This is a continuation-in-part of U.S. patent application Serial No. 07/287,890 filed 21 December 1988.

The present invention relates generally to the synthesis and purification of polynucleotideε, and more particularly, to automated techniques for solid phase synthesis and purification of polynucleotideε using phosphoramidite and/or hydrogen phosphonate chemistries.

g&CKGRQUϊJB

A key factor in the recent advances in molecular biology has been the development of reliable and convenient methods for synthesizing polynucleotideε, e.g. Itakura, gSJgn.S- Vol. 209, pgs. 1401-1405 (1980); and Wallace et al, pgs. 631-663, in Scouten, ed. gQl.jcl gfaas,g BJQghgiti gkEY (John Wiley & Sons, New York, 1982). As the use of synthetic polynucleotideε has increased, the demand for even greater convenience in the preparation of pure, ready-to-uεe polynucleotides has also increased. This demand has stimulated the development of many improvements in the basic procedures for solid phase syntheεiε, e.g. Sinha et al,

Kasleig Asiάs Bsse&Esb. vol. 12, pgs. 4539-4557 (1984)(beta-cyanoethyl in phosphoramidite chemistries); Froehler et al, _PetEahgcl£Qn etters. Vol. 27, pgs. 469- 472 (1986)(H-phosphonate chemistry); Ger ann et al, _. a_U BiQsbe ., Vol. 165, pgs. 399-405 (1987); and . Ikuta et al- &aal _Λ Ckes.., Vol. 56, pgs. 2253-2256 (1984)(rapid purification of synthetic oligonucleotides by way of trityl moieties); Molko et al, European patent publication 241363 dated 3 April 1987 (improved base-labile acyl protection groups for exocyclic amines), and the like.

In spite of such progress, difficulties are still encountered in current methods of polynucleotide synthesis and purification. For example, H-phosphonate and phoεphoramidite monomers readily degrade in the presence of even trace amounts of water. This contributes greatly to their very short in-solution shelf lives, and the need to use large molar excesseε of reactants to drive the coupling reactions to completion in reasonable times. The presence of water- degraded reactants leads to less pure final products and to more expensive syntheses. This problem is particularly acute for large scale (milligram and greater) syntheses, as well as for small scale (less than 1 umole) syntheses. In the case of the former, the cost of the mono eric reactants makes up ,the greatest portion of the overall cost of synthesis. Any reduction in the excess molar amount of reactantε needed to adequately drive the coupling reaction would lead to substantial cost reductions in the synthesis of large amounts of polynucleotideε, e.g. the milligram and gram quantities comptemplated for therapeutic use. In the case of the latter, degradation of the reactantε by trace amounts of water limits the efficiency that

can be achieved on currently available commercial synthesizers, and increases the cost of synthesis because greater amounts of reactant must be used to counter losses due to water and oxygen present in trace amounts in the solvents, tubing, and vessels, or due to water and oxygen leaking into the system from faulty connections, valves, and the like.

As a further example, derivatized controlled pore glass (CPG), the current support of choice in most solid phase methodologies, can be responsible for spurious indications of coupling yields, e.g. Pon et al, Ϊ TegbBigijeSr Vol. 6, pgs. 768-775 (1988). Moreover, CPG, like most glasseε, lackε chemical stability in some of the highly corrosive deprotection reagents, such as concentrated ammonia and trichloroacetic acid, used in polynucleotide synthesis. As a consequence, the CPG support itself can be degraded in the deprotection steps and can be a source of contamination of the final product. This problem is exacerbated by the relative long reaction times required to remove currently used protection groups for exocyclic amines. An extended period of deprotection is required to remove these groups after the polynucleotide has been cleaved from the solid phase support. Thus, complete automation of synthesis and purification has been impractical. Another problem with CPG is that itε surface supports chain growth at sites other than those associated with the 5' terminus of an attached nucleoside. Such "extraneous" chain growth gives rise to a heterogeneous population of 5 ¹ - blocked (usually tritylated) polynucleotides. Typically, the "extraneous" tritylated products lack one or more 3' nucleotideε. This, of course, preventε one from succeεεfully taking advantage of the

relatively high hydrophobicity of the trityl group to purify "correct sequence" polynucleotides. Incorrect- sequence extraneous chains are also tritylated. Finally, the hydrophilic nature of CPG causes it to absorb water present in trace amounts in the solvents, which leads to. the degradation of the highly water- sensitive monomeric reactantε.

In view of the above, the field of solid phase polynucleotide synthesis could be significantly advanced by the availability of alternative support materials (i) which have the favorable mechanical properties of CPG, but which also possess greater chemical stability under the reaction conditions of polynucleotide synthesis, (ii) which provide less opportunity for extraneous chain growth during synthesis, and (iii) which would permit more efficient syntheses, particularly under conditions of reduced molar excess of reactantε. The use of such materialε coupled with improved exocyclic protection groups would not only allow practical automation of polynucleotide synthesis and purification in a single inεtrument, but alεo would permit more efficient and leεε expenεive εyntheses of polynucleotideε and their derivatives.

The invention is directed to a system and method for solid phase polynucleotide synthesis on a nonswellable porous polystyrene support by H- phoεphonate and/or phosphoramidite chemistries. The use of the polystyrene support (1) significantly reduces the number of failure sequences caused by extraneous initiation of polynucleotide chain

growth on the support material, (2) permits deprotection of the polynucleotide product in the presence of the support material by virtue of its superior stability in the presence of deprotection reagents, and (3) permits more efficient syntheses, particularly under conditions of reduced molar excess of the monomeric reactants.

As used herein, "nonswellable" means that the porous polystyrene material remains substantially mechanically rigid, in particular does not increase in volume, when exposed to solvents, reactantε, and products of the phosphoramidite and/or hydrogen phosphonate chemistries. Preferably, "nonswellable" meanε that the volume of the porous polystyrene support increases in volume no more than 5-10% over its dry volume when exposed to the solvents, reactantε, and productε of the phoεphoramidite and/or hydrogen phoεphonate chemistries. Mechanical rigidity is desirable for efficient and uniform transfer of reagents to the polynucleotide chain during synthesis.

An important feature of the invention is efficient monomer coupling to the growing polynucleotide chains under conditions of reduced molar exceεε of monomer over polynucleotide. It iε believed that the increaεed efficiency reεultε from the hydrophobic nature of the nonswellable porous polystyrene support, which unlike CPG does not accumulate moisture, even over many coupling cycles. In particular, with the use of the nonswellable porouε polyεtyrene supports, much lower amounts of reactantε are required for achieving equivalent effective reactant concentrationε adjacent to the growing polynucleotide as those achieveable with CPG.

Preferably, the correct-sequence polynucleotideε

synthesized by the method of the invention are purified from the crude mixture cleaved from the reaction column by passage of the crude mixture over an adsorbent which preferentially adsorbs the 5'-blocking group of the correct-sequence polynucleotides. More preferably, whenever the 5'-blocking group is a trityl, the adsorbent is a nonswellable polystyrene solid.

BEie£ Bes ^g riBfc-ian 9l £_!£ gwsifigg

Figure 1 diagrammatically representε a preferred apparatus for implementing the method of the invention.

Figure 2 illustrates a densitometric εcan of an autoradiogra of electrophoretically separated components of the crude reaction productε of a 72-mer synthesis on a polystyrene support (A) and a CPG support (B).

Figures 3A and 3B are capillary electrophogra s of -mer syntheses on polystyrene (A) and CPG (B).

The invention is a method and system for producing oligonucleotideε and/or polynucleotides of a predetermined sequence. The method comprises the steps of (i) providing a nonswellable porous polystyrene support, (ii) providing a 5'-blocked protected nucleoside having a 3'-hydroxyl and a 5'-hydroxyl, the 5'-blocked protected nucleoside being attached to the nonswellable porous polyεtyrene support, usually by a base-labile linkage between the 3'-hydroxyl of the protected nucleoside and the support, such that the 5 ¹ - blocked protected nucleoside forms a protected correct- sequence chain; (iii) deblocking the 5'rbydroxyl of the correct-sequence chain; (iv) reacting with the 5'-

hydroxyl of the correct-sequence chain a 5'-blocked protected nucleoside monomer selected from the group consisting of 5'-blocked protected nucleoside-3'- phosphoramidites ahd 5'-blocked protected nucleoεide- 3'-H-phosphonateε to form either a protected correct-- sequence chain or a failure sequence, the failure sequence having a 5'-hydroxyl; (v) capping the failure sequence by reacting a capping agent with the 5'- hydroxyl of the failure sequence; (vi) repeating steps (iii)-(v) until the polynucleotide of the predetermined sequence is obtained; and (vii) deprotecting the exocyclic amines of the polynucleotide and cleaving the polynucleotide from the nonswellable porous polystyrene support to form a cleavage mixture.

Preferably, the method further includes the step of purifying the polynucleotide from the cleavage mixture. More preferably, the step of purifying includes the steps of exposing the cleavage mixture to a highly crosslinked polystyrene adsorbent, separating the blocked polynucleotide from the failure sequences by washing the polystyrene absorbent to preferentially remove the failure sequenceε, deblocking the 5'- hydroxyl of the polynucleotide, and eluting the polynucleotide from the polystyrene adsorbent.

Preferably, the step of reacting includes the step of oxidizing the internucleoside phosphorous from the trivalent to the pentavalent state in the phosphoramidite chemistry. Preferably, the method includes the further step of oxidizing the internucleotide phosphorous from the trivalent to the pentavalent state prior to the step of deprotecting in the H-phosphonate chemistry. In further preference, the step of reacting includes delivering the 5'-blocked protected nucleoside monomers to the nonswellable

porouε polystyrene support in less than or equal to ten fold molar excess of the correct-εequence chainε. In still further preference, in small scale syntheses less than or equal to 4 mg of monomer is reacted with the correct-sequence chain. As used herein, "small scale synthesis" meanε less than or equal to 1 umole of initial nucleoside being attached to the nonswellable porous polystyrene support and the reaction volume being between about 50-200 uL.

As used herein, the term polynucleotide refers to a single stranded chain of either deoxyribonucleotides or ribonucleotideε having from a few, e.g. 2-20, to many e.g. 20 to εeveral hundred or more, nucleotides. The term alεo includeε chains of nucleosideε linked by analogs of the phosphate bond, e.g. thiophosphates, and the like.

Detailed procedures for the phoεphoramidite and hydrogen phosphonate methods of polynucleotide synthesis are described in the following references, which are incorporated by reference: Carutherε et al, U.S. Patents 4,458,066 and 4,500,707; Koester et al, U.S. patent 4,725,677; Matteucci et al, ζj _Λ gteEA S ea SQS- _t , Vol. 103, pgs. 3185-3191 (1981); Caruthers et al,

Seaetis Bag-geer-jagr vol. 4, pgs. 1-17 (198 ); Jones, chapter 2, and Atkinson et al, chapter 3, in Gait, ed.,

Presε, Washington, D.C., 1984); Froehler et al, -ESfcEShectEe Le ers, Vol. 27, Pgs. 469-472 (1986); Garegg et al, get ahedrs Letters, Vol. 27, pgs. 4051- 4054 and 4055-4058 (1986); Froehler et al, HuS-l is Asϊ£ S sea s r Vol. 14, pgs. 5399-5407 (1986); Usman et al, J,., As Cbes S S./ Vol. 109, pgs. 7845-7854 (1987); Froehler, g t beάrQΩ Lettersr Vol. 27, pgs. 5575-5578 (1986); Andrus et al, letra eά Qa et ers ,

Vol. 29, pgε 861-864 (1988); Brill et al, J. Am. Chem. Soc, Vol. Ill, pg. 2321 (1989); and Mori et al, Nucleic Acidε Research, Vol. 17, pgε. 8207-8219 (1989).

An important feature of the invention is the use of a nonswellable porous polystyrene support for synthesis. Nonsweliability of the porous polystyrene depends directly on the degree of crosslinking among the styrene polymers. Such crosεlinking is usually measured by a crosslink ratio, which is the molar ratio of crosslinking material (e.g. divinylbenzene) and chain material (e.g. styrene). Preferably, the nonswellable porouε polystyrenes of the invention have a crosslink ratio in the range of about 10-60 percent; more preferably, they have- a crosεlink ratio of about 20-50 percent; and most preferably, they have a crosslink ratio of about 30-50 percent.

As used herein, "porous" meanε that the nonswellable polystyrene contains pores having εubεtantially uniform diameterε in the range of between 100-4000 x 10~ ⁸ cm. Preferably, the pore diameterε are about 1000 x 10~°cm. Several different means are available for manufacturing porous polyεtyrene εolidε. As used herein, the term "porouε" includes, but is not limited to, so-called macroporous polyεtyreneε and so- called acroreticular polystyrenes. These materials are widely available commercially in a variety of shapes and sizes, e.g. Polymer Laboratories, Ltd. (Shropshire, United Kingdom); Hamilton Co. (Reno, NV), or the like, and are described in U.S. patent 4,297,220, which is incorporated by reference. Preferably, the nonεwellable porouε polyεtyrene supports are used in the form of beads having a diameter in the range of 15-100 x 10 ^" "*cm, and more preferably, in the range of 50-70 x 10 cm.

Prior to its use in synthesis, the nonswellable porouε polystyrene must be linked to a 5'-blocked protected nucleoside, which forms the first nucleoside of the polynucleotide to be synthesized. The nature of this linkage, the 5'-blocking agent, and the protecting groups of the exocyclic amines and internucleoside phosphorous are important features of the invention. Preferably, the first 5*-blocked protected nucleoside is linked to the polystyrene support by way of a base- labile linkage. More preferably, this linkage is an aminomethylsuccinate group, as is commonly used in phosphite triester synthesis, e.g. Atkinson et al, pgε. 45-49, in Gait, ed. QligQj s.lgQ J-g-e & grastisal &BB g s (IRL Press, Oxford, 1984). The linkage is formed by reacting a 5'-blocked protected nucleoside-'3-0-succinate with an amino-derivatized polystyrene support. Synthesis of the 5'-blocked protected nucleoεide-3'-0-succinate iε well known in the art, e.g. Atkinson et al, pgs. 47-49 (cited above); and U.S. patent 4,458,066. Accordingly, these references are incorporated by reference.

As used herein, "5"-blocked" refers to a group attached to the 5'-hydroxyl of either the monomeric nucleoside intermediates used in the invention, or the correct-sequence chain of protected nucleosideε. (However, note that chains initiated at "extraneous" sites can be 5*-blocked and yet not be of the correct sequence). Selection of the 5'-blocking group is constrained by three criteria: (i) it must mask the 5'-hydroxyl of the monomer so that it does not compete with the 5'-hydroxyls of the correct-sequence chains during the monomer addition stepε, (ii) it must be acid-labile, in that it can be removed so as to expose the 5'-hydroxyl upon mild acid treatment, and (iii) it

must be sufficiently hydrophobic to allow 5'-blocked correct-sequence chains to be preferentially adsorbed onto a polystyrene adsorbent over unblocked failure sequences. Preferably, the 5'-hydroxylε are protected as trityl ethers. That is, the 5'-hydroxyl blocking agent, or group, is a trityl. Most preferably, the blocking group is 4,4'-dimethoxytrityl. Synthesis of 5'-blocked nucleoside intermediates is well know in the art, e.g. Froehler et al (cited above), and Carutherε et al (U.S. patents cited above). The trityl blocking groups are removed, that is, the 5'-hydroxylε are "deblocked" by exposure to a mild protic acid. Several trityl deblocking reagentε have been uεed in solid phase polynucleotide synthesis, e.g. Caruthers et al, Senetis Vol. 4, pgε. 1-17 (1984). Preferably, deblocking iε accompliεhed by expoεure to 2% trichloroacetic acid for about 3 inuteε at room temperature.

As used herein, "protected" in reference to the monomeric nucleoside intermediates and the correct- sequence chains means (i) that the exocyclic amines of either compound have been acylated with a protection group that prevents the exocyclic amine from participating in the synthetic reaction εtepε, and (ii) that the internucleoside phosphorous of phosphoramidite intermediates are masked by a baέe-labile protection group. Preferably, the phosphorous protection group iε beta-cyanoethyl, as disclosed by Koester et al, U.S. patent 4,725,677. Many acyl protection groups are available for use in the invention, e.g. Jones, pgε. 23-34, in Gait, ed. (cited above). Preferably, the exocyclic amine protection groups are εufficiently baεe-labile εo that the correct-εequence chainε can be deprotected and cleaved from the polystyrene support in

the same reaction step. Preferably, the exocyclic amines, of the monomeric nucleoside intermediates are protected by acyl protection groups of the form - C0CHR ₁ R _2r wherein R± is H or lower alkyl, and R ₂ is H, lower alkyl, lower alkoxy, lower aryloxy, or substituted-lower aryloxy. Preferably, the substituents of the lower aryloxy are electron- withdrawing, such aε nitro-, cyano-, or sulfonate.

As used herein, the term lower alkyl refers to straight-chained, branched, or cyclic alkyls containing from 1 to 6 carbon atoms. Preferably, the term lower alkoxy refers to methoxy, ethoxy, isopropoxy, tert- butyloxy, or the like. Preferably, the term lower aryloxy refers to phenoxy, napthoxy, biphenyloxy, or the like.

"Electron-withdrawing" denotes the tendency of a substituent to attract valence electronε of the molecule of which it iε apart, i.e. it is electronegative, March, Adxasseg gςgfrQJ-g S esμ.StK _Ϊ - pgε. 16-18 (John Wiley, New York, 1985).

These acyl protection groups, when uεed with the polyεtyrene supports of the invention, allow deprotection of the exocyclic amines and cleavage of the polynucleotide product from the solid phase support in a single step. Moreover, when beta-cyanoethyl protection groups are employed in the phosphoramidite approach, deprotection of the internucleoside phosphates can also be effected. The single deprotection/cleavage step occurs rapidly enough so that the entire synthesis-purification procedure can be practically automated.

Briefly, the groups are attached to the monomeric nucleoside intermediates by acylation of the exocyclic amino groups of the baseε (adenine, guanine, and

cytidine) of the deoxynucleosideε 2'-deoxyadenosine, 2'-deoxyguanoεine, and 2'-deoxycytoεine, or of the ribonucleosides, adenosine, guanosine, and cytosine. Thymidine and uridine do not need base protection. Acylation occurs by reacting the acid chloride, such as methoxyacety1 chloride, isobutyryl chloride, or phenoxyacetyl chloride, or the acid anhydride, such as methoxyacetic anhdride, isobutyric anhydride, or phenoxyacetic anhydride, with the 3',5' protected nucleoεides. The 3' and 5' protecting groups can be either trimethylsilyl or dimethoxytrityl. The trimethylsilyl groups are applied with either hexamethyldiεilazane or trimethylεilyl chloride and can be removed under mild conditions with a neutral aqueous treatment. The dimethoxytrityl group can be applied with dimethoxytrityl chloride either before or after acylation. After acylation protection of the amino group and 5' di ethoxytritylation, the 3' hydroxyl group iε converted to a phosphoramidite moiety. This phoεphitylation iε achieved typically with biε(diiεopropylamino)methoxyphosphine or bis(diiεopropylamino)cyanoethoxyphoεphine with catalysis by diisopropylammonium tetrazolide to give the methyl or cyanoethyl phosphoramidite nucleosideε, respectively, as shown by Formula I.

Eatmi-la £

Here, R- _j _ and R ₂ are as described above. DMT represents dimethoxytrityl. B represents adenine, guanine, thymidine, or cytidine. R' representε methyl or beta-cyanoethyl. And iPr is isopropyl. With the above protection group, deprotection and cleavage can be achieved by treatment in concentrated (29%) ammonia for 6 hours at 20°C, or for 1 hour at 55°C.

As used herein, "correct-sequence chain" refers to a chain of nucleosides which iε capable of reacting with an additional monomeric nucleoεide intermediate via its 5'-hydroxyl (i.e. it iε uncapped) and whose sequence corresponds, to that of the desired polynucleotide. The term includes the first nucleoside attached to the solid phase support (i.e. a nucleoside chain of one unit) as well as the completed polynucleotide product of the predetermined sequence. As uεed herein, "failure εequence" referε to chainε of nucleosides which have not reacted with a monomeric nucleoside intermediate during an addition step and which are subsequently capped. The term also includes polynucleotide chains whoεe growth waε initiated at an extraneous site of the solid phase support.

Thiophosphate analogs of polynucleotideε can be syntheεized in accordance with the invention following the thionization steps taught by Froehler, 5.etrafag-3rgR Letters , Vol. 27, 5575-5578 (1986), for H-phosphonate chemistry, or the thionizaton steps taught by Stec et al, J _A &π Cb s So .r Vol. 106, pgs. 6077-6079 (1984), for phosphoramidite chemistry.

The polystyrene support is amino-derivatized by εtandard procedureε, e.g. Wallace et al, pgs. 638-639, in Scouten, ed. §g_ £ gbase BJQ.g roAst Y (cited above). Briefly, hydroxymethylpthalimide iε reacted with the polystyrene support with a catalytic amount of

ethylsulfonic acid to form pthalimidomethyl polystyrene. This material is treated with hydrazine to remove the pthalimide protecting group and give aminomethylated polystyrene. The amino loading varies from 20-60 umoles of amino functionality per gram of nonswellable porous polystyrene. This level can be controlled by adjuεting the concentrationε of the reagentε and reaction time.

This material is then reacted with the S'-blocked protected nucleoside-3'-0-εuccinate. Unreacted amine of the polystyrene are rendered inactive by acylation with a moncarboxylic acid, e.g. as disclosed in U.S. patent 4,458,066. Preferably, the amines are rendered inactive by acetylation with acetic anhydride.

Aε outlined above, synthesis of the desired polynucleotide usually proceeds by repeated cycles of deblocking, monomer addition, and capping until εyntheεiε iε complete. Aε used herein, the term capping refers to reacting either the free 5' hydroxyl of a 3' to 5' growing nucleotide chain or the free 3' hydroxyl of a 5' to 3' growing nucleotide chain with a capping agent to render the chain incapable of participating in subsequent condensation steps. The preferred capping agents of the invention are phosphite monoeεterε of the form:

H

R- o - P - o ιι O

ggsπu -l II

wherein R, either alone or together with the oxygen to which it is attached, iε unreactive with the reagents used in solid phase oligonucleotide synthesis, particularly phosphoramiditeε or nucleoεide hydrogen phosphonates. Preferably, R represents a lower alkyl, an electron-withdrawing substituted lower alkyl, a lower alkyl- or halo-substituted aryl, or a heterocycle containing nitrogen, oxygen, or sulfur and from 5-8 carbon atoms. More preferably, R is methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n- pentyl, cyclopentylmethyl, isopentyl, neopentyl, n- hexyl, neohexyl, isohexyl, cyclohexylmethyl,beta- cyclopentylethyl, lower alkyl- or halo-substituted phenyl, lower alkyl- or halo-substituted benzyl, or lower alkyl- or halo-substituted phenylethyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, beta-electron-withdrawing-substituted ethyl, or the like. In further preference, the electron-withdrawing substituent of beta-electron-withdrawing-subεtituted ethyl is cyano, nitro, phenylsulphonyl, or phenylester. Most preferably, the beta-electron-withdrawing- substituted ethyl iε beta- cyanoethyl. In further preference, the lower alkyl- or.halo-substituentε of the lower alkyl- or halo-substituted phenyl and benzyl are methyl, chloro, or bromo. In further preference, morpholinyl, thiomorpholinyl, and piperidinyl are morpholino, thiomorpholino, and piperidino, respectively.

The chemical structures illustrated by Formula II are referred to in the literature as both phosphites and phosphonates. Reflecting the approximate usage in the literature, the structureε will be referred to herein as phoεphiteε, except when R iε a nucleoside. In such caεeε the structure will be referred to as a

hydrogen or H- phosphonate.

As illustrated by Formula III, the capping method of the invention compriεeε reacting a phosphite monoeεter defined by Formula II, i, with the free 5' or 3' hydroxyl of a failure sequence, j , in the presence of a εterically hindered acid chloride, ^, to form a phosphite diester, , between the failure sequence and a group which iε inert to subsequent reaction steps.

H-P^o

_j py iv ^x i ^' dlr .

r

Preferably, the capping agents of the invention (^ in Formula IV below) are prepared by alkaline hydrolysis of the symmetrical phosphite diesterε, , as described by Gibbε et al in gynt esA r P9-_- 410-413 (1984), which iε incorporated by reference. The phoεphite monoeεter ^ can be used directly as a salt

af ter evaporating volatile by products of the reaction or after purification by conventional meanε.

EQTOH-U IX

In the εterically hindered acid chloride , R' iε preferably tert-butyl, sec-butyl, cyclohexyl, adamantyl, norbornyl, phenyl, aryl, or the like. More preferably, R' is tert-butyl, norbornyl, or adamantyl. Moεt preferably, R' iε adamantyl.

Preferably, X ⁺ iε ammonium, lower alkylammonium, pyridinium, lutidiniu , cyclohexylammonium, 1,8- diazabicyclo.5.4.0]undec-7-ene ammonium, a metal salt cation such as Na ⁺ , K ⁺ , Li ⁺ , Ba ⁺ , Mg ⁺ , or the like. More preferably, X ⁺ is triethylammonium, tetrabutylammonium, diisopropylethylammonium, pyridinium, lutidinium, or cyclohexylammonium. Moεt preferably, X ⁺ is triethylammonium, tetrabutylammonium, or l,8-diazabicyclo[5.4.0]undec-7-ene ammonium.

Preferably, prior to delivery to the synthesis column bearing the oligonucleotide, a phosphite monoeεter of the invention and its cationic counter ion are dissolved in a solution comprising an aprotic polar solvent, such as acetonitrile, tetrahydrofuran, dichloromethane, or the like, or.some combination thereof, and a mild base εuch aε pyridine, picoline,

lutidine, collidine, or the like. Pyridine iε the moεt preferred mild base. Preferably, the concentration of the phosphite monoeεter iε between about .01 to .10 molar. Likewise, the sterically hindered acid chloride (^ in Formula III), prior to delivery to the synthesis column, is dissolved in a solution comprising an aprotic polar solvent, such as acetonitrile, tetrahydrofuran, dichloromethane, or the like, or some combination thereof, and a mild base such aε pyridine, picoline, lutidine, collidine, or the like. Pyridine is the most preferred mild base. The respective solutions are delivered concurrently to the synthesis column bearing the growing oligonucleotide so that the molar ratio of the phosphite monoeεter to the sterically hindered acid chloride present in the reaction mixture is about 1:5. This operation can be readily performed by an automated DNA synthesizer, such aε the Applied Bioεyεtemε models 380A, 380B, or 381A. The capping procedure of the invention iε performed as a step in each cycle, after the coupling reaction, to render the failure sequenceε inert. Preferably, the synthesis column iε immersed in the reaction mixture for about 20-120 seconds at room temperature, after which the reagents are flushed from the column with a solvent, such aε acetonitrile, tetrahydrofuran, dichloromethane, pyridine, or the like, or some combination thereof. All vessels within the instrument must be maintained rigorously free of moisture and oxygen under an atmosphere of an inert gas, such aε argon.

At the completion of εyntheεiε, the polynucleotide chains (both correct-sequence chains and failure sequences) are deprotected and cleaved from the polyεtyrene support to form a cleavage mixture.

Preferably, this is accomplished by treatment with concentrated ammonium hydroxide for 4-5 hours at room temperature or for about 1 hour at 55°C. The cleavage mixture is applied to a solid phase adsorbent that preferentially adsorbs the S'-blocked deprotected polynucleotideε. The preferential adεorption iε accomplished by selecting a blocking group and solid phase adεorbent εo that they have a strong mutual attaction relative to the rest of the polynucleotide. The attraction can be based on hydrophobicity, charge, or other phyεiochemical property. Trityl blocking groupε are preferentially adsorbed onto a hydrophobic solid phase adsorbent, such aε polyεtyrene. Preferably, whenever trityl blocking groupε are used the solid phase adsorbent iε a nonswellable porous polystyrene pretreated with a concentrated alkylammonitim salt solution. Preferably, the nonswellable porous polyεtyrene used aε the adsorbent is used in the form of beads having a diameter in the range of about 15-100 x 10~ ^"4 cm and pore size in the range of 100-1000 angstromε. More preferably, the bead diameter iε in the range of 50-70 x 10 ^""4 cm and pore diameter iε about 300 angεtromε.

Prior to applying the cleavage mixture to the polyεtyrene adεorbent, the adsorbent is flushed with an organic solvent, such aε acetonitrile, to wet the surface, then a concentrated alkylammonium salt solution, preferably a 2 M solution of triethylamine acetate, is applied to eεtablish a lipophilic counterion. After the cleavage solution, containing the polynucleotide εyntheεiε mixture in concentrated ammonia, iε applied to the polystyrene adsorbent, the adsorbent is then washed, preferably with dilute ammonia (a 3% solution) and water, to remove the

failure sequences and other contaminants, the correct- sequence chainε are deblocked, i.e. de-tritylated, and the de-tritylated polynucleotide iε eluted from the adsorbent. Preferably, the elution iε accomplished with a neutral solvent, e.g. 20% acetonitrile in water, or like solvent. By way of example, polystyrene adsorbent (50 milligrams per 5 OD units of oligonucleotide to be purified) is packed in a purification chamber, e.g. a standard flow through chromatography column, with an internal volume of about 500ul. The polystyrene adsorbent iε pre-waεhed with 5 ml of acetonitrile, then fluεhed with 5 ml of 2 M triethylammonium acetate. The cleavage mixture iε εlowly forced through the purification chamber manually with a 5 ml εyringe at about 2 ml per minute to load the tritylated sequences. After loading, the purification chamber iε washed first with 10 ml of 3% ammonia/water and then with 10 ml water. 3 ml of 2% trifluoroacetic acid in water is applied to the purification chamber and allowed to set for 3 minutes, afterwhich 15 ml of water iε flushed through the chamber to remove reεidual acid. Finally, the purified oligonucleotide iε eluted from the adsorbent with 1 ml of 20% acetonitrile/water.

Preferably, the method of the invention iε automated. The apparatus for automating can take several forms. Generally, the apparatuε comprises a series of reagent reservoirε, a synthesis chamber containing the nonswellable porouε polyεtyrene support, a purification chamber (which may be the same or different from the synthesis chamber) containing the solid phase adεorbent, and a computer controlled meanε for transferring in a predetermined manner reagents from the reagent reservoirs to and from the synthesis

cha ber and the purification chamber, and from the synthesis chamber to the purification chamber. The computer controlled meanε for transferring reagents can be implemented by a general purpose laboratory robot, such as that disclosed by Wilson et al, BJtQ3.ggbB3.sm8S- Vol. 6, pg.779 (1988), or by a dedicated system of tubing, and electronically controlled valves. Preferably, the computer controlled means iε implemented by a dedicated system of valves and tubing connecting the various reservoirs and chambers. In further preference, the reagents are driven through the tubing by maintaining a positive pressure in the reagent reservoirs by means of a pressurized inert gas, such aε argon, as iε used by many widely available automated εyntheεizerε, e.g. Applied Bioεystems, Inc. models 380B or 381A DNA syntheεizerε.

A diagrammatic representation, of a preferred embodiment of such an apparatuε iε illuεtrated in Figure 1. The apparatus of Figure 1 iε set forth aε if the phoεphoramidite chemistry were being employed. The same instrument can also be uεed to automate the H- phoεphonate εyntheεiε and purification with obvious modifications, e.g. different εyntheεiε reagentε are used, different reaction times are required, and the . •like. These modifications are readily implemented via programmable controller 48, or like means. 5'-blocked protected nucleoside intermediates are stored in reservoirs 2 through 10, one reservoir each for the four natural nucleoεideε. Optionally, an additional reεervoir 10 iε provided for a 5'-blocked protected nucleoside analog, e.g. deoxyinoεine, a linking agent, e.g. U.S. patent 4,757,141, or like intermediates. The reservoirε 2 through 10 containing the εyntheεiε intermediates are connected to synthesiε chamber 28 by

way of valve block 24 whose operation iε controlled by controller 48. Syntheεiε reagentε are stored in reservoirs 12 through 18. For example, in phosphoramidite chemistry these can be 12 trichloroacetic acid in dichloromethane for deblocking, 13 iodine/lutidine/water/tetrahydrofuran solution for oxidizing internucleoside phosphorouε, 14 tetrazole/acetonitrile solution for activating the nucleoεide intermediateε, 15 ammonium hydroxide for cleaving the completed chain from the εynthesiε support, 161-methylimidazole/tetrahydrofuran εolution and 17 tetrahydrofuran/lutidine/acetic anhydride εolution for capping, and 18 acetronitrile for washing. These reagent reservoirs are connected to synthesis chamber 28 by way of valve block 22 which iε controlled by controller 48. Synthesis proceeds under programmed control with each nucleotide being added to the growing chain by successive cycles deblocking, addition, capping, and oxidizing. Reagents removed from syntheεiε chamber 28 are directed to either trityl collection station 30, waste reservoir 38, or purification chamber 34 by way of valve block 32, which iε controlled by controller 48. During each cycle trityl blocking groupε releaεed by deblocking are monitored photometrically at trityl collection station 30 to track the efficiency of the syntheεiε.

When synthesiε iε complete, the synthesiε support is treated with concentrated ammonium hydroxide to deprotect and cleave the polynucleotide chains. Before the resulting solution (the cleavage εolution) iε transferred to purification chamber 34, the polystyrene adsorbent in the chamber is fluεhed firεt with acetonitrile and then with triethylammonium acetate, from reεervoirs 40 and 41, respectively. The cleavage

mixture iε transferred to purification chamber 34 via valve block 32 where it reacts with the polyεtyrene adεorbent. The polyεtyrene adεorbent is then treated with a series of purification reagents from reservoirs 40 through 46 to separate failure sequences and impurities from correct-sequence polynucleotides and to elute the correct-sequence polynucleotides from the adsorbent. Transfer of reagents from the reservoirs and to and from the purification chamber 34 are made by way of valve blocks 26 and 36, which are controlled by controller 48 First, dilute ammonium hydroxide from 40 fluεheε purification chamber 34 to remove capped failure sequences and other contaminants. Next, the tritylated polynucleotideε are detritylated by treatment with a 2% triflluoroacetic acid εolution in water, reεervoir 44, and the the detritylated polynucleotideε are eluted with a 20% acetonitrile solution in water, reεervoir 42, and are collected in product veεεel 50.

The following examples serve to illustrate the present invention and are not to be considered limitations thereof.

E- ELES

Example i. .Cg _B ar_lsΩn s£ t.aQ5_W5 S ΪΩ Initiation

A 5'-tritylated-thymidine-derivatized polystyrene support (4000 angstrom pore size), a 5'-tritylated- thymidine-derivatized CPG support (1000 angstrom pore size), and a 5'-tritylated-thymidine-derivatized CPG support (500 angstrom pore εize) were treated with 5'- trityldeoxyadenoεine-3'-phoεphoramidite (A) and

tetrazole activator for one hour and then detritylated. The nucleosidic material was then removed by treating the supports with ammonia, and separately analyzed by HPLC, the pertinent results of which are shown in Table I. Addition of A to the supports can only occur at ttøo types of siteε: (i) reactive εiteε on the support surface which were never capped or became uncapped, or (ii) detritylated thymidine. Reaction at the former sites leads to the detection of adenoεine monophoεphate (AMP) in the HPLC analyεiε, and in practice to a heterogeneous population of tritylated polynucleotideε cleaved from the εyntheεiε εupport (as discussed below). Reaction at the latter siteε leadε to the detection of an A-T dinucleotide in the HPLC analyεiε. The presence of the latter sites reflects instability of the trityl group during storage. Other compounds detected in the HPLC analysis are thymidine (the predominant component) and benzamide, a by-product of the removal of the protecting group of adenine.

Beiatiye &roQUPts £££_£ £hκaKft£gaKftK EsaHfi Support iMP thymidine benzamide A-T dimer tTMnP'i'-T'r-fcf'i. tf-

Polystyrene 1.9% — — 0.4%

CPG 5.7% — — 0.8%

(1000 angstrom)

CPG 4.1% — — 2.4%

(4000 angstrom)

The data indicate that the polystyrene support generates lesε than half the extraneouε εtart sites as does CPG. Therefore, polynucleotides made with the polystyrene support are.more pure and can be more easily purified than polynucleotides made on a CPG . support.

Example ii. Synthesis si ιa la-roer ftiUgaimfiAssfeA-te SB £2£ m E tøtϊi- sna

Three 18-mer oligonucleotideε of the same sequence were synthesized on an Applied Bioεystemε model 38OB εyntheεizer (which haε 3 reaction chamberε) programmed for using phoεphoramidite chemistry, two on CPG supports and the other on a polystyrene support in accordance with the invention (beads from Polymer Laboratories, Ltd., part number 1412-6602, derivatized aε described above). All three reaction chambers corresponded to 0.2 umole-scale εyntheεeε and the machine waε run in accordance with manufacturer'ε recommended protocolε. The crude product of each synthesis waε analyεed by capillary electrophoreεis on an Applie Biosyεtems Model 270A instrument. This analyεiε indicated that peaks corresponding to the correct sequence polynucleotide make up 63% and 73% of the crude product from the CPG-based εyntheseε, respectively, and 83% of the crude product from the polystyrene-base synthesis.

Example III. gyntbesis si a 12- sji EglY Hglea iae £B≤£Sϋ_l EQiYSt rene Two 72-mer polynucleotideε of the same sequence (baεe compoεition - _^ c^øG^c-T^g) were syntheεized on an Applied Biosystems Model 391 PCR-Mate using the manufacturer^ recommended protocolε. In one εyntheεis a

polyεtyrene εupport waε uεed aε deεcribed in Example I. In the other synthesis, CPG waε used as a εupport. The crude polynucleotides were radiolabeled with -^P-ATP, separated by polyacrylamide gel electrophoresis, and autoradiographed. The bandε of each lane on the film were quantitated by densitometry. The denεitometry profiles are shown in Figures 2A and 2B. The largest peak in both Figures corresponds to the 72-mer correct- sequence product. The profiles clearly show larger populations of failure sequences in the crude product of the CPG-baεed εyntheεiε.

Example iv. gyntheais si a 2^PiSK EataRH Asa£ 33 SH tø&ZKSRg, &£E&ϊύmmk kiKitiRg aw its

Si MfiRRKfiK 8SftS£3Qt Two sets of identical 21-mer polynucleotides (5'-

GTCAGTTCCATCAACATCATT-3' ) were syntheεized concurrently on separate .05 umole polystyrene, 0.2 umole polystyrene, and 0.2 umole CPG columns on an Applied Biosystems model 380B DNA syntheεizer uεing the manufacturers recommended protocol. In the firεt εet, 7.5 mg of phoεphoramidite (0.1 M) (the amount in the recommended protocol) in εolution for 21 days waε delivered to the reaction vesεel on each coupling cycle. In the εecond εet, 4.0 mg of freεhly disolved phosphoramidite monomer (in an approximate .05 M solution) was delivered to the reaction vessel on each coupling cycle. Otherwise, the synthesis of the two sets of polynucleotideε was identical. The crude reaction mixtures and polystyrene adsorbent-purified ("OPC purified") reaction mixtures were analyzed by capillary electrophoreεiε (CE) using an Applied Biosyεte ε model 270A instrument. Aε shown by the data in Table II, syntheseε on polyεtyrene reεulted in equal

or better yieldε in every caεe. Moreover, examination of the CE electrophorograms (Figure 3) εuggestε that undesired chain growth off the support may contribute to the comparatively high 20-mer (N-l er) contamination in the CPG samples. CE analysis of the purified samples indicates that these contaminants are trityl-bearing.

The foregoing disclosure of preferred embodiments of the invention has been presented for purposes of illuεtration and deεcription. It iε not intended tσ be exhauεtive or to limit the invention to the precise form disclosed. Obviously many modifications and variations are posεible in light of the above teaching.

The embodimentε were chosen and deεcribed in order to best explain the principleε of the invention and its practical application, to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modificationε as are suited to the particular use contemplated. It iε intended that the scope of the invention be defined by the claims appended hereto.