Note: Descriptions are shown in the official language in which they were submitted.
WO 94/22890 PCT/US94/02993
215~63~
-1 -
NOVEL 5' SUBSTITUTED NUCLEOSIDES AND
OLIGOMERS PRODUCED THEREFROM
Field of the Invention
The present invention relates to novel 5'-substituted
nucleosides and to oligonucleotide analogs prepared therefrom having from
2 to about 60 bases and having an internucleoside backbone containing one
or more 3'-O-PO2H-O-5'-CR1 R2 internucleoside linkages instead of the
10 naturally occurring backbone of phosphodiester internucleoside linkages.
The present invention also relates to a method of synthesizing
oligonucleotide compounds having from 2 to about 60 bases and having an
internucleoside backbone containing one or more 3'-O-PO2H-O-5'-CR1 R2
internucleoside linkages instead of the naturally occurring backbone of
15 phosphodiester internucleoside linkages, this process comprising
preparation of 5'-substituted nucleoside compounds, for example, as
illustrated in Scheme 1, and utilizing them as synthons in automated DNA
synthesizers. Oligonucleotide analogs of the present invention are useful as
nuclease resistant, sequence specific antisense compounds.
R~ck~round of the Invention
An antisense compound binds to or hybridizes with a nucleotide
sequence in a nucleic acid (RNA or DNA) to inhibit the function (or synthesis)
25 of the nucleic acid. Because they can hybridize with both RNA and DNA,
antisense compounds can interfere with gene expression at the level of
transcription, RNA processing or translation.
As discussed, e.g., in Klausner, A., Biotechnology. 8:303-304
(1990), the development of practical applications of antisense technology is
30 hampered by a number of technical problems. Thus, natural, phosphodiester-
linked antisense oligomer compounds are susceptible to rapid degradation
by nucleases that exist in target cells and elsewhere in the body; both
exonucleases, which act on either the 3' or the 5' terminus of the nucleic acid,and endonucleases, which cleave the nucleic acid at internal phosphodiester
35 linkages between individual nucleosides. As a result of such'nuclease action,the effective half life of many administered antisense compounds is very short,
necessitating the use of large, frequently administered, doses.
WO 94122890 _ 9 ~ 3 ~ -2- PCTIUS94/02993
The high cost of producing antisense DNA or RNA on currently
available DNA synthesizers is another problem. Armstrong, L., Rusiness
Week, March 5, 1990, page 89, estimated the cost of producing one gram of
antisense DNA to be about $100,000.
There is also a problem regarding delivery of antisense agents
to targets within the body (and cell). Thus, antisense agents targeted to
genomic DNA must permeate the plasma and the nuclear membrane to gain
~ccess to the nucleus. The consequent need for increased hydrophobicity to
enhance membrane permeability must be balanced against the need for
increased hydrophilicity (water solubility) in body fluids such as the plasma
and cell cytosol.
Also, oligonucleotide compounds such as antisense DNA are
susceptible to steric reconfiguration around chiral phosphorous centers. This
results in stability problems, too, whether the compounds are free within the
body or hybridized to target nucleic acids.
To overcome the stability and drug delivery limitations, various
oligonucleotide analogs have been investigated. In order to be of practical
utility, such analogs should have good cell penetration properties, be
resistant to nuclease degradation, have good sequence specific hybridization
to target nucleic acids, and be synthesized by chemical methods that are not
too difficult or costly.
Recent efforts to overcome the foregoing problems and prepare
antisense compounds that are stable, nuclease resistant, relatively
inexpensive to manufacture and which can be delivered to and hybridized
with nucleic acid targets throughout the body have involved synthesizing
oligonucleotide analogs that consist of oligonucleotide analog sequences
with internucleoside linkages that differ from the 'normal' internucleoside
phosphodiester linkage, either by introducing modifications in the
phosphodiester structure or by using non-phosphate internucleoside linkages
that approximate the length and orientation of the normal phosphodiester
internucleoside linkage. Uhlman, E. and Peyman, A., Chemical Reviews,
9(4):544-584 (1990).
Among the modified phosphodiester linkages that have been
reported are phosphorothioates, alkylphosphotriesters, methylphosphonates
and alkylphosphoramidates. Also, a variety of non-ionic oligonucleotide
analogs sequences containing non-phosphate internucleoside linkages, such
as carbonate, acetate, carbamate, sulfone, sulfoxide, sulfonamide and dialkyl-
or diaryl- silyl derivatives have been synthesized and reported. More
WO 94/22890 215 9 6 3 ~ PCT/US94/02993
-3 -
recently, chimeric oligonucleotide analogs comprising nucleoside linkages
containing two carbon atoms and one nitrogen atom or one oxygen atom, as
well as those containing three carbon atoms, have been reported. See, e.g.,
International Patent Publication WO 9202534.
The prior art describes 1-mononucleosides and
- mononucleotides substituted at the 5'- position by a variety of substituents, 2-
dinucleosides phosphates substituted at the 5'-position of the 3'-terminal
nucleoside by methyl, ethyl, propyl and allyl; it also discloses the
corresponding pairs of stereoisomers (Padyukov, A, et al. (1980), Collection
of C7echoslov~k Chemic~l Communic~tions. vol 45, 2550-2557). This
reference reports that no significant differences were observed between two
stereoisomers in pancreatic ribonuclease degradation. The prior art shows
that rates of cleavage of 5'-substituted dinucleotide phosphates by nuclease
vary unpredictably with the steric configuration at the 5'-position, the
nucleoside base and the nuclease used. The prior art is, however, devoid of
disclosure of the novel 5'-substituted oligonucleotides of this invention; nor
does it contain any suggestion of their excellent nuclease stability or
hybridization properties.
The present invention provides novel 5'-substituted nucleosides,
as well as oligonucleotide analogs of two bases and longer derived therefrom
which are resistant to nucleases and will bind in a sequence specific manner
to complementary nucleic acid sequences. Also provided is a method of
synthesizing such oligonucleotide analogs using the nucleoside derivatives
described in this specification. Another advantageous feature of the present
invention is the relative ease with which optically pure isomers of the 5'-
substituted nucleoside analogs of the invention as compared to
phosphorothioate, methylphosphonate and phosphoroamidate nucleosides
used in the prior art to synthesize oligonucleotide analogs.
Summ~ry of the Invention
The present invention provides novel 5'-substituted nucleoside
analogs and oligonucleotide analogs of 2 to about 60 bases containing 3~-O-
PO2H-O-5'-CR1 R2 substituted internucleoside linkages instead of the
naturally occurring phosphodiester internucleoside linkages.
More particularly, in one aspect, the present invention provides
novel nucleoside analogs having the structure of Formula I below:
WO 94/22890 PCT/US94/02993
9 6 3 ~ -4-
Q-(cHR)n~ o E3
RR2 ~
L(CHR)n R4 E
Fonnula
wherein:
Q is selected from the group consisting of H, OH, NHR, CHO,
phosphate, lower-alkyl, lower alkenyl, protected O-, protected N-, lower
alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl,
amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and
phosphonate;
L is selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2),
H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower
10 alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl,
phosphonium salt, phosphonate and t-butyldimethylsilyloxy;
each R is independently selected from the group consisting of H, OZ,
SZ and NHZ;
each R1 and R2 is independently selected from the group consisting of
15 H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower
alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy;
each R3 and R4 is independently selected from the group consisting of
H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy:
each Z is independently selected from the group consisting of H, lower
20 alkyl, lower alkenyl, aryl, acetyl and protecting groups for O-, S-, and N-;
each E is independently selected from the group consisting of
-OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, lower alkyl, lower alkenyl, lower
alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen,
epoxyethyl, phosphonium salt and phosphonate;
each n is independently 0 or an integer from 1 to 4; and
each B is independently select from the group consisting of adenine,
cytosine, guanine, thymine, uracil or a modification thereof that does not
substantially interfere with the affinity of an oligonucleoside or chimeric
oligonucleotide analog for its antisense counterpart wherein the bases are
30 selected from the group consisting of adenine, cytosine, guanine, thymine
and uracil;
or an optical isomer thereof or a pharmaceutically acceptable salt thereof.
WO 94/22890 PCT/US94102993
2 1 ~
-5 -
In another embodiment, the invention provides oligonucleotide
analogs having the structure of Formula ll below:
Q-(CHR)n~B
R4_ ~oYB
R3 ~
/ R4 E q O B
R3,~
L(CHR)n R4 E
Formub 11
wherein:
Q is selected from the group consisting of H, OH, NHR, CHO,
phosphate, lower-alkyl, lower alkenyl, protected O-, protected N-, lower
alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl,
amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and
1 0 phosphonate;
L is selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2),
H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower
alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl,
phosphonium salt, phosphonate and t-butyldimethylsilyloxy;
each R is independently selected from the group consisting of H, OZ,
SZ and NHZ;
each R1 and R2 is independently selected from the group consisting of
H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower
alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy;
each R3 and R4 is independently selected from the group consisting of
H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy:
each Z is independently selected from the group consisting of H, lower
alkyl, lower alkenyl, aryl, acetyl and protecting groups for O-, S-, and N-;
each E is independently selected from the group consisting of
-OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, lower alkyl, lower alkenyl, lower
alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen,
epoxyethyl, phosphonium salt and phosphonate;
each n is independently 0 or an integer from 1 to 4
W094/22890 '~5~6~ -6- PCT/US94/02993
each B is independently selected from the group consisting of adenine,
cytosine, guanine, thymine, uracil or a modification thereof that does not
substantially interfere with the affinity of an oligonucleoside or chimeric
oligonucleotide analog for its antisense counterpart wherein the bases are
5 selected from the group consisting of adenine, cytosine, guanine, thymine
and uracil;
each W is selected from 3'-(OPO2HO)C(R1 R2)-5' and a natural
phosphodiester internucleoside linkage with the proviso that at least one W is
3'-(OPO2HO)C(R1 R2)-5'; and
q is 0 or an integer from 1 to 60.
As used herein, the term 'oligonucleotide' means nucleic acid
compounds which contain only 'natural' phosphodiester internucleoside
linkages. On the other hand, the term 'chimeric oligonucleotide analogs'
means compounds that comprise sequences containing both 'synthetic'
15 oligonucleoside linkages and oligonucleotide linkages. By the term
'oligonucleotide analogs,' we mean both oligonucleotide analogs that contain
only synthetic (as opposed to the naturally occurring phosphodiester)
internucleoside linkages and chimeric oligonucleotide analogs.
The present invention also provides a method of synthesizing
20 oligonucleotide compounds having from 2 to about 60 bases and having an
internucleoside backbone containing one or more 3'-O-PO2H-O-5'-CR1R2
internucleoside linkages instead of the naturally occurring backbone of
phosphodiester internucleoside linkages, this process comprising joining a
5'-end nucleoside, a middle unit, and 3'-end nucleoside, by conventional
25 synthetic organic procedures known in the art.
WO 94/22890 21~ 9 ~ ~ ~ PCT/US94/02993
net~iled nescription of the Invention
The nucleoside analogs of the present invention have the structure
of Formula I below:
R1
Q-(CHR)n~B
L(cHR)n R4 E
Fomlula
wherein:
Q is selected from the group consisting of H, OH, NHR, CHO,
phosphate, lower-alkyl, lower alkenyl, protected O-, protected N-, lower
10 alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl,
amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and
phosphonate;
L is selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2),
H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower
15 alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl,
phosphonium salt, phosphonate and t-butyldimethylsilyloxy;
each R is independently selected from the group consisting of H, OZ,
SZ and NHZ;
each R1 and R2 is independently selected from the group consisting of
20 H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower
alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy;
each R3 and F4 is independently selected from the group consisting of
H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy:
each Z is independently selected from the group consisting of H, lower
25 alkyl, lower alkenyl, aryl, acetyl and protecting groups for O-, S-, and N-;
each E is independently selected from the group consisting of
-OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, lower alkyl, lower alkenyl, lower
alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen,
epoxyethyl, phosphonium salt and phosphonate;
each n is independently 0 or an integer from 1 to 4; and
each B is independently select from the group consisting of adenine,
cytosine, guanine, thymine, uracil or a modification thereof that does not
WO 94/22890 f~ J S 9 6 3 ~ PCT/US94102993
substantially interfere with the affinity of an oligonucleoside or chimeric
oligonucleotide analog for its antisense counterpart wherein the bases are
selected from the group consisting of adenine, cytosine, guanine, thymine
and uracil or a naturally occurring modification thereof;
or an optical isomer thereof or a pharmaceutically acceptable salt
thereof.
The oligonucleotide analogs of the invention have the structure of
Formula ll below:
Q-(CHR)n~B
W ,~ ~ Y
R3 ,~
/ R4 E q o B
R3,~
L(CHR)n R4 E
Fonnu~
wherein:
Q is selected from the group consisting of H, OH, NHR, CHO,
phosphate, lower-alkyl, lower alkenyl, protected O-, protected N-, lower
alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl,
15 amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and
phosphonate;
L is selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2),
H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower
alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl,
20 phosphonium salt, phosphonate and t-butyldimethylsilyloxy;
each R is independently selected from the group consisting of H, OZ,
SZ and NHZ;
each R1 and R2 is independently selected from the group consisting of
H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower
25 alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy;
each R3 and R4 is independently selected from the group consisting of
H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy:
WO 94/22890 ~ l $ ~ 6 3 ~ PCTIUS94/02993
each Z is independently selected from the group consisting of H, lower
alkyl, lower alkenyl, aryl, acetyl and protecting groups for O-, S-, and N-;
each E is independently selected from the group consisting of
-OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, lower alkyl, lower alkenyl, lower
alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen,
epoxyethyl, phosphonium salt and phosphonate;
each n is independently 0 or an integer from 1 to 4
each B is independently selected from the group consisting of adenine,
cytosine, guanine, thymine, uracil or a modification thereof that does not
substantially interfere with the affinity of an oligonucleoside or chimeric
oligonucleotide analog for its antisense counterpart wherein the bases are
selected from the group consisting of adenine, cytosine, guanine, thymine
and uracil or a naturally occurring modification thereof;
each W is selected from 3'-(OPO2HO)C(R1 R2)-5' and a natural
phosphodiester internucleoside linkage with the proviso that at least one W is
3'-(OPO2HO)C(R1 R2)-5'; and
q is 0 or an integer from 1 to 60.
As employed above and throughout the disclosure, the following
terms, unless otherwise indicated, shall be understood to have the following
meanings:
"Alkyl" means a saturated aliphatic hydrocarbon which may be
either straight- or branched-chain. Preferred groups have no more than
about 12 carbon atoms and may be methyl, ethyl and structural isomers of
propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl.
"Lower alkyl" means an alkyl group as above, having 1 to 7
carbon atoms. Suitable lower alkyl groups are methyl, ethyl, n-propyl,
isopropyl, butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, and n-heptyl.
"Aryl" means phenyl, naphthyl, substituted phenyl and
substituted naphthyl.
"Substituted phenyl (or naphthyl)" means a phenyl (or naphthyl)
group in which one or more of the hydrogens has been replaced by the the
same or different substituents selected from halo, lower alkyl, nitro, amino,
- acylamino, hydroxyl, lower alkoxy, aryl, heteroaryl, lower alkoxy, alkylsulfonyl,
and trifluoromethyl.
"Heteroaryl group" means, pyridyl, furyl, thienyl, or imidazolyl.
"Substituted heteroaryl" means a heteroaryl group in which one
or more of the hydrogens has been replaced by the the same or different
wo 94n2890 ~ ~ S ~ 6 ~ ~ PCT/US94/02993
-10-
substituents selected from halo, lower alkyl, nitro, amino, acylamino, hydroxyl,lower alkoxy, aryl, heteroaryl, lower alkoxy, alkylsulfonyl, and trifluoromethyl.
"Lower alkenyl" means an unsaturated aliphatic hydrocarbon
having 2 to 8 carbon atoms, such as ethylene, propylene, butylene, isobutylene,
5 etc., including all structural and geometrical isomers thereof.
"Halo" means bromo, chloro or fluoro.
An ~O-, S-, or N-protecting group" is a radical attached to an
oxygen, sulfur, or nitrogen atom, respectively, which radical serves to protect
the oxygen, sulfur, or nitrogen functionality against undesired reaction. Such
protecting groups are well known in the art; many are described in "The
Peptides." E. Gross and J. Meienhofer, Eds. Vol 3 Academic Press, NY
(1981). The N-protecting groups can be N-acyl, N-
alkoxycarbonyl, N-arylmethoxy-carbonyl, trifluoromethylacyl and N-
arylsulfonyl protecting groups. Suitable O-protecting groups include benzyl,
tert-butyl, methyl, tosyl, dimethoxytrityl, tert-butyl-dimethylsilyl, and
carbobenzoxy groups. S-Protecting groups include methyl, tert-butyl, benzyl,
and carbobenzoxy groups.
The abbreviation "iPr2" as used herein refers to diisopropyl.
The present invention provides a method of synthesizing
oligonucleotide compounds having from 2 to about 60 bases and having an
internucleoside backbone containing one or more 3'-O-PO2H-O-5'-CR1R2
internucleoside linkages instead of the naturally occurring backbone of
phosphodiester internucleoside linkages, this process comprising preparation
of 5'-substituted nucleoside compounds, for example, as illustrated in Figure
1, and utilizing them as synthons in automated DNA synthesizers.
Scheme 1 illustrates the preparation of various 5-substituted
nucleoside analogs of the invention which, in turn, are useful for the
preparation of 3'-O-PO2H-O-5'-CR1 R2 linked dinucleosides and
oligonucleotide analogs of the invention. As shown in Scheme 1, (when B is
thymidine) 3'-t-butyldimethylsilyloxy-2'-deoxy-5'-formyl-5'-deoxy-thymidine 2
is a key intermediate for the preparation of various 5'-substituted nucleoside
analogs of the invention, including ones containing the 5'-methyl group 5, the
5'-nitromethyl group 8, the 5'-aminomethyl group 9, the 5'-epoxy group 7,
and the 5'-azidomethyl group 13.
Thus, as shown in Scheme 1A, reaction of methylmagnesium
bromide with the aldehyde 2 yields the 5'-methyl thymidine 3 which, upon
treatment with dimethyltrityl chloride followed by desilylation and reaction with
chloro-2-cyanoethyl-N,N-diisopropyl-phosphoramidite, affords the 5'-
WO 94/22890 ~ 63~ PCT/US94/02993
-1 1 -
substituted nucleoside synthon 5. The synthon 5, may be utilized for the
synthesis of 5'-modified DNA employing a DNA synthesizer such as ABI 380B
Oligomer synthesizer. As shown in Scheme 1B, the 5'-epoxy compound 7
may be prepared by the reaction of the aldehyde 2 with diazomethane,
5 whereas the 5'-nitromethyl compound 8 may be prepared by the reaction of
the aldehyde 2 with nitromethane. The chemical reduction of the nitro group
with lithium aluminum hydride affords the desired 5'-aminomethyl compound
9, which may be acylated to yield the trifluoroacetyl amide 10. As illustrated
in scheme 1C, azide ring opening of the epoxy compound 7 provides the
10 corresponding azidomethyl analog 12. The amide 11 and azido analog 13
may be prepared, respectively, from compounds 10 and 12 via deprotection
of t-butyldimethylsilyloxy group followed by reaction with chloro-2-cyanoethyl-
N ,N-diisopropyl-phosphoramidite.
WO 94/22890 2 ~ 5 ~ 6 3 2 PCT/US94102993
-12-
Scheme 1 General Sy.lU-esls of Tar~et ~cn~ er (B = T)
A. 5'-Alkyl '~Q 1 ''sation
TBSO~ Et~N~ 7a2c ~ CH~M~Br ; ~ Dmt CI, 4;DMAP
CH3 CH3
DmtrO--~O B DmtrO~O B
\ Y CIP(OCH2CH2CN)~
HO 4-DAMP, EtNiPr2 OP(OCH 2CH2CN)(N-iPr2)
4 5
e. 5'-Amlnoalkyl '1c ~ ncallon
K2C02 ~ CH2N2 TBSO~
1. DmtCI
4-DMAP
2 LiAlH4
H2N ODmtr CF3CONH ODmtr CF3CONH ODmtr
~o B \~oyB 1. nBu4N F \~0 B
)_/Y (CF3C0)20 y 2. Cl(P(OCH2CH2CN)(N-iPr2) )~
TBSO TBSO OP(OCH2C H2CN)(N-ipr2)
11
C. 5'-~ o-'~yl Mo~iilitallon
O N 3 ODmtr ODmtr
1. LiN3 \~Y ,N3~B
2. DmtrCI 2. Cl(P(OCH 2C H2CN)(N -i Pr2)
TBSO DMAP/pyr TBSO Op(ocH2cH2cN)(N-ipr2)
12
7 13
WO 94/22890 2 I ~ ~ ~ 3 ~ PCT/US94/02993
-~3-
This invention also contemplates pharmaceutically acceptable
salts of the compounds of Formula 1. It is well known in the pharmacological
arts that nontoxic addition salts of pharmacologically active amine
compounds do not differ in activities from their free base. All stereoisomers as5 well as optical isomers related to the novel antisense agents described in this
disclQsure are also considered to be within the scope of this invention.
Pharmaceutically acceptable salts include both acid and base
addition salts. ~Pharmaceutically acceptable salt" refers to those salts which
retain the biological effectiveness and properties of the free bases and which
10 are not biologically or otherwise undesirable. Suitable pharmaceutically
acceptable acid addition salts can be formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid,
and the like, and organic acids such as acetic acid, propionic acid, glycolic
acid, pyruvic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
15 cinnamic acid, mandelic acid, methanesulfonic acid, and p-toluenesulfonic
acid, and the like.
Pharmaceutically acceptable base addition salts include those
derived from inorganic bases such as sodium, potassium, lithium, ammonium,
calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the
20 like. Particularly preferred are the ammonium, potassium, sodium, calcium
and magnesium salts. Salts derived from pharmaceutically acceptable
organic non-toxic bases include salts of primary, secondary, and tertiary
amines, substituted amines, including naturally occurring substituted amines,
cyclic amines and basic ion exchange resins, such as isopropylamine,
25 tripropylamine, ethanolamine, 2-diethylaminoethanol, 2-
dimethylaminoethanol, dicyclohexylamine, Iysine, arginine, histidine,
caffeine, procain, hydrabamine, choline, betaine, ethylenediamine,
glucosamine, methylglucamine, theobromine, purines, peperizines,
piperidine, polyamine resins and the like. Particularly preferred organic non-
30 toxic bases are isopropylamine, diethylamine, ethanol-amine and
dicyclohexylamine.
In one embodiment, the compounds of the present invention
comprise oligomeric antisense agents, as shown in Formula ll, of about 6 to
about 60 bases, preferably from about 9 to about 50 bases, more preferably
35 from about 12 to about 25 bases, most preferably from 15 to 18 bases. An
important feature of this invention is that the methyl group present at the 5'-
position of the sugar interferes with the hydrolysis of the phosophodiester
bond by nucleases. The methyl group present at the 5'-position unexpectedly
WO 94/22890 S 9 6~ PCT/US94/02993
-14-
enhanced the nuclease stability. Another important feature of this invention is
the discovery that the novel 5'-substituted nucleosides of the invention may
be incorporated at multiple sites within an oligonucleotide analog, and have
no appreciable effect on the hybridization stability of the resulting (antisense)
oligonucleotide analog to its natural 'sense' target oligonucleotide when
compared to the hybridization stability to the same 'sense' target of the
corresponding unmodified antisense oligonucleotide.
These antisense agents can be formulated into compositions
together with one or more non-toxic physiologically acceptable carriers,
adjuvants or vehicles which are collectively referred to herein as carriers, forparenteral injection or oral administration, in solid or liquid form, for rectal or
topical administration, or the like.
The compositions can be administered to humans and animals
either orally, rectally, parenterally (intravenous, intramuscularly or
subcutaneously), intracisternally, intravaginally, intraperitoneally, locally
(powders, ointments or drops), or as a buccal or nasal spray.
Compositions suitable for parenteral injection may comprise
physiologically acceptable sterile aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions and sterile powders for reconstitution
into sterile injectable solutions or dispersions. Examples of suitable aqueous
and nonaqueous carriers, diluents, solvents or vehicles include water,
ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like),
suitable mixtures thereof, vegetable oils (such as olive oil) and injectable
organic esters such as ethyl oleate. Proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersions and by the use of surfactants.These compositions may also contain adjuvants such as preserving,
wetting, emulsifying, and dispensing agents. Prevention of the action of
microorganisms can be ensured by various antibacterial and antifungal
agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the
like. It may also be desirable to include isotonic agents, for example sugars,
sodium chloride and the like. Prolonged absorption of the injectable
pharmaceutical form can be brought about by the use of agents that delay
absorption, for example, aluminum monostearate and gelatin.
Solid dosage forms for oral administration include capsules,
tablets, pills, powders and granules. In such solid dosage forms, the active
compound is admixed with at least one inert customary excipient (or carrier)
such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as
WO 94/22890 ~ PCT/US94/02993
-15-
for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, (b)binders, as for example, carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidone, sucrose and acacia, (c) humectants, as for example,
glycerol, (d) disintegrating agents, as for example, agar-agar, calcium
5 carbonate, potato or tapioca starch, alginic acid, certain complex silicates and
sodium carbonate, (e) solution retarders, as for example paraffin, (f)
absorption accelerators, as, for example, quaternary ammonium compounds,
(g) wetting agents, as for example, cetyl alcohol and glycerol monostearate,
(h) adsorbents, as, for example, kaolin and bentonite, and (i) lubricants, as,
10 for example, talc, calcium stearate, magnesium stearate, solid polyethylene
glycols, sodium lauryl sulfate or mixtures thereof. In the case of capsules,
tablets and pills, the dosage forms may also comprise buffering agents.
Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules, using such excipients as lactose
15 or milk sugar as well as high molecular weight polyethyleneglycols, and the
like.
Solid dosage forms such as tablets, dragees, capsules, pills and
granules can be prepared with coatings and shells, such as enteric coatings
and others well known in the art. They may contain opacifying agents, and
20 can also be of such composition that they release the active compound or
compounds in a certain part of the intestinal tract in a delayed manner.
Examples of embedding compositions which can be used are polymeric
substances and waxes.
The active compounds can also be in micro-encapsulated form,
25 if appropriate, with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions, suspensions, syrups and
elixirs. In addition to the active compounds, the liquid dosage forms may
contain inert diluents commonly used in the art, such as water or other
30 solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl
benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils,
particularly cottonseed oil, ground-nut oil, corn germ oil, olive oil, castor oil
and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and
35 fatty acid esters of sorbitan or mixtures of these substances, and the like.
Besides such inert diluents, the composition can also include adjuvants, such
as wetting agents, emulsifying and suspending agents, sweetening, flavoring
and perfuming agents.
WO 94/22890 PCT/US94/02993
$63~ -16-
Suspensions, in addition to the active compounds, may contain
suspending agents, as for example, ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose,
aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of
5 these substances, and the like.
Compositions for rectal or vaginal administration are preferably
suppositories which can be prepared by mixing the compounds of the present
invention with suitable non-irritating excipients or carriers such as cocoa
butter, polyethyleneglycol or a suppository wax, which are solid at ordinary
10 temperatures but liquid at body temperature and, therefore, melt in the rectum
or vaginal cavity and release the active component.
Dosage forms for topical administration include ointments,
powders, sprays and inhalants. The active component is admixed under
sterile conditions with a physiologically acceptable carrier and any
15 preservatives, buffers or propellants as may be required. Opthalamic
formulations, eye ointments, powders and solutions are also contemplated.
Actual dosage levels of the active ingredient in the compositions
may be varied so as to obtain an amount of active ingredient that is effective
to obtain a desired therapeutic response for a particular composition and
20 method of administration. The selected dosage level therefore depends upon
the desired therapeutic effect, on the route of administration, on the desired
duration of treatment and other factors.
The total daily dose of the active ingredients administered to a
host in single or divided doses may be in amounts, for example, of from about
25 0.5 mg to about 10 mg per kilogram of body weight. Dosage unit
compositions may contain such amounts or such submultiples thereof as may
be used to make up the daily dose. It will be understood, however, that the
specific dose level for any particular patient will depend upon a variety of
factors including the body weight, general health, sex, diet, time and route of
30 administration, rates of absorption and excretion, combination with other
dnugs and the severity of the particular disease being treated.
The present invention is further directed to a method of inhibiting
the expression of a gene that comprises administering to a host mammal in
need of such inhibition an inhibition-effective amount of a compound of
35 Formula ll, in which that compound hybridizes to a nucleotide sequence of
the gene.whose expression is to be inhibited. In a preferred embodiment, the
compound of Formula ll is dissolved or dispersed in a physiologically
tolerable carrier.
WO 94/22890 2 1 5 ~ ~ 3 ~ PCT/US94/02993
-17-
As discussed elsewhere herein, inhibition of the expression of a
gene can be effected by interfering with transcription, translation, or RNA
processing. Hence, the activity of an antisense molecule can be at the level
of messenger RNA or genomic DNA. So, for example, when an antisense
molecule hybridizes to messenger RNA, translation is inhibited. When an
antisense molecule hybridizes to genomic DNA, transcription is inhibited. An
antisense molecule may also bind to other nucleic acid species in a cell,
including heterogeneous nuclear RNA (hnRNA) and pre-messenger RNA.
A host mammal in need of the inhibition of the expression of a
gene suffers from a disease state in which the expression of the gene is
implicated. Such disease states include a variety of cancers, in which the
expression of an oncogene or oncogenes is implicated, cystic fibrosis,
Huntington's chorea, and other such disease states in which the aberrant
expression of a normal gene or the expression of an abnormal gene is
responsible, in whole or in part, for the disease condition.
As used herein, an "inhibition-effective amount" is the amount of
a compound of the present invention which is sufficient to inhibit the
expression of the gene whose expression is to be inhibited. Means for
determining an inhibition-effective amount will depend, as is well known in
the art, on the nature of the gene to be inhibited, the type of inhibition desired
(i.e., inhibition of translation or transcription or both), the mass of the subject
being treated, and the like.
It is to be understood that the compound of the Formula ll used
in the inhibition of the expression of a gene must hybridize to a sequence of
that gene in such a way as the expression of that gene is inhibited. That is,
the nucleotide bases used to make a compound of the Formula ll (B in
Formula ll as defined above) must hybridize to the nucleotide sequence of the
gene whose expression is to be inhibited. Such sequence can readily be
ascertained from the known sequence of that gene, and the appropriate
antisense molecule of Formula ll can therefore be prepared. Hybridization of
greater than about 90 percent homology (identity), and more preferably about
99 percent homology, is contemplated in the present invention.
The following examples further illustrate the invention and are
- 35 not to be construed as limiting of the specification and claims in any way.
WO 94/2~890 ~ 1~N594/02993
FxAMpl FS:
Example 1 N~-Ren7oyl-3'-t-butyldimethylsi~yloxy-~'-deoxy-5'(RS)-methyl-
~denosine
A solution of DMSO (2 mM, 142 ~I) in methylene chloride (0.5
ml) was added to a stirred solution of oxalyl chloride (2.~ ml) at -78C under
nitrogen. After 5 minutes, a solution of N6-benzoyl-3'-t-butyldimethylsilyloxy-
2'-deoxy-adenosine (1 mM, 469 mg) in DMSO/CH2CI2 (0.4 ml/1.16 ml) was
1 0 added over a ten minute period. Stirring was continued for 20 minutes and
then triethylamine (5 mM, 700 ~I) was added. The reaction mixture was
stirred for an additional 5 minutes. Methyl- magnesium bromide ( 3 M in
ether, 2 mM, 666 1ll) was added via syringe, and the reaction mixture was
stirred for 1 hour at -78C, followed by warming to room temperature
1 5 overnight. Water was added and the mixture was extracted into chloroform
(2x40 ml), washed with brine and dried over anhydrous sodium sulfate. The
crude product was purified by flash chromatography (silica gel; 90%
EtOAc/Hexane). Yield 390 mg (81%). Mol. Wt.:483.3 for C24H33NsoSi. FAB-
MS: (M+H)+ = 484.2.
Example 2 N6-Ben7Oyl-3'-t-butyldimethylsilyloxy-2'-deoxy-5'-
dimethoxytrityl-5'(RS)-methyl-~denosine
4,4-Dimethoxytrityl chloride (0.992 mM, 336 mg) was added to a
25 solution of N6-benzoyl-3'-t-butyldimethylsilyloxy-2'-deoxy-5'(RS)-methyl-
adenosine (0.824 mM, 400 mg), triethylamine (1.12 mM, 156 ~11) and 4-
dimethylaminopyridine (0.04 mM, 4.88 mg) in pyridine/ethylene chloride (1 :1;
32 ml) and stirred under nitrogen at 60C for 1 hour. Then the reaction
mixture was heated to 100C and progress of the reaction was monitored by
30 thin layer chromatography (TLC). As required, more dimethoxytrityl chloride
(2x200 mg) was added while maintaining the temperature at 100C. The
reaction was stopped after 18 hours by addition of water, extraction into ethyl
acetate and drying the pooled organic layers over anhydrous sodium sulfate.
The crude product was purified by flash chromatography (silica gel; 20%
35 EtOAc/Hexane). Yield: 307 mg (54%). Mol Wt: 785.3 for C4sHs1 NsO6Si.
FAB-MS: (M+H)+ = 786.3.
WO 94/22890 PCT/US94/02993
_19_
Example 3 N6-Ren7Oyl-~'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-
~denosine
Tetra-n-butyl ammonium fluoride (1 M in THF; 0.764 mM) was
5 added dropwise via syringe to a solution of 3'-t-butyldimethylsilyloxy-2'-
deoxy-5'-dimethoxytrityl-5'(RS)-methyl-adenosine (0.382 mM, 300 mg) in
anhydrous THF (4 ml). The reaction was stirred under nitrogen for 6 hours at
room temperature. After a standard aqueous work-up, the crude product was
purified by flash chromatography (silica gel; 100% EtOAc). Yield: 260 mg
(97%) Mol. Wt. 671.4 for C3gH37N~O6. FAB-MS: (M + H)+ = 672.4.
Example 4 ~16-Ren7Oyl-?'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-
~lenosine-3~-o-(Rs)-~-cyanoethyl-N .N-diisopropyl-phosphor~midite)
1 5 A solution of chloro-2-cyanoethyl-N ,N-diisopropyl-
phosphoramidite (0.345 mM, 66.2 ~I) in anhydrous THF (2 ml) was added to a
solution of N6-benzoyl-2'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-adenosine
(0.235 mM, 160 mg), diisopropylethylamine ( 0.92 mM, 200 ~I) and 4-
dimethylaminopyridine (8 mg) in anhydrous THF (2 ml), and stirred under
20 nitrogen for 3 hours. After an aqueous work-up, the crude product was
purified by flash chromatography (silica gel; 50% EtOAc/Hexane. Yield: 140
mg (68.4%) Mol. Wt.: 871.0 for C4gHs4N7O7P. FAB-MS: (M + H )+ = 872.3.
Example 5 3'-t-Butyldimethylsilyloxy-~'-deoxy-5'(RS)-methyl-thymidine
To a solution of 3'-t-butyldimethylsilyloxy-2'-deoxy-thymidine (14
mM, 5 9) in 140 ml of methylene chloride was added at room temperature
(8.98 9, 21 mmol) of Dess-Martin periodinane reagent. After 30 minutes of
reaction time, the mixture was diluted with 150 ml of ether, and a mixture of
30 Na2S2O3 (19.9 9, 126 mmol) in 150 ml of saturated sodium bicarbonate was
added, and stirring continued for 15 minutes. The reaction mixture was
poured over 1~0 ml of ethyl acetate and the organic layer was separated,
washed with saturated sodium bicarbonate solution (2x150 ml), dried over
anhydrous sodium sulfate and concentrated in vacuo. The crude aldehyde in
35 600 ml of benzene was azeotroped (Dean Stark) and the solvent was
removed in vacuo to afford 4 9 of the crude aldehyde.
To a solution of the above aldehyde (4 9) in 150 ml of THF at
-78C was added dropwise methylmagnesium bromide in ether (3.0 M, 90 ml,
WO 94/22890 3 % -20- PCT/US94/02993
20 eq.); after stirring for 1 hour, the reaction mixture was poured over a
mixture of 1000 ml of ice/saturated ammonium chloride and 10 ml of acetic
acid. The resulting mixture was extracted with ethyl acetate (5x200 ml) and
the organic layer was washed with saturated sodium bicarbonate (2x450 ml)
and brine (1x450 ml), dried over anhydrous sodium sulfate and concentrated
in vacuo. The product was purified by flash chromatography (100 9 silica gel;
50% EtOAc/Hexane). Yield: 1.93 9.
Example 6 3'-t-Rutyldimethylsilyloxy-2'-deoxy-5'-dimethoxytrityl-5'(RS)-
methyl-thymidine
To a solution of 3'-t-butyldimethylsilyloxy-2'-deoxy-5'(RS)-
methyl-thymidine (300 mg, 0.8 mmol) in 2.7 ml of pyridine at room
temperature was added 98 mg (1 eq.) of dimethylamino-pyridine, 0.6 ml (5
eq.) of triethylamine and 1.37 9 (5 eq.) of dimethoxytrityl chloride. The
reaction mixture was allowed to react at 75C overnight. The mixture was
diluted with ethyl acetate, washed with water, followed by saturated
ammonium chloride solution, and the organic layer was dried with anhydrous
sodium sulfate and concentrated in vacuo. The crude product was purified by
flash chromatography (silica gel; 30% EtOAc/Hexane). Yield: 225 mg.
Example 7 ~'-Deoxy-5'-dimethoxytrityl-5'(RS)-methyl-thymidine
Tetra-n-butyl ammonium fluoride (1.1 M in THF; 0.68 mM, 0.9 ml)
was added dropwise via syringe to a solution of 3'-t-butyldimethylsilyloxy-2'-
deoxy-5'-dimethoxytrityl-5'(RS)-methyl-thymidine (0.68 mM, 460 mg) in
anhydrous THF (3.44 ml). The reaction was stirred under nitrogen for 4 hours
at room temperature. After a standard aqueous work-up the crude product
was purified by flash chromatography (silica gel; 60 - 75% EtOAc/Hexane).
Yield: 340 mg (92%).
WO 94/22890 2 1 ~ ~ ~ 3 2 PCT/US94/02993
Example 8 ~'-Deoxy-5'-dimethoxytrityl-5'(RS)-methyl-thymidine-3'-O-(RS)-(2-
cy~noethyl-N .N-diisopropyl-phosphoramidite)
To a solution of 2'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-
5 thymidine (0.58 mM, 320 mg) in 6 ml of methylene chloride at -40C was
- added diisopropylethylamine (1.5 eq., 155 mg), followed by chloro-2-
cyanoethyl-N,N-diisopropyl-phosphoramidite (170 mg, 1.3 eq.). The reaction
mixture was stirred at -30C for 2 hours, and at 0C for two additional hours.
Methanol (0.1 ml) was added to the mixture and the resulting reaction mixture
10 was stirred at 0C for 1/2 hour, diluted with ethyl acetate, and washed with
saturated ammonium chloride solution. The organic layer was dried over
anhydrous sodium sulfate and concentrated in vacuo. The crude product was
purified by flash chromatography (silica gel; 40% EtOAc/Hexane). Yield: 376
mg (65%).
Example 9 3'-t-Rutyldimethylsilyloxy-~'-deoxy-5'(RS)-(1 -c~rbmethoxy- 1 -
phenylsulfonyl)methyl-thymidine
To the crude aldehyde from Example 5 (3 mmol) was added
20 dropwise at -78C a solution of sodium methyl phenylsulfonylacetate in 6 ml
of THF. After allowing the mixture to react at -78C overnight, the reaction
mixture was worked-up according to the procedure described in Example 5.
The product was purified by flash chromatography (silica gel; 50%
EtOAc/Hexane). FAB-MS: (M + H )+ = 569.
Example 10 3'-t-Butyldimethylsilyloxy-2'-deoxy-5'-epoxyethyl-5'-deoxy-
thymidine
The crude aldehyde from Example 5 (0.5 mmol) was added
30 dropwise via syringe at -78C to a solution of diazomethane (approx. 1.5
mmol) in ether. After 2 hours of reaction time, the mixture was allowed to
stand at -78C for overnight. The reaction mixture was worked-up according
to the procedure described in Example 5. The product was purified by flash
chromatography (silica gel; 50% EtOAc/Hexane). Yield: 100 mg. FAB-MS
35 shows the desired mol ion for the title epoxide.
wo 94,22890 2 ~ 6 ~ ~ -22- PCT/US94/02993
Example 11 Oli~omer Synthesis Usin~ 2'-Deoxy-5'-dimethoxytrityl-5'(RS)-
methyl-thymidine-3'-O-(RS)-(2-cyanoethyl-N .N-diisopropyl-phosphoramidite)
The synthesis of the oligomers listed in Table 1 was conducted
5 using an ABI 380B synthesizer in accordance with the manufacturer's
protocols. Synthesis of strands at one micromole scale using monomer (e.g
2'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-thymidine-3'-O-(RS)-(2-cyanoethyl-
N,N-diisopropyl-phosphoramidite) was carried out. Monomer was dried just
before DNA synthesis. The purification of DNA product was conducted with
10 DMT + reverse phase HPLC with slicing through major peak.
TABLE 1
Oligomer Sequence
number
1 5'-l l ~ T~T-3' (SEQ ID NO:1)
2 5'- 1 1 I m I I I TT-3' (SEQ ID NO:2)
3 5'-m 1 1 I TT'T~T-3' (SEQ ID NO:3)
4 5'- ~ TT-3' (SEQ ID NO:4)
5'-TT~T TT~T~ TT~T-3' (SEQ ID NO:5)
6 5'-GGG TGT GTG rTA GCG GG-3' (SEQ ID NO:6)
7 5'-GGG TGT GTG TT~A GCG GG-3' (SEQ ID NO:7)
8 5'-GGG TGT GTG~ T~T~A GCG GG-3' (SEQ ID
NO:8)
9 5'-CCC GC~T ~A~A CAC ACA CCC-3' (SEQ ID
25 NO:9)
In the above Table 1, the ~ in the sequence signifies the location
of a 5'-methyl-phosphodiester bond instead of a natural phosphodiester
bond.
Example 11 Analysis of Nuclease Stability
For the nuclease stability studies, oligonucleotides were
labelled at the 5' terminus with 32p by using T4 polynucleotide kinase and
35 standard end-labelling procedures. Unincorporated 32P-ATP was removed
by passing over a NucTrap column followed by purification over Sephadex G-
25. A trace (1-1011M) of 32P-labelled oligonucleotide was combined with
unlabelled oligomer at a concentration of 1 IlM. These conditions minimize
WO 94122890 21 5 9 63 2 PCT/US94/02993
-23-
any variation incurred due to differences in oligomer labelling efficiencies andsimulate concentrations used in typical antisense experiments. The oligomer
was added to Dulbecco's minimal essential medium (DMEM) cell culture
media containing 20% fetal calf serum (FCS) which serves as a source of 3'
5 exonuclease activity. After incubation at 37C for 2.5 hours, the mixture was
denatured for 2 minutes at 90C and analyzed by denaturing polyacrylamide
gel electrophoresis (PAGE; 20% polyacrylamide/8 M urea; 19:1
acrylamide:bis-acrylamide; 89 mM Tris/89 mM boric acid/2 mM EDTA). As a 0
hour control, the oligonucleotide was added to serum-free DMEM media prior
10 to PAGE analysis. Gels were dried between sheets of cellulose drying film,
imaged, and quantitative data obtained using a Phosphorimager. The gel
lanes demonstrate the effect observed for the various modified
oligonucleotides as compared to the control phosphodiester oligomer.
Multiple reactions were run for each oligonucleotide, and the results proved to
15 be reproducible. Furthermore, more dramatic results were obtained in
experiments in which only a trace of 32P-labelled oligonucleotide (i.e., 1-10
~M total oligomer) was added to media containing 10% or 20% FCS.
Oligodeoxynucleotide #1 (5'-TT I I I I I I TT~T-3'; SEQ ID NO:1;
containing a 3'-end cap modification with a methyl group at the 5' carbon of
20 the sugar at the penultimate thymidylate residue) was added to a final
concentration of 3 nM to tubes on ice containing RPMI 1640 media with L-
glutamine (GIBCO). Also added were: HEPES (GIBCO) to a final
concentration of 20 mM, and fetal bovine serum to 10%. Total reaction
volume was 400 ~11. Tubes which were incubated for 0 time (controls) did not
25 receive serum. Tubes were incubated at 37C for 5, 30, 60, and 120 minutes.
Time 0 tubes were kept on ice. An extra 120 min reaction lacking serum was
also incubated at 37C as a control to check for chemical degradation of the
oligomer. As a positive control, d(T)11 oligodeoxynucleotide (Oligomer #2
(SEQ ID NO:2), prepared in the same manner as the other, experimental
30 oligomers) was assayed at time 0 and at 120 minutes without serum.
Reactions were stopped by extraction with equal volumes of a
24:1 mixture of chloroform and isoamyl alcohol, five times. The final aqueous
layer was filtered through a Spin-X, 0.22 ~M cellulose acetate filter unit. Fifty
microliters of final sample, kept at 40C, was loaded onto a Gen Pak Fax
35 anionic exchange column by automatic injection, and eluted in 15 mM sodium
phosphate, 1 mM EDTA mobile phase, pH 8.0, with a 0-0.5 M NaCI gradient
utilizing a LKB HPLC system. The gradient went from 0 to 0.4 M NaCI in 55
WO 94n2890 2 ~ ~ 9 6 3 ?~ PCT/US94/02993
-24-
minutes, was held to 60 minutes, increase to 0.5 M NaCI at 65 minutes, and
held to 70 minutes. Elution was monitored at 269 nm.
Parent oligomer and reaction product peaks were integrated for
peak areas. Peak retention times were compared to controls and d(T)
5 oligomer standards that had been previously analyzed on this HPLC system.
The results of the assay show that the parent oligomer (Oligomer #1, an 11-
mer (retention time of 41.76 min)), was rapidly digested, by 3'-~5'
exonucleolytic activity present in the 10% fetal bovine serum. The product
was an extremely stable, N-1 reaction product, a 10-mer (retention time of
10 38.64 min), the result of cleavage of the 3'-terminal thymidylate residue by the
enzyme. The 10-mer remained undigested for up to 120 minutes in the
presence of serum, based on the resulting peak areas.
Thus, the 3'-modification provides protection to the remaining
oligomer against further digestion by inhibiting the activity of the 3'->5'
15 exonuclease present in the serum. It is concluded that the methyl group
present at the 5' position of the sugar interferes with the hydrolysis of the
phosophodiester bond by the nuclease enzyme.
Example 13 Hybridiz~tion (Tm) Testin~
Two nanomoles of each of the 1 7-mer antisense
oligonucleotides of Table 1 (oligomer numbers 6, 7, 8 and 9) were combined
with two nanomoles of their respective complementary sense strands in a
buffer containing 50 mM Na2HPO4/100 mM NaCI/1 mM EDTA, denatured at
25 85C, and annealed by slow cooling to room temperature. The annealed
DNA was heated from 35C to 85C and absorbance data at 260 nM
collected at 0.2 C intervals. From this data, melting curves were generated
and values for melting temperature (Tm) derived.
The Tm for the sense and antisense control (normal, unmodified
30 phosphodiester [PDE]) strands annealed together is 68 +/- 0.5 degrees. The
Tm for each of Oligomers 6 (SEQ ID NO:6) and 7 (SEQ ID NO:7) was 68C.
The Tm observed for Oligomer # 8 (SEQ ID NO:8) annealed with sense PDE
strand was 67.8 degrees. The Tm for Oligomers 8 (SEQ ID NO:8) and 9 (SEQ
ID NO:9) annealed together is 66.8 degrees. The results show that there is
35 virtually no loss of duplex stability when the normal PDE bond is replaced
with this substituted PDE bond. The results further show that the methyl-PDE
bond does not at all interfere in Watson Crick base paring even when both
members of the pair that anneal to each other are methyl-PDE modified (i.e.,
WO 94/22890 PCT/US94/02993
215~6:~
-25-
Oligomers 8 and 9). This result indicates that the racemic 5'-methyl analogs
of nucleosides can be incorporated at multiple sites within an oligonucleotide,
and have essentially no effect on hybridization stability.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
10 (i) APPLICANT: Saha, Ashis
(ii) TITLE OF INVENTION: NOVEL 5' SUBSTITUTED NUCLEOSIDES AND
OLIGOMERS PRODUCED THEREFROM
15 (iii) NUMBER OF SEQUENCES: 9
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sterling Winthrop, Inc.
(B) STREET: 9 Great Valley Parkway
(C) CITY: Malvern
(D) STATE: PA
(E) COUNTRY: USA
(F) ZIP: 19355
25 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
WO 94/22890 2 ~- ~ 9 5 3 PCT/US94/02993
-26-
(viii) ATTORNEY/AGENT INFORMATiON:
(A) NAME: Newman, Irving
(B) REGISTRATION NUMBER: 22,638
(C) REFERENCE/DOCKET NUMBER: PRF 161
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (215) 889-8824
10 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
20 (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (10A11)
(D) OTHER INFORMATION: /note= "The bases are linked by a
5'-methyl-phosphodiester bond."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
I I I I I I I I I I T 11
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
WO 94/22890 ~ 1 5 9 ~ ~ 2 PCT/US94/02993
-27-
(ii) MOLECULE TYPE: DNA (genomic)
5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
I I I I I I I I I I T 11
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
20 (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (8^9)
(D) OTHER INFORMATION: /note= "The bases are linked by a
5~-methyl-phosphodiester bond."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (9^10)
(D) OTHER INFORMATION: /note= "The bases are linked by a
5'-methyl-phosphodiester bond."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
1 1 1 1 1 1 1 1 1 1 10
W O 94/22890 æ 1 ~ 9 6 ~ ~ PCTrJS94/02993
-28-
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (6^7)
(D) OTHER INFORMATION: /note= nThe bases are linked by a
5'-methyl-phosphodiester bond."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
l l l l l l l l l I T 11
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (2^3)
(D) OTHER INFORMATION: /note= "The bases are linked by a
5'-methyl-phosphodiester bond."
WO 94/22890 2 I ~ ~ B ~ 2 PCTIUS94/02993
-29 -
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (5^6)
(D) OTHER INFORMATION: /notee "The bases are linked by a
5'-methyl-phosphodiester bond."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (6^7)
(D) OTHER INFORMATION: /note= nThe bases are linked by a
5'-methyl-phosphodiester bond."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
1 5 (B) LOCATION: (9^10)
(D) OTHER INFORMATION: /note= "The bases are linked by a
5'-methyl-phosphodiester bond."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (10A11)
(D) OTHER INFORMATION: /note= "The bases are linked by a
5'-methyl-phosphodiester link."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (11^12)
(D) OTHER INFORMATION: /note= "The bases are linked by a
5'-methyl-phosphodiester bond."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
l l l l l l l l l I TT 12
(2) INFORMATION FOR SEQ ID NO:6:
WO 94/22890 PCT/US94/02993
2~ ~963~ -30-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (10^11)
(D) OTHER INFORMATION: /note= ~The bases are linked by a
5'-methyl-phosphodiester bond."
15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GGGTGTGTGT TAGCGGG 17
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (11^12)
(D) OTHER INFORMATION: /note= "The bases are linked by a
5'-methyl-phosphodiester bond."
35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GGGTGTGTGT TAGCGGG 17
WO 94/22890 2 I S 9 6 3 2 PCT/US94/02993
-31 -
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
1 0 (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (9^10)
(D) OTHER INFORMATION: /note= nThe bases are linked by a
5'-methyl-phosphodiester bond."
(ix) FEATURE:
(A) NAMEtKEY: misc_feature
(B) LOCATION: (10^11)
(D) OTHER INFORMATION: /note= "The bases are linked by a
5'-methyl-phosphodiester bond."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (1 1 ^12)
(D) OTHER INFORMATION: Inote= "The bases are linked by a
5~-methyl-phosphodiester bond."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GGGTGTGTGT TAGCGGG 17
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
WO 94/22890 2t /1 5 9 6~ ~ PCT/US94/02993
-32-
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (5~6)
(D) OTHER INFORMATION: /note= "The bases are linked by a
5'-methyl-phosphodiester bond."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (6^7)
(D) OTHER INFORMATION: /note= "The bases are linked by a
5'-methyl-phosphodiester bond."
1 5
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: (7^8)
(D) OTHER INFORMATION: /note= "The bases are linked by a
5'-methyl-phosphodiester bond."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CCCGCTAACACACACCC 17
