(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`14 March 2002 (14.03.2002)
`
`
`
`(10) International Publication Number
`WO 02/20537 A2
`
`G1)
`
`International Patent Classification’:
`
`C07H 19/00
`
`@1)
`
`International Application Number:
`
`=PC1/CA01/01263
`
`(22)
`
`International Filing Date:
`10 September 2001 (10.09.2001)
`
`(74)
`
`(25)
`
`Filing Language:
`
`(26)
`
`Publication Language:
`
`English
`
`English
`
`(81)
`
`(30)
`
`Priority Data:
`60/231,301
`
`8 September 2000 (08.09.2000)
`
`US
`
`(71)
`
`Applicant (for all designated States except US): UNI-
`VERSITY TECHNOLOGIES
`INTERNATIONAL
`INC. [CA/CA]; Suite 130, 3553-31st Street N.W., Calgary,
`Alberta T2L 2K7 (CA).
`
`(72)
`(75)
`
`Inventors; and
`Inventors/Applicants (for US only): PON, Richard, T.
`
`[CA/CA]; 108 Ranch Estates Road N.W., Calgary, Alberta
`T3G 2B4 (CA). YU, Shuyuan [CA/CA]; 53 Hawktree Cir-
`cle N.W., Calgary, Alberta T3G 3M1 (CA).
`
`Agents: NASSIF, Omar, A.et al.; Gowling Lafleur Hen-
`derson LLP, Suite 4900, Commerce Court West, Toronto,
`Ontario MSL 1J3 (CA).
`
`Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
`GM,HR, HU,ID,IL, IN,IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NO, NZ, PH, PL, Pl, RO, RU, SD, SE, SG, SI,
`SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU,
`ZA, ZW.
`
`(84)
`
`Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`
`[Continued on next page]
`
`(54) Title: LINKER PHOSPHORAMIDITES FOR OLIGONUCLEOTIDE SYNTHESIS
`
`Synthesis of linker phosphoramidites (Exam
`
`les 1-3
`
`DMTO
`
`ocHton
`
`DMTO
`
`or
`
`9HOCCH,O
`
`o
`
`Oo
`
`2a X= CHeCH2
`b X = CH20CH2
`© X= CH2OPhOCH,
`
`HOCHsCH20H
`
`3a X = CHoCH,
`xyoo
`b X = CH20CHe
`o
`© X= CHsOPhOCH; ToOo 0
`
`
`JOCH2CH2CN
`
`CI-Rex
`
`DMTOft5
`
`OCH2CHCN
`
`oN o-Ra
`
`4a X = CH2CH2
`bX= CHsOCHs
`© X= CH2OPhOCHs
`
`A novel approach for
`(57) Abstract:
`ease of
`cleavage of
`combining the
`carboxylic acid linker arms with the single
`phosphoramidite coupling chemistry of
`the universal supports useful in oligonu-
`cleotide synthesis. There is disclosed a
`new class of phosphoramidite reagents,
`linker phosphoramidites, which contain a
`bifunctional
`linker arm with a protected
`nucleoside linked through a 3’-ester bond
`on une end and a reactive phosphoramidite
`group or other phosphate precursor group
`on the other end. The phosphoramidite
`group on the linker phosphoramidite may
`be activated under the same conditions
`
`and has similar reactivity as conventional
`nucleoside-3’-phosphoramidite
`reagents
`lacking the intermediate linker arm. The
`3’-ester linkage contained withinthe linker
`phosphoramidite has similar properties to
`the linkages on prederivatized supports.
`Theester linkageis stable to all subsequent
`synthesis steps, but upon treatment with
`a cleavage reagent,
`such as ammonium
`hydroxide, the ester linkage is hydrolyzed.
`This releases the oligonucleotide product
`with the desired 3’-hydroxyl
`terminus
`and leaves the phosphate portion of the
`reagent attached to the support, which is
`subsequently discarded.
`
`WO02/20537A2
`
`

`

`WO 02/20537 A2
`
`—__MNTIATININNNNNNTANIAMIN ITIAMAT AT
`
`patent (AI, BE, CH, CY, DE, DK, ES, Fl, FR, GB, GR, IE,—Hor two-letter codes and other abbreviations, refer to the "Guid-
`IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
`ance Notes on Codes andAbbreviations" appearing at the begin-
`CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD,
`ning ofeach regular issue of the PCT Gazette.
`TG).
`
`Published:
`
`—_without international search report and to be republished
`uponreceipt ofthat report
`
`

`

`WO 02/20537
`
`PCT/CA01/01263
`
`
`CROSS-REFERENCE TO RELATED APPLICATION
`The present application claims the benefit under 35 U.S.C. §119(e) ofprovisional
`[0001]
`patent application S.N. 60/231,301, filed September 8, 2000,
`the contents of which are
`hereby incorporated by reference.
`
`
`LINKER.PHOSPHORAMIDITESFOR OLIGONUCLEOTIDE SYNTHESIS
`BACKGROUND OF THEINVENTION -
`
`.
`FIELD OF THE INVENTION
`(0002
`In one of its aspects the present invention relates to a novel series ofphosphorus-
`containing compounds useful in oligonucleotide synthesis.
`In another of its aspects, the
`present invention relates the use of these compoundsin oligonucleotide synthesis.
`
`.
`
`DESCRIPTION OF THE PRIOR ART
`
`[0003]
`
`Oligonucleotides have become widely used as reagents for biochemistry and
`
`molecular biology (G. M. Blackburn and M. J. Gait, Nucleic Acids in Chemistry and
`
`Biology, 1990, IRL Press, Oxford). These materials are used as DNA sequencing primers (C.
`
`J. Howe and E. S. Ward, Nucleic Acids Sequencing: A Practical Approach, 1989, IRL Press,
`
`Oxford), polymerase chain reaction or “PCR” (N. Smyth Templeton, 1992, Diagnostic
`
`Molecular Pathology 1, 58-72) primers, DNA probes (L. J. Kricka, Nonisotopic DNA Probe
`Techniques, 1992, Academic Press, San Diego) and in the construction of synthetic or
`modified genes (S. A. Narang, Synthesis and Applications of DNA and RNA, 1987,
`Academic Press, San Diego). Modified oligonucleotides are also finding widespread use as
`diagnostic and therapeutic agents - sec one or moreof:
`
`(a)
`(b)
`(c)
`
`(d)
`
`S.L. Beaucage and R. P. Iyer, 1993, Tetrahedron 49, 6123-6194;
`S. L. Beaucage and R. P. Iyer, 1993, Tetrahedron 49, 1925-1963;
`S. Verma and F. Eckstein, 1998, Annu. Rev. Biochem. 67, 99-134; and
`
`R. P. Iyer, A. Roland, W. Zhou and K. Ghosh, 1999, Curr. Opin. Molec.
`
`Therap. 1, 344-358.
`
`Particularly important has been the development of high density DNA arrays (M. Schena,
`
`DNA Microarrays: A Practical Approach, 1999, Oxford University Press, Oxford), which
`
`SUBSTITUTE SHEET(RULE 26)
`
`

`

`WO 02/20537
`
`PCT/CA01/01263
`
`can contain thousands or tens of thousands of different DNA sequences. Consequently,
`demand for chemicallysynthesized oligonucleotides has been increasing steadily and many
`
`millions of oligonucleotides per year are now required.
`[0004]
`Solid-phase chemical synthesis is the only method capable of producing the
`number of
`synthetic
`oligonucleotides
`required
`and
`automated «synthesis
`using
`
`phosphoramidite coupling chemistry (S. L. Beaucage and R.P. Iyer, 1992, Tetrahedron 12,
`
`2223-2311) has become the preferred synthetic method. The first step in solid-phase
`synthesis is attachment of a nucleoside residue to the surface of an insoluble support,-such as
`a controlled pore glass or polystyrene bead, through a covalent linkage (R. T. Pon, “Solid-
`phase supports for oligonucleotide synthesis”, Unit 3:1 in Current Protocols in Nucleic Acid
`Chemistry, eds., S. L. Beaucage, D. E. Bergstrom, G. D. Glick and R.A. Jones, 2000, John
`
`—
`
`Wiley & Sons, New York). This linkage must be resistant to all of the chemical steps
`required to synthesize the oligonucleotide on the surface of the support. Furthermore, the
`linkage must be cleavable after synthesis is complete to release the oligonucleotide product
`from the support.
`
`[0005]
`
`It is also important that the product released from the support have a terminus
`
`which is well defined and can participate in subsequent enzymatic reactions,
`
`i.c. be
`
`recognized by enzymes such as polymerases. The preferred strategies for solid-phase
`
`oligonucleotide synthesis all attach the 3'-terminal residue to the support and assemble the
`
`oligonucleotide sequence in the 3'- to 5'- direction. After cleavage from the support, a 3'-
`hydroxyl group is desired since this is identical with the structure created by enzymatic
`cleavage. A 3'-terminal phosphate is not as satisfactory since this is not extendable by
`
`polymerases and such oligonucleotides cannot function as PCR or sequencing primers.
`
`[0006]
`
`The above linker requirements are satisfied by using a carboxylic or dicarboxylic
`
`acid linker arm to attach the first nucleoside residue by means of an ester linkage to the 3'-
`
`hydroxyl group. After synthesis, hydrolysis of this ester linkage with ammonium hydroxide
`
`releases the oligonucleotide from the support with the desired 3'-OH functionality. Methods
`
`for attaching nucleosides to supports by such means are well known,asillustrated by the
`
`prior art shown in Figures 1-1 and 1-2.
`
`In this approach dicarboxylic linker arms such as
`
`succinic acid, hydroquinone-O,O-diacetic acid, diglycolic acid, oxalic acid, malonic acid,
`
`etc. are frequently used.
`
`SUBSTITUTE SHEET(RULE 26)
`
`

`

`WO 02/20537
`
`PCT/CA01/01263
`
`the chemistry required to form the carboxylic ester or amide
`However,
`[0007]
`attachments to the supports is different from the phosphoramidite chemistry required to build
`up the oligonucleotide sequence. Therefore, the nucleoside attachment step is usually done
`
`separately from the automated synthesis. The correct prederivatized supports, containing
`either A, C, G, T or other minor nucleosides, must be selected in advance of automated
`synthesis. This is satisfactory when producing small numbers of oligonucleotides but
`becomes tedious and a potential source of error when large numbers ofdifferent sequences
`are synthesized, such as in 96 well plates. ‘Although fast coupling reagents have been
`developed, which allow automation ofthe esterification/amidation steps immediately prior to
`the phosphoramidite synthesis cycles (see R. T. Pon, S. Yu and Y. S. Sanghvi, 1999,
`Bioconjugate Chemistry 10, 1051-1057 and R. T. Pon and S. Yu, 1999, Synlett, 1778-1780),
`these reagents require specially modified DNA synthesizers to perform the esterification
`chemistry as well as the phosphoramidite chemistry.
`
`It is more desirable to have a method which uses only a single coupling chemistry
`[8008]
`commercially
`available
`automated
`instrumentation
`is
`only
`designed
`for
`since
`phosphoramidite synthesis. A variety of “universal” solid-phase supports containing a diol
`moiety, which have one hydroxy group free and one hydroxy group either protected or linked
`to the support, have been developed to meet this need (R. T. Pon, "Solid-phase supports for
`
`oligonucleotide synthesis", Unit 3.1 in Current Protocols in Nucleic Acid Chemistry, eds., S.
`
`L. Beaucage, D. E. Bergstrom, G. D. Glick and R.A. Jones, 2000, John Wiley & Sons, New
`
`York) - see Figure 1-3. In this approach, the same nucleoside-3'-phosphoramidite reagents
`
`used to synthesize the oligonucleotide sequence are used to attach the first nucleoside residue
`
`to the support. However, this results in the oligonucleotide being attached to the support
`through a 3'-phosphate and not a 3'-ester linkage. Therefore, cleavage from the support
`initially produces a 3'-phosphorylated product. Formation of the desired 3'-OH terminus
`requires either additional reagents or prolonged deprotection time to remove the 3'-phosphate
`group. The dephosphorylation reaction is also not quantitative and so a mixture of products
`is produced, Therefore, this approach is unsatisfactory because of the longer processing time,
`the reduced yield of desired 3'-OH product, and the mixture of 3'-phosphorylated and non-
`
`phosphorylated sequences in the final product.
`
`SUBSTITUTE SHEET(RULE 26)
`
`

`

`WO 02/20537
`
`PCT/CA01/01263
`
`Thus, despite the advances made to date there is still room for improvement.
`[0009]
`Specifically, it would be desirable to have a new approach to oligonucleotide synthesis which
`combines the advantages of using phosphoramidite coupling chemistry with the advantages
`of efficient automated synthesis without the need to resort to the “correct prederivatized
`supports” referred to above.
`
`.
`SUMMARYOF THE INVENTION
`Tt is an object of the present invention to obviate or mitigate at least one of the
`[0010]
`above-mentioned disadvantagesofthe priorart.
`
`It is an object of the present invention to provide a novel phosphorus-containing
`[0011]
`compound useful in oligonucleotide synthesis.
`
`is another object of the present invention to provide a novel process for
`It
`[0012]
`oligonucleotide synthesis.
`
`Accordingly, in one of its aspects, the present invention provides a compound
`[0013]
`having FormulaI:
`
`x'_Q-z!
`
`)
`
`wherein:
`X' comprises a protected nucleoside moiety selected from the following structures:
`
`RO
`
`"BY
`
`R’0
`
`O
`
`Be
`
`O
`
`B*
`
`R!
`
`OR?
`
`1
`
`R
`
`B*
`
`OR?
`
`wherein:
`
`R'is hydrogen, fluorine or -OR’;
`
`SUBSTITUTE SHEET(RULE 26)
`
`

`

`WO 02/20537
`
`PCT/CA01/01263
`
`R? and R’ are the sameor different and each is selected from hydrogen,
`methyl and a protecting group; and
`B’is a nucleic acid base;
`
`Q is amoiety selected from:
`
`ie
`
`| by
`
`A2
`
`A’ R
`
`6
`
`q
`
`oshatgh°
`
`0
`
`pL
`
`lm
`
`qT
`
`n
`
`and.
`
`I
`
`4
`
`LR
`
`LEE
`
`I
`
`ILA
`
`1
`
`Pp
`
`.
`
`wherein:
`
`—R*—;
`
`0GA G0Fe
`
`RAR
`|
`
`4 AS
`
`6
`
`O
`
`I
`
`Q' is an organic moiety;
`Q’is selected from —O—, —N(H)~, —N(R”)— and —S—;
`Q? is selected from —S(O)y—, —S(O)—, —C(O)—-, —O—, —O—(R*)—O— and
`
`A’ and A’ may be the sameordifferent and eachis selected from hydrogen,
`halogen, a Cy.:9 alkyl group, a Cs.19 aryl group, a C3.19 cycloalkyl group, -COOR’, —CONH,
`—CONR’, —CN, —NOQ2, —SR’, —S(O)R’, —S(O)2R’, —SC(CoHs)3, a Cy-19 alkylsulfonyl
`group, a Cs.19 aryl group, a Cy.19 alkylthio group, —Si(R")s, a C119 haloalkyl group, naphthyl,
`9-fluorenyl, 2-anthraquinonyl,
`
`oN
`x
`
`-wherein G is C or N with at least one G being N, and
`
`O-Z
`
`SUBSTITUTE SHEET(RULE 26)
`
`

`

`WO 02/20537
`
`PCT/CA01/01263
`
`A? and A* may be the same or different and each is selected from hydrogen,
`halogen, a Cy-19 alkyl group, a Cs.i9 aryl group, a C3.1q cycloalkyl group and an electron
`withdrawing group, provided that at
`least one of A°® and A‘ comprises an electron
`withdrawing group;
`.
`R3, R*, R° and R®° are the same or different and each is selected from
`
`hydrogen, halogen, a Cj-i9 alkyl group, a Cs.39 aryl group and a C3.19 cycloalkyl group;
`,
`R’ is selected from a C10 alkyl group, a Cs.19 aryl group and a C3.19
`' cycloalkyl group;
`Rois a Cho alkyl group or a Cs.19 aryl group;
`R° is a Cs10 aryl group or —CH)—; and
`
`1, m, n and p are independently 0 or 1;
`
`o is an integer in the range 0-30; and
`
`q is an integer in the range 0-50; and
`Z' is a phosphorylation moiety.
`In another of its aspects, the present invention provides a process for producing a
`. (0014)
`compound having Formula J:
`
`x'-Q-Z!
`
`@
`
`wherein:
`X' comprises a protected nucleoside moiety selected from the followingstructures:
`
`R’0-~
`
` *iB*
`
`O
`
`R!
`
`Be
`
`R!
`
`‘
`
`-~Q
`
`R70
`
`R!
`
`7
`
`]
`
`R
`
`oO
`
`oO
`
`B*
`
`Be
`
`OR?
`
`wherein:
`
`R'is hydrogen, fluorine or -OR’;
`
`SUBSTITUTE SHEET(RULE 26)
`
`

`

`WO 02/20537
`
`PCT/CA01/01263
`
`R? and R?are the sameordifferent and eachis selected from
`
`hydrogen, methyl and a protecting group; and
`B’ is a nucleic acid base;
`
`Q is a moiety selected from:
`
` q
`
`and
`
`R AAR
`bot
`
`—o-¢-tah e006o—
`
`O
`
`Lo
`
`Rt At RO
`
`wherein:
`
`Q' is an organic moiety;
`Q’is selected from —O—, —N(H)—, —N(R’)— and —S—;
`Q? is selected from —S(O).—, —S(O)—-, —C(O)—, —O—, —O—(R°)—O— and
`
`A' and A? may be the sameor different and each is selected from hydrogen,
`halogen, a Cy-19 alkyl group, a C549 aryl group, a C3.19 cycloalkyl group, —COOR’,
`—CONH, —CONR’, —CN, —NO:, —SR’, —S(O)R’, —S(O)2R’, —SC(CéHs)3, 2 Cr10
`alkylsulfonyl group, a Cs.19 aryl group, a C119 alkylthio group, —Si(R”)s, a Cj-19 haloalkyl
`
`group, naphthyl, 9-fluorenyl, 2-anthraquinonyl,
`
`G=\
`
`G4y
`
`7
`
`SUBSTITUTE SHEET(RULE 26)
`
`

`

`WO 02/20537
`
`PCT/CA01/01263
`
`wherein Gis C or N with at least one G being N, and
`
`O-Z,
`
`A? and A‘ may be the sameor different and each is selected from-hydrogen,
`halogen, a Cj.;9 alkyl group, a Cs.19 aryl group, a C3.19 cycloalkyl group and an electron
`withdrawing group, provided that at least one of A? and A* comprises and an electron
`
`withdrawing group;
`R°, R*, R° and R° are the same or different and each is selected from
`hydrogen, halogen, a C;.19alkyl group, a Cs.;9 aryl group and a C3.19 cycloalkyl group;
`R’ is selected from a Ci-10 alkyl group, a Cs.19 aryl group and a C3.19
`cycloalkyl group;
`R8 is a Cyto alkyl group or a Cs.19 aryl group;
`R?is a Cs.19 aryl group or —CHz—; and
`
`1, m, n and p are independently 0 or 1;
`
`o is an integer in the range 0-30; and
`
`q is an integer in the range 0-50; and
`Z' is a phosphorylation moiety;
`
`the process comprising the step of reacting compounds of FormulaII, II] and
`
`IV:
`
`x-_on
`Qi)
`
` HQo-R*
`Ca)
`
`2
`(IV)
`
`wherein R'is a protecting group and Z” is a phosphorus containingprecursor to
`Z' or activated phosphorylatoin moiety.
`
`In another of its aspects, the present invention provides a process for producing a
`[0015]
`derivatized nucleoside having Formula Va or FormulaVb:
`
`SUBSTITUTE SHEET(RULE26)
`
`

`

`WO 02/20537
`
`PCT/CA01/01263
`
`Te
`R—0-Gra str? citbel
`
`O
`
`1
`
`OL} wn
`
`n
`
`4
`R
`
`I
`
`A
`
`2
`6
`AUR
`
`(Va)
`
`.
`
`or
`
`1
`x!-o-c-+-Q
`ut
`Oo
`
`Ll
`
`R? A? R
`25
`|
`lt
`1
`C-O-C~C—C— OR’
`ly vi V6
`iI
`1 o
`R* At R'
`
`(Vb)
`
`wherein:
`X' comprises a protected nucleoside moiety selected fromthe followingstructures:
`
`R20—
`
`O
`
`B*
`
`R!
`
`RO
`
`R!
`
`B*
`
`O
`
`R} is hydrogen, fluorine or -OR? ;
`R’ and R°are the sameor different andeach is selected from hydrogen, methyl
`
`and a protecting group; and
`B’is anucleic acid base;
`Q'is an organic moiety;
`
`SUBSTITUTE SHEET(RULE 26)
`
`

`

`WO 02/20537
`
`PCT/CA01/01263
`
`Q* is selected from —O—, —N(H)—, —N(R”)— and —S—;
`Q?is selected from —S(O).—, —S(O)—, —C(O)—, -O—, —O—-(R°)—O— and —R°-;
`A! and A? may be the same or different and cach is selected from hydrogen, halogen, a
`C}-10 alkyl group, a Cs.19 aryl group, a C3.19 cycloalkyl group, —COOR’, —CONH, —CONR’,
`"CN, —NO:, —SR’, —S(O)R’, —S(O)},R’, —SC(CHs)3, a C110 alkylsulfonyl group, a Cs-19 aryl
`group, a Cj.49 alkylthio group, —Si(R’)3, a C19 haloalkyl group, naphthyl, 9-fluorenyl, 2-
`
`anthraquinonyl,
`
`G=\as
`
`wherein G is C or N with at least one G being N, and
`
`A? and A‘ may be the same or different and each is selected from hydrogen, halogen, a
`
`C1-19 alkyl group, a Cs-19 aryl group, a C3-10 cycloalkyl group and an electron withdrawing group,
`providedthat at least one ofA’ and A* comprisesan electron withdrawing group;
`|
`R?, R*, RS and R° are the sameor different and each is selected from hydrogen, halogen, a
`C1-19 alkyl group,a Cs10aryl group and a C3.;9 cycloalkyl group;
`R’is selected from a Cy-19 alkyl group, a Cs.19 aryl group and a C3.19 cycloalkyl group;
`R$is a Cy40 alkyl group or a Cs.;9 aryl group;
`R’ isa Cs.10 aryl group or —CH2—;
`
`1, m, n and p are independently 0 or 1;
`
`o is an integer in the range 0-30;
`
`q is an integer in the range 0-50; and
`R”is hydrogen or a protecting group;
`
`the process comprising the step of reacting together compounds having Formula IT and
`
`VI:
`
`10
`
`SUBSTITUTE SHEET(RULE 26)
`
`

`

`WO 02/20537
`
`PCT/CA01/01263
`
`QD)
`
`~
`
`R”*is hydrogen or a protecting group, with a compound having Formula VIIa (in the case where
`the nucleoside of Formula Vais being produced) or VIIb (in the case where the nucleoside of
`Formula Vb is being produced):
`
`e|
`H-Q21-G
`R
`
`-
`
`aa
`Q?_-c—c-tor®
`on
`Al
`A
`
`q
`
`|e
`
`Ay
`(Va)
`
`par
` H-Q2--C—-C-—Go”
`I, a te
`R’ A’ R'
`.
`(VUb)
`
`Thus, the present inventors have developed a novel approach for combining the ease
`[0016]
`of cleavage of carboxylic acid linker arms with the single phosphoramidite coupling chemistry of
`the universal supports. This entails synthesis of a new class of phosphoramidite reagents, linker
`phosphoramidites, which contain a bifunctional linker arm with a protected nucleoside linked
`through a 3'-ester bond on one end and a reactive phosphoramidite group or other phosphate
`precursor group on the other end - see Figures 2 and 3. The phosphoramidite group on the linker
`phosphoramidite is activated under
`the same conditions and has
`similar
`reactivity as
`conventional nucleoside-3'-phosphoramidite reagents lacking the intermediate linker arm. The 3'-
`ester linkage contained within the linker phosphoramidite has similar properties to the linkages
`on prederivatized supports. The ester linkageis stable to all subsequent synthesis steps, but upon
`treatment with a cleavage reagent, such as ammonium hydroxide, the ester linkage is hydrolyzed.
`
`11
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`This releases the oligonucleotide product with the desired 3'-hydroxyl terminus and leaves the
`
`phosphate portion of the reagent attached to the support, which is subsequently discarded.
`[0017]
`As used throughout this specification, the term "oligonucleotide" is intended to have a
`broad meaning . and
`encompasses
`conventional
`oligonucleotides,
`backbone-modified
`oligonucleotides (e.g., phosphorothioate, phosphorodithioate and methyl-phophonate analogs
`
`useful as oligotherapeutic agents), labeled oligonucleotides, sugar-modified oligonucleotides and
`
`oligonucleotide derivatives such as oligonucleotide-peptide conjugates.
`[0018]
`Throughout this specification, when reference is made to a substituted moiety, the
`nature of the substitution is not specification restricted and may be one or more members
`selected from the group consisting of hydrogen, a C)-C2g alkyl group, a Cs-C39 aryl group, a Cs~
`Cao alkaryl group (each of the foregoing hydrocarbon groups may themselves be substituted with
`one or more of a halogen, oxygen and sulfur), a halogen, oxygen and sulfur. Further, the term
`
`“alkyl”, as used throughout this specification, is intended to encompass hydrocarbon moieties
`having single bonds, one or more doubles bonds, one or more triples bond and mixtures thereof.
`[0019]
`The compound of Formula I is useful
`in producing oligonucleotides of desired
`sequence on a support material.
`In the present specification, the terms “support” and “support
`
`material” are used interchangeably and are intended to encompass a conventional solid support.
`
`The nature of the solid support is not particularly restricted and is within the purview of a person
`
`skilled in the art. Thus, the solid support may be an inorganic substance. Non-limiting examples
`
`of suitable inorganic substances may be selected from the group consisting of silica, porous
`
`iron oxide, nickel
`glass, aluminosilicates, borosilicates, metal oxides (e.g., aluminum oxide,
`oxide) and clay containing one or more of these. Alternatively, the solid support may be an
`organic substance such as a cross-linked polymer. Non-limiting examples of a suitable cross-
`
`linked polymer may be selected from the group consisting of polyamide, polyether, polystyrene
`
`and mixtures thereof. One preferred solid support for use herein is conventional and may be
`
`selected from controlled pore glass beads and polystyrene beads.
`
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0020]
`
`Embodiments of the present
`
`invention will be described with reference to the
`
`accompanying drawings, wherein like numerals designate like elements, and in which:
`
`Figure 1a illustrates a prior art synthesis of attaching a nucleoside to a support, -
`
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`Figure 1b illustrates a prior art approach for synthesizing oligonucieotides in tandem;
`Figures 2 and 3 illustrate preferred embodiments of the present process;
`
`Figure 4 illustrates a preferred embodiment of the present process for synthesizing
`oligonucleotides in tandem;
`Figure 5 illustrates the synthetic routes used in Examples 1-3 below
`Figure 6 illustrates the synthesis of a preferred reagent for tandem synthesis.
`
`.
`
`DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
`Phosphoramidite reagents are usually prepared by reacting an alcohol with a trivalent
`[0021]
`phosphite, such as 2-cyanoethyl diisopropylchlorophosphoramidite, N,N-diisopropylmethyl-
`
`phosphonamidic chloride, or bis-(diisopropylamino)-2-cyanoethoxyphosphine. Protected 2'-
`
`deoxyribonucleosides, ribonucleosides, or other nucleoside compoundswith either free 3'- or 5'-
`
`hydroxyl groups are the most commonsubstrates for this reaction since the resulting nucleoside
`phosphoramidite reagents can be used to assemble oligonucleotide sequences. However, many
`
`other reagents such as amino or thiol end-modifiers, non-nucleotide spacers, fluorescent dyes,
`
`lipophilic groups such cholesterol or Vitamin E, and non-isotopic labels, such as biotin have also
`
`been converted into alcohols and then into phosphoramidite reagents. In these reagents, the
`
`phosphoramidite group is used as a reactive group to permanently attach the reagent to the
`
`oligonucleotide sequence through a stable phosphate linkage.
`{0022]
`In an aspect of the present invention, a reagent such as a protected nucleoside or a
`
`non-nucleoside end modifier with a free hydroxyl] group is esterified to a carboxylic acid linker
`
`arm. The resulting ester linkage will become the site of subsequent cleavage when exposed to
`ammonium hydroxide or other cleavage conditions. This internal cleavage site differentiates the
`linker phosphoramidites of this invention from previous phosphoramidite reagents which never |
`
`separate the phosphate group from the product. The carboxylic linker arm should have a second
`
`site (e.g., hydroxyl) which can react with a trivalent phosphite to convert the reagent into a
`phosphoramidite reagent. Thus the linker can be any compound with both a carboxylic acid
`group and an alcohol - see Figure 2. Examples of possible linkers include, but are not limited to:
`4-hydroxymethylphenoxyacetic acid (HMPA); 4-hydroxymethylbenzoic acid (HMBA); 4-(4-
`hydroxymethyl-3-methoxyphenoxy)-butyric
`acid
`C(HMPB);
`3-(4-hydroxymethylphenoxy)-
`propionic acid; glycolic acid;
`lactic acid; 4-hydroxybutyric acid; 3-hydroxybutyric acid; 10-
`
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`hydroxydecanoic acid; 12-hydroxydodecanoic acid; 16-hydroxyhexadecanoic acid; or 12-
`hydroxystearic acid.
`|
`[0023]
`Traditionally,
`linker arms for solid-phase oligonucleotide synthesis have been
`dicarboxylic acids such as succinic acid, hydroquinone-O, O'-diacetic acid, diglycolic acid, oxalic
`acid, malonic acid, etc. and it is desirable to maintain these types of linker arms in the invention
`because their useful properties have been well established. Therefore, a second route towards
`
`synthesis of linker phosphoramidite reagents (Figure 3) which uses well-known dicarboxylic
`acids is also possible. In this procedure the cleavable ester linkage is produced by attaching one
`end of the dicarboxylic acid linker to a nucleoside. The other end of the dicarboxylic acid is then
`
`coupled through an ester or amide linkage to a second diol or amino-alcohol which serves to
`
`convert
`the carboxyl group into an alcohol or amino group capable of forming the
`phosphoramidite portion of the linker phosphoramidite. Examples of possible compoundsfor the
`second portion of the linker arm include, but are not limited too: ethylene glycol; diethylene
`glycol; triethylene glycol; tetraethylene glycol, pentacthylene glycol; hexaethylene glycol; 2-
`aminoethanol; 1,2-diaminoethane; 1,3-propanediol; 3-amino-l-propanol; 1,3-diaminopropane;
`1,4-butanediol; 4-amino-l-butanol; 1,4-diaminobutane; 1,5-pentanediol; 1,6-hexanediol; 6-
`
`amino-1-hexanol; 1,6-diaminohexane; or 4-amino-cyclohexanol.
`
`[0024]
`
`The phosphorus containing group on the end of the linker may be any type of
`
`precursor which can be activated and react under oligonucleotide synthesis conditions. A variety
`of chemistries are known for oligonucleotide synthesis, such as the phosphodiester method, the
`
`phosphotriester method, the modified phosphotriester method, the chlorophosphite or phosphite-
`triester method, the H-phosphonate method, and the phosphoramidite method. However, at the
`present time, only the last two methods are used regularly and the phosphoramidite method is by
`
`the far the most popular.
`
`“activated
`term “activation” or
`the
`specification,
`this
`As used throughout
`[0025)
`phosphorylation moiety" is intended to have broad meaning and refers to the various ways in
`
`which a phosphorus group can be attached through either a phosphite ester, phosphate ester, or
`phosphonate linkage. Phosphorus moieties containing either trivalent (P™) or pentavalent (PY)
`oxidation states are possible and the oxidation state of the phosphorus may change (usually from
`p'to PY) during the course of the coupling reactions. Thus, reagents which are precursors to the
`desired products may have a different oxidation state than the product. The reagents used for
`
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`phosphorylation may be inherently reactive so that no external activating or coupling reagents
`are required. Examples of this type include chlorophosphite, chlorophosphate, and imidazole,
`triazole, or tetrazole substituted phosphite and phosphate reagents. Phosphorylation reagents
`which are stable until activated by the presence of a separate activating agent are more
`* convenient and are widely used. Examples of these reagent include phosphoramidite and. bis-
`phosphoramidite reagents such as 2-cyanoethyl-N,N'-diisopropylphosphoramidite derivatives
`
`and bis-(N,N'-diisopropylamino)-2-cyanoethylphosphine. Reagents with reactive groups may
`also be substituted with other reactive groups to make for more desirable coupling properties. An
`example of this is the conversion of highly reactive phosphorustrichloride into phosphorustris-
`
`(imidazolide) or phosphorustris-(triazolide) species before use. Phosphorylation reagents may
`
`also require in situ conversion into activated species by additional coupling reagents. This may
`
`be similar to the formation of carboxylic esters and amides where carbodiimide coupling
`
`reagents, such as dicyclohexylcarbodiimide or 1-[3-(dimethylamino)propy1]-3-ethylcarbodiimide
`
`hydrochloride and similar reagents; uronium coupling reagents, such as O-benzotriazol-1-yl-
`
`N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-benzotriazol-1-yl-N,N,N',N'-
`
`tetramethyluronium tetrafluoroborate
`
`(TBTU)
`
`or O-(7-azabenzotriazol-1-yl)-N,N,N',.N-
`
`tetramethyluronium hexafluorophosphate (HATU) and similar reagents; and phosphonium
`
`coupling
`
`reagents,
`
`such
`
`as
`
`benzotriazol-1-yloxytris(dimethylamino)phosphonium
`
`hexafluorophosphate
`
`(BOP)
`
`or
`
`benzotriazol-1-yloxytripyrrolidinophosphonium
`
`hexafluorophosphate (PyBOP) and similar reagents are possible. It may also require coupling
`reagents which produce mixed anhydride intermediates such as pivaloyl chloride, especially
`useful for coupling H-phosphonate reagents: and substitutedarylsulphonyl chloride, imidazolide,
`triazolide, and tetrazolide reagents which are especially useful for coupling of phosphate
`reagents. Phosphorylation reagents may also have protecting groups which allow them to be
`
`more easily handled as neutral, uncharged species. These protecting groups are removable to
`
`allow the charged species to be produced in situ without isolation and then this charged species
`
`participates in the coupling reaction. An example of this approach is known as the modified
`
`phosphotriester approach. Thus, there is a broad and diverse range of reagents and reaction
`conditions for introducing phosphorus groups and for coupling them to produce phosphite,
`phosphate, and phosphonate linkages. However, these methods are all knownto those skilled in
`
`the art.
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`Linker phosphoramidite reagents of the four commonbases (A, C, G, and T) or other
`[0026]
`minor bases can be prepared and installed on automated DNA synthesizers in the same manner
`as the four conventional nucleoside-3'-phosphoramidite reagents (Figures 2 and 3). Inexpensive
`
`and readily available underivatized amino or hydroxyl] solid-phase supports can then be used as
`"universal" supports in either column or plate formats. Standard phosphoramidite coupling
`cycles can then be used to attach the linker phosphoramidite in the first synthesis cycle before
`
`switching to conventional phosphoramidite reagents for the subsequent chain extension steps. No
`
`additional coupling reagents are required since the activator (usually tetrazole) remains the same
`
`for both types of phosphoramidite reagent. Automated synthesizers which can support eight
`
`different phosphoramidite reagents at one time are already widely available and so having a set
`of
`four
`linker phosphoramidites
`and
`four
`conventional
`phosphoramidites
`installed
`simultaneously is not a problem. The fact that only four additional linker phosphoramidites are
`required is a significant advantage over our previous method of automatically attaching the first
`
`nucleoside through an ester or amide linkage, since this method required five extra reagents (four
`nucleosides and a coupling reagent) and synthesizers with this much extra reagent capacity are
`
`not readily available.
`[0027]
`After completion of the synthesis, cleavage of the product can be performed using the
`same reagents and conditions as previously used with prederivatized supports and the products
`will be released with the desired 3'-hydroxyl ends. The phosphate moiety of the linker
`
`phosphoramidite will remain attached to the support and is discarded. Depending on the linker
`
`arm used in the