthymine is DreDared (see: Kim, C.
U., et al., T~tran~dron L~tts., 1992, -_, 2~-28) and coupled
with N-hydroxyphthalimide to provide 117 which on exposure to
hydra2ine in ethanol should give 118. Treatment o 118 with
DMT-protected glycerol epoxide 119 provides 120. The
intermediate 120 is then transformed to the phospAoroamidite
121 using s,andard procedure. In second synthesis, compound
118 is coupled with amino acid aldehyde 122 under reduct~ve
amination conditions .o provlde 123. Droteclion c- the
secondary amino functionali_y with FMOC-' followed by
hydrolysis shouid a,~fo-d 125.
Example 15
In Figure 15, 1,2-dihydroxyproDanoic acid 126 is coupled
with N-hydroxylamine thymine 118 to give 127, which is then
transformed into phophoramidite syntAon 129 under standard
conditions. The compound 118 is also coupled with adipic acid
and transformed into nucleic acid building block 133.
Example 16
In Figure l~, first the building block 136 is synthesi2ed
frorr, 118 and 134 in 2 siI~lil2r manne- described in figure 1.
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Coupling of 139 with 118 provided 140. Treatment of 137 with
118 should pro~ide 138 which on condensation wilh 140 gives
the dimer 141.
Example 17
In Flgure ~7, an aidehyde 142 and an glycine benzylesler
is coupled to give 143. Trea,men. of 143 with 7 should p~ovide
14~ which on exposure to acelic acid gives 148. Mitsunobu
alkylation o 148 with Boc-NH-0-acetylhydroxylamine should
10 give 147 which on hydrogenation the building block 150 could
be obtained. Simil~rly cou? i-.g ~~ 143 wi.h 13 and following
-h~ same reactions as above s.suld y~ 2~ e synlhon 149.
Exa~ple 18
In Figure 18, reduc~ive amination of the aldehyde 142 and
Boc-N~-0-benzylhydroylamine ga~Je 1~1. Hydrogenation of 1~1
followed by alylation o~ 152 with glycolic acid 1~3 ( B. C.
Borer and D. C. BalogA, Tet~2nedron Letts., 1991, 3~., 1039)
should yield 1~4. Treatment ^, 154 wi~h TFA willl remove the
20 Bo- protecting srou?, whicr. c-. coupling would result in 1~.
The hydroxyl p-otec-ir,g grou? _ 15~ could sele~_ively be
removed with ace.ic 2C' d tc give 1~6. The ccmpound 1~6 ~ill
then be transformed to the bu~lding block 1~7 using standard
reaction conditions. Similarly the buildins ~lock 1~8 will be
2~ produced by coupling of 1~4 wlth 13 and following the steps
used for the preparation of 1~7.
Example 19
In Figure 19, alkylation of thymine-N-hydroxylamine 160
30 with alcohol 162 w ll yield 163. Tne compour.d 163 could be
transformed to the phosphoroamidite building block 166 by
following the steps used ir. ~_gure l.
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Example 20
In Figure 20, first the intermedia_e 169 is synthesized
from glutamic acid using standard react on conditions.
ALkylation of .hymine wi~h 1~9 would glve 170 which on
treatment with TFA should produce 171. The intermediate 171
could be coupled with Boc-glycine to pro~ide 173 which on
hydrolysis would afford the monomer synthon 174. Similarly 172
could be prepared by coupling of 118 and Boc-aminoacetic
aldehyde rollowed by hydrolysis of the benzylester.
Example 21
I ,n r- su~e 2-, the nterm.-dia_e 177 is p-epared from Boc-
NH-0-ben_vlhvdroxylamine and 175 usinS s_anda~ rezction
conditions. Hyàrogen-~ti_. o 177 foliowed by coupling with N-
1~ hydroxythymine 116 would produce 178. Removal of the THPprotecting group followed-by dimethoxytritylation and
phosphitylation should give the building block syntnon 181.
Similarly 182 could be prepared ~y following all the above
reactions and using TH~-Hydroxyacetic aldehyde instead of THP-
20 Hyd~oxyacetic acid.
Example 22
In Figure 22, the building block 191 could be preparedusing the known starting material 183 and following the
reaction conditions depicted at the bottom of figure 22.
2~
Example 23
In Figure 23, synthesis of the building block 199 could
be acco~?lished utilizing the starting materi21 183 and
rollowins the reaction conditions depicted at the bottom o
30 figu~ 23.
Example 24
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W O 96/14330 PCT~US95/14599
In Figure 24, the st2rtins ma~eri21 200 is tran~ormed ~o
the building block 207 by following the reaclion conditions
shown at the bottom or figure 2~.
r xample 2~
The compounds used and generat3d -n this ~xample are
shown in Figure l.
~ hymine acetic acid (1): Thvmine (37.8 g, 300 mmol) was
10 dlssolved in a solution of po.assium hy~roxide (64.5 g, 1150
mmol) in 200 ml OL- water. While .his solution was warmed in a
~0C wa.er ~ath, a solu.ion o ~-o~oace_ic ac-- (62.5 g, 450
mmol) in 100 ml o,~ wa~e was added ov3r 1 h psrioA. The
reac.ion was s~irr3d of an~ther l~ a. Ih~s _emp3ratur2. I. was
allowed to cool to room ~emDer2t~lr- and .he p; was adjusted -o
1~ 5 5 with conc. HCl. The solution was th3~ cooled in a
refrigeralor for 2 h. Any precipitate (unreacted thymine)
formed was removed by filtration. The solution was then
adjusted to p~ 2 wilh conc. HCl and DU_ in a freezer for 2h.
The white precipit~-e was collec~3d by -iltra.ion and dried in.
20 a vacuum oven at 40C for 6 h. The yield was .~g (88%).
N-Boc-~-Serine methyl ester (2): L-Serine methyl ester
(15.6 g, 100 mmol) was suspended in TH.-/DMF(100 ml each)
mixture at room temp~rature. To tnis stirred mixture was added
2~ triethylamine (11.13 g, 110 mmol) followed by di-tert-butyl
dicarbonate (24.0 g, llO mmol) and the stirring continued at
room temperature for 30 minutes. Water (20 ml) was added and
tr.e solution was stirred at room ~-mperature ror 8 h. The
solution was evaporated to dryness. The residue was suspended
in ethyl acetate (250 ml) and trealed with potassium hydrogen
sulfate (0.25 N soluLion, lOOml). The product was extracted
immediately with ethyl aceta~e solution. The organic extract
was washed with water (100 ml), ~rine (100 ml) and dried over
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anhydrous sodium sulfate. Evaporation of the organic solvent
provided an oily residue of 26g (90%).
N-Boc-L-Serine(OT~) methyl ester (3): The compound 2 (15
g, 68.49 mmol) was dissolve~ d-y CH,Cl, (100 mi) and ~reated
with 3,4-dihydro-2H-pyran (8.4 g, 100 mmol) and catalytic
amount of p-toluene sulfonic acid (100 mg) at room
temperature. The reaction mixture w2s allowed to stir at room
temperature for 12 h and evaporated to dryness. The residue
was dissolved in ethyl acetato (200 ml), washed with 5% NaHCO3
solution (100 ml), water (50 ml) and brine (50 ml). The
~organic ex~ract was dried ova_ anhydrous Na~SO, and evaporated
to d-yness. The residue was pure enough fo~ the next step and
used as such. Yleld 15g (72~).
. .
N-Boc-L-Serinol(OTHP) ~4): Serine(OTHP) methyl ester (10
g, 33 mmol) was dissolved ir. d-y THE (lOO ml) and cooled to
0C in an ice bath under argon atmos?here. To this cold
stirred solution was added Dorar.e-methyl sulfide complex (2 M
solution in THF, 100 ml 200 mmol) d~ring 1 h period at 0C
20 temperature. After the addition of borane, the reac~ion
..ixture was warmed .o room .emDeratura and heatad aL 40C for
6 h. The reaction mix~ure was cooled to 0C, neutraiized with
water and acetic acid to pH 6-7 and extracted with ether
`' (3xlOO ml). The ether extract was washed with water (2xlOO ml)
2~ and brine (lOOml), dried over anhydrous Na,SO4 and evaporated
to dryness to give a crude ?roduc~ as an oil. The oil on
purification by îlash coiumn cf silica gel using Aexane ->
acetone as the eluent gave 8g (88%) of pure product.
N-Boc-L-Serine(OT~P) OIb (~): To a stirred solution of
the compound 4 (8 g, 29.09 mmol) in dry CH2Cl2 (100 ml) at 0C
was added TEA ( 3.54 g, 35 mmol) followed by isobutyryl
chloride (3.71 g, 3; mmol) during 30 mins period. Then, the
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reaction mixture was s~irred at room .emperature ror ~ h anà
evaporated to dryness. The residue was dissolved in EtOAc (200
ml), washed with 5% NaHCO3 solution (50 mi), water (50 ml) and
brine (50 ml). The organic ex~ract W2S dried over anhydrous
Na~SO and evaporated to dryness to give a crude product 25 an
oil. The oil on puri~ication Dy flasr. column o~ silica gel
using hexane -> acetone as the eluer.t gave 7.9g (79%) of pure
product.
L-Serinol(OIb) .(6): Compound ~ (10 g, 28.98 mmol) was
dissolved in CH7Cl, (100 ml) zllowed ~o s~ir at room
temperature with TFA (50 ml) ~or 1 h and evaporated to
dryness. Th- resiaue was dissolved in me~nanol (50 ml) and
evaporated agai... TAe residue was dissolved in CH,Cl, (200
ml), washed with sz.. NaHCO3 solution (2x100 ml), water (100
15 mlJ and brine (50 ml). The organic ex~ract was dried over
anhydrous Na~SO and evaporated to dryness .o give 4.5g (96%)
of the ?roduct as an oil.
N-(T~yminylacetyl)-~-Serinol(OI~) (8): T.hymine acetic
20 acid 7 (7.3 g, 0 mmoi) and N-metAylmor?Aoiine (~.4 ml, ~0
mmol) were dissoived in 100 r,i of DM~. The solutlon was
allowed to cool _o -20C under argon a.mosphere. To this cold
stirred solution, isobutyl chloroformate (5.2 ml, 40 mmol) was
added in one portion. After 15 minutes, a solution of 6 (6.44
2~ g~ 40 mmol) in 30 ml of DMF (chilled to the same temperature)
was added. The reaction mixture was s_irred at -20C for 30
minutes, warmed .o room temperature and the stirring continued
for 1 h. The reaclion mixtu-e was evaporated to dryness and
the residue dissolved in CH2Cl- (200 ml). Tr.e organic solution
was washed wi.h 5% NaHCO3 solution (100 ml), water (100 ml)
and ~rine (50 ml). The organic extract was dried over
anhydrous Na.SO and evaporated to dryness to give a crude
produc~ as foam. The crude ?roduct was puriried by flasA
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column of silica gel using CH2Cl2-> acetone as the eluent to
give 12g (92~) of pure product.
4,4'-Dimeth~y L~ityl-N-tThyl~nylacetyl)-~-Serinol(OI~)
(9): The compound 8 (10 g, 30.58 mmol) was coevaporated with
dry pyridine (3x~0 ml) and dlssolved in d~y pyridine (100 ml).
To this solution was added TEA (3.54 g, 35 mmol) followed by
DMTCl (11.83 g, 35 mmol) at room temperature under argon
atmosphere. The reaction mixlure was sl~rred for 12 h,
10 quenched with methanol (20 ml) and stirred for 30 minutes. The
solution was evaporated IO dryness and dissolved in CH~Cl,
(2~0 ml). The organic e~tract was washed with 5% NaHCO3
solutior. (100 ml), waler (100 ml) and b-ine (50 ml). The
CH2Cl, iayer was dried over anhydrous Na,SO~ and evaporated ~o
dryness to give a crude product as foam. The crude product was
1~ purified by flash column of silica gel using CH2Cl2-> acetone
as the eluent ~o give 17g (88~) of pure product.
1-0-(4,4'-D~metho~yLlityl)-2-t~no(thyminylacetyl)]-~-
propan-1,3-diol (lO): The compound 9 (10 g, 15.89 mmol) was
20 dissolved in melhanol (20 ml). To this solution was added 1~'
NaOH soiu~ion (20 ml, 20 ~mol) a. 0C t-mperalure. The
reaction mixture was stirred ror 1 h, quenched with acetic
acid to pH 7. The solution was extracted with EtOAc (2x100
ml). The organic extract was washed with 5% NaHCO3 solution
25 (100 ml), water (100 ml) and brine (50 ml). The EtOAc layer
was dried over anhydrous Na2SO and evaporated to dryness to
give a crude product as foam. The crude ?roduct was purified
by flash column of silica gel using CH~Cl2-> acetone as the
eluent to give 8.2g 92%) OL- pure p-oduct.
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WO 96/14330 PCTIUS95/14599
1 ~ ~0~ (4 ~ 4 ~ -D~ metho~y ~ r ityl ) -2 - ~m; n~(thys~inylacetyl)-~-
p2:opan- 3'-O-tN,N-diis~ -cyanoeth~l~hophor~m;dite
(11): The compound 10 (8.00 g, 14.31 mmol) was coevapora.ed
with dry pyridine (3x50 ml) and dried over solid NaOH
overnight under vacuum. The d_i-~d material was dissolved in
dry CH,C12 (100 ml) and cooied to 0C under argon atmosphere.
To this cold solution was added N,N-diisopropylethylamine
(5.23 g, 25 mmol) followed by 2-cyanoetnyl-N,N-
diisopropylchlorophosphoramidite (4.72 5, 20.00 mmol) under
10 argon atmosphere. The reac~_n mixt~re was s~irred at 0C for
1 h and at room temperature -~r ~ h. Th~e reac-ion mixture was
diluteà with CH2Cl7 (100 m ,. T:ne CH,Cl~ solut~on was washed
with 5% NaHCO3 solution ('~0 m',, water (100 ..;) and ~rine (50
ml). The CH,Cl, layer was dried over anhydrous Na,SO~ and
evaporate-,d to dryness IO give a crude ?roduct as foam. The
15 crude product was purified by flash column of si~ica gel using
C~,Cl,-> ace~one containing 0.1~ TEA as the eluent to give 10
g(x%) of pure producl. Th.e form was dried over solid NaOH in
vacuum overnight. The ro-m was dissolved in CH,Cl, (15 ml) and
dropped into s~irred sol~uti~ of dry hexanes (2000 ml) ~nder
20 argon d~ ing l h period. A~te~ the addi_ion c_ CH,Cl,solution,
tne Drecipitale that ormed was stirreà fo- additional h and
filtered, washed witn dry hexanes (200 ml~ and dried over
soiid NaOH overnight. Yieid: 9.5g (87~).
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Example 26
(See igure 23)
N-(te~t-3utylosy~ h~nyl) -O-~enzyl-I,-Serine (2):
.O-Benzyl-L-Serine 1 (10 g, 51.28 mmol) was suspended in
THF/H70 (8:2, 100 ml) mixture at room temperature. To this
stirred miXtUrQ was added triethylamine (6.06 g, 60 mmol)
followed by di-te~t-butyl dicarbona~e (13.08 g, 60 mmol), and
10 the stirring continued at room temperature overnight. The
homogenous solution was eva?orated to d-yness and the residue
dissoived in ethyl acetate (300 ml). ~he organ~c exrra_~ was
washed with 0.5N soiution o DO_ZSSlU:lt hydrogen sulfate (100
ml), water (lO0 ml) and brine (50 ml). The ethyi acetate
extract was dried over anhydrous sodium sulfate and evaporated
1~ to dryness to give 14 g (93~) of an oily residue.
N-(tert-3utyloxy~-hQnyl)-O-Benzyl-L-Serinol(3):
N-(t rt-butyloxycarbonyl)-O- benzyl-L-serine 2 (6.0 g, 20.34
mmol) was dissolved in dry THF and cooled to -20C under argon
20 atmcsphere. To tAis cold s~irred solution W25 added T~A (2.32
g, 23 mmol) and isobutyi chloro orma~e (3.13 g, 23 mmol). The
stirring was continued for 30 min at -20C under argon
atmosphere. The reaction mixture was filtered immediately
under a blanket of argon, the precipitate was washed with dry
25 THF (50 ml). The combined filtrate was added slowly into a
cold (0C) solution of NaBH; (7.4 g, 200 mmol) in THF/wate-
(80:20, 200 ml) during 10 min period. After the addition, the
reaction mixture was stirred for 2 h at 0C and the pH
adjusted to 7 with acetic acid. Tne solution was evaporated to
dryness, pzrtitioned between ethyl acetate/water (300:150 ml)
and extracted in ethyl acetate. The organic extr2ct was washed
with brine (lO0 ml), dried over anhydrous sodium sulfate and
evaporated ~o drvness. The crude ?,oduct was purified by flasn
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W ~ 96/14330 P~liU~35l1459~
column chromatograpny over silica gel using CH2Cl~-->EtOAc 2S
the eluent. The pure product was pooled together and
evaporated to dryness to give ..7 g (82~) of the Dure D-oduct
2s an oil. lHNMR (CDC13): ~ 1. Al (5, 9H, Boc~, 3.60 - 3.70 (m,
4H), 3.82 ~d, 2H), 4.53 ~s, 2.~, OCH,Ph), 5.20 (bs, lH, NH) and
7.30 - 7.40 (m, 5H, Ph).
N-(tert-Butyloa~y~h~nyl)-O-Benzyl-~-Serinol-O-Ib (4): To
a dried sol~tion of ~-(te~t-~utyloxycar~onyl)-O-benzyl-L-
10 serinol 3 (4.3 g, 1~.3 mmol) n dry Dyridine (50 ml) was addedTEA (2.02 g, 20 mmol) at room lemperature. To tnis stirred
solution was added isobu~vric anhydride (3.16 g, 20 mmcl) and
Ihe s_i~ring continued ove-niy;~t unde- crgon atmospher~. The
reaction mixLur2 was eV2DO-2-ea to d vness, part-.ioned-
between E~OAc (100 m') and NaHCO3 (5~ solution, lOO ml), and
extracted in EtOAc. The organic e.Ytr2ct was washed with water
(100 ml), brine (50 ml), and dried over anhydrous Na2SO . The
dried solution was evaDoraled to dryness to give a crude
residue. The r~sidue was puri~ied by flash chromatograp;~y over
silica gel using hexane --> ~tOAc as the eiuent. The pure
20 fractions were pooled .ogethe~ and evaDorated to ~ive ar. oily
product 4.5 g (84%). HNMR (^DC13): ~ 1.04 (d, 6.:, IbCH3),
1.39 (s, 9H, Boc), 2. 6 (m, 1.~, IbCH), 3.40 (m, 2H~, 3.92 (m,
2H), 4.12 (m, lH), 4.46 (s, 24, OCH,Ph), 6.84 (d, lH, NH) and
7.24 - 7.40 (m, 5H, Ph).
2~
N-(Thyminylacetyl)-O-Benzyl-~-Serinol-O-I~ (6):
N-(te~t-Butyioxycarbonyl)-3- b~nzyl-~-serinol-O-Ib 4 (~.3 g,
12.25 mmol) was allowed to stir at roo~ temDerature in
trifluoro acetic acid (20 ml) and CH~Cl,(20 ml) for 30 min.
30 The reaction mixture was evaporated to dryness, dissolved in
dry CH30H (10 ml) and evaporated again to dryness. The residue
was dried over solid KOH under vacu~im for 12 h. Tr.e dried
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WO96/14330 PCT~S95/14599
residue was used as such for further reaction wlthout
characterization.
Thymine acetic acid ~ (2.76 g, 15 mmol) was dissolved in
-~ dry DMF (75 ml) and cooled ~o -20~ under argon. To this cold
stirred solution was added N-me~hylmorpholine (1.72 g, 17
mmol) followed by isobutyl chloro ormate (2.31 g, 17 mmol).
After 15 min of stir~ing, z solution of the above TFA salt in
dry DMF (50 ml) was neutralized wi.h N-methylmorpholine (1.72
g, 17 mmol) and added into the cold stir~ed solution of
thymine acetlc acid at once. The reac~ion mixture was s~irred
at -20C for l h, warmed .o room _emperzture and the s~irring
continued overnigh'. ~he solu-i_n was evaporated to d~yness
and the residue dissolved in CH~Cl, (250 ml) and water (100
ml), and extracted in CH,Cl,. The o-ganic extract was washed
1~ with 5~ NaHCO. solution (100 ml), water (100 ml) and brine (50
ml). The CH,Cl.extra_ was d-ied ar.d evaporated to dryness to
give crude produc~. The cruae product was purified by flash
chromatography over s-lica gel using CH,Cl, --> acetone as the
eluen.. The necessary fraclions were collected and evaporated
20 to give 4.8 g (94%) of the pure product. The pure produc. was
crys.a lized from CH~Cl /hexane. m-: 122-124C. ;HNMR (C3Cl3):
1.04 (~, 6.~, IbCH3), 1.72 (s, 3.~, CH3), 2.44 (m, lH, IbCH),
3.42 (m, 2H), 4.06 (m, 2H), 4.18 (m, lH), 4.30 (s, 2H), 4.46
(s, 2.~;, OCH~Ph), 7.24 - 7.40 (m, 6;i, C6H and Ph), 8.22 (d, lH,
2~ NH) and 11.22 (s, lH, NH).
N-(Thyminylacetyl)-L-Serinol-O-I~ (7): N-(Thyminyl-
acetyl)-O-Benzyl- L-Serinol-O-Ib 6 (2.08 g, 5 mmol) was
dissolved in ethanol (50 ml). To this solution Pd(OH), (0.6 g)
and c~clohexene (5 ml) were added at room temperature. The
reaction mixture was heated at 70C for 12 h. The catalyst was
filtered, washed with methanol (20 ml). The filtrate was
evaporated to dryness ~o give a white solid. The white solid
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WO96/14330 PCT~S95114599
was dissolved in minimum amount OL- MeOH and cooled to room
temperature. The product crystallized as fine Dowde~. mp:
196-198C. Yield: 1.48 g (91%). HNMR (L~e,SO-c;): ~ 1.04 (d,
6H, IbCH3), 1.72 (s, 3~, CH3), 2.42 (m, l.i, IbCH), 3.40 (m,
2H), 3.94 (m, 2H), 4.06 (m, 1~ . 28 (s, 2r.), 4 . 90 (t, lH,
OH), 7.20 (s~ lH, C~H), 8.12 (d, i.~, NH) and ;1.22 (s, 1~,
NH).
4~4~-D~etho~yLlityl-N-(Thy~ nylacetyl)-~-Serinol-O-I~
10 (8~: N-(Thymlnylacetyl)-L-S7~inol-O-Ib 7 (1.48 g, 4.5 mmol)
was dissolved in dry pyridir.e (50 mi) under a gon. To this
stirred soluti~n w2s added T-A (C.51 g, 5 mmc`) and
N,N-dimethyiamino pyridlne (0.10~). Af_~r 10 min,
4,4'-dimethoxvtrityl c~lorid_ (1.69 g, ~ mmo') was added and
the stlrring continued at room remperzture under argon for
overnigh~. The reaction mixlure was quenched with MeOH (10
ml), stirred for 10 min and evapo~aled to dryness. The residue
was dissolved in EtOAc (200 ml), washed with 5~ NaHCO3
solution (100 ml), water (lC0 m') and b,in- '50 ml). The
crganic extrac~ was dried ove- annyd~ous Na7SO~ and evapo-ated
20 ~o dryness. Th- crud~ ?rodl_. W2S p~rified by flash column
ehromatography over silica a-l using CH,Cl,--> EtOAc as th~
eiuent. The pure fractions were pooied and eva~orated to give
2.5 g (88%) of foam. iHNMR (CDC13): ~ 1.04 (d, 6H, IbCH3), 1. 72
(s, 3H, CH3), 2.40 (m, iH, IbCH), 3.38 (m, 2H), 3.72 (s, 6H,
2~ 2 . 0CH3), 4.12 (m, 2H), 4.20 (m, lH), 4 . 32 (d, 2H), 6. 84 (m,
4H, Ph), 7.20 - 7.40 (m, 12H, C H and Ph), 8.30 (d, lH, NH)
and 11. 28 (s, lH, NH) .
30 1-0- (4, 4 ' -Dimetho~y L ityl) -2- ~amino (thyminylacetyl) ] -~-propan-
1, 3-diol (9):
4,4'-~imethoxytrityl-N-(Thvminylacetyl)-L-Serinol-O-Ib 8 (3.4
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W O96114330 PCTrUS95/14599
g, 5.41 mmol) was dissolved in MeOH (30 ml) and c0012d to 0C
in an ice bath. To this cold sLirred solution was added 2N
NaOH (10 ml, 20 mmol) and the sti-ring continued for 30 min at
0C. The pH of the solution was adjusted to 7 wi~h acetic acid
and evaporated to dryness. The residue was part_-ioned between
water (50 ml) and CH2Cl (1;0 ml) and extracted in CH.Cl,. The
aaueous layer was extracted again with CH.Cl, (SO ml). The
combined organic extrac- was washed with brine (SO ml), dried
and evaporated to dryness. Th~ residue was puri~ied bv ~lash
column chromatosraphy over s lica gel using CH,Cl --> acelone
2S the eluent. Yield: 3.0 g (99%). :HNMR (CDCl3): 1.72 (s, 3H,
CH3), 3.0 (m, 2H), 3.42 (m, 2H), 3.72 (s, 6H, 2.0CH3), 3.9a
(m, lH), 4.32 ~d, 2H), 4.68 (-., '.;, OH), 6.84 ~ , 4H, Dh),
7.20 - 7.40 (m, 12H, C5H and P.~), 8.06 (d, lH, N.~) and 11.28
(bs, lH, NH).
0-(4,4~-Dimethoxytrityl)-2-tamino(thyminylacetyl)]-~-pr
opan-3-0-(N,N-- diisopropyl)--,B-cyanoethyl~hosphor~m;dite (10):
1-0-(4,4'-Dimethoxytrityl)-2-~amino(thyminylacetyl)]-L-
propan-1,3-diol 9 (3.1 g, 5.~ mmol) was dried over sol-d NaOH
under vacuum overnight and Ai ssolved in dry CH~C_ (100 ml).
20 The solution W25 cooled to 0_ under argon atmos?here. To this
coid stirred solution was added N,N-diisopropyle_~ylaminQ
(1.29 g, 10 mmol) followed by 2-cyanoethyl-N,N-diisopropyl-
chlorophophoramidite (1.96 g, 8.3 mmol). The reaction mixture
was stirred at 0C for 1 h and at room temperature ror 2 h.
2~ The reaction was diluted with CH,CH, (100 ml) and the organic
layer was washed with 5% NaHCO3 solution (100 ml), water (100
ml) and brine (50 ml). The CH,Cl, extract was dried and
evaporated to dryness to give an oily residue. ThQ residue was
purified by flash chromatosrapny over silica gel using
CH.Cl~-->EtOAc containing 0.1~ TEA as the eluent. The pure
30 fractions were pooled together ar.d evaporated to give a foam.
The foam was dried over solid NaOH under vacuum overnight. The
dr1ed foam was dissolved in d-v CH~Cl. (20 ml) and dropped
CA 02202274 1997-04-09
WO96/14330 PCT~S95/14599
into a stirred solution of dry hexane (2000 ml) under argor.
during lh period. After the addi~ion, the precipitate rormed
was stirred for addit~onal lh and flltered, washed with dry
hexane (100 ml) and the solid was dried over solid NaOH under
vacuum for 4 h. Yield: 3.5 g (83%).
~ .
ExamDle 27
(See Figur2 24)
N - ( te~t - ~utyloa~y~nyl )-0-3enzyl-D-Serine (12):
lO O-Benzyl-D-Serine 11 (5 g, 2~.64 m~ol) was suspended in
THF/H,O (8:2, 70 ml) mix~ure a_ room ~emperature. To this
stirred mixture was added ~-iethviamin~ (4.04 g, 40 mmol)
followed by dl---rt-butyl ~ ona_e (6.54 g, 30 mmol), and
the stirring conlinu2d at roo~. temper2ture overnigh~. The
homogenous solu.ion was evaporated _o dryness and the residue
1~ dissolved in ethyl acetate (150 mi). The organic extract was
washed with 0.5N solution o~ potassium hydrogen sulfate (100
ml), water (100 ml) and brine (50 ml). The ethyl acetate
extract was dri-d over annvsrous sodium sulfate and evaporated
to d-yr.ess .o give 7.;6 g (100~) o- an oily residue.
~-(tert-B~tylo~ycarDonyl)-O-Benzyl-D-Serinol (13):
N-(te-t-But~'ox~carbonyi)- O-~enzyl-D-serine 10 (7.56 g, 25.63
mmo7) was dissolved in dry T'iF and cooled to -20C under argon
atmosphere. To this cold stirred solution was added TEA (3.03
2~ g~ 30 mmol) and isobu~yl chlo-orormate (4.08 g, 30 mmol). The
stirring was continued for 30 min a. -20C under argon
atmosphere. The reacllon mixlure w2s filtered immediately
under a blanke- of argon, the preciDitate was washed with d-v
THF (~0 ml). The com~ined f ltrate was added slowly into a
Y cold (0C) solulion o- NaBH: (7.4 g, 200 mmol~ in THF/water
(80:20, 200 m7) during 10 min peri-d. After the addition, tAe
reaction mixture was stirre~ for 2 h at 0C and the pH
adjusted to 7 w tr. a~etic 2_' d. The solution was evaporaled to
-- 91 --
CA 02202274 1997-04-09
W O96/14330 PCTrUS95/14599
dryness, partitioned Detwe^n ethyl aceta.e/wa~er (300:150 ml)
and extracted in ethyi ace~a.e. The organic extract was washed
with brine (100 ml), dried over anhydrous sodium sulfate and
evaporated to dryness. The crude product was purified by rlash
column chromatography over sllica gel using CH,Cl2-->EtOAc as
the eluent. The pure ?roduct was pooled together and
evaporated to dryness ~o give 6.68 g (92%) of he pure product
as an oil. IHNMR (CDCl3): ~ 1.41 (s, 9H, Boc), 3.60 - 3.70 (m,
4H), 3.82 (d, 2H), 4.53 (s, 2H, OCH~Ph), 5.20 (bs, lH, NH) and
10 7 30 - 7.40 (m, 5H, Ph).
N-(tert-Butyloxycar~onyl)-O-Benzyl-D-Serinol-O-I~ (14):
To a dr'~d solution o-
N-(tert-Butyloxycarbonyl)-O-benzyl-D-serincl I3 (6.6 g, 23.5
mmol) in dry pyridine (50 ml) was added TEA (3.03 g, 30 mmol)
at room .emperature. lo this stirred solution was added
isobutyric anhydride ( .74 g, 30 mmol) and the stirring
continued overnight under argon atmosphere. The reaction
mixture was evaporated .o dryness, partitioned between EtOAc
(200 ml) and NaHCO3 (5% solu~ion, 100 ml), and ex~ractsd in
20 EtO~c. The organic ex~ra~ was washed wi.h water (100 ml),
brine (50 ml), and dried over anhydrous Na2SO . The dried
solution was evaporated to dryness to give a crude residue.
The residue was puriried by flash chromatography over silica
gel using hexane --> EtOAc as the eluen.. The pure fractions
25 were pooled together and evaporated to give an oily product
8.0 g (97%). lHNMR (CDCl3): ~ 1.04 (d, 6H, IbCH3), 1.39 (s,
9H, Boc), 2.46 (m, lH, IbCH), 3.40 (m, 2H), 3.92 (m, 2H), 4.12
(m, lH), 4.46 (s, 2H, OCH,Ph), 6.84 (d, lH, NH) and 7.24 -
7.40 (m, 5.Y, Ph).
N-(Thyminylacetyl)-O-Benzyl-D-Serinol-O-I~ (1~):
N-(tert-Butyloxycarbonyl)- O-benzyl-D-serinol-O-Ib 14 (5.0 g,
- 92 -
CA 02202274 1997-04-09
W O96/14330 PCTrUS95/14599
.
14.25 mmol) was allowed to s_-r 2t room temperature in
trifluoro acetic acid (20 ml) and CH7Cl, (20 ml) for 30 min.
The reaction mixture was e-~zoorat2d to dryness; dissoived in
dry CH30H (10 ml) and evapor2ted again .o dryness. The residue
5 was dissolved in CH,Cl, (150 ml), the pH was zdjusted to 7
with 5% NaHCO3 solution and ex~racted in CH.C;2. The organic
layer was washed with wate~ (~0 ~.1) and ~rine (50 ml). The
CH,Cl.extract was dried ana e-~aporated to dryness. Th~ residue
that obtained was dried ove~ solid KOH under vacuum ror 12 h.
Ine dried residue was used 2S such for rurthe~ reaction
without characterization.
Tnv~ne ace~ic acid 5 (2.57 y, 14 mmol) ~as dissolved in
~y DM~ (50 ml) and cooled -~ -20C under aroon. To this cold
stirr_d solution was added ~i-metAylmorpnolin2 (1.52 g, 1~
1~ mmol) f~llowed by isobutyl chloroforma~e (2.04 g, 15 mmol).
.~fter 1; min of stirring, a solution o' the above amine in
d-y DMF (50 ml) was added into th~ cold stirred solution of
-hvmine acetic acid at once. The r action mixture was stirred
al -20C for 1 h, warmed to room temperature and the stir-ing
20 c~~.tinued overnight. Tne solution was evaDora_ed to d-yness
a..~ ,he residue dissolved n CH.Cl~ (250 ...1) and wate~ (100
m,`, and ex~racted in CH7Cl . Th- organic extract was w~shed
with 5% NaHCO3 solution (100 ml), water (100 ml) and ~rine (50
ml). The CH2Cl~extract was dried and evaporated to dryness to
give crude product. The crude product was puriried by flash
chromatography over silica gel using CH,Cl.--> acetone as the
eluen_. The necessary fractions were collected and evaDorated
~o give 2.8 g (54%) of the pure produc~. HNM~ (CDC13): ~ 1.04
(d, 6H, lbCH3), 1.72 (s, 3H, CH3), 2.44 (m, lH, IbCH), 3.42
(m, 2H), 4.06 (m, 2H), 4.18 (m, lH), 4.30 (s, 2H), 4.46 (s,
2.::, OCH.Pn), 7.24 - 7.40 (m, 6H, C H and Ph), 8.22 (d, lH, NH)
and 11.22 (s, lH, NH).
- 93 -
CA 02202274 1997-04-09
W O96114330 PCT~US95/14599
The titled compound was aiso prepared by using the method
described ror the preparation "~" isome~ Reagents Used:
Thymine acetic acid (2.2 g, 12 mmol)i Isobutyl chloroformate
(1.77 g, 13 mmol); N-methyimorpholine (1.52 g, 15 mmol); TFA
salt (3.65 g, 10 mmol)i N-methylmorpholine (1.5 g, 15 mmol)
and dry DMF (100 ml). Yield: 3.5 g (84%).
N-(Thyminylacetyl)-D-Serinol-O-Ib (16): N-(Thyminyl-
acetyl)-O-Benzyl-D-Serinol-O-Ib 1~ (3.5 g, 8.39 mmol) was
1~ dissolved in ethanol (50 ml). To this solution Pd(OH), (1.00
g) and cyclohexene (10 ml) we~e added at room temperature. The
reaction mixture was heated a. 7CC for 12 h. The catalyst was
filtered, washed witn methanol (20 ml~. The ~iltrate was
evaporated to dryness to give an white solid. Yield: 2.7 g
(98%). iHNMR (Me,SO-d,): ~ 1.04 (d, 6H, IbCH3), 1.72 (s, 3H,
CH3), 2.42 (m, lH, IbCH), 3.40 (~.., 2H), 3.94 (m, 2H), 4.06 (m,
lHJ, 4.28 (s, 2H), 4.90 (t, lH, OH), 7.20 (5, lH, C,H), 8.12
(d, lH, NH) and 11.22 (s, lH, NH).
4,4'-D~metho~yL~ityl-N-(Thyminylacetyl)-D-Serinol-O-I~
20 (17) N-(Thyminylacetyl)-D-Serinol-O-Ib 16 (2.7 g, 8.26 mmol)
was dissolved in dry pyridine (50 ml) under argon. To tnis
stirred solution was added TEA (1.01 g, 10 mmol) followed by
4,4'-dimethoxytrityl chloride (3.38 g, 10 mmol) and the
stirring continued 2- room temperature under argon for
2~ overnight. The reaction mixture was quenched with MeOH (10
ml), stirred for 10 min and evaporated ~o dryness. The residue
was dissolved ir. EtOAc (250 ml), washed with 5% NaHCO3
solution (100 ml), water (100 ml) and brine (50 ml). The
organic exlract was dried over annydrous Na2SO4 and evaporated
30 to dryness. The crude product was purified by flash column
chromatography over silica gel using CH,Cl,--> EtOAc as the
eluent. The pure fractions were pooled and evaporated to give
5.0 g (96%) o~ foam. lHNMR (CDCl3): ~ 1.04 (d, 6H, IbCH3), 1.72
- 94 -
CA 02202274 1997-04-09
W O96/14330 PCTrUSgS/14~99
(s, 3H, CH3), 2.40 (m, lH, IbC~), 3.38 (m, 2H), 3.72 (s, 6H,
2.0CH3), 4.12 (m, 2H), 4.20 (m, lH), 4. 32 (d, 2H~, 6.84 ~m,
4H, Ph), 7.20 - 7.40 (m, 12H, C6H and Ph), 8.30 (d, lH, NH)
and 11.28 (s, lH, NH).
1-O-(4,4'-D ~ et~ay ltyl)-2-~ino(thy~linyl-
acetyl)]-D-propan-1,3-diol (18): 4,4'-Dimethoxytrityl-N-(-
Thyminyiacetyl)-D-Serinol-O-Ib 17 (5.0 g, 7.95 mmol) was
dissolved in MeOH (30 ml) a~d cooled to 0C in an ice bath. To
10 this cold stirred solution was added 2N NaO~ (10 ml, 20 mmol)
and the stirring continued ror 30 min at 0C. The pH o~ the
solution was adjusted to 7 wi~h acetic ac~d znd evaDora~d to
aryness. The r~sidue was par~__' oned belween wate~ (50 ~1) and
CH,Cl, (250 ml) and extracted in CH,Cl~. The aqueous layer was
extracted again with CH,Cl, (~0 ml). The combined organic
15 extract was washed with brine (50 ml), dried and evapora~ed to
dryness. The residue was pu-~îied by flash column
chromatog_apny over silica gel using CH.Cl --> acetone 2S the
eluent. Yield: 4.0 g (90%). :HNMR (CDCl3): 1.72 (s, 3H, C;~3),
3.0 (m, 2H), 3..2 (m, 2H), 3.72 (s, 6~, 2.0CH3), 3.94 (r.., lH),
20 4.32 (d, 2~), a.68 (m, lH, OH), 6.8~ (m, 4~, Ph), 7.20 - 7.40
(m, 12.-', C H and Ph), 8.0~ , N~) and 11.28 (bs, l.., NH).
1-0-(4,4'-D~meth~yL-ityl)-2-t~no(thyminylacetyl)~-D-
propan-3-0- (N,N-~;isor-opyl)-~-cyanoethyl~hosphoramidite
2~ (~2): 1-0-(4,~'-Dimethoxyt-ityl)-2-[aminolthyminyl-
acetyl)]-D-propan-1,3-diol 18 (2.79 g, 5.0 mmol) was dried
o~er solid NaO~; unde- vacuum o~ernigh~ and dissolved in dry
CH2C1. (100 ml). The solutio~ was cooled to 0C under argon
atmosphere. To this cold st~rred solution was added
30 N,N-diisopropylethylamine (1.29 g, 10 mmol) followed by
2-cyanoetAyl-N, N-diisopropylchloropAospnoramidite (1.96 g,
8.3 mmol). The reaction mix.ure was s~irred at O~C for 1 h and
at room temperature ror 2 h. The reaction was diluted with
- 95 -
CA 02202274 1997-04-09
W O96/14330 . PCTrUS95/14599
. .
CH2Clz (100 ml) and the organic layer w2s washed with 5g6 NaHCO3
solution (100 ml), water (100 ml) and brine (50 ml). The
CH~Cl2extract was drieà and evaporated to d_yness to give an
oily residue. The residue was purified by flash chroma~ography
over silica gel using CH~Cl,-->EtOAc containing 0.1% TrA as
the eluent. The pure rractions were pooled together and
evaporated to give a roam. The roam was dried over solid NaOH
under vacuum overnigh_. The dried foam was dissolved in dry
CH2Cl~ (20 mi) and dropped into a s.irred solution of dry
hexane (2000 ml) und?r argon during lh period. After the
addit-on, the precipitate rormed was stirred for additional ih
and fil.ered, washed with dry hexane (100 ml) and the solid
was ~ried over solid NaO~; un~er vacuum for 4 h. Yield: _.3 g
(87%).
.
1~ Example 28
(Figure 25)
l-O-Benzyl-2--[ ( tert-}:utylo~y~ hnnyl) ~ no] -3-
tN3-~enzoyl (thyminyl) -~-propanol (21): To a stirred solution
of N.-benzoylthymine 20 (5.75 g, 2~ mmol) in dry THF (200 ml)
20 under arson was added ~-iphenyl pAos?hine (10.48 g, 40 mmol)
and N~-tert-butyloxyc2rDonyl-~-benzyloxy-L-serinol 3 (5.3 g,
18.86 mmol) at room temperature. After l~ min, diethylazo-
dicarboxylate (6.96 g, 40 mmol) was added slowly durins 30 min
period. The reaction mixture was covered with aluminum foil
25 and allowed to stir at room temperature under argon for 24 h.
The solvent was evaporated to dryness and the residue
dissoived in EtOAc (300 ml). The organic extract was washed
with 5% NaHCO3 solution (100 ml), water (100 ml) and brine
(100 ml), and dried over anhydrous Na,SO~. The dried EtOAc
extrz-~ was evaporated to dryness .o give an orange oil. The
crude p_oduct was purlfied by flash chromatography over silica
gel using hexane--~ EtOAc as the eluent. The fraction having
the -equired product was pooled and evaporated to give a pale
- 96 -
CA 02202274 1997-04-09
WOg6/14330 PCT~S95/14S9g
pink oil. Yield: 8.0 g (86~ HNMR (CDCl3): 1.41 (s, 9H,
Boc), 1.72 (s, 3H, CH3), 3.56 (m, 2H), 4.20 (m, 2H), 4.32 (m,
lH), 4,52 (d, 2H, OCH2Ph), 5.20 (d, lH, NH), 7.06 (s, lH, C6H)
and 7.20 - 7.60 (m, 10H, Ph).
2--~(te~t-3utyloxy~honyl)~no]-3-rN3-~enzoyl
(thyminyl ~ -~-propan-1-ol (22): ~-O-Benzyl-2-[(tert-~utyloxy-
car~onyl)amino]-3-[N3-benzoyl('hvminyl)-L-propanol 21 (4.93 g,
10 mmol) was dissolved in MeOH (100 ml) and ~reated with Pd/C
10 (10~, 1 g). The reaction mixtur2 was hydroaenated at 50 psi of
hydrogen for 12 h. The ca-aiys~ was filtered, washed with MeOH
(50 ml) and the fi'lrate was e~aDorated .o dryness. The
residue was c_ystallized f-om ~ce~one/hexane to give 3.70 g
(92%) of pure product. mp: 1~6-lS9~C. -HNMR (CDCl3): 1.42 (s,
9H, ~oc), 1.94 (s, 3H, CH3), 3.64 (m, 4~), 3.84 (m, 1~), 4.14
1~ (m, lH), 5.22 (d, l.Y, NH), 7.18 (s, lH, C6H), 7.48 (t, 2H,
Ph), 7.62 (t, lH, Ph) and 7.98 (d, 2H, Ph).
l-O-Iso~utyry1-2- t (tert-~utylo~yr~o~yl) ~m~no~ -3-
~N3-~enzoyl (thyminyl ) -~-propanol (23): 2-[(T~rt-3utyloxy-
20 carbonyl)amino]-3-[N3-benzoyl !~hyminyl)-L-propan-1-sl 22
(1.60 g, 3.97 mmol) was dissol-~;e~ in d-y p~_idine (30 ml) and
allowed to stir at room ~empe_ature under argon. To this
stirred solution was added TEA (0.51 g, 5 mmol) and isobutyric
anhydride (0.79 g, ~ mmol). The reaction mixture was stirred
2~ a. room temperature for 12 h and e~aporated to dryness. The
residue was dissolved in EtOAc (150 ml) and washed with 5%
NaHCO3 solution (100 ml), wate_ (100 ml) and brine (50 ml).
The organic extract was firied and e~aporated to dryness. The
residue was purified by flash column chromatography over
silica gel using CH~Cl2--> EtOAc as the eluent. The pure
fractions were coll~cted together and evaporated to give 1.6 g
(85~) of foam. The pure produot was crystallized from
acetone/hexane. mp: 16~-167C. -HNM~ (CDCl3~: 1.16 (d, 6H,
CA 02202274 1997-04-og
W O96/14330 PCTrUS95114599
IbCH3), 1.42 (s, 9H, Boc), 1.94 (s, 3H, CH3), 2.52 (m, lH),
3.64 (m, 4H), 3.84 (m., lH), 4.14 (m, lH), 5.22 (d, lH, NH),
7.18 (s, lH, C?H), 7.48 (t, 2.Y, Ph), 7.62 (t, lH, Ph) and 7.98
(d, 2H, Ph).
1-O-Iso~utyryl-2-~(~-hyd~ydcetyl)~m~no]-3-
tN3-~enzoYl(t~ ny~ ~anol (24): 1-O- Isobutyryl-2-
[(tert-butyloxycarbonyl)aminoj-3-[N3-benzoyl (thyminyl)
-L-propanol 23 (1. 6 g, 3.38 mmol) was allowed to stir in a
10 mixture of TFA (5 ml) and CH,Cl? (10 ml) at room temperature
for 30 min and evapo~ated _o dryness. The resldue was
dissolved in d-y MeO;~ (10 ml) an~ eva?orated again. The
~esidue that ob~ained was ~_ied O'Jer solid NaOH under vacuum
overnight. The dried ma~erial was used as such for the next
reaction.
To a stirred solution of glycolic acid (0.53 g, 7 mmol)
in dry DMF (50 ml) was added 1-hydroxybenzotriazole (0.67 g, 5
mmol) and l-ethyl-3-(3-dimetAylaminopropyl)-carbodiimide
hydrochloride (EDC) (1.91 g, 10 mmoi). After sti-ring for 15
20 min, TEA (1.01 g, 10 mmol) and the above TFA salt in DM~ (20
ml) were added 2- room temperature. The react on mixture was
stirred for 12 h and evaporated to dryness. The -esidue was
partitioned between CH2Cl, (lS0 ml) and water (100 ml), and
extracted in CH~Cl,. The organic exlract was washed with brine
2~ (50 ml), dried and evaporated to dryness. The residue was
purified by flash chromatography over silica gel using CH2Cl?
-->acetone as the eluent. The fractions having the required
product were collected and evaporated to give 1.35 g (92~) of
foam. iHNMR (CDCl3): 1.16 (d, 6H, IbCH3), 1.94 (s, 3H, CH3),
2.52 (m, lH), 3.20 (bs, lH), 3.80 - 4.30 (m, 6H), 4.56 (m,
lH), 7.14 (d, 2H, CsH and NH), 7.50 (t, 2H, Ph), 7.64 (t, lH,
Ph) and 7.94 (d, 2H, Ph).
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W O96/14330 . PCT~US~5/14599
l-O-IsoL~Ly yl~2~t(~-(4/4~-dimeth~yL ityl)-O-acetyl)
A~no]-3- rN3-~enzoyl(thyminyl)-~-propanol ~25): 1-0-Iso-
butyryl-2-[(~-hydroxyacetyl)amino]-3- [N3-benzoyl(thyminyl)
-L-propanol 24 (1.2 g, 2.78 mmol) was dissolved in dry
5 pyridine (~0 mi) and allowed to s~ir at room temperature under
argon atmosphere. To this slirred solution was adde~ TEA (0.35
g, 3.5 mmol) and 4,4'-dimethoxytrityl chloride (1.18 g, 3.5
mmol). The reaction mixture was stirred at room temperature
for 12 h, quenched with MeOH (10 ml) and evaporated to
10 dryness. The residue was dissolved in EtOAc (150 ml), washed
with 5~ NaHCO3 solution (100 ml), water (100 ml) and ~rine (50
ml). The organic extract was drieà over Na,SO4 and evaporated
to dryness. Th- rGsidu w2s ?l-ifi~d by flash chromatography
over silica ~el using CH,Cl -->EtCAc eS the eluen.. The DUr2
rractions were pooled and evaporated to give 1.7 g (83%~ of
1~ pure product. :HNMR (CDC13): 1.16 (d, 6H, IbCH3), 1.94 (s, 3H,
CH3), ~.52 (m, lH), 3.74 (s, 6H, 2.0CH3), 3.80 - 4.30 (m, 6H),
4.56 (m, lH), 6.82 (d, 4H, Ph), 7.14 (d, 2H, C~H and NH) and
7.26 - 8.00 (m, 14H, Ph).
2~ -(4,4'-Dimetho~y- ityl)-O-acetyl)~no~-
3-thyminyl-L-psopanol (26): 1-0-Isobutyryl-2-[(~-
(4,4'-dimethoxytrityl)-O-acetyL)aminoj-3-[N3-benzoyl(thyminyl)
-L-propanol 25 (1.55 g, 2.05 mmol) was dissolved in MeOff (20
ml) and cooled to O~C in an ice bath. To this cold stirred
2~ solution was added 2N NaOH (5 ml, 10 mmol) and the stirring
continued for 30 min at 0C. The pH of the solution was
adjusted to 7 wiLh acetic acid and evaporated to dryness. The
residue was partltioned between water (50 ml) and CHzCl2 (150
ml) and extracted in CH2C12. The aqueous layer was extracted
30 again with CH,Cl2 (50 ml). The combined organic extract was
washed with brir.e ~50 ml), dried and evaporated to dryness.
The residue was Durified by flash column chromatography over
silic2 gel u5in.g CH2C12--> acetone as the eluent. Yield: 1.0 g
_ 99 _
CA 02202274 1997-04-09
. .
WO96/14330 PCT~S95/14599
, .
(99%). iHNMR (CDC13): 1.94 (s, 3H, CH3), 3.74 (s, 6H, 2.OC~3),
3.80 - 4.30 (m, 6H), 4.56 (m, lH), 6.82 (d, 4H, Ph), 7.1~ (d,
2H, C6H and NH) and 7.26 - 8.00 (m, 14H, Ph).
2-t(~-(4,4'-D~methG~yL~ityl)-O-acetyl)amino~-
3-~hyminyl-~-~ropan- l-O-(N,N-diiso~ropyl)-~-cyanoethyl-
~osphor~dite tz): 2-[(~-(4,4'-Dimethoxytrityl)-
O-acetyl)amino]-3-thyminyl-L-propanol 26 (1.00 g, 2.09 mmol)
was dried over solid NaOH under vacuum overnight and dissolved
10 in dry CH,Cl, (50 ml). The solution was cooled to 0C under
argon atmospnere. To thls cold stirred solu~ion was added
N,N-diisopropylethylamine (0.54 g, ~.2 mmol) followed by
2-cyanoethyl-N,N-diisopropylchloropnosphoramidi~e (0.73 g, 3.1
mmol). The reaction mixture was stirred at 0C for 1 h and at
room temperature or 2 h. The reacrion was dilu~ed with CH,Cl-
(100 ml) and the organic layer was washed with 5% NaHCO3
solution (100 ml), water (100 ml) and brine (50 ml). The
CH7Cl7ex~ract was dried and evapora~ed to dryness to give an
oily residue. The residue was pu~ified by ~lash chromatography
over silica gel using CH2Cl -->EtO~.c containing 0.1% TEA as
20 the eluent. The pure frac.ions were pooled together and
evaporaled to give a foam. The foam was dried over solid NaOH
under vacuum overnight. The dried foam was dissolved in dry
CH2Cl2 (10 ml) and dropped into a stirred solution of dry
hexane (800 ml) under argon during 30 min period. After the
2~ addition, the precipitate formed was stirred for additional 30
min and filtered, washed with dry hexane (100 ml) and the
solid was dried over solid NaOH under vacuum for 4 h. Yield:
1.3 g (82%).
-- 100 --
CA 02202274 1997-04-09
W096/14330 PCT~S95/14599
Exampie 29
(Figu_e 26)
~ --t2rt--~utylosyr~--honyl--O--~enzylh~ ~ o~y7:~m~r~o (28):
O-Benzyl hydroxylamine hydrochloride (15.9 g, 100 mmol) was
suspended in THF (150 ml) ana waLer (50 ml) mixtu_e. To this
stirred mixture was added T~A (15.15 g, 150 mmol) followed by
di-tert-butyldicarbonate (23.98 g, 110 mmol). The reaction
mixture was s~irred 2t room .emperature for 12 h and
evaporated to dryness. The ~esidue was partitioned between
O EtOAc (250 ml) and water (200 m~), and extracted in EtOAc. The
EtOAc extracl was washed witn potassium hydrogen sulfate (100
ml) and brine (100 ml), dried and evaporated .o dryness tO
give 15 g (91~) o- clear OLl.
1-Chloro-2-(tetr~.y~L~y dnyl) oxy-ethane (29): l-Chloro
15 etnanol (8.06 g, 100 mmol) was dissolved in dry CH,C1, (100
ml) and c0012d to 0C in an ice bath under argon. To this
stirred solution was added dihydro?yran (12.6 g, 150 mmol)
fo'lowed by pyridinium -p-toluene -4-sulfonate (1.25 g, 5
mmol) and the stirring con_inued for overnight. Tne reaction
20 m~x~ure was evaDorated tO àryness and dissolved i.. EtOAc (200
mi). The rtOAC extract was washed with 5% NaHCO~ solutior. (100
ml), water (100 ml) and brine (100 ml). The organic extract
was dried and e~aporated to dryness. The crude material was
purified by flash chromatography over silica g~l using hexane
25 ~~> CH,Cl2 as the eluent. The pure fractions collected
together and e~aporated to give 11 g (67%) of pure product.
N-te~t-~utylosy~ ~nyl-N-~(tetr~ydL~yy dnyl)oYy]
ethyl-O-~enzyl~y~y1~m~n~ (30): To a stirred solution of
N-tert-butyloxycarbonyl-O-benzylhydroxylamine 28 (5.79 g,
25.96 mmol) in dry DMF (50 ml) was added NaH (60%, 1.2 g, 30
mmol) slowly during 15 min period under argon atmosphe-e at
0C. The reaction was allowed to stir at 0 C for 30 m n and
-- 101 -
CA 022022i4 1997-04-09
W O96/14330 PCTrUS95114599
. t
at room temperature for 1 h. 1-Chloro-2-(tetrahydro-
pyranyl)oxy- ethane 29 (4.95 g, 30 mmol) was added and the
reaction mixture was heated at 80C for 12 h. The reaction was
cooled and evaporated to dryness. The residue was suspended in
water (50 mi), pH of the soluti~n adjusted to 7 and extracted
in EtOAc (150 ml). The EtOAc eY.tra_t was washed with water and
brine, drieà and evaporated to dryness. The residue was
purified by rlash chromatography over silica gel using
hexane--> CH.Cl,as the eluen.. The required fractions were
collected and eva~orated to give 6.0 g (66%) of an oily
product. lHNMR (CDCl3): ~ 1.48 (s, 9H, Boc), 1.49 - 1.84 (m,
6H, 3.CH,), 3.48 - 3.70 (m, 4H, 2.CH,), 3.86 (m, 2H, CH2),
4.60 (., lH, CH), 4.84 (s, 2.., CH.~h) and 7.32 - 7.42 (m, 5;{,
Ph).
N-tert-3utylo~y~~~honyl-N- ~ (2-hy~ ~y) ethyl] -O-
benzyl}ly~l~G~yl:lmin~ (31): A stirred solution of
N-tert-Butyioxycarbonyl-N-[(tetrahydropyranyl)oxy]ethyl-
O-benzylhyàroxylamin- 30 (3.51 g, ~O mmol) in T.4F:water: AcOH
(1:1:1, 100 ml) was heated at 7Cr ror 3 h. The reaction was
20 cooled to C_ and the pH adjus~ed ~o 7 with solid NaHCO3. The
reaction mixlure was extracted wiLh EtOAc (2x75 ml~. The
combined organic extract was washed with wa~er (100 ml) and
brine (100 ml), dried and evaporated to dryness. The residue
was purified by flash chromatography over silica gel using
2~ CH2C12 --> EtOAc as the eluent. The pure fractions were pooled
and evaporated to give 2.5 g (94%) of foam. lHNMR (CDCl3): ~
l.a8 (s, 9H, Boc), 3.60 (t, 2H, CH,), 3.74 (m, 2H, CH2), 4.84 a
(s, 2H, CH,Ph) and 7.32 - 7.42 (m, 5H, Ph).
N- tert-Butyloxy~7~honyl-N- ~ ~ (2-iso}:~utyryl) oxy] ethyl] -
O-~enzyl~.y~,Lyl ~m~nF~ (32): To a stirred solution of
N-tert-butyloxycarbonyl-N-[(2-hydroxy)ethyl]-O-benzylhydroxyla
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. .
mine 31 (4.2 g, 16.6 mmol~ in dry pyridine (50 ml) was added
TEA (2.02 g, 20 mmol) followed by isobutyric anhydride (3.16
g, 20 mmol) at room temperature under argon atmosphere. The
reaction mixture was stirred at room temperature for 12 h and
evaporated to dryness. the residue was dissolved in EtOAc (200
ml), washed with 5~ NaHCO3 solution (l00 ml), water and b-ine
(50 ml). The organic extract was dried and evaporated to
dryness. The residue was puriried by flash chromatography over
silica gel using CH,Cl, as the eluenr. The pure fractions
collected and evaporated to give ..5 g (80~) of pure compound.
iHNMR (CDCl3): ~1.04(d,6H, IbCH3), 1 . 48 ~s, 9L~, 30c), 2 . 44 (m, lH,
IbCH), 3.60 (t, 2~., CH,), 3.74 (m, 2:~, CH,), 4.84 (s, 2H,
CH~Pn) and 7 . 32 - 7 . 42 (m, 5r., Ph) .
N- (Thyminylacetyl) -~--~ t (2-iso~utyryl) o~ et~yl] -0-
~enzyl}.y~L~yl~m~nr~ (33): N-tert-Butyloxycarbonyl-N-
[[(2-isobutyryl)oxy]ethyl]-O-benzylhydroxylamine 32 (5.0 g,
l4.84 mmol) was dissolved n CH,Cl (l0 ml) and allowed to
stir in TFA (12 ml) for 30 min. The reaction mixture was
evaporated to dryness and dissolved in dry methanol (l0 ml).
20 I. was evaporated again to dryness and àried under vacuum over
solid NaOH overnight. The dried material used as such fo- the
next reaction without characterization.
Thymine acetic acid ~ (3.13 g, 17 mmol) was dissolved in
25 dry DMF (75 ml) and cooled to -20C under argon. To this cold
stirred solution was added N-methylmorpholine (2.02 g, 20
mmol) followed by isobutyl chlororormate (2.72 g, 20 mmol).
After lS min of stirring, 2 solution of the above TFA salt in
dry DMF (50 ml) was neutraLized with N-me~hylmorpholine (2.02
30 g, 20 mmol) and added immediately into the cold stirred
solution of thymine acetic acid at once. The reaction mixture
was stirred at -20CC ~or l h, warmed to room temperature and
the stirring continued overnlght. The solution was evaporated
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to dryness and the residue dissolved in CH2C12 (250 ml) and
water (100 ml), and extracted in CH2C17. The organic extract
was washed with 5% NaHCO3 solution (100 ml), water (100 ml)
and brine (50 ml). The CH2Cl2extract was dried and evaporated
5 to dryness to give crude product. The crude product was
purified by flash chromatography over silica gel using CH2C1,
--> acetone as the eluent. The necessary fractions were
collected and evaporated to give 4.0 g (70%) of the pure
product. The pure product was crystallized from CH7Cl2/hexane.
10 mp: 185-188C. lHNMR (Me2SO-dO): ~ 1.00 (d, 6H, IbCH3), 1.74
(s, 3H, CH3), 2.44 (m, lH, IbCH), 3.92 (m, 2H), 4.18 (t, 2H),
4.68 (bs, 2H), 4.98 (s, 2H), 7.34 (s, lH, C6H), 7.40 - 7.50
~m, 5H, Ph) and 11.32 (bs, lH, NH).
Example 30
1~ (Figure 27)
(2~,4~) -2-C~-h~ ?tho~cy-4-hyd~o~y~y- olidine (35): In a
250 ml round bottom flask equipped with a magnetic stir bar
and a reflex condenser were placed dry methanol (40 ml) and
cooled in ice bath under argon atmosphere. To this stirred
20 solution was added acetyl chloride (4.32 g, ~5 mmol) followed
by cis-4-hydroxy-D-proline 34 (5.00 g, 38.17 mmol). The
resulting solution was heated at reflex for 7-8 h and cooled
to room temperàture. The solution was diluted with ether, and
the resulting white solid was collected by suction, was with
25 ether and dried under vacuum over solid NaOH. Yield; 6.9
g(100~ HNMR (CDCl3): 2.09 (2 dd, lH), 2.34 (m, lH), 3.49 -
3.73 (m, 3H), 3.79 (s, 3H, CH3), 4.34 (m, 2H).
(2~,4~)-1-(tert-Butyloxyo~-honyl)-2-~-~ h- -tho~y-
4-hydLo~y~y-.olir~; n~ (36): To a stirred solution of
(2R,4R)-2-Carbomethoxy-4-hydroxypyrrolidine 35 (6.9 g, 38.12
mmol) in THF/water (8:2, 150 ml) was added TEA (10.1 g, 100
mmol) followed by di-tert-butyldicarbonate (10.9 g, 50 mmol)
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096/14330
at room temperature. The reaction was stirred at room
temperature for 6 h and evaporated to dryness. The residue was
dissolved in EtOAc (200 mI) and washed with 0.5N potassium
hydrogen sulfate (50 ml), water (100 ml) and brine (50 ml).
The organic extract was dried over Na2SO4 and e~aporated to
dryness to give 7.8 g (84%) of an oily product. The oily
product on drying gave colorless solid: mp: 75-77C. lHNMR
(CDCl3): 1.45 (s, 9H, Boc), 2.09 (2 dd, lH), 2.34 (m, lH),
3.49 - 3.73 (m, 3H), 3.79 (s, 3H, CH3), 4.34 (m, 2H).
~2~,4~)-1-(tert-3utyloxy~rh~nyl)-2-hyd~oay~ethyl-
4-hydlo~y~y r O~ n~ (37): (2R,4R)-1-(tert-Butyloxycarbonyl)-
2-carbomethoxy-4-hydroxypyrrolidine 36 (7.0 g, 28.6 mmol) W2S
dissolved in dry THF (100 ml) and cooled in ice salt bath
under argon atmosphere. To this cold solution was added
lithium borohydride (1.88 g, 85.8 mmol) in small portions
during 15 min period. After the addition of lithium
borohydride, the reaction mixture was allowed to stir at 0C
for 1 h followed by 15 h at room temperature under argon. The
solution was cooled to C and diluted with water (50 ml) and
the pH was adjusted with AcOH to 6. The reaction was
evaporated to dryness and dissolved in EtOAc (200 ml), washed
with water (100 ml) and brine (100 mlJ. The EtOAc extract was
dried and evaporated to dryness. The residue was purified by
flash column chromatography over silica gel using CH2Cl2-->
EtOAc as the eluent. The pure fractions were collected and
evaporated to dryness to afford 5.00 g (81~) of clear oil. The
oil on standing gave colorless solid. mp: 95-97C. lHNMR
(CDCl3): 1.45 (s, 9H, Boc), 1.90 ( dd, lH), 2.34 (m, lH), 3.40
- 3.62 (m, 3H), 4.00 (m, 2H), 4.28 (bs, lH), 4.44 (m, lH).
(2~,4~)-1-(terf-Butyloxy~-honyl)-2-(4,4'-
Dimet}lGd~y L ityl) o~cymethyl-4-}.ydLo~y~y ~ol;~1; n~ (38):
(2R,4R)-1-(tert- Butyloxycarbonyl)-2- hydroxymethyl-
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W 096/14330 P~~ a/14599
4-hydroxypyrrolidine 37 (4.4 g, 20.28 mmol) was-dissolved in
dry pyridine (50 ml) and allowed to stir under argon
atmosphere. To this stirred solution was added TEA (2.53 g, 25
mmol) followed ~y 4,4'-dimethoxytrityl chloride (7.45 g, 22
mmol). The reaction mixture was stirred at room temperature
for 12 h and quenched with MeO~ (10 ml). The solution was
evaporated to dryness and dissolved in EtOAc (200 ml). The
EtOAc layer was washed with 5% NaHCO3 solution (100 ml), water
(100 ml) and ~rine. The organic extract was dried over
anhydrous Na7SO4 and evaporated to dryness. The residue was
purified by flash chromatography over silica gel using hexane
--> EtOAc as the eluent. The required fractions were pooled
together and evaporated to give 8.09 g ~100%) of an orange
foam. lHNMR (CDC13): 1.45 (s, 9H, Boc), 1.90 ( dd, 1~), 2.3
(m, lH), 3.40 - 3.62 (m, 3H), 3.74 (s, 6H, 2.0CH3), 4.00 (m,
1~ 2H), 4.28 (bs, lH), 4.44 (m, lH), 6.82 (d, 4H, Ph), and 7.26 -
8.00 (m, 9~, Ph).
(2~,4~)-1-(tert-B~tylo~y~-h~nyl)-2-(4,4'-
DimethG~yL~ityl)oYymethyl-4-[(p-toluenesulfonyl)oYy~pyrrolidin
20 e (~2): (2R,4R)-1-(tert-Butyloxycarbonyl)-2-(4,9'-Dimethoxy-
~rityl) oxymethyl-4-hydroxypyrrolidine 38 (8.09 g, 20.27 mmol)
was dissolved in dry pyridine/ CH2Cl (2:1, 200 ml) and
chilled in an ice bath under argon atmosphere. To this cold
solution was added TEA (3.03 g, 30 mmol) followed by
2~ p-toluenesulphonyl chloride (5.7 g, 30 mmol). The reaction
mixture was allowed to stir at 0C fo- 3 h and below 30C for
8 h. The reaction mixture was evaporated to dryness,
partitioned ~etween EtOAc (200 ml) and 5% NaHCO3 solution (100
ml), and extracted in EtOA_. The EtOAc extra~t was washed with
water (100 ml) and brine (100 ml), dried and evaporated to
r
dryness. TAe crude produc. was purified by flash
chromatography over silica gel using hexane --> EtOAc zs the
eluen.. The pure fractions were pooled together and evaporated
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to give 12.2 g (89%) of an orange oil.lHNMR (CDC13): 1.45 (s,
9H, Boc), 1.90 ( dd, lH), 2.3a (m, lH), 2.40 (s, 3H, CH3),
3.40 - 3.62 (m, 3H), 3.74 (s, 6H, 2.0CH3), 4.00 (m, 2H), 4.28
(bs, lH), 4.44 (m, lH), 6.82 (d, 4H, Ph), and 7.26 - 8.00 (m,
5 13H, Ph)
.. .
(2~,4S)-l-(tert-Butylo~y~rhonyl)-2-(4,4'-
D~eth~yL.ityl) o~nethyl-4-azido ~y~l;~;n~ (40)
(2R,4R)-1-(tert- Butyloxycarbonyl)-2-(4,4'-
10 Dimethoxytrityl)oxymethyl- 4-[(p-toluenesulfonyl)oxyy-
pyrrolidine 39 (5.1 g, 7.58 mmol) was dissolved in
dimethylformamide (50 ml) and diluted with water (5 ml). To
this stirred solution was added sodium azide ( 0.65 g, 10
mmol) and heated at 80~C for 8 h. It was cooled and
evaporated to dryness. The residue was partitioned between
CH7Cl2 (200 ml) and water (100 ml), and extracted in CH2Cl2.
The organic extract was washed with ~rine (50 ml), dried over
Na2SO4 and evaporated to dryness. The crude product was
purified by flash chromatography over silica gel using hexane
--> EtOAc as the eluent. The pure fractions were pooled
20 together and evaporated to give 3.8 g (92~) of a clear oil.
lHNMR (CDCl3): 1.45 (s, 9H, Boc), 1.90 ( dd, lH), 2.34 (m,
lH), 3.40 - 3.62 (m, 3H), 3.74 (s, 6H, 2.0CH3), 4.00 (m, 2H),
4.28 (bs, lH), 4.44 (m, lH), 6.82 (d, 4H, Ph), and 7.26 - 7.80
(m, 9H, Ph).
2~
(2~,4S)-1-(te~t-Butyloxy~nyl)-2-},y~k~y thyl-
4-aminG ~yr ol;~ine (41): (2R,4S)-1-(tert-Butyloxycarbonyl)-
2-(4,4'-Dimethoxytrityl)oxymethyl-4-azido-pyrrolidine 40 (2.72
g, 5 mmol) in methanol (75 ml) was hydrogenated in the
7 30 presence of 10% palladium on charcoal (0.3 g) at room
temperature and 5 atm pressure. After 12 h, the catalyst was
filtered, washed with methanol (20 ml) and the solvent removed
under vacuum. Yield 1.0 g (93%). HNMR (CDCl3~: 1.45 (s, 9H,
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W O96/14330 PCTrUS95/14599
Boc), 1.90 ( dd, lH), 2.34 (m, lH), 3.40 - 3.62 (m, 3H), 4.00
(m, 2H), 4.28 (bs, lH) and 4.44 (m, lH).
(2~, 4S)--1--( tert--Butylo~ h~nyl)--2--l~y~.l~G~y -thyl--
5 4-ph~h~ ~y~,ol;~3;ne (42): (2R,4S)-l-(tert-Butyloxy-
carbonyl)-2-hydroxymethyl-4-amino-pyrrolidine 41 (1.00 g, 4.63
mmol) was dissolved in dry methanol (20 ml) and treated with
N-ethoxycarbonyl phthalimide (1.09 g, 5 mmol) at room
temperature. The reaction mixture was stirred for 6 h and
10 evaporated to dryness. the residue was purified by flash
chromatography over silica gel using CH2Cl2--> EtOAc as the
eluent. The pure fractions were collected and evaporated to
give 1.5 g (94%) of pure compound as foam. lHNMR (CDCl3): 1.45
(s, 9H, Boc), 1.90 ( dd, lH), 2.34 (m, lH), 3.40 - 3.62 (m,
3H), 4.00 (m, 2H), 4.28 (bs, lH), 4.44 (m, lH) and 7.3 - 7.6
1~ (m, 4H, Ph).
(2~,4S)-l-(tert-ButyloY~y~-hnnyl)-2-tN3-~enzoyl
(thymin-l-yl)] methyl--4-~h~h=~lim~c~o ~yl r o~ n~ (43): To a
stirred solution of N3-benzoylthymine 20 (1.15 g, 5 mmol) in
20 dry THF (70 ml) under argon was added triphenyl phosphine
(2.62 g, 10 mmol) and (2R,4S)-1-(tert-Butyloxycarbonyl)-
2-hydroxymethyl-4-phthalimido- pyrrolidine (1.4 g, 4.05 mmol)
at room temperature. After 15 min, diethylazodicarboxylate
(1.74 g, 10 mmol) was added slowly during 10 min period. The
2~ reaction mixture was covered with aluminum foil and allowed to
stir at room temperature under argon for 24 h. The solvent was
evaporated to dryness and the residue dissolved in EtOAc (150
ml). The organic extract was washed with 5% NaHCO3 solution
(100 ml), water (100 ml) and brine (100 ml), and dried o~er
30 anhydrous Na2SO4. The dried EtOAc extract was evaporated to
dryness to give an orange oil. The crude product was purified
by flash chromatography over silica gel using hexane--> EtOAc
as the eluent. The fraction having the required product was
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W O96/14330 PCT~US95114599
pooled and evaporated to give a pale pink oil. Yield: 2.0 g
(89%). lHNMR (CDC13): 1.41 (s, 9H, Boc), 1.72 (s, 3H, CH3),
1.90 ( dd, lH), 2.34 (m, lH), 3.40 - 3.62 (m, 3H), 4.00 (m,
2H), 4.28 (bs, lH), 4.44 (m, lH), 7.06 (s, lH, C6H) and 7 20 -
5 7-60 (m, 9H, Ph).
Example 31
Synth~e; ~ Of Oligonucleotides: Oligonucleotides
containing modified amino acid nucleic acid backbones were
synthesized on an automated DNA synthesizer (Applied
Biosystems model 394) using standard phosphoramidite
chemistry. ~-Cyanoethyl phosphoramidities, synthesis reagents
and CPG polystyrene colums were purc~ased from Applied
Biosystems (Foster City, CA). For phosphorothioate
oligonucleotides, the standard oxid~tion bottle was replaced
15 with tetraethylthiuram disulfide/acetonitrile, and the
standard ABI phosphorothiate program was used for the stepwise
thiation of the phosphate linkages. After cleavage from the
controlled pore glass column, the protecting grou?s were
removed by treating the oligonucleotides with concentrated
20 ammonium hydroxide at 55C for 8 hrs. The oligonucleotides
(DMT-on) were purified by HPLC using a reverse phase semiprep
C3 column (ABI) with a linear gradient of 5% acetonitrile in
O.lM triethylammonium acetate (buffer A) and acetonitrile
(buffer B). The DMT protecting group was cleaved by treatment
with 80% acetic acid and the product was ethanol precipitated.
2~
The purity of the products were checked by HPLC using an
analytical Cl~ column (Beckman). The amino acid nucleic acid
~ monomers were incorporated at the 3'-end, 5'-end and in the
middle or a DNA sequence with a coupling efficiency of 100%. A
t homo polymer containing 16 amino acid modified thymine was
30 also prepared with out any problem.
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WO96/14330 PCT~S95114599
Example 32
~ y~ridization analysis: The ability of the amino acid
modified oligonucleotides of the invention to hybridize to
their complementary RNA and DNA sequences is determined by
thermal melting analysis. The RNA complement is synthesized by
Genset corporation (La Jolla, CA) and purified by denaturing
urea PAGE. Natural antisense oligonucleotides or those
containing functionalized at specific locations are added to
either the RNA or DNA complement at stoichiometric
concentrations to form hybrid duplexes. The absorbance (260
nm) hyperchromicity dependence on temperature upon duplex to
random coil transition is monitored using Varian Cary lE
UV-Visible spectrophotometer. The measurements are performed
in a buffer of 10 mM Na-phosphate, pH 7.4, 0.1 mM EDTA, and
NaCl to yield an ionic strength of either 0.1 M or 1.0 M.
15 Data are analysed by a graphic representation of 1/Tm vs
ln[Ct], where [Ct] is the total oligonucleotide concentration.
From this analysis the thermodynamic parameters are
determined. Based on the information gained concerning the
stability of the duplex or hetero-duplex formed, the placement
20 Of modified pyrimidine into oligonucleotides is assessed for
its effects on helix stability. Modifications that drastically
alter the stability of the hybrid exhibit reductions or
enhancements in the free energy (delta G) and decisions
concerning their usefulness in antisense oligonucleotides are
made.
Hybridization studies were conducted with
oligonucleotides containing amino acid nucleic acid backbone
at 3'-end as well as at the 5'-end. Preliminary studies showed
that the modified oligonucleotides form duplex with their
complementary RNA and DNA sequences like unmodified
30 oligonucleotides.
Example 33
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O96114330 PCTrUS95tl4599
N~ ~a~e ~esistance. Natural, phosphorothioate and
modified oligonucleo~ides of the invention are assessed for
their resistance to serum nucleases by incubation of the
oligonucleotides in media containing various concentrations of
fetal calf serum or adult human serum. Labeled
oligonucleotides are incubated for various times, treated with
protease K and then analyzed by gel electrophoresis on 20%
polyacrylamide-urea denaturing gels and subsequent
autoradiography or phosphor-imaging. Autoradiograms are
quantitated by laser densitometry. Based upon the location of
the modifications and the known length of the oligonucleotide
it is possible to determine the effect of the particular
modification on nuclease degradation. For the cytoplasmic
nucleases, a HL60 cell line is used. A post-mitochondrial
supernatant is prepared by difrerential centrifugation and the
labeled oligonucleotides are incubated in this supernatant for
various times. Following the incubation, oligonucleotides are
assessed for degradation as outlined above for serum nucleo-
lytic degradation. Autoradiography results are quantitated for
comparison of the unmodified i.e., phosphorothioate and the
modified oligonucleotides.
Preliminary studies on the amino acid modified
oligonucleotides showed that they are resistant to Snake Venom
Phosphodiesterase.
Incorporation ~ Rererence
All patents, patents applications, and publications cited
are incorporated herein by reference.
Equivalents
The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. Indeed, various modifications of the above-
described makes for carrying out the invention which are
-- 111 --
