AU2003257499A1 - Reusable solid support for oligonucleotide synthesis - Google Patents

Description

Our Ref:12183421 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): University Technology International, Inc.

Suite 204, 609 14 Street N.W.

Calgary Alberta T2N 2A1 Canada Address for Service: Invention Title: DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Reusable solid support for oligonucleotide synthesis The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 WO 00/01711 PCT/CA99/00600 -1- REUSABLE SOLID SUPPORT FOR OLIGONUCLEOTIDE SYNTHESIS TECHNICAL FIELD In one of its aspects, the present invention relates to a reusable solid support for oligonucleotide synthesis. In another of its aspects, the present invention relates to a process for production of such a reusable solid support. In yet another of its aspects, the present invention relates to a process for use of such a reusable solid support.

BACKGROUND ART The art of organic chemistry on solid supports is generally known. A useful review article on this topic may be found in "Organic Chemistry on Solid Supports" by Friichtel et al., Angew. Chem. Int. Ed. Engl., 1996, 35, pgs. 17-42, the contents of which are hereby incorporated by reference.

As discussed in Friichtel et al., the art has developed automated solidphase synthesis of polypeptides, oligonucleotides and oligosaccharaides. Of particular interest here is solid-phase synthesis of oligonucleotides. The following are useful review articles/textbooks on this topic: Beaucage et al., Tetrahedron, 1992, 48, 2223; Davis et al., Innovation and Perspectives in Solid Phase Synthesis R. Epton), Intercept, Andover, 1992, pg. 63; Montserra et al., Tetrahedron, 1994, 50, 2617; and S. L. Beaucage et al., Tetrahedron, 1993, 49, 6123-6194; the contents of each of which are hereby incorporated by reference.

In the solid-phase synthesis ofoligonucleotides, it is known to synthesize the oligonucleotide on an inorganic solid support bearing a succinyl linker arm see, for example, any of the following references: WO 00/01711 PCT/CA99/00600 -2- Caruthers et al., Genetic Engineering, Plenum Press, New York (1982), Vol. 4, pgs. 1-17; Letsinger et al., Genetic Engineering, Plenum Press, New York (1985), Vol. 5, pg. 191; Froehler et al., Nucleic Acids Research, 14:5399-5407 (1986); and Matteucci et al.. Journal of American Chemical Society, 103:3185-3186 (1981); the contents of each of which are hereby incorporated by reference.

Typically, the succinyl linker arm has the following general formula: DMTO-

B

II I

O-C-CH

2

,-CH,-C-N-[SUPPORT]

II

Thus, the succinyl group links the growing oligonucleotide from its terminal 3' hydroxyl group by an ester bond to a primary amine on the support, which may be, for example, conventional controlled pore glass (CPG) or silica, by an amide bond. Once the desired oligonucleotide has been synthesized, it is freed or cleaved from the succinyl linker arm hydrolyzing the ester carbonyl group. The hydrolysis agent is usually concentrated ammonium hydroxide. Typically, this reaction can take from 1-4 hours to complete. With improvements to current solid-phase oligonucleotide synthesizers, this cleavage step can represent 50% or more of the total time require to synthesize the desired oligonucleotide.

Another type of linker arm is disclosed in United States patent 5,112,962 [Letsinger et al. (Letsinger)], the contents of which are hereby incorporated by WO 00/01711 PCT/CA99/00600 reference. Letsinger teaches a linker arm for solid support synthesis of oligonucleotides and oligonucleotide derivatives have the following formula: DMTO

B

H

N-[SUPPORT]

II II 0 0 Thus. Letsinger teaches an oxalyl linker arm which purportedly release the synthesized oligonucleotide or oligonucleotide derivate in a period of 1-30 minutes in a manner that leaves the oligonucleotide fully protected. The oxalyl linker arm purportedly can be rapidly cleaved by 5% ammonium hydroxide in methanol, ammonium hydroxide, wet tertiary amine, triethylamine/alcohol, triethylamine/methanol, triethylamine/ethanol, aqueous trimethylamine and other bases. Unfortunately, the oxalyl linker arm of Letsinger suffers from its purported advantage. Specifically, the present inventors have discovered that the oxalvl linker arm ofLetsinger is susceptible to significant spontaneous hydrolysis spontaneous hydrolysis of 10-40% per month) which renders it difficult to use in commercial operations. The oxalyl arm is also difficult to prepare because it requires using oxalyl chloride, which is highly reactive, toxic and therefore dangerous.

Regardless of the specific nature of the linker arm, it is generally accepted in the art that the linker arm is not reusable after production and cleavage of the desired oligonucleotide. Thus, conventional linker arms may be regarded as nonrecyclable. This is illustrated in Figure 1 which illustrates the conventional use of a succinyl linker arm for the production of an oligonucleotide. Thus, as illustrated, after cleavage of the desired oligonucleotide, the support is irreversibly linked to the linker compound the succinyl moiety) and cannot be reused.

WO 00/01711 PCT/CA99/00600 -4- The art is in need of a linker arm for solid support oligonucleotide synthesis, which linker arm is recyclable. More specifically, the art is in need of a linker arm capable of repeated oligonucleotide synthesis/cleavage.

In published International patent application WO 97/23496 [Pon et al.], the contents of which are hereby incorporated by reference, there is reported the first recyclable linker arm. This linker arm is based on a derivatized solid support having the following formula: 0 0 II

II

C C HO- R s Y X- [SUPPORT] wherein: R 8 is selected from the group consisting of a substituted or unsubstituted Ci-C 20 alkyl group, a substituted or unsubstituted C, C 30 aryl group and a substituted or unsubstituted C,-Co 4 alkylaryl group; X 3 and X 4 are the same or different and are selected from the group consisting of-O-, and

-N(R

1

R

1 2 is selected from the group consisting of a substituted or unsubstituted CI-C,2 alkyl group, a substituted or unsubstituted C 5

-C

3 0 aryl group and a substituted or unsubstituted C 5 -C4o alkylaryl group; and Y is selected from the group consisting of:

-CH,-CH,-CH

2 -CH=CH-; -CH=C(CH 3

-C(CH

3

)=C(CH

3 and While a linker arm based on the solid support described by Pon et al. is a significant advance in the art, there is still room for improvement. Specifically, the solid support described by Pon et al. has the following disadvantages.

WO 00/01711 PCT/CA99/00600 First, prior to attachment of the linker moiety, the solid support must be derivatized by a process comprising the step of reacting together the compounds of Formulae I, II and III: HO-R X 3 H HX4-[SUPPORT]

Y

(II) (In) wherein R 8

X

3

X

4 and Y are as defined above. Practically, this involves two steps reaction of the compound of Formula III with one of the compounds of Formulae I and II and subsequent reaction with the other of compounds of Formulae I and II. Thus, the disadvantage is additional labour required to effect a two-step derivatization of the solid support.

Second, each step of the derivatization described in the previous paragraph has the potential of incompletely derivatizing each HX 4 moiety on the support thereby increasing the likelihood of a heterogeneous surface. Practically, it becomes necessary to block or cap underivatized HX 4 moieties so that the linker moiety does interact with them. Thus, the disadvantage is additional labour and cost required to effect derivatization of the solid support.

Third, a linker arm based on the derivatized support described by Pon et al. is not as resistant to partial cleavage during regeneration as a derivatized support having a more fully saturated moiety.

In light of these disadvantages, it would be desirable to have an improved recyclable solid state support material useful in the oligonucleotide synthesis. It would be especially desirable if the the linker moiety could be attached to the support material with little or no derivatization required of the latter.

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide a novel solid support for oligonculeotide synthesis which obviates or mitigates at least one of the abovementioned disadvantages of the prior art.

WO 00/01711 PCT/CA99/00600 -6- It is another object of the present invention to provide a novel process for producing the solid support.

It is an object of the present invention provide a novel linker arm for solid support oligonucleotide synthesis which obviates or mitigates at least one of the above-mentioned disadvantages of the prior art.

It is another object of the present invention to provide a novel process for producing a linker arm for solid support oligonucleotide synthesis.

Accordingly, in one of its aspects, the present invention provides a reusable linker arm for solid support oligonucleotide synthesis, the linker arm comprising the following formula: T [SUPPORT] wherein Z is a linker moiety and T is an organic radical.

In another of its aspects, the present invention provides a reusable linker arm for solid support oligonucleotide synthesis, the linker arm comprising the following formula: NUCLEOSIDE-Z-O-T-

[SUPPORT]

wherein Z is a linker moiety and T is an organic radical.

In yet another of its aspects, the present invention provides a process for production of a reusable linker arm for oligonucleotide synthesis having the following formula: SZ-0- [SUPPORT] WO 00/01711 PCT/CA99/00600 -7wherein Z is a linker moiety and T is an organic radical, the process comprising the step of reacting together the compounds of Formulae I and II:

Z-OH

(I)

HO- [SUPPORT]

(II)

wherein Z and T are as defined above.

In another of its aspects, the present invention provides a process for production of a reusable linker arm for oligonucleotide synthesis having the following formula: NUCLEOSIDE-Z- O T--[SUPPORT] wherein Z is a linker moiety and T is an organic radical, the process comprising the step of reacting together the compound of Formulae I, II and III:

HO-Z-OH

(I)

HO- T- [SUPPORT]

(II)

NUCLEOSIDE- OH (In) wherein Z and T are as defined above.

WO 00/01711 PCT/CA99/00600 -8- In yet another of its aspects, the present invention provides a process for producing an oligonucleotide having a desired sequence comprising the steps of: reacting a linker arm having the formula: NUCLEOSIDE-Z-O- T- [SUPPORT] wherein Z is a linker moiety and T is an organic radical, with at least one oligonucleoside base until an oligonucleotide having the desired sequence is produce; (ii) cleaving the oligonucleotide having the desired sequence to produce a free oligonucleotide have the desired sequence; and a used linker arm; and (iii) recycling the used linker arm to Step As used throughout this specification, the term "oligonucleotide" is intended to have a broad meaning and encompasses conventional oligonucleotides, backbone-modified oligonucleotides phosphorothioate, phosphorodithioate and methyl-phophonate analogs useful as oligotherapeutic agents) and oligonucleotide derivatives such as oligonucleotide-peptide conjugates.

Throughout this specification, when reference is made to a substituted moiety, the nature of the subsitution is not specification restricted and may be selected from the group consisting of a C,-C20 alkyl groups, aC 5

-C

3 0 aryl group a C,-Co 4 alkaryl group.

BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the present invention will be described with reference to the accompany drawing in which: Figure 1 illustrat-s a specific process pathway for conventional oligonucleotide synthesis; and Figures 2 and 3 illustrate specific preferred embodiments of the present invention.

WO 00/01711 PCT/CA99/00600 -9- BEST MODE FOR CARRYING OUT THE INVENTION Initially, to facilitate an understanding of the invention, reference will be made to Figure 1, which illustrates a conventional process for solid support oligonucleotide synthesis.

Thus, the initial step of the process illustrated in Figure 1 comprises reacting a linking compound, such as succinic acid (while succinic acid is illustrated, succinic anhydride may also be used), with a conventional amineterminated support. The reaction results in the formation of an amide linkage between the linking compound and the support to produce succinyl-support conjugate.

Next, the succinyl-support conjugate is reacted with a desired initial nucleoside to produce a linker arm. In the illustrated nucleoside, DMT is dimethyoxytrityl, B is the nucleobase and R' is H (for deoxyribonucleosides) or OR (for ribonucleosides) wherein R is H or a conventional blocking/ protecting group. The reaction results in the formation of an ester linkage between the linking compound and the desired initial nucleoside at the 3' position of the latter.

The linker arm is then used in conventional oligonucleotide synthesis (e.g.

in a conventional automated synthesizer) to produce an oligonucleotide of desired sequence attached to the linker arm.

The oligonucleotide is then cleaved from the linker by hydrolysis. This serves to cleave the ester bond thereby freeing the oligonucleotide and an amineterminated, non-reusable linker arm.

The present inventors have surprisingly and unexpectedly discovered that a support having a hydroxy-terminated functionality may be combined with a conventional linking compound to produce linker arm which may used to synthesize an oligonucleotide of desired sequence. A key feature of the invetion is that the linker arm may be regenerated or recycled after cleavage of the oligonucleotide of desired sequence. To the inventors' knowledge, this is the first discovery of a derivatized support which may be used repeatedly in oligonucleotide synthesis.

The reusable linker arm of the present invention has the following formula: WO 00/01711 PCT/CA99/00600 Z-O- T-[SUPPORT] wherein Z is a linker moiety and T is an organic radical.

Preferably, T contains at least one carbon.

Preferably, T is a C 1

-C

30 0 organic moiety, more preferably a C-C200 organic moiety, most preferably a C,-Cioo organic moiety.

As will be appreciated by those of skill in the art, T may be a saturated or unsaturated organic moiety. Further, T may contain one or more heteroatoms.

For example, T may comprise at least one heteroatom selected from N and 0.

In one preferred embodiment, the organic moiety in T comprises at least one moiety having the formula:

O

I I

-C-

In another preferred embodiment, the organic moiety in T comprises at least one moiety having the formula: In yet another preferred embodiment, the organic moiety in T comprises at least one moiety having the formula: WO 00/01711 PCT/CA99/00600 0 -11-

II

N(H)C- In yet another preferred embodiment, the organic moiety in T comprises at least one moiety having the formula: In yet another preferred embodiment, the organic moiety in T comprises at least one moiety having the formula: 0

II

Further. those of skill in the an will recognize that the organic moiety in T may be unsubstituted or substituted. For examples, the organic moiety of T may be substituted by at least one moiety selected from the group comprising a

C,-CO

4 alkyl group, a C 5

-C

40 aryl group, a C,-C 4 o alkoxy group, a Ci-C 4 0 ester group,a C,-C4o hydroxy group, a C,-C, 4 acrylate group and a C 5

-C

4 0 alkylaryl group.

In one preferred embodiment, T has the formula:

-ECH

2 o- CH2-CH 2 -O CH2 L 1, WO 00/01711 PCT/CA99/00600 -12wherein q and s are the same or different and each is an integer having a value of 0-40 and r is an integer having a value of 1-200. In this embodiment, it is further preferred that q and s are the same or different and each is an integer having a value of 1-20 and r is an integer having a value of 1-150.

In another preferred embodiment, T has the formula:

OR

8 -O -EQaeCH-CH-CH 2 -OfCH 2 wherein a is 0 or 1, Q is an organic moiety, R 8 is hydrogen or a protecting group and b is an integer having a value of 0-40. In this embodiment, a may be 0 and R8 may be hydrogen. Further, a may be 1 and R 8 may be a protecting group.

Non-limiting examples of protecting groups may be selected from the group comprising acetyl, chloroacetyl, methoxyacetyl, t-butyl phenoxyacetyl, phenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl (DMT), dialkylphosphite, pivalyl-isobutyloxycarbonyl, r-butyldimethylsilyl, phenoxyacetal, 9phenylxanthen-9-yl (pixyl), tetrahydropyranyl, methoxytetrahydropyranyl, methoxymethyl, benzyloxymethyl, methoxyethoxymethyl, methylthiomethyl, dialkylphosphate, levulinyl, dimethylphenylsilyl, trimethylsilyl, isopropyldimethylsilyl, diisopropylmethylsilyl, diethylisopropylsilyl, triisopropylsilyl, acetyl, benzoyl, pivaloyl, trifluoroacetyl, allyl, benzyl, o-nitrobenzyl, ohydroxystyryldimethylsilyl. 2-oxo- 1,2-diphenylethyl, allyloxycarbonyl, monomethoxymethyl, nitroveratryloxycarbonyl, dimethoxybenzoin, dimethoxybenzoin carbonate, methylnitropiperonyl carbonate, fluorenylmethoxycarbonyl, 2-phenylsulfonylethoxycarbony, fluorophenylmethoxypiperidinyl and mixtures thereof.

In this embodiment, Q may be a moiety having the formula: WO 00/01711 PCT/CA99/00600 -13- Ra -CHE, CH ECH 2 ±CHa2 O-CH 2 wherein q, r, s, t and u are the same or different and each is an integer having a value of 0-40 and R" is selected from the group comprising hydrogen, hydroxyl, a C,-Co alkyl group, a C 5

-C

4 0 aryl group, a CI-C 4 0 alkoxy group, a Ci-C 40 ester group,a C,-C 4 0 hydroxy group, a C,-C 40 acrylate group and a C 5

-C

4 0 alkylaryl group. Preferably, s is 0, q, r and u are the same or different and each is an integer having a value of 1-10, t is an integer of 1-5 and R" is hydroxyl.

In yet another preferred embodiment, T has the formula:

OR

8 -O EQ CH-CHI-O--CH3wherein a is 0 or 1, Q is an organic moiety, R 8 is selected from the group comprising hydrogen, hydroxyl, a C,-C, 4 alkyl group, a C 5 -C4o aryl group, a C,-

C,

0 alkoxy group, a C,-C 4 0 ester group,a C,-C 4 0 hydroxy group, a C,-C 40 acrylate group and a C 5

-C

4 0 alkylaryl group, and b is an integer having a value of 0-40.

Preferably, Q is a C,-Co 00 organic moiety. As will be appreciated by those of skill in the art, Q may be a saturated organic moiety or an unsaturated organic moiety.

It is preferreed that Q is a C,-Co 00 organic moiety comprising at least one heteroatom selected from N and 0.

In one preferred embodiment, the organic moiety Q comprises at least one moiety having the formula: WO 00/01711 PCT/CA99/00600 0 I I ii In another preferred embodiment, the organic moiety Q comprises at least one moiety having the formula: In yet another embodiment, the organic moiety Q comprises at least one moiety having the formula: 0

II

-N(H)C-

In yet another embodiment, the organic moiety Q comprises at least one moiety having the formula: In yet another embodiment, the organic moiety comprises at least one moiety having the formula: 0

II

WO 00/01711 PCT/CA99/00600 As will be appreciated by those of skill in art, the organic moiety Q may unsubstituted or substituted. For example, the organic moiety Q may be substituted by at least one moiety selected from the group comprising a C,-C4o alkyl group, a C 5 -Co 4 aryl group, a C,-C, 4 alkoxy group, a C,-C4o ester group,a C,-

C

4 0 hydroxy group, a C 2

-C

4 0 acrylate group and a C,-Co alkylaryl group.

In one preferred embodiment, Q has the formula: O

O

NH-C-{CH,]-

CHC-NH Hf NH- CH z X Z wherein each of x, y and z is an integer having a value of 1-40.

In the above formula for the present linker arm, Z is a linker moiety. As will be discussed below, Z is derived from a linker compound have the general formula HO-Z-OH (Formula I below). The nature of the linker compound is not particularly restricted.

In one preferred embodiment, linker moiety Z has the formula: 0 0 II

II

HO-C--C-CH

2 As will be apparent to those of skill in the art, this linker moiety may be derived from succinic acid or succinic anhydride.

In another preferred embodiment, linker moiety Z has the following formula: WO 00/01711 PCT/CA99/00600 -16- 0 0 II II

HO-C-CH-O-CH-C-

As will be apparent to those of skill in the art, this linker moiety may be derived from diglycolic acid or diglycolic anhydride.

In yet another preferred embodiment, linker moiety Z has the following formula: 00 II II

HO-C-C-

As will be apparent to those of skill in the art, this linker moiety may be derived from oxalic acid or oxalyl chloride.

In yet another, and most, preferred embodiment, linker moiety Z has the following formula:

O

II 4 5 1 HO-C(R R C)X' wherein: R 2 and R 3 are the same or different and are selected from the group consisting of hydrogen, halide, a substituted or unsubstituted CI-C 20 alkyl group, a substituted or unsubstituted C,-C 3 0 aryl group and a substituted or unsubstituted WO 00/01711 PCT/CA99/00600 -17-

C

5

-C

40 alkylaryl group; R 4 and R 5 are the same or different and are selected from the group consisting of hydrogen, a substituted or unsubstituted CI-Co alkyl group, a substituted or unsubstituted

C

5

-C

30 aryl group and a substituted or unsubstituted

C,-C

40 alkylaryl group; X' is selected from the group consisting of

-S(O)

2 and R is selected hydrogen, a substituted or unsubstituted

C,-C

20 alkyl group, a substituted or unsubstituted

C,-C

3 0 aryl group and a substituted or unsubstituted

C

5 -Co alkylaryl group; n is 0, 1 or 2; and one of A' and B' is selected from the group consisting of hydrogen, halide, a substituted or unsubstituted

C,-C

2 0 alkyl group, a substituted or unsubstituted

C,-

C

30 aryl group and a substituted or unsubstituted

C,-C

40 alkylaryl group, and the other of A' and B' has the formula: 0 X (CR 6 R )mCp wherein p is 0 or 1, X 2 is selected from the group consisting of-0-, -S(0) 2 and R is selected from the group comprising hydrogen, a substituted or unsubstituted C,-C20 alkyl group, a substituted or unsubstituted Cs-

C

30 aryl group and a substituted or unsubstituted

C

5

-C

40 alkylaryl group, R 6 and

R

7 are the same or different and are selected from the group consisting of hydrogen, a substituted or unsubstituted C,-C,2 alkyl group, a substituted or unsubstituted

C

5

-C

30 aryl group and a substituted or unsubstituted

C

5

-C

4 0 alkylaryl group, and m is 0, 1 or 2. In this embodiment, B' preferably is selected from the group consisting of hydrogen, halide, a substituted or unsubstituted

C,-C

2 0 alkyl group, a substituted or unsubstituted

C

5

-C

30 aryl group and a substituted or unsubstituted

C

5

-C

40 alkylaryl group. Preferably, at least one, more preferably each, of R, R 4

R

5 R' a.id R 7 is hydrogen and preferably at least, more preferably both, of m and n are 1. It is further preferred that each of R 2 and R 3 is hydrogen and that X' and X 2 are both Thus, in this embodiment, the most WO 00/01711 PCT/CA99/00600 -18preferred form of linker moiety Z is derived from hydroquinone-O,0'-diacetic acid.

In yet another preferred embodiment, linker moiety Z has the following formula: 0 0 II 67 1 HO-C(RR'C)n----Y-(CRR wherein R 4

R

5

R

6 and R 7 are the same or different and are selected from the group consisting of hydrogen, a substituted or unsubstituted

C,-C,

0 alkyl group, a substituted or unsubstituted C 5

-C

30 aryl group and a substituted or unsubstituted

C

5

-C

4 0 alkylaryl group, Y is selected from the group consisting of O, S, SO, and I is an integer less than or equal to 60, q is an integer in the range of 1-1000, n and m are the same or different and are 1 or 2, with the proviso that, when Y is O, at least one ofn and m is 2. Preferably, 1 is an integer in the range of 1-10, and q is an integer in the range of 1-1000. In this embodiment, the most preferred form of linker moiety Z is derived from thiodiglycolic acid R 4

=R

5 n=m=l and Y=S).

The SUPPORT in the above formula is 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 glass, aluminosilicates, borosilicates, metal oxides aluminum oxide, iron oxide, nickel 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. The preferred solid support for use herein is conventional and may be selected from controlled pore glass bead or polystyrene beads. Further, the support may be either in particle WO 00/01711 PCT/CA99/00600 -19form beads), three-dimensional slabs polymeric inserts and foams) or in a flat two-dimensional like format plastic sheets, glass chips, silicon wafers, etc.). The material used for the support may also be soluble in certain solvents liquid-phase supports), but can be precipitated or crystallized from other solvents.

The reusable linker of formula: Z- O- T- [SUPPORT] (again, Z is a linker moiety and T is an organic radical), may then be reacted with a conventional nucleoside-linker compound to produce another linker arm according to the present invention. This other linker arm has the following formula:

NUCLEOSIDE-Z-O-T-[SUPPORT]

wherein Z is a linker moiety and T is an organic radical. The discussion herein above with respect to Z and T applies equally here. Preferably, in the above formula, NUCLEOSIDE is a moiety selected from one of the following formulae: WO 00/01711 PCT/CA99/00600 R0 S 0 B* B RO B 0 0 S R 9

OR

1 0

O

-0

B

0 OR R 9 wherein R' and R' are the same or different and are hydrogen or a protecting group, R 9 is hydrogen (for deoxyribonucleosides or DNA) or -OR" (for ribonucleosides or RNA) wherein R" is hydrogen or a protecting group, and B' a nucleic acid base. Thus, in the case of RNA, there are two hydroxyl groups which may be protected. Also, the linker can be attached to either the or (if ribose) hydroxyl positions. Indeed, for RNA sequences, it makes little difference whether the ester linker formed between the nucleoside and the linker compound is at the or hydroxyl position of the nucleoside. Thus, those of skill in the art will recognize that the nucleoside may be protected or blocked at the various of its hydroxyl moieties.

Non-limiting examples of useful protecting groups may be selected from the group consisting of acetyl, chloroacetyl, methoxyacetyl, t-butyl phenoxyacetyl, phenoxyacetyl, trityl, methoxvtrityl, dimethoxytrityl (DMT), dialkylphosphite, pivalyl-isobutyloxycarbonyl, t-butyldimethylsilyl, phenoxyacetal, 9-phenylxanthen-9-yl (pixyl), tetrahydropyranyl, methoxytetrahydropyranyl, methoxymethyl, benzyloxymethyl, methoxyethoxymethyl, methylthiomethyl, dialkylphosphate, levulinyl, dimethylphenylsilyl, trimethylsilyl, isopropyldimethylsilyl, n. P B I so a- ^92e% WO 00/01711 PCTCA99/00600 -21diisopropylmethylsilyl, diethylisopropylsilyl, triisopropylsilyl, acetyl, benzoyl, pivaloyl, trifluoroacetyl, allyl, benzyl, o-nitrobenzyl, ohydroxystyryldimethylsilyl, 2-oxo-1,2-diphenylethyl, allyloxycarbonyl, monomethoxymethyl, nitroveratryloxycarbonyl, dimethoxybenzoin, dimethoxybenzoin carbonate, methylnitropiperonyl carbonate, fluorenylmethoxycarbonyl, 2-phenylsulfonylethoxycarbony, fluorophenylmethoxypiperidinyl and the like.

As is known in the art, the main prerequisite for the protecting group used on the 5'-hydroxyl position is its ability to be selectively removed without causing cleavage of the linker arm. Thus, the preferred protecting group for desired 5'-hydroxyl position(s) is the acid labile dimethoxytrityl group. The main prerequisite for protecting groups on other hydroxyl positions, is stability to the conditions used for removal of the above protecting group. These latter protecting groups may be removed by the same conditions used to cleave the linker (discussed below) or separate conditions. The preferred protecting groups for these positions are trialkylsilyl t-butyldimethylsilyl) or acetyl. Additional information may be obtained from the following references: 1. T. W. Greene and P. G. M. Nuts, "Protecting Groups in Organic Synthesis". Second Edition (1991), John Wiley and Sons, Inc., NY; 2. M. Schelhaas and H. Waldman, "Protecting Group Strategies in Organic Synthesis", Angew. Chemie Int. Ed.

Engl. 35, 2056-2083 (1996); 3. M. J. Gait, ed., "Oligonucleotide Synthesis A Practical Approach", IRL Press, Oxford (1984); 4. S. A. Narang, ed., "Synthesis and Applications of DNA and RNA", Academic Press, Inc., Orlando (1987); and S. Agrawal, ed., "Methods in Molecular Biology, Vol. Protocols for Oligonucleotides and Analogs", Humana Press, Totowa, NJ (1993);

I

WO 00/01711 PCT/CA99/00600 -22the contents of each of which are hereby incorporated by reference, for a discussion of other possible hydroxyl protecting groups.

The manner by which the desired nucleoside may be protected is conventional and within the purview of a person skilled in the art. See, for example United States patent 3,400,190 (Melby), United States patent 4,458,066 (Caruthers et the contents of each of which are hereby incorporated by reference.

A preferred method for production ofdeoxyribonucleosides in the context of the present invention is to use a nucleoside with a 5'-dimethoxytrityl protecting group and an appropriate exocyclic amino protecting group, N 6 dimethoxytrityl-2'-deoxyadenosine. N'-benzoyl-5'-dimethoxytrityl-2'deoxycytidine, 5'-dimethoxytrityl-N -isobutyryl-2'-deoxyguanosine, or dimethoxytritylthymidine.

A preferred method for production ofribonucleosides in the context of the present invention is to use a 5'-dimethoxytrityl protected nucleoside, with appropriate exocyclic amino protection, and no protecting groups on either of the or hydroxyl positions. The linker can then react with either one of the two adjacent hydroxyl groups (it doesn't matter which) to give a mixture of and 3'linkages. The unreacted hydroxyl groups may then be acetylated by treatment of the immobilized nucleoside with acetic anhydride. Alternatively, ribonucleosides which have a 5'-dimethoxytrityl group, appropriate exocyclic amino group protection, and either a 3'-hydroxyl protecting group or a mixture of and 3'protecting groups can be used. The 3'-protected compounds are generally unwanted isomers which are simultaneously produced when the 2'-hydroxyl position is protected and having little other use.

The reusable linker arm having the formula: Z-O- T- [SUPPORT]

I

WO 00/01711 PCT/CA99/00600 -23may be produced by a process comprising the step of reacting together the compound of Formulae I and II:

Z-OH

(I)

HO- T- [SUPPORT]

(II)

wherein Z and T are as defined above.

The reusable linker arm having the formula: NUCLEO SIDE- Z-0- T-[SUPPORT] comprises the step of reacting together the compounds of Formulae I, II and III:

HO-Z-OH

(I)

HO-T-[SUPPORT]

(II)

NUCLEOSIDE- OH (mI) wherein Z and T are as defined above.

The compounds of Formulae I and II or of Formulae I, II and III (depending on which version of the present linker arm is being produced) are preferably reacted in the presence of an activating agent. As used throughout this specification, the term "activating group" is intended to have a broad meaning and is intended to encompass electrophilic reagents capable of activating a WO 00/01711 PCT/CA99/00600 -24carboxyl moiety on the linking compound of Formula II) by attachment of a leaving group to the acyl carbon of the carboxl moiety see, for example,

M.

Bodanszky, "Principles of Peptide Synthesis", Second Edition, Springer-Verlag, Berlin (1993), the contents of which are hereby incorporated by reference. Thus, the activating agent should be capable of initiating at least one of the following: formation of a reactive acylating agent (this is an example ofa derivate) from the carboxyl moeiy in a separate step or steps, followed by immediate treatment with the amino component (in this case, for example, an amino-terminated support) to form an amide linkage or a hydroxy component (in this case a hydroxy-terminated support or a hydroxyl group on the desired nucleoside) to form an ester linkage; formation of an isolable acylating agent, separately, optionally with purification prior to treatment with the amino or hydroxy component as discussed in and formation of an acylating intermediate in the presence of the amino/hydroxy component, by the addition of an activating agent to a mixture of the two components. Thus, each of and are applicable to the formation of both carboxylic esters and amides and all three routes can be used to attach nucleosides to supports.

For example, the Letsinger method, which first reacts oxalyl chloride with triazole, and then adds a nucleoside to the resulting oxalyl triazolide is an example of route Conversion of the carboxylic acid group into an "active" ester using either p-nitrophenol, or di-, tri-, tetra-, or penta- chlorinated or fluorinated phenols, or N-hydrosuccinimide are common examples of route Route has been the most commonly used method in recent years and both the carbodiimide reagents (dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)ethylcarbodiimide, and diisopropylcarbodiimide) and uronium reagents azabenzotriazol-1 11,3,3-tetramethyluronium hexafluorophosphate

(HATU),

2-(1H-benzotriazol-l-yl)- .1,3,3-tetramethyluronium hexafluorophosphate, (HBTU)) may be used in this approach.

In a preferred elmbodiment, in addition to an activating reagent, the reaction of the compounds of Formulae I, II and III is conducted in the presence of a nucleophilic catalyst or additive (typically 4-dimethylamino pyridine (DMAP), 1-hydroxybenzotriazole (HOBt), or 1-hydroxy-7-azabenzotriazole WO 00/01711 PCT/CA99/00600 (HOAt)) to speed up the reaction and a tertiary amine base (typically triethylamine, pyridine, or diisopropylethylamine) to ionize the carboxylic acid group.

Thus, those of skill in the art will recognize that the precise nature of the activation agent is not particularly restricted provided, of course, that the activated carboxylic acid group is capable of initiating formation of the ester or amide linkage, as appropriate, and the activating reagent does not have any deleterious effect on the desired nucleoside.

Thus, activation of the carboxylic acid 'by conversion into an acid chloride, an active ester nitrophenyl, nitrophenylthio, trichlorophenyl, trifluorophenyl, pentachlorophenyl, pentafluorophenyl, or 3-hydroxy-2,3dihydro-4-oxo-benzotriazine esters); an active hydroxylamine ester Nhydroxyphthalimide or N-hydroxysuccinimide); acid anhydride; or mixed anhydride will produce derivates which will form the desired linkage, and thus, these strategies are encompassed herein.

Non-limiting examples of activating agents may be selected from the group consisting of arylsulfonyl chlorides benzenesulfonyl chloride (BS- Cl), mesitvlenesulfonyl chloride (MS-C1), triisopropylsulfonylchloride

(TPS-CI));

active arvlsulfonyl esters imidazole. triazole, nitrotriazole, ortetrazole esters ofBS-CI. MS-Cl or TPS-C1); 2-ethoxy- 1 -(ethoxycarbonyl)-1 ,2-dihydroquinoline (EEDQ); acyl carbonates; 1,1 '-(carbonyldioxy)dibenzotriazoles; chlorotrimethylsilane; carbodiimides dicyclohexylcarbodiimide (DCC), 1-(3dimethylaminopropyl)-ethylcarbodiimide (DEC), diisopropylcarbodiinmide

(DIC))

either alone or in combination with auxillary nucleophiles 1hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), Nhydroxysuccinimide (HOSu), or 3-hydroxy-3,4-dihydro- 1,2,3-benzotriazin-4-one (HOObt)) and/or catalysts 4-dimethylaminopyridine (DMAP) or Nmethylimidazole or uronium salts tetramethyluronium chloride (TMU-CI), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3tetramethyluronium tetrafluoroborate (TBTU), 2-succinimido-1,1,3,3tetramethyluronium tetrafluoroborate (TSTU), 2-(3,4-dihydro-4-oxo- 1,2,3- WO 00/01 71 1 PCT/CA99/00600 -26benzotriazin-3-y)- 1,1 ,3),3-tetramethyluroiun tetrafluoroborate (TDBTU), 2-(2oxo-1(2H)-pyridYl-l ,L3--3-tetraJTethyluronium tetrafluoroborate (TPTU), norbornene-2,3-dicarboximido)- 1,1,3 ,3-tetramethylurolium tetrafluoroborate (TNTU), O-(7-azabenzotriazol- 1-yl)-l ,3-dimethyl- 1,3 -dimethyleneuron urnhexafluorophosphate (HAMDU), O-(7-azabenzotriazol- l-yl)-l ,3 -dimethyl- 1,3-trimethyleneuroflium hexafluorophosphate (HAMTU), O-(7-azabenzotrazol- I1-yl)- 1,1,3 ,3-bis(pentamethylele)uroflium hexafluorophosphate (HAPipU), 0-(7azabenzotriazol- 1-vl)-l .bis(.tetramethylele)urolium hexafluorophosphate (HAPyU), O-(7-azabelzotriazol- l-yl)-l ,1,3 .,3-tetramethylurofliurn hexafluorophoSphate (HATU)) either alone or in combination with auxiliary nucleophiles I -hvdroxybenzotriazole (HOBt), 1 -hivdroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), or 3-hydroxy-3 ,4-dihydro- 1,2,3benzotriazin- 4 -ofle (HOObt)) and/or catalysts 4-dimethylaminopyridifle (DMA-P) orN-methylimidazole (NMI)) orphosphonium salts benzotriazol- 1-yI- oxytris(dimethylamilo)phosphoflium hexafl uo ropho sp hate (BOP), benzotriazole- 1 .yl-oxy-trispyrrolidiflophosphoflium hexafluorophosphate (PyBOP), 2-(benzotriazol-1 -yI)oxy- 1,3-dimethylimfidazoidiliuml hexafluorophosphate (BOL), bromo, tris(pyrrolidino)phOSphofliUm hexafluoropho sp hate (PyBroP), 7-azabenzotriazol-l -yloxytris- (dimethylamino)Phosphoflium hexafluorophosphate (AOP), and 7azabenzotriazol- 1 vIoxytris(pyrrolidino)phOSPhornium hexafluorophosphate (PyAOP)) either alone or in combination with auxiliary nucleophiles and/or catalysts (discussed above) will also produce the desired linkag-e.

Other examples of suitable activating reagents may be found in any of the following references: M. Bodanszky, "Principles ofPeptide Synthesis", Second Edition, Springer-Verlag, Berlin (1993); J. Jones, "Amino Acid and Peptide Synthesis", Oxford University Press, Oxford (1992); C! Grant, "Synthetic Peptides: A Users Guide", W. H. Freeman Co., NY (1992); WO 00/01711 PCT/CA99/00600 -27- E. Haslam, Tetrahedron, 36, pg. 2409, (1980); and M.-A-Ogliaruso and J. F. Wolfe, "Synthesis of Carboxylic Acids, Esters and Their Derivatives", John Wiley Sons, Chicester (1991); the contents of each of which are hereby incorporated by reference.

In producing the present linker arm, the order of reaction is not particularly restricted. Thus, in one embodiment (this is the preferred embodiment), the compounds of Formulae I and III are initially reacted to form a conjugate which is reacted with the compound of Formula II. In another embodiment, the compounds of Formulae I and II are initially reacted to form a conjugate which is reacted with the compound of Formula III.

The addition of compounds of Formulae I and III to Formula II, usually will not result in the quantitative conversion of each immobilized hydroxyl group into a derivatized ligand. Therefore, it is preferred that unreacted hydroxyl groups on the surface of the support be protected (capped) by reaction with a capping reagent. This will mitigate the free hydroxyl group participating in subsequent oligonucleotide chain extension reactions, resulting in defect sequences lacking the terminal nucleoside. Preferably, the capping reagent should be reversible so that the capping agent can be removed to regenerate the hydroxyl sites prior to the next round of support derivatization. Capping of the unreacted sites is conventional and can be performed by reaction with an activated carboxylic acid or anhydride to form an ester, or by addition of a protecting group, as described hereinabove. Thus, for example, t-butylphenoxyacetic anhydride, methoxyacetic anhydride or preferably chloroacetic anhydride, combined with 2,6-lutidine and N-methylimidazole in THF solution are useful examples of capping reagents.

With reference to Figure 2 there is illustrated a preferred pathway illustrating the use of the present linker arm in a recycled/regenerated manner.

In Figure 2, DMT refers to dimethoxytrityl and B refers to a nucleobase as described hereinabove. As will be apparent to those of skill in the art, the support is recycled after oligonucleotide cleavage and support regeneration to a point in WO 00/01711 PCT/CA99/00600 -28the reaction scheme where it may again be coupled with the HQPD-nucleoside conjugate for further oligonucleotide synthesis.

With further reference to "Oligo Synthesis" (Step in Figure 2, once the present linker arm has been produced, it may be used in the conventional manner to synthesize an oligonucleotide see, for example, United States patent 5,112,962 (Letsinger), incorporated by reference hereinabove. Once the oligonucleotide has been synthesized, it may be cleaved from the solid support to yield the free oligonucleotide and the support may then be regenerated see Step #4 of Figure 2.

The cleavage step comprises hydrolysis at the point of attachment of the initial nucleoside to the linking compound. The regeneration of the support involves the removal of two moieties: the removal of the structure represented by Formula I (above) from Formula II (above), which occurs simultaneously with the release of the oligonucleotide product, and (ii) the removal of the moiety used to protect (cap) unreacted hydroxyl sites of Formula II (above) on the support. Removal of these two moieties can occur simultaneously or separately to regenerate the support. Simultaneous removal of both moieties using only a single reagent is simpler but care should be taken to use reagents which will not deleteriously affect the oligonucleotide product. A two-step regeneration involving the removal of the oligonucleotide using one reagent (typically ammonium hydroxide) and then treatment of the support with a second reagent (which may be faster but otherwise damaging to the oligonucleotide product thereby necessitating use of a two-step regeneration) allows flexibility in the choice of capping and regeneration reagents.

The reagent used to effect cleavage is not particularly restricted and is within the purview of a person skilled in the art. Preferably, the reagent is a base mild enough not to damage the oligonucleotide product but sufficiently strong to effect rapid cleavage. Non-limiting examples of suitable reagents for this purpose may be selected from the group consisting of ammonium hydroxide, ammonium hydroxide/methanol, ammonia/methanol, ammonium hydroxide/methylamine, potassium carbonate/methanol, t-butylamine, ethylenediamine, methylamine, dimethylamine, trimethylamine/water and the like. Cleavage may also be PCT/CA99/00600 WO 00/01711 -29performed under neutral conditions using fluoride ion 1M tetrabutylammonium fluoride/THF ortriethylamine trihydrofluoride). The reagent used to remove the capping reagent from unreacted sites may consist of the above reagents or other stronger bases such as sodium or potassium hydroxide. In our preferred embodiment, ammonium hydroxide can be used to cleave the oligonucleotide product from the support, remove the HQPD linker arm, and cleave chloroacetyl protected hydroxyl groups in a single regeneration step. The preferred temperature for the cleavage and regeneration is room temperature, but higher or lower temperatures can be employed, subject to the limitations of the apparatus used.

With reference to Figure 3. there are illustrated specific preferred examples of hydroxyl reusuable linker arms falling within the scope of the present invention.

Embodiments of the invention will be illustrated in the following Examples which should not be construed as limiting the scope of the invention.

In the Examples, the following materials were used: 1. Long chain alkylamine (LCAA) or glycerol (Gly) derivatized controlled pore glass (CPG) beads (120/200 mesh) were obtained from CPG Inc (Lincoln Park, NJ); 2. Toyopearl AF-amino-650M and HW65F supports were obtained from TosoHaas (Montgomeryville,

PA);

3. Other supports were obtained from the manufacturers listed in Tables 1 and 2; 4. HQPD, Hydroquinone-O,O'-diacetic acid, commercially available from Lancaster Synthesis Ltd. (Lancashire, England); Ammonium hydroxide solutions (28-30%) and solvents were obtained from VWR Canlab (Edmonton, Alberta, Canada); 6. Capping solutions were formulated as either Cap A (acetic anhydride/2, 6-lutidine/THF in a volume ratio of 1:1:8) and Cap B (N-methylimidazole and THF in a volume ratio of 16:84) or Cap A (chloroacetic anhydride and THF, 17% by weight) and Cap B 6-lutidine and N-methylimidazole in THF in a volume ratio of 12:16:72);

I

WO 00/01711 PCT/CA99/00600 7. Anhydrous pyridine and acetonitrile, distilled from CaH,; 8. DIEA, diisopropylethylamine, reagent grade; 9. MeCN, acetonitrile, low water DNA synthesis grade; DMAP, 4-dimethylaminopyridine, reagent grade; 11. DEC, 1-(3-dimethylaminopropyl)-ethylcarbodiimide, reagent grade; 12. Sulfurizing reagent, Beaucage thiolating reagent, from Pharmacia Biotech, was used as a 0.05M solution in acetonitrile; and 13. HBTU, 2-(1H-benzotriazol- 1-yl)- 1,1,3,3-tetramethyluronium hexafluoro-phosphate, reagent grade; In the following Examples the amount of nucleoside (loading) on the insoluble supports was determined by spectrophotometric trityl analysis. In this procedure, a sample of support (4-5 mg) was accurately weighed directly into a 10 mL volumetric flask. A solution of dichloroacetic acid in 1,2-dichloroethane in a volume ration of 5:95 was then added to fill the flask. The contents were then thoroughly mixed and the absorbance of the orange coloured solution was measured at 503 nm using a Philips UV/Vis spectrophotometer. The nucleoside loading (in pmol/g of CPG) was then calculated as: Loading (Ao 03 x Vol x 1000) (Wt x 76) wherein As, 0 absorbance at 503 nm, Vol solution volume in mL, and Wt amount of CPG tested in mg. The accuracy of the trityl determination was approximately 2-3%.

Example 1 SYNTHESIS OF NUCLEOSIDE-3'-O-HODA

HEMIESTERS

deoxyribonucleoside (10 mmol), hydroquinone-O, O'-diacetic acid (15 mmol, 3.39 4-dimethylaminopyridine (1 mmol, 122 mg), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15 mmol, 2.88 g) were combined in a 100 mL round bottom flask equipped with a magnetic stir bar. Triethylamine (0.8 mL) and anhydrous

I

WO 00/01711 PCT/CA99/00600 -31pyridine (50 mL) were added to the flask and the contents were stirred at room -temperature overnight.

The reaction was checked by TLC methanol/chloroform). If more than a trace of starting nucleoside was visible, more 1-(3-dimethylaminopropyl)- 3-ethylcarbodiimide hydrochloride (2-5 mmol) was added to the reaction and stirring was continued for another day. When TLC showed complete disappearance of the starting nucleoside, the solution was concentrated by evaporation until a thick oil was formed. The oil was redissolved in chloroform 200 mL) and transfer to a separatory funnel. The chloroform solution was washed with aqueous sodium bicarbonate 100 mL x 2) and then water 100 mL x The funnel was slowly inverted to mix the two phases. The chloroform phase was collected and the aqueous phase was discarded. If an inseparable emulsion was formed, then either centrifugation (for small volumes) or (for large volumes) precipitation by addition of hexanes followed by filtration and redissolving the sticky precipitate back into chloroform can be performed.

The chloroform solution was added to anhydrous magnesium sulfate and mixed to remove residual moisture from the solution. The magnesium sulfate was filtered off, the filtrated was washed with a small amount of chloroform and then the chloroform solution was evaporated to dryness. A light brown foam, containing a mixture of diester and nucleoside hemiester sodium salt, was formed and solidified.

The hemiester sodium salt was converted into a more soluble pyridinium salt by dissolving the foam in pyridine 50-100 mL) and then adding AG X4 HI cation exchange resin (2 The mixture was stirred for approximately 5 minutes and then the ion exchange resin was filtered off. The pyridine solution was evaporated to dryness. A light brown foam formed and solidified. The sold was dried under vacuum overnight to remove excess pyridine.

I

WO 00/01711 PCT/CA99/00600 -32- Example2 PREPARATION OF 12-DIMETHOXYTRITYL- HYDROXYDO-DECANOIC ACID DERIVATIZED

SUPPORTS

This example describes the synthesis of a C 12 linker arm within the scope of the present invention and how it can be used to convert commercially available amino-derivatized supports into reusable hydroxyl-derivatized supports.

12-Hydroxydodecanoic acid (9.25 mmol) was coevaporated to dryness with pyridine Then pyridine 40 mL) and dimethoxytrityl chloride (10.2 mmol) were added. After stirring overnight, the solution was concentrated (to mL), diluted with CHC13 (50 mL), washed with aq. NH4HCO3 (3x) and water The crude material was then purified on a silica gel column by elution with a 1% TEA/CHC1, 4% MeOH/1 %TEA/CHCl1 gradient. The prodcut yield was 6.7 mmol of 12-dimethoxytrityl-hydroxydodecanoic acid as a brown oil.

An amino functionalized support (0.5 12-dimethoxytritylhydroxydodecanoic acid (0.2 mmol), 4-dimethylaminopyridine (0.1 mmol), 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.6 mmol), triethylamine (0.1 mL), and pyridine (7 mL) were shaken at room temperature (16 The support was filtered off, washed, and dried. Linker loading was determined by trityl analysis and the results are provided in Table 1. Unreacted amino and hydroxyl groups on the derivatized support (if present) were then acetylated by treating the support with equal volumes 1 M acetic anhydride/2,6lutidine/THF (Cap A) and 2M N-methylimidazole/THF (Cap B) reagents for 3 hours. The support was then filtered off, washed, and dried.

WO 00/01711 PCT/CA99/00600 -33- Table 1- Loading Results Using 12-Dimethoxvritvlhvdroxvdodecanoic Acid Linker Arm Linker arm Experiment Support loading (mol/g) 1 Pharmacia HL-30 amino primer support 216 2 Long chain alkylamine CPG 87 3 Amino Tentagel, Millipore 107 4 Tovopearl AF-amino-650M 217 Aminoethyl polystyrene, Hamilton 73 6 Aminnimethvl nnlvstvrene Applied Biosystems 8 s Example 3- DERIVATIZATION OF TOYOPEARL HW-65F SUPPORT WITH 1.4-BUTANEDIOL DIGLYCIDYL

ETHER

This Example describes how hydroxyl surface groups on commercially available Toyopearl HW65 supports are extended with a butane diglycidyl linker to create a reusable support.

Toyopearl HW-65F vinyl alcohol/methacrylic acid copolymer was obtained as a slurry in 500 ml 20% ethanol/water. This slurry was evaporated to dryness to yield of 90 g of dry support. The hydroxyl content of the dry support was determined, in triplicate, by derivatization with dimethoxytrityl chloride/tetrabutylammonium perchlorate and trityl analysis, to be 1,095 pmol/g.

The dry HW-65F support (25 1.0 M aqueous NaOH solution containing 1 mg/mL NaBH 4 (100 mL) and 1,4-butanediol diglycidyl ether mL) were shaken at room temperature (3.5 The support was filtered off and washed with water, acetonitrile, and then chloroform. After drying, DMT derivatization and analysis Reddy and P.J. Voelker, 1988, Int. J. Peptide Protein Res. 31, 345-348, the contents of which are hereby incorporated by reference) of a sample indicated 902 imol/g of remaining hydroxyl groups.

Therefore, the epoxide loading was estimated to be 193 gmolg.

WO 00/01711 PCT/CA99/00600 -34- The epoxide derivatized support (25 benzoic anhydride (51 4dimethylaminopyridine (6.6 g) and anhydrous pyridine (180 mL) were shaken at room temperature (overnight) to benzoylate unreacted hydroxyl groups. The support was filtered off, washed (methanol, then chloroform), and dried. DMT derivatization and analysis indicated that the residual hydroxyl group loading had decreased to only 5 imol/g.

The benzoylated support (25 THF (140 mL), and 2.9 N aqueous 4 (16.6 mL, 48 mmol) were shaken at room temperature (13 Trityl derivatization and analysis of an aliquot showed an hydroxyl loading of 98 pmol/g. Additional 2.9 N HCIO1 (34 mL) was added and shaking continued for another 3 h. The support was filtered off, washed, and dried and a final trityl derivatization and analysis indicated an hydroxyl loading of 103 tmol/g.

Example4- SYNTHESIS OF OLIGONUCLEOTIDE PHOSPHOROTHIOATES AND SUPPORT RECYCLING USING CHLOROACETIC ANHYDRIDE CAPPING.

This Example provides experiments which illustrate the suitability of a variety of different supports for repetitive oligonucleotide synthesis.

The following reagents were installed on a Perkin-Elmer/Applied Biosystems 394 4-column. 8-base position DNA synthesizer: Ports dABz, dG'

B

dCBz, and T phosphoramidites (0.2 M solutions).

Port 0.15 M 5'-dimethoxytrity-N 6 -benzoyl-2'-deoxyadenosine-3'-O-hydroquinone-O.O'-diacetyl hemiester pyridinium salt and 0.15 M diisopropylethylamine in anhydrous acetonitrile.

Port 0.15 M HBTU and 0.15M DMAP in anhydrous acetonitrile.

Port 0.45 M Tetrazole/acetonitrle.

Port #10: 28% Ammonium hydroxide.

I

WO 00/01711 PCT/CA99/00600 Port #11: 1 M Chloroacetic anhydride in THF (Cap A reagent).

Port #12: 1 M 2,6-Lutidine and 2 M N-methylimidazole in THF (Cap B reagent).

Port #14: 5% Dichloroacetic acid/1,2-dichloroethane.

Port 15: 0.05 M Beaucage reagent in acetonitrile.

Up to four synthesis columns, each containing one of the supports listed in Table 2, were installed on the synthesizer and, if necessary, manually detritylated to deblock the hydroxyl linker arm.

The synthesizer was then programmed to automatically execute the following steps: 1: A "Begin" procedure consisting of a column wash, nucleoside coupling to the support by simultaneous addition (4.0 sec) of nucleoside hemiester (port and coupling reagent (port and a 600 sec wait, column wash, capping of unreacted hydroxyl sites (Cap A B reagents, 300 sec), column wash. and priming of ports #1, 2, 3, 4, and 9.

2: Synthesis of the 20-base phosphorothioate oligonucleotide sequence dGCCCAAGCTGGCATCCGTCA (Trityl-off).

3: A 15 minute ammonium hydroxide hydrolysis step to cleave the oligonucleotide from the support.

After completion of the ammonium hydroxide hydrolysis, the columns were removed from the synthesizer, manually treated with 0.05 M potassium carbonate/methanol solution (5 min), rinsed with methanol, dried by aspiration WO 00/01711 PCT/CA99/00600 -36min), re-installed on the synthesizer, and rinsed with anhydrous acetonitrile.

The automated synthesis was then repeated Steps 1,2, and 3 above) using the same synthesis column a total of twelve times.

The amount of trityl color released after the first detritylation step was collected and quantitated to determine the amount of nucleoside added to the support the results are reported in Table 3. The released oligonucleotide solution was deprotected (55 0 C. 16 evaporated to removed ammonia, and quantitated by UV at 260 nm the results are reported in Table 4. The correct identity of the products, obtained from each of the results shown Table 4, was verified by electrophoresis and comparison to authentic material. Furthermore, no unusual impurities, attributable to the support recycling were present. These results confirmed that each of the nine supports used in this experiment could be reused and in several cases satisfactory results (comparable to new supports) were obtained, even after six or more uses.

SYNTHESIS OF OLIGONUCLEOTIDE PHOSPHOROTHIOATES AND SUPPORT RECYCLING USING METHOXYACETIC ANHYDRIDE

CAPPING

This Example illustrates the use of methoxyacetic anhydride as the capping reagent instead ofchloroacetic anhydride used in the previous Examples.

The automated DNA synthesizer was set-up with reagents, as described in Example 4, with the exception of the Cap A and B reagents, which were as follows: Port #10: 0.5 M Methoxyacetic anhydride and 0.5 M 2,6-lutidine in acetonitrile (Cap A).

Port #12: 1 M N-Methylimidazole in acetonitrile (Cap B).

The automated nucleoside derivatization, oligonucleotide synthesis, and support recycling procedure was then performed using the supports listed in WO 00/01711 PCT/CA99/00600 -37- Table 5 and the procedure described in Example 4. However, because of the greater stability of the methoxyacetyLgroup themanual column regeneration step with 0.05M potassium carbonate/methanol was increased from 5 min to 15 min.

The amount of trityl color released after the first detritylation step was collected and quantitated to determine the amount of nucleoside added to the support the results are reported in Table 6. The released oligonucleotide solution was deprotected (55°C, 16 evaporated to removed ammonia, and quantitated by UV at 260 nm the results are reported in Table 7. The composition of the products obtained in Table 7 was examined by gel electrophoresis and the expected products were obtained in each case. This indicated that methoxyacetic anhydride could also be used as a satisfactory capping reagent during the support recycling.

0 Table 2 Supports Used For Phosphorothioate Synthesis and Support Recycling [Experiment ISupport Linker Arm Amount used (mg) 1 Long chain alkylamine C PG Hydroxyhiexylsuicciinyldiamiide 16.2 2 Long chain alkylainine CPG Ilydroxydodecanoic acid 21.1 3 Glycerol CPG 13.3 4 Toyopearl AF-amnino-650M Hydroxydodecanoic acid Aminoethyl polystyrene, Hamilton Hydroxydodecanoic acid 21.5 6 Aminomnethyl polystyrene, Applied Biosystemis llydroxydodecanoic acid 34. 1 7 Pharmacia hydroxyl primer support': Butanediol diglycidyl 14.5 8 Toyopearl H-IW65F Butanediol diglycidyl 15.4 9 Hydroxyethyl polymvethac ry Iate/poly sty re ne, Hamilton 27.3 *proprietary material supplied by Phiarmiacia Table 3 Tab 3 -N cleoside loading obtained after repetitive synthesis on the same support FExuoeritnent1 Synthesis II and First Nucleoside Loading Level (ltrnol/g)

I

2 13 1 4 j1 1 6 1 7 8 9

I

10 1 11 I t I L I r r I r I I I 2 3 62 60 57 58 54 56 54 54 54 62 51 49 56 46 72 68 65 6 7 64 64 62 61 60 57 55 54 4 6 75 7 76 7 70 57 47 40 43 6 46 30 35 35 3 38 35 35 3 1 3 7 3 7 8 155 111 j 107 97 122 1115 95 1, 122187 1 110 1 01 8 III 104 102 71 66 Ii I I '1 I I 1 1 9 1 34 I 1 48 I 48 49 51 67 9_ _I Table 4 Amount of Crude Oligonucleotide Produced From Repetitive Syntheses on the Same Support Synthesis and amount of crude product produced units) Experiment 1 2 3 4 5 6 7 8 9 10 11 12 1 7530 7590 7530 7650 6790 6850 5740 5930 4750 3640 2780 2350 2 7630 7680 6590 6970 6680 6680 6110 5170 3460 2420 2700 1850 3 8350 7740 8270 7970 8120 8120 7440 7370 6690 6170 5190 4660 4 12200 11500 12600 11700 11600 11800 9880 6710 5850 4750 3540 2550 8010 8340 7990 7960 7810 6720 5690 3900 3280 3780 2550 2050 6 3610 4300 4220 4540 4830 4560 4630 4620 4300 3730 3390 2750 7 9300 8190 7460 7620 8550 7390 5900 4900 2280 4180 2430 1750 8 11520 11100 10600 11400 11500 11400 9980 n.d. n.d. 8390 5060 6270 9 2360 3260 4520 4630 5290 6420 6610 n.d. n.d. 8570 6420 6830 Table 5 Supports Used For Oigonucleotide Phosphorothioate Synthesis and Support Reccin Experiment Support Linker Arm Amount used (mg) I Glycerol CPG 14.5 2 1Toyovearl 11W65F IB utanediol diglycidyl 14.6 Table 6 NuIcleoside Loadiig Obtainecd Afte r Renetitive Syntheisis oil thle Samle Supoport Table 6 Table 7 Amount of Crude Oligonticleotide Produced From Repetiive Synthecses on the Same Support I Synthesis ft and amount of crude product produced, (A 260 units)

II

1 1 I I I I1 Exp~ien I I 1 4 5 16 7 8 10 1It 12

II

-1I750 7570 7850 _6910 -17500 }_7440 )7400 _680 6810 61 2 848 18201 10200 10900 I9490 110200_]130__0700_j 10000 9190 8450 16170' Documentl5-20/10/03 -41A- Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims (171)

1. A reusable linker arm for solid support oligonucleotide synthesis, the linker arm comprising the following formula: Z-O- T- [SUPPORT wherein Z is a linker moiety and T is an organic radical.

2. The reusable linker arm defined in claim 1, wherein T contains at least one carbon.

3. The reusable linker arm defined in claim 1, wherein T is a CI-C 300 organic moiety.

4. The reusable linker arm defined in claim 1, wherein T is a CI-C 20 0 organic moiety. moiety. The reusable linker arm defined in claim 1, wherein T is a C,-Co 00 organic

6. The reusable linker arm defined in claims 1-5, wherein T is a saturated organic moiety.

7. The reusable linker arm defined in claims 1-5, wherein T is an unsaturated organic moiety.

8. The reusable linker arm defined in claim 1, wherein T is a C -C 30 0 organic moiety comprising at least one heteroatom selected from N and 0. PCT/CA9900600 WO 00/01711 -43-

9. The reusable linker arm defined in claims 1-8, wherein the organic moiety comprises at least-one-moiety having the formula: O SII The reusable linker arm defined in claims 1-8, wherein the organic moiety comprises at least one moiety having the formula:

11. The reusable linker arm defined in claims 1-8, wherein the organic moiety comprises at least one moiety having the formula: O I I N(H)C-

12. The reusable linker arm defined in claims 1-8, wherein the organic moiety comprises at least one moiety having the formula: -C-0-C

13. The reusable linker arm defined in claims 1-8, wherein organic moiety comprises at least one moiety having the formula: PCT/CA99/00600 WO 00/01711 -44- 0 -C-0-

14. The reusable linker arm defined in claims 1-13, wherein the organic moiety is unsubstituted.

15. The reusable linker arm defined in claim 14, wherein the organic moiety is substituted by at least one moiety selected from the group comprising a alkyl group, a C,-C 4 o aryl group, a CI-C 40 alkoxy group, a C,-C4o ester group,a C,- Co hydroxy group, a C,-C 4 o acrylate group and a C 5 -C 40 alkylaryl group.

16. The reusable linker arm defined in claims 1-15, wherein T has the formula: CH2O-CH2-CH2- OCH2s wherein q and s are the same or different and each is an integer having a value of 0-40 and r is an integer having a value of 1-200.

17. The reusable linker arm defined in claim 16, wherein q and s are the same or different and each is an integer having a value of 1-20 and r is an integer having a value of 1-150.

18. The reusable linker arm defined in claims 1-15, wherein T has the formula: WO 00/01711 PCT/CA99/0060 0 Ra EQCH 2 CHCHOC2 wherein a is 0 or 1, Q is an organic moiety, W~ is selected from -OH, -Nil 2 -NPR and -OR wherein R is a protecting group and b is an integ5er having a value of 0-

19. The reusable linker arm defined in claim 18, wherein a is 0 and R 8 is- OH.. The reusable linker arm defined in claim 18, wherein a is I and R' is -NR or -OR.

21. The reusable linker arm defined in claims 18-20, wherein the protecting group is selected from the group compri sing acetyl, chioroacetyl, methoxyacetyl, t-butyl phenoxyacetyl, phenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl (DMT). dialkyiphosphite, pivalyl-isobutyloxvcarbonyl, t-butyldimethyIsilyl, 9- phenylxanthen- 9 -yl (pixyl), tetrahydropyranyl, methoxytetrahydropyranYl, methoxymethyl, benzyloxymethyl, methoxyethoxynethyl, methyithiomethyl, dialkyiphosphate, levulinyl, dimethyiphenylsilyl, trimethylsilyl, isopropyl- dimethylsilyl, diisopropylmethYl5ilYl, diethylisopropylsilYl, triisopropylsilyl, benzoyl, pivaloyl, trifluoroacetyl, allyl, beuzyl, o-nitrobenzyl, o- hydroxystyryldmethylsilyl, 2-oxo-1I,2-diphenylethyl, allyloxycarbonyl, monomethoxymethyl, nitroveratryloxycarbonyl, dimethoxybelzolf, dimethoxybenzoin carbonate, methylnitropiperoflYl carbonate, fluorenyl- methoxycarbonyl, 2-phenylsulfonylethoxycarbony, fluoropheflyl- methoxypiperidinyl and mixtures thereof. WO 00/01711 PCT/CA99/00600 -46-

22. The reusable linker arm defined in claim 18, wherein Q comprises a moiety having the formula: R a -ECH24CH ECH2-O-CH2j O-CH- 2 wherein q, r, s, t and u are the same or different and each is an integer having a value of 0-40 and R" is selected from the group comprising hydrogen, hydroxyl, a Ci-C 40 alkyl group, a aryl group, a CI-C4 alkoxy group, a C,-C 40 ester group,a C,-C4 hydroxy group, a C,-C 4 0 acrylate group, a C;-Co alkylaryl group, -NH 2 -NHR and -OR, wherein R is a protecting group.

23. The reusable linker arm defined in claim 22, wherein s is 0, q, r and u are the same or different and each is an integer having a value of 1-10, t is an integer of 1-5 and R a is hydroxyl.

24. The reusable linker arm defined in claims 1-15, wherein T has the formula: -E QaCH-CH-CH 2 -O-CH 2 wherein a is 0 or 1, Q is an organic moiety, R' is selected from -OH, -NR and -OR wherein R is a protecting group and b is an integer having a value of 0-

25. The reusable linker arm defined in claim 24, wherein a is 0 and R 8 is -OH. PCT/CA99/00600 WO 00/01711 -47-

26. The reusable linker arm defined in claim 24, wherein a is 1 and R a is -NR

27. The reusable linker arm defined in claim 18, wherein Q is a C,-Coo organic moiety.

28. The reusable linker arm defined in claim 18, wherein Q is a saturated organic moiety.

29. The reusable linker arm defined in claim 18. wherein Q is an unsaturated organic moiety. The reusable linker arm defined in claim 18, wherein T is a CI-Coo organic moiety comprising at least one heteroatom selected from N and 0.

31. The reusable linker arm defined in claims 27-30, wherein the organic moiety comprises at least one moiety having the formula: 0 I1 -C-

32. The reusable linker arm defined in claims 27-30, wherein the organic moiety comprises at least one moiety having the formula:

33. The reusable linker arm defined in claims 27-30, wherein the organic moiety comprises at least one moiety having the formula: I PCT/CA9900600 WO 00/01711 -48- 0

34. The reusable linker arm defined in claims 27-30, wherein the organic moiety comprises at least one moiety having the formula: The reusable linker arm defined in claims 27-30, wherein organic moiety comprises at least one moiety having the formula: O II -C-O-

36. The reusable linker arm defined in claim 27-35, wherein the organic moiety is unsubstituted.

37. The reusable linker arm defined in claim 27-35, wherein the organic moiety is substituted by at least one moiety selected from the group comprising a C,-C 4 0 alkyl group, a C 5 -C 40 aryl group, a Ci-Co 4 alkoxy group, a C,-C 4 0 ester group,a CI-C 40 hydroxy group, a C,-C 4 0 acrylate group and a C 5 -C 4 0 alkylaryl group.

38. The reusable linker arm defined in claim 18, wherein Q has the formula: PCT/CA99/00600 WO 00/01711 -49- 0 0 -ECH3-C-NH CH2 NH-CCH2 wherein each of x, y and z is an integer having a value of 1-40.

39. The reusable linker arm defined in claims 1-38, wherein Z has the following formula: 0 0 II it HO C H-CH 2 C-- The reusable linker arm defined in claims 1-38, wherein Z has the following formula: 0 0 II II HO-C-CH2 -O-CHkC--

41. The reusable linker arm defined in claims 1-38, wherein Z has the following formula: WO 00/01711 PCT/CA99/00600 0 0 II II HO-C-C-

42. The reusable linker arm defined in claims 1-38, wherein Z has the following formula: R R 2 0 HO-C(R R C)nX A R 3 B 1 wherein: R 2 and R 3 are the same or different and are selected from the group consisting of hydrogen, halide, a substituted or unsubstituted C,-C, 0 alkyl group, a substituted or unsubstituted C 5 -C 30 aryl group and a substituted or unsubstituted C 5 -C 4 o alkylaryl group; R 4 and R 5 are the same or different and are selected from the group consisting of hydrogen, a substituted or unsubstituted C,-C, 0 alkyl group, a substituted or unsubstituted C 5 -C 3 0 aryl group and a substituted or unsubstituted C 5 -C 4 0 alkylaryl group; X' is selected from the group consisting of -S(0) 2 and R is selected from the group comprising hydrogen, a substituted or unsubstituted C,-C 2 0 alkyl group, a substituted or unsubstituted C 5 -C 3 0 aryl group and a substituted or unsubstituted C.-C 4 alkylaryl group; n is 0, 1 or 2; and one of A' and B' is selected from the group consisting of hydrogen, halide, a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted C 5 -C 30 aryl group and a substituted or unsubstituted C 5 -C4o alkylaryl group, and the other of A' and B' has the formula: WO 00/01711 PCT/CA99/00600 -51- SX (CR R )mC-- O JP wherein p is 0 or 1, X 2 is selected from the group consisting of-O-, -S(O) 2 and R is selected from the group comprising hydrogen, a substituted or unsubstituted C,-C 2 0 alkyl group, a substituted or unsubstituted C 5 C 30 aryl group and a substituted or unsubstituted C,-Co alkylaryl group, R 6 and R 7 are the same or different and are selected from the group comprising hydrogen, a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted C 5 -C,3 aryl group and a substituted or unsubstituted C 5 -C 4 0 alkylaryl group, and m is 0, 1 or 2.

43. The reusable linker arm defined in claim 42, wherein p is 0.

44. The reusable linker arm defined in claims 42-43, wherein B' is selected from the group consisting of hydrogen, halide, a substituted or unsubstituted C,- C 2 o alkyl group, a substituted or unsubstituted C,-C 3 0 aryl group and a substituted or unsubstituted CS-Co 4 alkylaryl group. The reusable linker arm defined in claims 42-44, wherein each ofR 4 R 5 R 6 and R 7 is hydrogen.

46. The reusable linker arm defined in claims 42-45, wherein each ofm and n are 1.

47. The reusable linker arm defined in claims 42-46, wherein each ofR', R 2 and R 3 is hydrogen. PCT/CA99/00600 WO 00/01711 -52-

48. The reusable linker arm defined in claims 42-47, wherein X' and X 2 are both

49. The reusable linker arm defined in claims 1-48, wherein SUPPORT is an inorganic substance. The reusable linker arm defined in claim 49, wherein the inorganic substance is selected from the group consisting of silica, glass beads, porous glass, aluminosilicates, borosilicates, metal oxides, clays and mixtures thereof.

51. The reusable linker arm defined in claims 1-48, wherein SUPPORT is an organic substance.

52. The reusable linker arm defined in claim 51, wherein the organic substance is a cross-linked polymer.

53. A reusable linker arm for solid support oligonucleotide synthesis, the linker arm comprising the following formula: NUCLEO SIDE- Z-O- T-[SUPPORT] wherein Z is a linker moiety and T is an organic radical.

54. The reusable linker arm defined in claim 53, wherein T contains at least one carbon. The reusable linker arm defined in claim 53, wherein T is a C,-C300 organic moiety. WO 00/01711 PCT/CA99/00600 -53-

56. The reusable linker arm defined in claim 53, wherein T is a Ci-C200 organic moiety.

57. The reusable linker arm defined in claim 53, wherein T is a C,-CIoo organic moiety.

58. The reusable linker arm defined in claims 53-57, wherein T is a saturated organic moiety.

59. The reusable linker arm defined in claims 53-57, wherein T is an unsaturated organic moiety. The reusable linker arm defined in claims 53-57, wherein T is a C,-C3oo organic moiety comprising at least one heteroatom selected from N and 0.

61. The reusable linker arm defined in claims 53-60, wherein the organic moiety comprises at least one moiety having the formula: 0 II C-

62. The reusable linker arm defined in claims 53-60, wherein the organic moiety comprises at least one moiety having the formula:

63. The reusable linker arm defined in claims 53-60, wherein the organic moiety comprises at least one moiety having the formula: PCT/CA99/00600 WO 00/01711 -54- 0" 1I

64. The reusable linker arm defined in claims 53-60, wherein the organic moiety comprises at least one moiety having the formula: The reusable linker arm defined in claims 53-60, wherein organic moiety comprises at least one moiety having the formula: O I I -C-O-

66. The reusable linker arm defined in claims 53-65, wherein the organic moiety is unsubstituted.

67. The reusable linker arm defined in claims 53-65, wherein the organic moiety is substituted by at least one moiety selected from the group comprising a C,-C4o alkyl group, a C 5 -C 4 0 aryl group, a C 1 -C 4 0 alkoxy group, a C,-C4o ester group,a C,-C 40 hydroxy group, a C,-C 40 acrylate group and a C,-C 4 0 alkylaryl group.

68. The reusable linker arm defined in claims 53-67, wherein T has the formula: PCT/CA99/00600 WO 00/01711 -CH 23 -fO-CH 2 -CH 2 03-C1H2 qr wherein q and s are the same or different and each is an integer having a value of 0-40 and r is an integer having a value of 1-200.

69. The reusable linker arm defined in claim 68, wherein q and s are the same or different and each is an integer having a value of 1-20 and r is an integer having a value of 1-150. The reusable linker arm defined in claims 53-67, wherein T has the formula: R b Q aCHf-CH-CH2-0- CH2] wherein a is 0 or 1, Q is an organic moiety, R" is selected from -OH, -NR and -OR wherein R is a protecting group and b is an integer having a value of 0-

71. The reusable linker arm defined in claim 70, wherein a is 0 and R 8 is OH..

72. The reusable linker arm defined in claim 70, wherein a is 1 and R" is -NR or -OR. WO 00/01711 WO 0001711PCT/CA99/00600 -56m-

73. The reusable linker arm defined in claims 70-72, wherein the protecting group is selected from the garoup comprising acetyl, chioroacetyl, methoxyacetyl, t-butyl phenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl (DMT), dialkyiphosphite, pivalyl-isobutyloxycarboyll t-butyldimethylsilyl, phenoxyacetal, 9-phenylxanthen-9-yl (pixyl), tetrahydropyranyl, methoxytetrahydropyranyl, methoxymethyl, benzyloxymethyl, methoxyethoxymethyl. methyithiomethyl, dialkyiphosphate, levulinyl, dimnethylpheflylsilyl, trirnethylsilyl, isopropyldimethylsilyl, dilsopropylmethylsilyl, diethylisopropylsilyl, triisopropylsilyl, benzoyl, pivaloyl, trifluoroacetyl, allyl, benzyl. o-nitrobenzyl, o-hvdroxystyryldimethylsilyl, 2-oxo- 1,2 -diphenyl ethyl, allyloxycarbonyl, mono-methoxymethyl, nitroveratryloxycarbonyl, dimethoxybenzoin, dimethoxybenzoin carbonate, methylnitropiperonyl carbonate, fluorenylmethoxycarboflyl, 2"-phenylsulfonyl- ethoxycarbony, fluoropheny-methoxypiperldilyl and mixtures thereof.

74. The reusable linker arm defined in claim 70, wherein Q comprises a moiety having the formula: Ra ECH 2 3 YECH t ECH2]-0±__Il oH]- -cH 2 wherein q, r, s, t and u are the same or different and each is an integer having a value of 0-40 and R' is selected from the group comprising hvdrogen, hydroxyl, a CI-C 40 alkyl group, a C.-C, 4 aryI group, a CX- 4 alkoxy group, a C 1 -C 4 ester group,a C 1 -C 40 hydroxy group, a CI-C 40 acrylate group, aC,-C 4 0 alkylaryl group, -NH 2 -NI{R and -OR, wherein R is a protecting group. The reusable linker arm defined in claim 74, wherein s Is 0, q, r and u are the same or different and each is an integer having a value of 1 -10. t is an integer of 1-5 and R' is hydroxyl. WO 00/01711 PCT/CA99/00600 -57-

76. The reusable linker-arm-defned in claim 70, wherein T has the formula: R a L fCH2TCH2-iCH2-H wherein a is 0 or 1, Q is an organic moiety, R' is selected from -OH, -NR and -OR wherein R is a protecting group and b is an integer having a value of 0-

77. The reusable linker arm defined in claim 76, wherein a is 0 and R" is -OH.

78. The reusable linker arm defined in claim 76, wherein a is 1 and R" is -NR or -OR.

79. The reusable linker arm defined in claims 53-78, wherein Q is a C,-C,0o organic moiety. The reusable linker arm defined in claims 53-78, wherein Q is a saturated organic moiety.

81. The reusable linker arm defined in claims 53-78, wherein Q is an unsaturated organic moiety.

82. The reusable linker arm defined in claims 53-78, wherein T is a C-Ctoo organic moiety comprising at least one heteroatom selected from N and 0.

83. The reusable linker arm defined in claims 76-82, wherein the organic moiety comprises at least one moiety having the formula: WO 00/01711 PCT/CA99/00600 0 II -C-

84. The reusable linker arm defined in claims 76-82, wherein the organic moiety comprises at least one moiety having the formula: The reusable linker arm defined in claims 76-82, wherein the organic moiety comprises at least one moiety having the formula: 0 II -N(H)C-

86. The reusable linker arm defined in claims 76-S2. wherein the organic moiety comprises at least one moiety having the formula:

87. The reusable linker arm defined in claims 76-82. wherein organic moiety comprises at least one moiety having the formula: 0 II WO 00/01711 PCT/CA99/00600 -59-

88. The reusable linker arm defined in claims 76-87, wherein the organic moiety is unsubstituted----

89. The reusable linker arm defined in claims 76-87, wherein the organic moiety is substituted by at least one moiety selected from the group comprising a C,-C 40 alkyl group, a C 5 -C 40 aryl group, a C,-C 40 alkoxy group, a C,-C 40 ester group,a C,-C 4 0 hydroxy group, a C,-C 4 o acrylate group and a C 5 -C 4 0 alkylaryl group.

90. The reusable linker arm defined in claim 53, wherein Q has the formula: O O CH 2 3 NHCH2-,NH-E CH2E- wherein each of x, y and z is an integer having a value of 1-40.

91. The reusable linker arm defined in claims 53-90, wherein Z has the following formula: O O II II -C-CH2-CH2-C-

92. The reusable linker arm defined in claims 53-90, wherein Z has the following formula: WO 00/01711 PCT/CA99/00600 O 0 II II -C-CH 2 -O-CH 2 -C-

93. The reusable linker arm defined in claims 53-90, wherein Z has the following formula: 0 0 II II -C-C-

94. The reusable linker arm defined in claims 53-90, wherein Z has the following formula: O II 4 -C(R R C)nX wherein: R 2 and R 3 are the same or different and are selected from the group consisting of hydrogen, halide, a substituted or unsubstituted Ci-C20 alkyl group, a substituted or unsubstituted C 5 -C 30 aryl group and a substituted or unsubstituted C5-C 4 0 alkylaryl group; R 4 and R 5 are the same or different and are selected from the group consisting of hydrogen, a substituted or unsubstituted C,-C20 alkyl group, a substituted or unsubstituted C,-C 30 aryl group and a substituted or WO 00/01711 PCT/CA99/00600 -61- unsubstituted C 5 -C 4 0 alkylaryl group; X' is selected from the group consisting of -S(C)--and R is selected from the group comprising hydrogen, a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted Cs-C 3 0 aryl group and a substituted or unsubstituted C,-C 40 alkylaryl group; n is 0, 1 or 2; and one of A' and B' is selected from the group consisting of hydrogen, halide, a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted C5-C 3 0 aryl group and a substituted or unsubstituted C,-C 40 alkylaryl group, and the other of A' and B' has the formula: 0 \X2(CR R)mC- p wherein p is 0 or 1, X 2 is selected from the group consisting of-0-, -S(0) 2 and R is selected from the group comprising hydrogen, a substituted or unsubstituted C,-C 2 0 alkyl group, a substituted or unsubstituted C 5 C 30 aryl group and a substituted or unsubstituted C,-C 4 0 alkylaryl group, R 6 and R' are the same or different and are selected from the group comprising hydrogen, a substituted or unsubstituted C,-C,o alkyl group, a substituted or unsubstituted C,-C 30 aryl group and a substituted or unsubstituted C 5 -C 40 alkylaryl group, and m is 0, 1 or 2. The reusable linker arm defined in claim 94, wherein p is 0.

96. The reusable linker arm defined in claims 94-95, wherein B' is selected from the group consisting of hydrogen, halide, a substituted or unsubstituted CI- C 20 alkyl group, a substituted or unsubstituted C 5 -C 3 0 aryl group and a substituted or unsubstituted C,-Co alkylaryl group. WO 00/01711 PCT/CA99/00600 -62-

97. The reusable linker arm defined in claims 94-96, wherein each ofR 4 R 5 R 6 and R 7 is hydrogen--

98. The reusable linker arm defined in claims 94-97, wherein each ofm and nare 1.

99. The reusable linker arm defined in claims 94-98, wherein each ofR', R 2 and R 3 is hydrogen.

100. The reusable linker arm defined in claims 94-99, wherein X' and X 2 are both

101. The reusable linker arm defined in claims 53-100, wherein SUPPORT is an inorganic substance.

102. The reusable linker arm defined in claim 101, wherein the inorganic substance is selected from the group consisting of silica, glass beads, porous glass, aluminosilicates, borosilicates, metal oxides, clays and mixtures thereof.

103. The reusable linker arm defined in claims 53-100, wherein SUPPORT is an organic substance.

104. The reusable linker arm defined in claim 103, wherein the organic substance is a cross-linked polymer.

105. The reusable linker arm defined in claims 53-104, wherein NUCLEOSIDE is a moiety selected from one of the following formulae: WO 00/01711 PCT/CA99/00600 -63- R'O B* RO- B* 0 0 O R 9 OR 10 O -0 B 0 B OR R wherein R 8 and R' 0 are the same or different and are hydrogen or a protecting group, R 9 is hydrogen or -OR" wherein R" is hydrogen or a protecting group, and B' is a nucleic acid base.

106. A process for production of a reusable linker arm for oligonucleotide synthesis having the following formula: Z-O- T-[SUPPORT] wherein Z is a linker moiety and T is an organic radical, the process comprising the step of reacting together the compound of Formulae I and II: WO 00/01711 PCT/CA99/00600 -64- Z-OH HO- T- [SUPPORT] (II) wherein Z and T are as defined above.

107. The process defined in claim 106. wherein T contains at least one carbon.

108. The process defined in claim 106, wherein T is a CI-C 300 organic moiety.

109. The process defined in claim 106, wherein T is a C,-C 2 00 organic moiety.

110. The process defined in claim 106, wherein T is a C,-C 0 oo organic moiety.

111. The process defined in claims 106-110, wherein T is a saturated organic moiety.

112. The process defined in claims 106-110, wherein T is an unsaturated organic moiety.

113. The process defined in claims 106-112, wherein T is a C 1 -C 3 00 organic moiety comprising at least one heteroatom selected from N and 0.

114. The process defined in claims 106-113, wherein the organic moiety comprises at least one moiety having the formula: PCT/CA99/00600 WO 00/01711 0 II -C-

115. The process defined in claims 106-113, wherein the organic moiety comprises at least one moiety having the formula:

116. The process defined in claims 106-113, wherein the organic moiety comprises at least one moiety having the formula: O II -N(H)C-

117. The process defined in claims 106-113, wherein the organic moiety comprises at least one moiety having the formula:

118. The process defined in claims 106-113, wherein organic moiety comprises at least one moiety having the formula: WO 00/01711 PCT/CA99/00600 -66- II

119. The process defined in claims 106-118, wherein the organic moiety is unsubstituted.

120. The process defined in claims 106-118, wherein the organic moiety is substituted by at least one moiety selected from the group comprising a C,-C 40 alkyl group, a C,-C 4 o aryl group, a alkoxy group, a C,-C4o ester group,a C- C 4 0 hydroxy group, a C,-Co 4 acrylate group and a C,-C 4 0 alkylaryl group.

121. The process defined in claims 106-120, wherein T has the formula: -CH 2 -fO-CH-CH 2 o I-CH23- q 0 -r 21 s wherein q and s are the same or different and each is an integer having a value of 0-40 and r is an integer having a value of 1-200.

122. The process defined in claim 121, wherein q and s are the same or different and each is an integer having a value of 1-20 and r is an integer having a value of 1-150.

123. The process defined in claims 106-120, wherein T has the formula: I WO 00/01711 WO 0001711PCTICA99/00600 -67- 4 -Q+]CHI)-CHCHYfCHI+ wherein a is 0 or 1, Q is an organic moiety, R' is selected from -OH, -N'IR and -OR wherein R is a protecting group and b is an integer having a value of 0-

124. The reusable linker arm defined in claim 123, wherein a is 0 and R' is OH..

125. The reusable linker arm defined in claim 123 wherein a is 1 and R a is -NR or -OR.

126. The process defined in claims 123-125. wherein the protecting group is selected from the group comprising acetyl, chioroacetyl, methoxyacetyl, t-butyl phenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl (DMT), dialkyiphosphite. pivalyl-isobutyloxycarboflyl. t-butyldimethyisilyl. phenoxyacetal, 9- phenylxanthen-9-yl (pixyl), tetrahydropyranyl, methoxytetrahydropyrany 1, methoxvmethyl, benzyloxymethyl, methoxyethoxvmethyl, methyithiomethyl. dialkyiphosphate, levulinyl, dimethylphenylsilyl, trimethylsilyl, isopropyl- dimethylsi lyl, dilsopropylmethylsilyl, diethylisopropylsilyl, triisopropylsilyl, benzovl. pivaloyl, trifluoroacetyl, alvl, benzyl, o-nitrobenzyl, o- hydroxystyryldimethylsilyl, 2-oxo- 1,2-diphenylethyl, allyloxycarbonyl, monomethoxymethyl, nitroveratryloxycarbonyl, dimethoxybenzoin, dimethoxybenzoin carbonate, methylnitropiperonyl carbonate, fluorenyl- methoxycarbonyl, 2-phenylsulfonylethoxycarbony, fluorophenyl- methoxypiperidinyl and mixtures thereof. PCT/CA99/00600 WO 00/01711

127. The process defined in claims 106-126, wherein Z has the following formula:_ 0 0 II II HO--C- 2 -CH 2 -C-

128. The process defined in claims 106-126, wherein Z has the following formula: O O II II HO-C-CH 2 -O-CH2-C-

129. The process defined in claims 106-126, wherein Z has the following formula: 00 II II HO-C-C-

130. The process defined in claims 106-126, wherein Z has the following formula: I WO 00/01711 PCT/CA99/00600 -69- 1 2 R R 0 11R R 2 HO-C(RR C)nX -A B' R 3 \B 1 wherein: R 2 and R 3 are the same or different and are selected from the group consisting of hydrogen, halide, a substituted or unsubstituted Ci-C20 alkyl group, a substituted or unsubstituted C,-C o aryl group and a substituted or unsubstituted C,-C 4 0 alkylaryl group; R 4 and R 5 are the same or different and are selected from the group consisting of hydrogen, a substituted or unsubstituted C,-C 2 0 alkyl group, a substituted or unsubstituted C,-C 30 aryl group and a substituted or unsubstituted C,-Co alkylaryl group; X' is selected from the group consisting of -S(O) 2 and R is selected from the group comprising hydrogen, a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted C 5 -C 30 aryl group and a substituted or unsubstituted C 5 -C 4 0 alkylaryl group; n is 0, 1 or 2; and one of A' and B' is selected from the group consisting of hydrogen, halide, a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted C 5 -C30 aryl group and a substituted or unsubstituted C-C 4 o alkylaryl group, and the other of A' and B' has the formula: 0 7II X-(CR 6 R )mC- p wherein p is 0 or 1, X 2 is selected from the group consisting of-O-, and R is selected from the group comprising hydrogen, a substituted or unsubstituted C,-CO, alkyl group, a substituted or unsubstituted C,- Co aryl group and a substituted or unsubstituted C 5 -C 4 0 alkylaryl group, R 6 and 1 WO 00/01711 PCT/CA99/00600 R 7 are the same or different and are selected from the group comprising hydrogen, a-substituted or unsubstituted C,-C, 0 alkyl group, a-ubstituted or unsubstituted Cs-Co 3 aryl group and a substituted or unsubstituted C 5 -C 40 alkylaryl group, and mis0, 1 or2.

131. The process defined in claim 130, wherein p is 0.

132. The process defined in claims 130-131, wherein B' is selected from the group consisting of hydrogen, halide, a substituted or unsubstituted C,-C,o alkyl group, a substituted or unsubstituted C,-C, 3 aryl group and a substituted or unsubstituted alkylaryl group.

133. The process defined in claims 130-132, wherein each ofR 4 R 5 R 6 and R 7 is hydrogen.

134. The process defined in claims 130-133, wherein each ofm and n are 1.

135. The process defined in claims 130-134, wherein each ofR', R 2 and R 3 is hydrogen.

136. The process defined in claims 130-135, wherein X' and X 2 are both

137. The process defined in claims 106-136, wherein SUPPORT is an inorganic substance.

138. The process defined in claim 137, wherein the inorganic substance is selected from the group consisting of silica, glass beads, porous glass, aluminosilicates, borosilicates, metal oxides, clays and mixtures thereof.

139. The process defined in claims 106-136, wherein SUPPORT is an organic substance. WO 00/01 711 PCT/CA99/00600 -71-

140. The process defined in claim-39-Twherein the organic substance is a cross-linked polymer.

141. The process defined in claims 106-140, wherein the process is conducted in the presence of an activating agent.

142. The process defined in claim 141, wherein the acitivatirig agent comprises at least one member selected from the group comprising an acid chloride; an active ester nitrophenyl, nitrophenylthio, trichiorophenyl, trifluorophenyl, pentachiorophenyl. pentafluorophenyl, or 3-hydroxy-2,3-dihy uro-4-oxo- benzotriazine esters); an active hydroxylamine ester N-hydroxyphthalinlide or N-hydroxysuccinimide); acid anhydride and mixed anhydride.

143. The process defined in claim 141, wherein the activating agent comprises at least one member selected from the group comprising arylsulfonyl chlorides benzenesulfonyl chloride (BS-Cl), mesitylenesulfonyl chloride (MS-Cl), triisopropylsulfonylchloride (TPS-CI)); active arylsulfonyl esters imidazole, triazole. nitrotriazole. or tetrazole esters of BS-C1, MS-Cl or TPS-CI); 2-ethoxy- 1 -(ethoxvcarbonyl)1,2-dhvdroquino lne (EEDQ); acyl carbonates; 1LI'- (carbonyldioxy)dibenzotnlazoles; chiorotrimethylsi lane; carbodjimides dicvclohexylcarbodiimide (DCC), I -(3-dimethylamninopropyl)-ethylcarbodiimide (DEC), diisopropylcarbodiimide (DIC) either alone or in combination with auxiliary nucleophiles 1 -hydroxybenzotriazole (HOBt), I -hydroxy-7- azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), or 3-hydroxy-3,4- dihydro- 1,2,3 -benzotriazin-4-one (HOObt)) and/or catalysts 4- dimethylaminopyridine (DMAP) or N-methylimidazole or uronium salts tetramethyluroniumn chloride (TMU-Cl), 1H-benzotriazol- 1-yl)- 1,1,3',3- tetramethyluronium hexafluorophosphate (I-BTU), IH-benzotriazol-1I-yl)- 1,1,3 ,3-tetrainethyluronium tetrafluoroborate (TBTU), 2-succinimido- 1,1,3,3- tetramethyluronium. tetrafluoroborate (TSTU), 2-(3 ,4-dihydro-4-oxo- 1,2,3- benzotriazin-3-yl)- 1,1,3 ),3-tetramethyluronium tetrafluoroborate (TDBTU), 2-(2- WO 00/0 1711 PCT/CA99/00600 -72- oxo-1I(2H)-pyridyl- 1,1,3 ,3-tetramethyluroflium tetrafluoroborate (TPTU), norbomene-2,3-dicarboximido)- 1,1,3,3 )-tet-ramethyluronium tetrafluoroborate (TNTU), O-(7-azabenzotnlazol- l-yl)-1 ,3-dimethyl- 1,3-dimethyleneurofliwmhexa- fluorophosphate (HAMDU), O-(7-azabenzotriazol- l-yl)-l ,3-dimethyl-1I,3-tri- methyleneurofliUm hexafluorophosphate (HAMTU), O-(7-azabenzotflazol- 1 -yl)- 1,1,3 ,3-bis(pentamethylefle)uronium hexafluorophosphate (HAPipU), O-(7- azabenzotniazol- I1-yl)-l 1,1,3 ,3-bis(tetramethyele)uroflium hexafluorophOSphate (HAPyU), O-(7-azabenzotriazol-1I-yl)- 1,1 ,3,3')tetrarnethyi-ofium hexafluorophosphate (HATU)) either alone or in combination with auxiliary nucleophiles I -hydroxybenzotriazole (HOBt), I1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), or 3-hydroxy-3 ,4-dihydro- 1.2.3 benzotriazin-4-ofle (HOObt)) and/or catalysts 4-dimethylamilopyridifle (DMA-P) or N-methylimidazole (NMI)) or phosphonium salts (egbenzotriazol- I -yl-oxytris(dimethylamfino)phosphonium hexafluorophosphate (BOP), benzotriazole- 1 yl-oxy-trispyrrolidinophosphonium hexafluoropho sp hate (PyBOP), 2-(benzotriazol-1 -yl)oxy-1I,3-dimethylimidazolidiniUm hexafluorophosphate (B301), bromo tris(pyrrolidino)phosphonium hexafluorophoSPhate (PyBroP?), 7-azabenzotriazol-l -yloxytris- (dimethylamilo)PhOSPhoflium hexafluorophosphate (AOP), and 7- azabenzotri azol-1I yloxytris(pyrrolidilo)phosphonium hexafluorophosphate (PyAOP)) either alone or in combination with auxillary nucleophiles and/or catalysts.

144. A process for production of a reusable linker arm for oligonucleotide synthesis having the following formnula: NUCLEOSiDE- Z-O- T-SUPPORTIl WO 00/01711 PCT/CA99/00600 -73- wherein Z is a linker moiety and T is an organic radical, the process comprising the step of reacting together the-compounds of Formulae I, II and III: HO-Z-OH HO-T-[SUPPORT] (II) NUCLEO SIDE- OH (l) wherein Z and T are as defined above.

145. The process defined in claim 144, wherein T contains at least one carbon.

146. The process defined in claim 144, wherein T is a C,-C 3 00 organic moiety.

147. The process defined in claim 144, wherein T is a C,-Czoo organic moiety.

148. The process defined in claim 144, wherein T is a CI-Co 00 organic moiety.

149. The process defined in claims 144-148, wherein T is a saturated organic moiety.

150. The process defined in claims 144-148, wherein T is an unsaturated organic moiety.

151. The process defined in claims 144-148, wherein T is a C -C 300 organic moiety comprising at least one heteroatom selected from N and 0. PCT/CA99/00600 WO 00/01711 -74-

152. The process defined in claims 144-151, wherein the organic moiety comprises at least one moiety having-the formula: O I I

153. The process defined in claims 144-151, wherein the organic moiety comprises at least one moiety having the formula:

154. The process defined in claims 144-151, wherein the organic moiety comprises at least one moiety having the formula: O I1 N(H)C-

155. The process defined in claims 144-151, wherein the organic moiety comprises at least one moiety having the formula:

156. The process defined in claims 144-151, wherein organic moiety comprises at least one moiety having the formula: WO 00/01711 PCT/CA99/00600 0 II -C-O-

157. The process defined in claims 144-156, wherein the organic moiety is unsubstituted.

158. The process defined in claims 144-156, wherein the organic moiety is substituted by at least one moiety selected from the group comprising a CI-C 40 alkyl group, a C,-Co aryl group, a C,-C 40 alkoxy group, a C 1 -Co 4 ester group,a C C 4 o hydroxy group, a C 2 -Co acrylate group and a C,-C 4 0 alkylaryl group.

159. The process defined in claims 144-158, wherein T has the formula: -ECH23-O-- CH 2 -CH 2 CH2 wherein q and s are the same or different and each is an integer having a value of 0-40 and r is an integer having a value of 1-200.

160. The process defined in claim 159, wherein q and s are the same or different and each is an integer having a value of 1-20 and r is an integer having a value of 1-150.

161. The process defined in claim 144-158, wherein T has the formula: WO 00/01711 WO 0001711PCT/CA99/00600 -76- Q-]dH 2 -CH-CH 2 -OfcHI]- wherein a is 0 or 1, Q is an organic moiety, R' is selected from -OH, -NR and -OR wherein R is a protecting group and b is an integer having a value of 0-

162. The reusable linker arm defined in claim 161, wherein a is 0 and R 8 is OH..

163. The reusable linker armn defined in claim 16 1, wherein a is 1 and R is -NR or -OR.

164. The process defined in c laims 16 1-16'3, wherein the protecting group is selected from the group comprising acetyl, chloroacetyl, methoxyacetyl, t-butyl phenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl (DMT), dialkylphosphite, pivalvl-isobutyloxycarbonyl, t-butyldimethvlsilyl, phenoxvacetal. 9- phenylxanthen-9-yl (pixyl), tetrahydropyranyl, methoxytetrahydropyranyl, methoxymethyl, benzyloxymethyl, methoxyethoxymethyl, methyithiomethyl, dialkylphosphate, levulinyl, dimethylphenylsilyl, trimethylsilyl, isopropyl- dimnethylsilyl, diisopropylmethylsilyl, diethylisopropylsilyl, triisopropylsilyl, benzoyl, pivaloyl, trifluoroacetyl, allyl, benzyl, o-nitrobenzyl, o- hydroxystyryldimethylsilyl, 2 -oxo 1 ,2 -diphenyl ethyl, allyloxycarbonyl, monomethoxymethyl, nitroveratryloxycarbonyl, dimethoxybenzoin, dimethoxybenzoin carbonate, methylnitropiperonyl carbonate, fluorenyl- methoxycarbonyl, 2-phenylsulfonylethoxycarbony, fluorophenyl- methoxypiperidinyl and mixtures thereof. WO 00/01711 PCT/CA99/00600 -77-

165. The process defined in claims 144-164, wherein Z has the following formula: O O II 1I HO-C-CH 2 -CH 2

166. The process defined in claims 144-164, wherein Z has the following formula: O O II 11 HO-C-CH--O-CH2 -C-

167. The process defined in claims 144-164, wherein Z has the following formula: 0 0 II II HO-C-C-

168. The process defined in claims 144-164, wherein Z has the following formula: WO 00/01711 PCT/CA99/00600 -78- R R2 0 11 4 5 1 -C(R RC)nX A R 3 B l wherein: R 2 and R 3 are the same or different and are selected from the group consisting of hydrogen, halide, a substituted or unsubstituted C,-Co alkyl group, a substituted or unsubstituted C,-Co aryl group and a substituted or unsubstituted C 5 -C 40 alkylaryl group; R 4 and R 5 are the same or different and are selected from the group consisting of hydrogen, a substituted or unsubstituted C,-C,0 alkyl group, a substituted or unsubstituted C 5 -C 3 0 aryl group and a substituted or unsubstituted C1-C 4 0 alkylaryl group; X' is selected from the group consisting of -S(O) 2 and R is selected from the group comprising hydrogen, a substituted or unsubstituted C,-C20 alkyl group, a substituted or unsubstituted C,-C 30 aryl group and a substituted or unsubstituted CS-Co 4 alkylaryl group; n is 0, 1 or 2; and one of A' and B' is selected from the group consisting of hydrogen, halide, a substituted or unsubstituted C,-Co alkyl group, a substituted or unsubstituted C,-Co 3 aryl group and a substituted or unsubstituted C,-C 4 0 alkylaryl group, and the other of A' and B' has the formula: 0 X2(CR6R )mC p wherein p is 0 or 1, X 2 is selected from the group consisting of-0-, -S(O) 2 and R is selected from the group comprising hydrogen, a substituted or unsubstituted C,-C 2 0 alkyl group, a substituted or unsubstituted C 5 C 3 0 aryl group and a substituted or unsubstituted C 5 -C 4 0 alkylaryl group, R 6 and WO 00/01711 PCT/CA99/00600 -79- R 7 are the same or different and are selected from the group comprising hydrogen, a-substituted or unsubstituted C,-Co alkyl group, a substituted-or-unsubstituted aryl group and a substituted or unsubstituted C 5 -C 40 alkylaryl group, and mis0. 1 or 2.

169. The process defined in claim 168, wherein p is 0.

170. The process defined in claims 168-169, wherein B' is selected from the group consisting of hydrogen, halide, a substituted or unsubstituted C,-C,o alkyl group, a substituted or unsubstituted C5-C 30 aryl group and a substituted or unsubstituted C,-C 40 alkylaryl group.

171. The process defined in claims 168-170, wherein each ofR 4 R 5 R 6 and R 7 is hydrogen.

172. The process defined in claims 168-171, wherein each of m and n are 1.

173. The process defined in claims 168-172, wherein each ofR', R 2 and R 3 is hydrogen.

174. The process defined in claims 168-172, wherein X' and X 2 are both

175. The process defined in claims 144-174, wherein SUPPORT is an inorganic substance.

176. The process defined in claim 175. wherein the inorganic substance is selected from the group consisting of silica, glass beads, porous glass, aluminosilicates, borosilicates, metal oxides, clays and mixtures thereof.

177. The process defined in claims 144-174, wherein SUPPORT is an organic substance. WO 00/01711 WO 0001711PCT/CA99/00600

178. The process defined in claim 177, wherein the organic substance is a cross-Alinked-polymer.

179. The process defined in claims 144-178, wherein the process is conducted in the presence of an activating agent.

180. The process defined in claim 179, wherein the acitivating agent comprises at least one member selected from the group co.-Ppnsing an acid chloride; an- active ester nitrophenyl, nitrophenylthio, trichlorophenyl, trifluorophenyl, pentachiorophenyl, pentafluorophenyl, or 3-hydroxy-2,3 -dihydro-4-oxo- benzotriazine esters); an active hydroxylamine ester N-hvdroxyphthalimide or N-hydroxysuccinimide); acid anhydride and mixed anhydride.

181. The process defined in claim 179, wherein the activating agent comprises at least one member selected from the group comprising arylsulfonyl chlorides benzenesulfonyl chloride (BS-Cl), mesitylenesulfonyl chloride (MS-Cl), triisopropylsulfonylchloride (TPS-Cl)); active arylsulfonyl esters imidazole, triazole, nitrotniazole. or tetrazole esters of BS-Cl, MS-Cl or TPS-Cl); 2-ethoxy- I -(ethoxycarbonyl)-lI.2-dihydroquinoline (EEDQ); acyl carbonates; 1,1'- (carbonyldioxy)dibenzotnlazoles; chiorotri methylsi lane; carbodiimides dicyclohexylcarbodiimide (DCC), I -(3-dimethylamninopropyl)-ethylcarbodiimide (DEC), diisopropylcarbodiirnide (DIC)) either alone or in combination with auxillary nucleophiles 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7- azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), or 3-hydroxy-3 ,4- dihydro- 1,2,3-benzotriazin-4-one (HOObt)) and/or catalysts 4- dimnethylaminopyridine (DMA-P) or N-methylimidazo le or uronium salts tetramethyluroniurn chloride (TMU-Cl), 2-(1 H-benzotriazol- 1-yl)-l ,1,3,3- tetramethyluronium hexafluorophosphate (HBTU), IH-benzotriazol- l-yl)- 1,1,3 ,3-tetramethyluronium tetrafluoroborate (TBTU), 2-succinimido- 1,1,3,3- tetramethyluroniumn tetrafluoroborate (TSTU), 2-(3 ,4-dihydro-4-oxo- 1,2,3- benzotriazin-3-yl)- 1, 1,3,3-tetramethyluronium tetrafluoroborate (TDBTU), 2-(2- oxo- I (2H)-pyridyl- 1, 1,3 ,3-tetramethyluronium tetrafluoroborate (TPTU), WO 00/01711 WO 0001711PCT/CA99/00600 norbomene-2,3-dicarboximido)- 1,1,3 ,3-tetramethyluronium tetrafluoroborate O-(7-azabenzotriazol- l-yl)-l ,3-dimethyl- 1,3-diniethyleneuronium hexa- fluorophosphate (HAMDU), O-(7-azabenzotriazol- l-yl)-l ,3 -dimethyl- 1,3-tni- methyleneuronium hexafluorophosphate (HAMTUI, O-(7-azabenzotriazol- 1 -yl)- 1,1,3 ,3-bis(pentamethylene)uronium hexafluorophosphate (HAPipU), O-(7- azabenzotriazol- l-yl)-l ,l,3 ,3-bis(tetramethylene)uronium hexafluorophosphate (HAPyU), O-(7-azabenzotriazol- 1-yl)-l, 1,3,3-tetramethyluronium hexafluorophosphate (HATUL)) either alone or in combination with auxiliary nucleophiles 1 -hydroxybenzotriazole (HOBt), I -hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), or 3-hydroxy-3,4-dihydro-1,2,3- benzotriazin-4-one (HOObt)) and/or catalysts 4-dimethylamninopyridine (DMAP) or N-methylimidazole (NMI)) or phosphonium salts benzotriazol- I -yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazole- 1-yI-oxy-trispyrrolidinophosphonium hexafluorophosphate (PyBOP), 2 -(benzotri azoI-1I -yl)oxy- 1, ,3-dim ethylimidazolidinium hexafluorophosphate (BOL), bromo tris(pyrrolidino)phosphoniulT hex afl uoropho sp hate (PyBroP), 7-azabenzotriazol- 1 -yloxytris- (dimethylamino)phosphonium hexafluorophosphate (AOP), and 7- azabenzotriazol- 1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP)) either alone or in combination with auxiliary nucleophiles and/or catalysts.

182. The process defined in claims 144-181, wherein NUJCLEOSIDE is a moiety selected from one of the following formulae: WO 00/01711 PCT/CA99/00600 -82- -0 B 0 -OB OR R wherein R 8 and R 1 0 are the same or different and are hydrogen or a protecting group, R 9 is hydrogen or -OR" wherein R" is hydrogen or a protecting group, and B* is a nucleic acid base.

183. The process defined in claims 144-182, wherein the compounds of Formulae I and II are initially reacted to form a conjugate which is reacted with the compound of Formula III.

184. The process defined in claims 144-182, wherein compounds of Formulae I and III are initially reacted to form a conjugate which is reacted with the compound of Formula II.

185. A process for producing an oligonucleotide having a desired sequence comprising the steps of: reacting a linker arm having the formula: WO 00/01711 PCT/CA99/00600 -83- NUCLEOSIDE-Z-O- T- [SUPPORT] wherein Z is a linker moiety and T is an organic radical, with at least one oligonucleoside base until an oligonucleotide having the desired sequence is produce; (ii) cleaving the oligonucleotide having the desired sequence to produce a free oligonucleotide have the desired sequence; and a used linker arm; and (iii) recycling the used linker arm to Step

186. The process defined in claim 185, wherein the used linker arm produced in Step (ii) has the formula: Z- O- T- [SUPPORT] wherein Z is a linker moiety and T is an organic radical.

187. The process defined in claims 185-186, wherein Step (iii) comprises the step of converting the used linker arm to a linker arm having the formula: NUCLEOSIDE- Z- O-T-[SUPPORT] wherein Z is a linker moiety and T is an organic radical. DATED this 22nd day of October 2003 UNIVERSITY TECHNOLOGIES INTERNATIONAL, INC. By its Patent Attorneys DAVIES COLLISON CAVE WO 00/01711 PCT/CA99/00600 0 0 NH 2 -SUPPORT ISUC CINYL- SUPPORT CONJUGATE PRODUCTION 0 0 HOCC HIHC-N -SUPPORT H r1i LINKER ARM PRODUCTION DMTO-- DMTOB+ -0 0 R ~CC H,CF12C-N -SUPPORT 11 I 11 0 O H OLIGONUCLEOTIDE SYNTHESIS 2 0OLIGONUC LEOTIDEO0-CC H 2 C H2CN -SUP PORT 0 0H ICLEAVAGE OLIGONUCLEOTIDE-OH H 2 N-CCH-,CH,C-N-SUPPORT 11 II 0 0 FIGURE 1 (PRIOR A RT) PCT/CA99/00600 WO 00/01711 HO B 0 -0 Hydroxyl DMTO )k OCH 2 ,C-OH HO Functionalized HBTUDMAPDIEA HOISupport Step Nucleoside Derivatization HO B 0 0 D M TO O C C H O HC O I THFS)0/MI/uinj Step Capping ID Capping r D etritylat Oligo Synthesis Oxidation! Coupling Suiphurization ion NH 4 OH, 5-15 min D eprotection Purification/ Oligonucleotide HN- Product Step Support Regeneration 0.05 M K-7C0 3 MeOH, 5 Min Step Cleavage 0 11 CI-CHC-0, FIGURE 2 PCT/CA99/00600 WO 00/01711 A, OHS Linker Arm HO -(CH 2 6 N H 0 0 N -w Support H (Preferred example) O 0 R'Oj 7 Y Support 9 B, C 12 Linker Arm 0 DMTO 1 C 19-Support H 0 R 0-R ROC XS Support (General example, R 9 H or protecting group, X 3 and X4= NH, NR, or 0) (Preferred example) (General example, X 5= NH, NR,or 0) C, BDG Linker Arm 0 OH 0 HO 0 ,41 dOSupport (Preferred example) OH OR' HO (CH) 1 0 X-Support (General example, X 0, NH, or NR, R' protecting group) D, GLY Linker Arm (Gly-CPG) O H O H HO 0 (Preferred example) (General example) FIGURE 3