CA2058632C

CA2058632C - Exonuclease-resistant oligonucleotides and methods for preparing the same

Info

Publication number: CA2058632C
Application number: CA002058632A
Authority: CA
Inventors: Brian C. Froehler
Original assignee: Isis Pharmaceuticals Inc
Current assignee: Ionis Pharmaceuticals Inc
Priority date: 1989-06-05
Filing date: 1990-06-05
Publication date: 2004-08-24
Anticipated expiration: 2010-06-05
Also published as: EP0476071A1; WO1990015065A1; CA2058632A1; EP0476071A4; KR920701230A; JPH05500799A

Abstract

A method is provided for making 3' and/or 5' end-capped oligonucleotides so as to render the oligonucleotide resistant to degradation by exonucleases. The exonuclease degradation resistance is provided by incorporating two or more phosphoramidate and phosphoromonothioate and/or phosphorodithioate linkages at the 5' and/or 3' ends of the oligonucleotide, wherein the number of phosphoramidate linkages is less than a number which would interfere with hybridization to a complementary oligon-ucleotide strand and/or which would interfere with RNAseH activity when the oligonucleotide is hybridized to RNA.

Description

~' WO 90/ 15065 ~ ~ ~ ~ PCT/US90/03138 EXONUCLEASE-RESISTANT
OLIGONUCLEOTIDES AND
METHODS FOR PREp,~RING THE SAME
' Technical Field The present invention is directed to oligonucleotides containing a 3'- and/or 5~-capped terminal and which are thereby rendered resistant to degradation by exonucleases. The exonuclease-resistant oligonucleotides have two or more phosphoramidate internucleotide linkages at one or both termini which render the oligonucleotides resistant to degradation.' Background DNA molecules contain internucleotide phosphodiester linkages which are degraded by exonucleases present in cells, culture media and human serum. For example, degradation by exonucleases in tissue culture media of DNA may be observed within about minutes to about six hours. Synthetic oligodeoxy-nucleotides with phosphodiester linkages are routinely used in genetic engineering, for example, to locate specific RNA or DNA fragments from a library. The long-30 term stability of an oligonucleotide for this utility is not a major concern, since the oligonucleotide is usually not exposed to the relatively stringent environment of the culture medium, therefore exonuclease degradation is not a substantial problem.
~ 35 However, it is in fact frequently desirable to produce oligodeoxynucleotides which are stable (i.e., for more than several hours or days) for long-term uses. For ;~,~: -2-WO 90/ 15065 ' PCT/US90/03138 example, a oligodeoxynucleotide with phosphodiester linkages can be used to block protein synthesis by hydrogen bonding to complementary messenger RNA thereby providing a tool for use in an antisense fashion.
Exonuclease-stable oligodeoxynucleotides could also be utilized to form triple-helix DNA which would interfere with the transcription process or with DNA replication, by competing with naturally'.occurring binding factors or by gene destruction. However, in order to utilize synthetic oligonucleotides in this manner, they must be stable to exonucleases, the major activity of which in cells and serum appears to be 3'.~0 5', i.e.:, digestion of oligonucleotides begins starting at the 3~ end.
The present invention is accordingly directed to such exonuclease-stable oligonucleotides.
Related Art:
The following references relate .to one or more aspects of the presently claimed..invention:~
Froehler, Tet. Lett. x,7(46):5575-5578 (1986), describes polymer-bound deoxynucleoside H-phosphonate diesters as precursors to phosphoramidate, thiophosphate and phosphate triester analogs of DNA.
Froehler et al. , Nuc Acids R_~s ,~,ø(il) :4831-4839 (1988), describe the synthesis o~ a 15-mar containing 12 phosphoroamidate linkages derived from primary and secondary amines. The chemistry of the process is summarized in the figure shown on page 4833 of the reference.
Froehler et al., Nuc. Acids Res ,~(13):5399-5407 (1986), describe the synthesis of.deoxyoligo-nucleotides via deoxynucleoside H-phosphonate intermediates. The chemistry of this process is essentially shown in scheme 2 on page 5401 of the 3 5 ref erence .

-3- ~a~< r ~'WO 90115065 PCT/US90/03138 Froehler, European Patent Publication No.

219342-7~.2, published 2 April 1987, is similar to the teachings of the latter two references in that the synthesis of DNA via deoxynucleoside H-phosphonate intermediates is shown.

Letsinger et al., Nuc. Acids Res. x(8):3487-3499 (1986), describe complexes of polyuridylic acid (poly U) and polythymidylic acid (poly dT) with oligonucleotides possessing different pendant groups that are linked to the oligonucleotide chain at the internucleotide phosphodiester linkages.

Stein et al., Nuc. Acids Res. ~(8):3209-3221, (1988) present a study of oligodeoxynucleotides modified so as to contain phosphorothioate linkages. The authors, in addition to evaluating a number of other physico-chemical properties of such oligonucleotides, study the susceptibilities of the compounds to a number of endonucleases and exonucleases. The authors found a significant decrease in the Tm of fully substituted phosphorothioate oligodeoxyn~zcleotides compared to diester controls (Figure 3), ~i.e., a 15-20C decrease in Tm and a 30-40 Kcal/mole decrease in DH for fully substituted molecules (p. 3215).

Brill et al., Tet. Lett. X9(43):5517-5520 (1988) describe the preparation of dinucleoside phos-phorodithioates by sulfur oxidation of thiophosphate triesters.

Agrawal, Tet. Lett. x(31):3539-3542 (1987) describe the automated synthesis of oligodeoxynucleosides containing methylphosphonate linkages, using nucleoside methylphosphonamidites as starting materials. The authors conclude that two adjacent methylphosphonate linkages at the 3~ end provides protection against degradation by snake venom phosphodiesterase and spleen phosphodiesterase (and, like Stein et al., the authors do not evaluate nuclease stability of the oligonucleotides in serum, tissue culture medium or cells).
PCT publication W089/05358, inventors Walder et al., describe oligodeoxynucleotides modified at the 3' terminus so as to render the oligonucleotide chain resistant to degradation within cells and body fluids.
Disclosed modifications at the 3' -terminal phosphodiester linkage include replacement of that linkage with an alkyl or aryl phosphotriester, hydrogen phosphonate, an alkyl or aryl phosphonate, an alkyl or aryl phosphoramidate, a phosphorothioate, or a phosphoroselenate, although the preferred modification is stated to be the incorporation of a 3' terminal phosphotriester linkage.
Disclosure of the Invention Accordingly, it is a primary object of the invention to address the above-mentioned need in the art and to provide exonuclease-resistant oligonucleotides.
According to a first aspect of the invention, there is provided an oligonucleotide resistant to degradation under physiological conditions, comprising at feast one phosphoramidate internucleotide linkage at the 5' termini and wherein at least one of the remaining internucleotide linkages is not a phosphoramidate linkage.
The oligonucleotide may have the formula selected from the group consisting of:

~:.~ , O B
T-O B O-x- O B O-P- O ' OH
~ Y. Jn ~ N Js Ri R~
(O
B O B Xi B
II
T-O O-p-O O- i - O OH
N s Yi n Ri ~Rz (In B O B X~ B O B
T-O O-i-O O_i-O O-~-O OH
N ~s ~ Yi ~m ~ N ~s Ri 'R2 RI RI
El '" 2058632 wherein each n, m, i, j and s is independently an integer and each s is in the range of about 2 to 10; each n and m is independently from 1 to about 50; s + n in formulas I and II is less than 100; and s + s + m in formula III is less than about 100; each i varies from 1 to n; each j varies from 1 to m; T is hydrogen or a hydroxyl-protecting group; R' and R2 are moieties independently selected from the group consisting of hydrogen, hydrocarbyl substituents of 20 carbon atoms or less, and E

_?_ 2~0~~6 '"'~'WU 90/15065 PCT/U890703138 oxyhydrocarbyl of 20 carbon atoms or less and 1-3 oxy groups, wherein said hydrocarbyl and oxyhydrocarbyl substituents are linear or branched alkyl of 1 to 20 carbon atoms, linear or branched alkenyl of 2 to 20 carbon atoms, cycloalkyl or cycloalkenyl of 3 to 20 carbon atoms, linear or branched alkoxy of 1 to 20 carbon atoms, or aryl of 6 to 18 carbon atoms, with the proviso that R1 and R2 are not both hydrogen;
each B is independently a protected or unprotected heterocyclic base;
each;Xi and.X~ is independently O or S; and each Yi and Y~ is independently R, -SR or -OR, where R is as defined for R1 and R2.
The present invention also provides methods for preparing such end-capped oligonucleotides.
Modes for Carryina Out the Invention As used herein the terms "polynucleotide" and "oligonucleotide" shall be generic to polydeoxyribo-nucleotides (containing 2'-deoxy-D-ribose or modified forms thereof), to polyribonucleotides (containing D-ribose or modified forms thereof), and to any other type of polynucleotide which is an N-glycoside of a purine or pyrimidine bases, or modified purine or pyrimidine bases.
The term "nucleoside" will similarly be generic to ribonucleosides, deoxyribonucleosides, or to any other nucleoside which is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base.
,There is no intended distinction in length between the term "polynucleotide" and "oligonucleotide" and these terms will be used interchangeably.:
It will be appreciated that as used herein the terms "nucleoside" and "nucleotides" will include those moieties which contain not only the known purine and pyrim~.dine bases, i.e., adenine, thymine, cytosine, zo~~~.
-s- _.
WO 90/15065 PCTfUS90/0~138 guanine and uracil, but~hlso other heterocyclic bases which contain protecting groups or have been otherwise modified or derivatized.
By ~~modified nucleosides" or "modified nucleotides" as used herein are intended to include those compounds containing one or more protecting groups such as acyl, isobutyryl, berizoylor the like, as well as any of the wide range of modified and derivatized'bases as known in the: art. Examples of such modified or deriva-tized bases include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxy-methylaminomethyluracil, dihydrouracil, beta-D-galacto-sylqueosine, inosine, N6-isopentenyladenine, 1-methyl-adenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-ethylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylamino-methyluracil, 5-methoxyamino~tethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-.methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, u~acil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid ~ethylester, uracil-5-oxyacetic acid (v), 3-(3-amino-3-N-2-carboxypropyl) urac~fl, and 2,6-diaminopurine.
Modified nucleosides o'r nucleotides can also include modifications on the sugar moiety, for example, wherein one or .wore of :'the hydroxpl groups are replaced - with halogen or aliphatic groups, or functionalized as ethers, amines,:etc.
.The polynucleotides'according to the present invention may be of any length, but lengths of about _g_ three to about fifty nucleotides are particularly useful for most genetic engineering applications. According to the present invention, the 3' and and/or the 5~ and of th~ polynucleotide will contain nt least two 5 phosphoramidate internucleotids linkages. The remaining intarnuclaotide linkages may bo phosphodiester linkages, phosphorothioate linkages or phosphorodithioate linkages, or any other internucleotide linkage, other than a phosphoramidate, or combinations of thane other linkages.
1o Method: for preparing such non-phoephoramidata linkagos era known in the art, e.g., as taught by Froahlar~at al., Nuc. Ac' s Rss. x;5399-5467 ~(1986~, and Froehler, B., fat. Lett~ x:5575-5578 (1986 ) _ 15 Internucleotide phoephodiester linkages are prepared from hydrogen phosphonat4 linkages preferably by oxidation with, e.g., aqueous iodine. A typical -procedure involves treatment of tho hydrogen phosphonate in 0.1 M iodine in~Pyr/NMI/Ha0/T~iF (5:1:5:90) for about 20 2-3 minutes, rollowed by treatment with 0.1 M iodine in Et3/820/T~IF (5:5:90) for another approximately ~-3 minutes.
Phoaphoromonothioate linkages are formed from the initially present hydrogen phosphonate linkages by 25 treatment with sulfur. Tho reaction is carried out at approximntely room temperature for on the order of 20 minutes ~n a solvent system which typically includes a sulfur 4olvant such a: CSa along with a basic solvent such as pyridine. Other suitable solvent systems include 3o CSZ/lutidina and CsZ/triathylamino;'C92 is preferred as the sulfur solvent because it acts to dissolve elemental sulfur. Tha follo~ring schema illustrates the postulated roaction:

O Basa:~ O 0 (RO)2_p_g ___~ (RO)2_p ~ _g ~___~ (RO)2 p 5 S~~ S_ , /~ ~
Sn Sn Sn (See, e.g., Stein et al., cited ab~o-ve.) To form hydrogen phospharodithioate linkages, sulfurization of the~hydrogen phosphoromonothioate linkages is effected using conditions identical to those just described for the preparation of the phosphoromono-thioate moiety. (Note: the terra "phosphorothioate" as used herein is intended to encompass both "phosphoro-monothioate" and "phosphorodithioate" linkages.) Structure of the End-Capped Oligonucleotides:
The~oligonucleotides of the invention, as noted above, are resistant to degradation under both physio-logical and tissue culture conditions, and in particular are resistant to degradation by exonucleases.
In order that the oligonucleotide be resistant 'to such enzymatic degradation, it is modified so that phosphodiester linkages initially present at the 3' terminus are replaced with a selected number of phos-phoramidate linkages, that number being at least one and less than a number which would cause interference with hybridization to a complementary oligonucleotide strand, and/or less than a number which would interfere with RNAseH activity when said the oligonucleotide is hybridized to RNA. Such a modification may additionally or alternatively be made at the 5' terminus.
It is preferred that the number of phosphorami-date linkages be selected such that the melting temperature of any duplex formed with complement is i -11- 2p'58~~2 'WO 90/15965 PGT/US90/03138 lowered by less than about 10°C~~elative to that obtained with an oligonucleotide containing only th~ainitial phosphodiester linkages. Prefe~ra~ly, the n~ber of phosphoramidate linkages is sutah that the malting temperature of a duplex formed-;is lowered by less than about 5°C. The number of phosghoramidate linkages present is typically and preferably between--about 2 and 10, more preferably between about 2 and 8, and most preferably between about 2 and 6.
The phosphoramidats linkage has the formula . ~ '~ Rt .
-p-p-N~ R2 wherein the Ri and R2 moieties are substituents which must be selected so as not to interfere with hybridization with complement. ~n most cases, the groups R1 and R2 are independently sel~ed from the group consisting of hydrogen, hydrocarbyl substitu~nts of ,.
20 carbon atoms or less, and oxy~ydrocarbyl ~ubstituents~
of 20 carbon atoms or less containing 1-3 oxy groups, with the proviso that R1 and R2 are not both hydrogen, i.e., the phosphoramidate linkages herein are always N-substituted. In this case, it~is preferred that one of the two substituents be hydrogen~~~ Suitable hydrocarbyl and oxyhydrocarbyl substituents include, for example, linear or branched alkyl of 1-20 carbon atoms, linear or branched alkenyl of 2-20 carbon atoms, cycloalkyl or cycloalkenyl of 3-20 carbon atoms, linear or branched alkoxy of 1-20 carbon atoms, or aryl of 6-18 carbon atoms. The hydrocarbyl substitusnt may be, for example, an alkoxy substituent having the formula CH30-(CH2)x- or a straight chain alkyl group having the formula -12- _.
'~V0 9015065 PCT/US90i0f~3$
CH3(CH2)y- where x is an integer in the range of 1-20, inclusive, preferably in the range of 1-10, inclusive, and y is an integer in the range of 0-15, inclusive.
Examples~of preferred oligonucleotide linkages within the aforementioned groups are wherein one of ~tl and R2 is H
and the other is either 2-methoxyethyl, dodecyl, or n-propyl. (The 2-methoxyethyl and dodecyl linkages art sometimes referred:to herein as "MEA" and "C12", respectively.) The R1 and R2 grd~ps maly,,'al~o be, in addition to the foregoing, macromolecular;species such as sugars, polypeptides, chromophoric groups, lipophilic groups, polymers, steroid hormones, or the like.
"Lipophilic" groups refer to moieties which are chemically compatible with the outer cell surface, i.e., so as to enable the oligonucleotide to attach to, merge with and cross the cell membrane. Examples of such .
lipophilic groups are fatty acids and fatty alcohols (in addition to the long chain hydrocarbyl groups described above).
Examples of preferred polypeptides that can be used for Ri and/or R2 include translferrin and epidermal growth factor (EGF), while suitable non-polypeptide polymers include ionic, nonionic and zwitterionic polymers. Examples of a particularly preferred polymer is polyethylene glycol.
Steroid substituents include any of the general family of lipid compounds which comprise sterols, bioacids, cardiac glycosides, seponans, and sex hormones, which include the following basic structure:

,".,,. -13 - ~ 0 10 Examples of steroids include natural corticosteroid hormones (produced by the adrenal glands), sex hormones (progesterone, androgens, and estrogens).
These various R1 and R2 groups can confer any of a variety of desired properties to the oligo-nucleotide. For example, if R1 or R2 is a polymer such as polyethylene glycol, a polypeptide or a lipophilic group such as a long-chain hydrocarbyl moiety, such a group may facilitate transport or permeation of the oligonucleotide through cell membranes, thus increasing the cellular uptake of the oligonucleotide. The R1 or R2 group may also be a group which affects target DNA or RNA
to which the oligonucleotide will bind, such as providing covalent linkages to the target strand to facilitate cleavage or intercalation of the oligonucleotide to the target strand. The R1 and R2 groups may additionally serve a cutting function (e.g., a site for cutting the complementary strand), or a receptor function (e.g., a receptor ligand).
It will be appreciated by those skilled in the art that the oligonucleotides of the present invention can include other phosphoramidate N-substituents not explicitly disclosed herein so long as those substituents confer exonuclease resistance and do not interfere with hybridization to a complementary oligonucleotide strand.

n..
=14-WO 90/15065 pCT/US~90/03138 The invention also encompasses oligonucleotide compositions containing oligonucleotides of the following formula I, II or III, i:e., wherein phosphoromonothioate and/or phosphorodithioate linkages are incorporated in addition to the phosphi~ramidate linkages:
B Xi B .O B
T-O O-P- O '0 ~-P- O OH
~ Yi Ri R2 (I) 35 B O B Xi B
T-O O-P-O . O-P- O OH
L 'N J S ~. yi J n ( I I) B O B
y ~i B ~ B
T-O O- i -p O-P- O O-P- 0 OH
N ~S ~ Y' ~~ ~ N . JS
(III) RI \RZ
in which g,, T, R1~ R2, Xi, X~, Y1, Y~, n, m~ i, j and s are as defined above. In these Structures, it is preferred that "s," which defines the number of phosphoraunidate linkages;- be in the range of 2-8, more preferably in the range of 2-6. It is also preferred that m and n be within the aforementioned ranges.
Synthetic-Methods:
According'to one embodiment of the present invention, the 3'-capped oligonucleotides may be prepared .. -15-by first~praparing a polymeribottnd polynucleoside With the formula IV
B O ~

~
H
wherein P is -a solid state pa~leric support,~or other type of solid support, and B the base port~~on of a nucleoside, i.e., a purine or .~yrimidine base, or any modified purine or pyrimidine:b~se. As isconventional in oligonucleotide syntheses,ath~e functional groups on the base, i.e., the amine grot~pdawill be appropriately protected during the course ~'f the synthesis and removed after the completed poiynucl~bti~de is removed from the polymer support. As is the convention, in the formula shown above in IV, the linkage ~C~o the polymer support is through the 3' hydroxy group,-the free hydr~xy group is the 5' group of the nucleoside 'The group ~ is a conventional hydroxy-protecti'igroup used'=in oligonucleotide synthesis, pri~f~irably the DMT group (dimethoxytrityl) or MMT groups (monomethoxytrityl).
The polymer-bound polynucleo~~id~'.hydrogen phosphonate ( IV ) is preferably prepared by treating the' DBU
(1.8-diazabicyclo[5.4.0]under-7-ere ammonium salt) of a 5'-protected (preferably, 5 D'l~T;~°~ ~hucleoside hydrogen phosphonate with a polymer-ba~n~ nucleoside, linked to support through its 3'-hydroxyl~group in the presence of an activating agent, as is khri in the art. Methods for preparing such polymer-bound~pi~lynucleosideAhydrogen phosphonates are disclosed, fi~.~~-example, by°- Froehler, 8. , et al., Nuc. Acids Res. ,~:4833:~4839 (1988j; Froehler, 8., et al., Nuc. Acids Res. ~;s5399-5467 (1986); and ~'~.6-WO 90/15065 PG'I'/US90~03138 Froehler, 8. , at al . , ,Nucleosides :-;, . d Nt~~~ p~~ i r~eQ ø: 287-291 (1987). Then, one or more nucleoside hydrogen phosphonates may be added (to make the two or more internucleotide linkages at the 3' end of the polynucleotide) by sequentially deprotecting the 5'-hydroxyl group of the polymer-bound polynucleotide, and condensing with the next nucleoside hydrogen phosphonate. The oligonucleotide chain elongation will proceed in conformance with a predetermined sequence in a series of condsna~ations, each ~or~e vbf which -results in the addition of another nucleoside to the oligomer. The condensation is typically accomplished with dehydrating agents, which are suitably phosphorylating agents or acylating agents such as isobutylchloroformate, diphenylchlorophosphate, organic acid anhydrides (such as acetic anhydride, isobutyric anhydride or trimethyl acetic anhydride) and organic acid halides such as pivaloyl chloride, pivaloyl bromide, ~1-adamantyl-carboxylic chloride or benzoyl chloride. The preferred condensing agent is pivaloyl chloride in pyridine acetonitrile. Prior to the addition of each successive nucleoside hydrogen phosphonate, the 5'~-protecting group or the carrier bound nucleotide is removed. Typically, for removal of the-DMT group, this is done by treatment with 2.5~ volume/volume dichioroacetic acid/CIi2C12, although l~ weight/volume trichloroacetic acid/CH2C12 or .ZnBr2-saturated nitromethane ars also-usefuiOther deprotection procedures suitable for ether known protecting groups will be apparent to those of ordinary skill in the art.
The carrier is preferably wa~ehed with anhydrous pyridine/acetonitrile (1/i,v/v) and the condensation reaction is completed in as many cycles as are required to form the desired number_of 3~--end internueleotide bonds which will be converted to.phosphoramidates. After ~l~r ' the required number of synthetic cycles, the carrier-bound polynucleotide hydrogen phosphonate is oxidized to convert the hydrogen phosphonata internucleotide linkages to phosphoramidate linkages, preferably by treatment with 5 the desired amine NHRIRa with Rl and Ra as defined earlier and CC14 as described in Froehler, et al., Nucleic Acidg Research xø:4831-4839 (1988). Although carbon tetrachloride is preferred, other mild oxidising agents may be utilized.
l0 After the oxidation to form the phosphoramidata internucleotide linkages, the oligonuclaotide is then completed by methods which foim nonphosphoramidate iinkageo, ouch as phosphodiester linkages, phosphorothioats linkages or phosphorodithioate linkages, 15 by methods known in the art referenced above..
The preferred method for completing the oligonucleotida is to continue then sequence using 5'-protected nucleoside hydrogen-phosphonatas. In the instance whera the 5~ end will not 20 be napped, after the last 5'-protected nucleoside hydrogen phosphonate has bean added, all of the hydrogen phosphonata linkages are oxidized to produce diastar linkages, preferably by aqueous iodine oxidation or oxidation using other oxidizing agents, such as 25 N-chlorosuccinimide, N-bromoauccinimide.or salts or periodic acid. This vial result in all of the intsrnucleotide linkages, except for the 3~-end. capped linkages which are phosphoramidata linkages, being phosphodiestar linkages. Thereafter, the oligonucleotide 30 may ba separated from the carrier; using conventional mBthoda, which in the_prafarrad instance is incubation ~rith concentrated ammonium hydroxide. Any protecting groups may be removed as described above using about a8 dichloroacetic acid/CfiaCla, or about 808 acetic acid, or 35 by other conventional methods, depending on the nature of z~.:5~gz~ 32 -~8-the protecting groups,; The desired oligonucleotide is then purified by HPLC, polyacrylamide gel electro-ghoresis or using other conventional techniques.
The following schemes illustrate various synthetic processes within the scope of the invention:
;~ ,v .

.~., -19- 258632 WO 90/15065 PCT/tJS90/03138 B O B HNRiR2~
io p TO O-P- 0~ . (;ji-.~p~ oxidizing agent A ~~ s (s z 2) r, -. ,.~
;~
B O B
To o-P- o o-I
N s 2 5 T: protecting group ~: protecting group or solid state carrier i: varies from 1 to 5 Q: hydmg~n or -Nit,lR2 (with the proviso that at least one Qi is hydrogen) 3 0 B: a purine or pyninzidine base R1,R2: sec text ~05863~ -20-WO, 90/ 15065 PCT/US90/03138 ..
heme 2a to 1 ) 5' - blockui nucleoside H - phosphonate and pivaloyl chloride as activator B 2) remove S' - blocking group O O- ' 3) ~1R2/oxidizing agent ~ O O--Yi J n 4) optional repetition of steps 1-3 B O B Xi B
HO O-P- O O- P- O O-N Js L Yi Jn Ri R2 T,'~; X;~'Yi, Rt, R2 = as defined in text and in Scheme 1 I
2~5a632 B

O
HO O-P- O ~ + TO O-p- p O-p-OU
I
Yi ~ n N s H
Ri R2 5' - blocked nucleoside 1 ) activating agent B O B
Xi B
2) oxidation TO O..".P- O O. p_ O O-2 5 ~ J s ~ Y; J n f ~
Ri R2 T, Xi, Y;, R1, R2, s, n, = as defined in text and in Schemes 1 and 2a 2, -22_ Sch m g O B .. HNR1R2, TO O-P- O p-~ o~~~ng agent I
io H ~s (s_>2) B O B
TO O-P- O O-Js ,.
Ri R2 2 0 1 ) remove T
2) condense 5' - blocked nucleoside H-phosphonate, H-phosphorothioate or H-phosphorodithioate with pivaloyl chloride as activator 3) remove 5' - blocking group 4) repeat steps 2) & 3) 5) oxidize to form phosphodiester, phosphorothioate and/or phosphorodithioate linkages 6) condense with additional 5' - blocked nucleoside phosphonates, optionally react with HNR1R2 in the presence of an oxidizing agent g O ,B
II Xi B p B
TO O- i - O O-P- O O-P- O O-0P
N Jp ~ Yi Jr ~ N ~ s Ri R2 Ri R2 z o ~$$~~~:
,r., -2 3 -The foregoing discussion has revolved around the consecutive addition of mononucleoside hydrogen phosphonates, but it will be understood that one or more nucleotides can~be added in a given cycle by using a polynucleotide, such as a di- or trinucleotide.
It will also be understood that while the above method has been described in connection with use of a solid state carrier if the object oligonucleotide is small, i.e., containing, for example, only five nucleotides (therefore having only four internucleotide linkages, two of which are phosphoramidate linkages) it is feasible to conduct the synthesis without the use of a solid state support. In such an instance a conventional 3'-hydroxy protecting group may be used which is different from the 5'-protecting group used in the synthesis, so that the 5'-protecting group may be selectively removed while the 3'-protecting group remains intact.
It will also be appreciated that the two or more phosphoramidate linkages need not each contain the same R1 and R2 groups. This may be accomplished by generating the first internucleotide hydrogen phosphonate linkage, and then oxidizing it with a first amine, generating the second hydrogen phosphonate internucleotide linkage, and then oxidizing it in the presence of a second (different) amine. This would result in a capped oligonucleotide having mixed phosphoramidate internucleotide linkages.
In another embodiment of the present invention, a 5'-capped oligonucleotide may be made. In such an instance, the above method may be modified by first forming a polymer-bound oligonucleotide having only hydrogen phosphonate internucleotide linkages which may then be oxidized to form phosphodiesters (or phosphorothioate or phosphorodithioate linkages). Then ' -24-w0 9,ousobs ~ PCT/US90/03138 forthe last two (or more) cycles, the 5'-end cap is formed when the last two or more nucleosides are added, followed by reaction with the amine NHR1R2:
Alternatj,,vely, tla,e 5' end may be added by adding a polynucleotide, such as a tri- or tetranucleotide containing the desired phosphoramidate internucleotide linkages.
In still another embodiment; a combination of both of the above methods for making a 5' and~a 3' end-capped oligonucleotide may ,~ utilized. The first two (or more) internucleotide linkages on the 3'-bound oligonucleotide may be oxidized to form the " phosphoramidate linkages then the ;non-teaninal portion of the oligonucleotide may be made (having phosphodiesters, phosphorothioate or phosphorodithioate internucleotide linkages), with the final two (or more) linkages being phosphoramidates, formed as described above.
~ Methods of Use:
Tie uses of 5'- or 3'-phosphoramidate-capped oligonuc~.eotides as made in accordance with the present invention may be as therapeutic agents against viral diseases (such as HIV, hepatitis B, cytomegalovirus), cancers (such as leukemias, lung cancer, breast cancer, colon cancer); or metabolic disorders, immune modulation agents, or the like, since the present end-capped oligonucleotides are stahla within the environment of a F ,, cell as well as in extracellular fluids such as serum, and can be used to selectively block protein synthesis, transcription, replication of RNA and/ox flNA which is uniquely associated with the disease or disorder. The end-capped oligonucleotides Qf the invention may also be used as therapeutics in animal health care, plant gene regulation (such as plant growth promoters) or in human WO 90/1565 PCT/US90/03'l38 diagnostics, such as to stabilize DNA probes to detect microorganisms, oncogenes, gens,c defects, and the like, and as research reagents to study gene functions in animal cells, plant cells,.micganisms, and viruses.
There may also be dermatologic p~ications for treatment of diseases or for cosmetic puree: There are many other potential uses which der$from the stability of the oligonucleotide to exonucls degradation, thus prolonging oligonucleotide intity within the relatively stringent environmerdof the cells.
It is to be understoc~d~~t~at whip the invention has been desc=ibed in conjunction with the preferred specif is embodiments thereof , #~t the foregoing description and the examples w~tit~h P fo~.lo~r are intended to illustrate and not limit the sc~~pe of the invention.
Other aspects, advantages and m~dificatiuns within the scope of the invention will beagparent to those skilled to which the invention pertains.
.~_~ry:

~~~~a6~~ ~~6-WO 90/15pf5 PCT/IJS90/03138 ~,. ~ Exhmble ~
- Polymer-bound polynucleoside H-phosphonates were prepared as described by Froehler et al., supra, on control pore glass using the DBU salt of the protected nucleoside H-phosphonate~ The diester linkages were generated by aqueous I2 oxidation and the a~idate linkages by amine/CCl4 oxidation. After two 'couplings the polynucleoside H-phosphonate was oxidized with a solution of 2-methoxyethylamine in Pyr/CC14 (1:5:5) (20 min.) followed by twelve more couplings and oxidation with aq. I2 (0.1 M in N-methyl morpholine/water/THF, 5:5:90) to generate a 15-mer containing two phosphor-amidate linkages at the 3' end and twelve diester linkages. The oligomer was removed-f'~rom the solid support, deprotected with cono. NH40H (45°C/18 hr.), and purified by HPLC (PRP~ using an acetonitrile (CH3CN) gradient in 50 mM aqueous TEAP. The DMT was removed from the product fraction (80% acetic acid/R:T./2 hrs.), evaporated, desalted arid evaporated. Approximately 1 ug of purified product was 5' end-labeled with T4 poly-nucleotide kinase and y-32P ATP for further characterization.
Example 2 Polymer-bound polynucleoside H-phosphonates were prepared as in the preceding example on control pore glass using the DBU salt of the protected nucleoside H-phosphonate. After twelve couplings the polynucleoside H-phosphonate was oxidized with aq. I2 (0.1 M in N-methyl morpholine/water/THF, 5:5:90) followed by two more couplings and oxidation with a solution of 2-methoxy-ethylamine in Pyr/CC14 (1:5:5) (20 min.) to generate a 15-mer containing twelve diester linkages at the 3' end and two phosphoramidate linkages at the 5' end. The oligomer was removed from the solid support, deprotected ,,.,, -27-with conc. NH~;OH (45°C/18 hr.) and purified by HPLC (PRP) using an acetonitrile (CH3CN) gradient in 50 mM aqueous TEAP. The DMT was removed from the product fraction (80%
acetic acid/R.T./2 hrs.), evaporated, desalted and evaporated.
~a~
Polymer-bound polynucleoside H-phosphonates were prepared as described as in the preceding examples on control pore glass using the D8U salt of the protected nucleoside H-phosphonate. The d°iester linkages were generated by aqueous I2 oxidation and the amidate linkages by amine/CC14. After two couplings the polynucleoside H-phosphonats was oxidized with a solution of 2-methoxyethylamine in Pyr/CC14 (1:5:5) (20 min.) followed by ten more couplings and oxidation with aq. I2 (0.1 M in N-methyl morpholine/water/THF, 5:5:90) to generate a 13-mer containing two phosphoramidate linkages at the 3' end and ten disster linkages. This was followed by two more couplings and oxidation with a solution of 2-methoxyethylamine in Pyr/CC14 (1:5:5) (20 min.) to generate a 15-mer containing two phosphoramidate linkages at the 3' end, ten diester linkages, and two phosphoramidate linkages at the 5' end. The oligomer was removed from the solid support and deprotected with conc.
NH4oH (45°C/18 hr.) and purified by HPLC (PRP) using an acetonitrile (CH3CN) gradient in 50 mM aqueous TEAP. The DMT was removed from the product fraction (80% acetic acid/R.T./2 hrs.), evaporated, desalted and evaporated.
Examnl~ 4 The procedure of Example 1 was repeated using dodecylamine to generate a 15-mer containing two phos-phoramidate linkages at the 3' end and twelve diester linkages, wherein the phosphoramidate linkages are such ~4~~~3~ ., ~28-WO 90/15065 : PCT/US90/03138 that one of R1 andaR2 as definea'earlier herein is hydrogen and the other is dodecyl.
~. Example 5 The procedure of Example 2 was repealed using dodecylamine in place of 2-methoxyethylamine, so as to yield a 15-mer cpxitaining twelve diester linkages at the 3'. end and two phoaphora~tidate linkages at the 5' end, wherein the phosphoramidate linkages are substituted as in the preceding example, i:e., one of R1 and R2 is hydrogen and the other is dodecyl:
Example 6 The procedure of ~cample 3 was repeated using dodecylamine in place of 2-methoxyethyla~iine, to give rise to a 15-mer containing two phosphoramidate linkages at the 3' end, ten diester linkages, and two phosphor-amidate linkages at the 5' end, wherein the phosphor-amidate is N-substituted as in the preceding two examples. ,, Example 7 The px~c~iure'of Example 1 was repeated using propylamine to generate a 15-mer containing two phos-phoramidate linl~ages at he 3' end and twelve diester linkages, wherein the-.phosphoramidate linkages are such that one of R1 and R2 as defiMed earlier herein is hydrogen_and the other is ,~-propyl.
Example 8 The proceduze of Example 2 was repeated using .. ~ ppylamine in place of 2-methoxp~ethylamine, so as to ~.y yield_a 15-mer containing twelve diester linkages at the 3' end and two phosphoramidate linkages at the 5' end, wherein the phD~phoramidate linkages are substituted as i -29- ~ 2058b3~
W~ ~I1'~5 PCT/US90/03138 in the preceding example, i.e., one of R1 and R2 is hydrogen and the other is ~-propyl.
Examp~~,e- 9 The procedure of Example 3 was repeated using propylamine in place of 2-methoxyethylamine, to give rise to a 15-mer containing two phosphoramidate linkages at the 3' end, ten diester linkag~e~, and two phosphor-amidate linkages at the 5' end, wherein the phosphor-amidate is N-substituted as in the preceding two examples.
Example 10 The following Example describes hybridization stability studies performed using end-capped oligo-nucleotides as described and~~~laimed herein.
Oligonucleotides containing end-caps were tested for their ability to form stable duplexes with complementary single-stranded DNA sequences; the various oligonucleotides tested were outlined below in Table 1.
Duplex stability was measured by determining the melting temperature Tm in solution ov~~,a range of temperatures.
The experiment was conducted in a solution containing 150 mM NaCl, 5 mM Na2HP04 and 3'°~CM DNA at a pH of 7.1.
The results obtained and set forth in Table 1 show that binding to complementary sequences is not materially affected by 3'-end-cap modification.

~ c~'~ -3 0-WO 90!15065 pCT/US9~0/03138~
Compound Tm (oC) I~ ~~
5' TCCA~TGATTTTTTTCTCCAT-O-P.;-O-T-O-P-O-T-OH 61.0 (~? HN ~1 OCH3 0~3' .
O O
rw 5' TC4CAGTGATTTTTTTCTCCAT-O-P-O-T-O-P-O-T-OH ' 60.5 (C12) (CH2)~~CR3 (CHZ)~~CN3 5' '~CCAGTGATTTTTTTCTCCAT-O-IP-O-T-O-PI-OH 61.5 (diester contro~)G p- p-Example 11 Several additional oligonucleotides also end-capped at the 3' terminal two internucleotide linkages were tested for their ability to form stable duplexes with complementary single stranded DNA
sequences, as described in the preceding example.
Results are set forth in Table 2.

_31_ 2058632 'WU 90/15065 PGT/US90/03138 Compound ,, ' Tm (°C) O O
5' TCTCCCTCTCTTT-O-IP-O-T-O-P-Qr-T~OH 58.5 iu~ethoxyethylawine) ~ ~
OCH3 p~3 2 0 a.
O o 5' TCTCCCTCTCTTT-O-IP-O-T-O-~P-O~'I~~C~H ~ 59.5 2 5 ~ ( a x.
O- O-..,%.n:
5' TCTCCCTCTCTTT-OH 56.5 35 (diester) Example 12 40 The following example was used to determine the efficacy of end-capped oligodeaxynucleotides virus inhibition and cellular toxicity using oligonucleotides capped at two terminal 3'-end internucleotide linkages with 2-methoxyethylamine and dodecylamine.
45 The acute infection assay used the MOLT-4 cell line which is susceptible to HIV infection. Measurement of HIV p24 was used to assay for inhibition of virus _32_ WO 90!15065 PGT/US90/03138 replication 7 day~~.~tter infection with virus at a multiplicity~of infection of approximately 0.1.
Approximately 1 x 106 cells were preincubated with oligonucleotide, washed, infected with virus stock and then incubated for 7 days in oligonucleotide. HIV p24 levels in the supernatant were measured by radioimmunoaseay and compared with control infections lacking oligonucleotide. Results are expressed as the percent of control p24 found in cultures containing oligonucleotide. Seguences of antisense oligonucleotides were complementary to HIV targets listed in Table 3.
Toxicity data was obtained by incubation of 3~-end-capped oligonucleotides with uninfected cells, followed by a comparison with cell numbers with control cultures incubated in the absence of aligonucleotide. Toxicity results are expressed as the percent reduction of cell numbers obtained by incubation in oligonucleotide for 7 days compared to controls. The effective inhibition of HIV replication using low levels (0.5 to 5 ACM) of capped oligodeoxynucleotides supports the conclusion that significant nuclease degradation of the oligonucleotides of the invention does not occur either extracellularly or intracellulary.

~~.. -33- ~:~ ..

Ta-.~
t SeQUll~l~'-~= HIV Inh i h i t i nn Toxicity PBS
5'AGAGATTTTCCACAC3~
-methoxyethylamine 0.5 ACM 70% 0%
5 . 0 ~,cM 9 0 % 0 %
50.0 ~M -- ' 4%

0.5 ~tM 0% 0%

5.0 ~M 90% 2%

50 ~M --* 5%

NEF

5'TTGCCACCCATCTTA3' -methoxysthylamine 2.5 ~tM 75% 0%

5.0 ~M 80% 0%

10.0 ~M 90% 0%

,, 50 ~I 0%

100 ~j='' 0%

propyl~~'~,rie , .

2.5 ~~M 65% 0%

F.
5 . p~ 80% 0%

50 =--* 3 %

100 ,. - * 3$

* -- not done under HIV inhibition column.
~w

Claims

34

1. An oligonucleotide resistant to degradation under physiological conditions, comprising at least one phosphoramidate internucleotide linkage at the 5' termini and wherein at least one of the remaining internucleotide linkages is not a phosphoramidate linkage.

2. The oligonucleotide of claim 1, wherein more than one of the remaining internucleotide linkages is modified.

3. The oligonucleotide of claim 2, wherein the modifications to the remaining internucleotide linkages do not interfere with hybridization to a complementary oligonucleotide.

4. The oligonucleotide of claim 2, wherein the modifications to the remaining internucleotide linkages do not interfere with RNAseH activity.

5. The oligonucleotide of claim 4, wherein said modifications include at least one phosphoromonothioate internucleotide linkage.

6. The oligonucleotide of claim 4, wherein said modifications include at least one phosphorodithioate internucleotide linkage.

7. The oligonucleotide of claim 4, wherein all of the remainder of the internucleotide linkages are phosphoromonothioate linkages.

8. The oligonucleotide of claim 2, wherein the phosphoramidate has the formula:
wherein R1 and R2 are independently selected from the group consisting of hydrogen, hydrocarbyl substituents of 20 carbon atoms or less, and oxyhydrocarbyl substitutes of 20 carbon atoms or less containing 1 to 3 oxy groups, and wherein said hydrocarbyl or oxyhydrocarbyl substituents are linear or branched alkyl of 1 to 20 carbon atoms, linear or branched alkenyl of 2 to 20 carbon atoms, cycloalkyl or cycloalkenyl of 3 to 20 carbon atoms, linear or branched alkoxy of 1 to 20 carbon atoms, or aryl of 6 to 18 carbon atoms, with the proviso that R' and R2 are not both hydrogen.

9. The oligonucleotide of claim 8, wherein one of R1 and R2 is hydrogen.

10. The oligonucleotide of claim 8, wherein one of R1 and R2 is hydrogen and the other is an oxyhydrocarbyl substituent having the structure (CH2)x, wherein x is an integer in the range of 1 to 20, inclusive.

11. The oligonucleotide of claim 10, wherein x is 2 and the oxyhydrocarbyl substituent is 2-methoxyethyl.

12. The oligonucleotide of claim 8, wherein one of R1 and R2 is hydrogen and the other is a straight-chain alkyl moiety having the formula CH3-(CH2)y wherein y is an integer in the range of 0 to 15, inclusive.

13. The oligonucleotide of claim 12, wherein y is 2 and said alkyl substituent is n-propyl.

14. The oligonucleotide of claim 1, further comprising a 3' terminal phosphoramidate internucleotide linkage.

15. The oligonucleotide of claim 1, having the formula selected from the group consisting of:
wherein each n,m,i,j and s is independently an integer and each s is in the range of about 2 to 10; each n and m is independently from 1 to about 50; s+n in formulas I and II is less than 100; and s + s + m in formula III is less than 100; each i varies from 1 to n; each j varies from 1 to m; T is hydrogen or a hydroxyl-protecting group; R1 and R2 are independently selected from the group consisting of hydrogen, hydrocarbyl substituents of 20 carbon atoms or less, and oxyhydrocarbyl substituents of 20 carbon atoms or less containing 1 to 3 oxy groups, and wherein said hydrocarbyl or oxyhydrocarbyl substituents are linear or branched alkyl of 1 to 20 carbon atoms, linear or branched alkenyl of 2 to 20 carbon atoms, cycloalkyl or cycloalkenyl of 3 to 20 carbon atoms, linear or branched alkoxy of 1 to 20 carbon atoms, or aryl of 6 to 18 carbon atoms, with the proviso that R1 and R2 are not both hydrogen, each B is independently a protected or unprotected heterocyclic base; each Xi and Xj is independently O or S; and each Yi and Yj is independently R, --SR or --OR, where R
is as defined for R1 and R2.

16. The oligonucleotide of claim 15 wherein Xi and Xj are O and Yi and Yj are - OH.

17. The oligonucleotide of claim 15 wherein Xi and Xj are S and Yi and Yj are - OH.

18. The oligonucleotide of claim 15 wherein Xi and Xj are S and Yi and Yj are - SH.

19. The oligonucleotide of claim 15 wherein each n, m, i, j and s are integers in the range of about 2 to 10 and may be the same or different.

20. The oligonucleotide of claim 15 which is of formula (I) or (II).

21. The oligonucleotide of claim 20 wherein Xi and Xj are O and Yi and Yj are - OH.

22. The oligonucleotide of claim 20 wherein Xi and Xj are S and Yi and Yj are - OH.