PROCESS FOR PRODUCING POLYNUCLEOTIDES

Abstract: In a transverse phosphite process for preparation of polynucleotides, a 5' reactive resin nucloside is reacted with a bifunctional phosphorus (III) reagent to produce a monofunctional resin nucleosidophosphorus (III) intermediate. This intermediate is condensed with a 3' reactive 5' blocked nucleoside to produce a dinucleosidophosphite. The latter is 5' deblocked and the cycle of reaction with bifunctional intermediate and condensation with 5' blocked 3' reactive nucleoside repeated to build a chain corresponding to the desire polynucleotide. Phosphite is converted to phosphate by oxidation to produce the polynucleotide. Improved structures and processes for linking the nucleotide chain to the resin, and improve techniques for detaching the final nucleotide from the resin are also disclosed.

Description: PROCESS FOR PRODUCING POLYNUCLEOTIDES Background of the Invention This invention relates to the field of synthesis of ribo and deoxyribopolynucleotides and, more particularly, to an improved synthesis process which utilizes a bifunctional phosphorus (III) reagent. Because deoxy ibonucleic acid (DNA) and ribonucleic acid (RNA) are genetic informational macro olecules of cen¬ tral importance in bioengineering, methods for their synthesis are of major scientific and commercial interest. Synthe¬ tically produced RNA and DNA can be used in the synthesis of genes which specify small p ptide hormones and which can be engineered into recombinant DNA plasmids for the manufacture of such hormones in bacteria. RNA and DNA polynucleotides of specific sequence have been used in the identification and isolation of commercially interesting genes. These polynu¬ cleotides may also be used for the modification of genes at specific sites and in the correction of genetic defects or for the improvement of animal or plant genes. Various methods are known to the art in which poly¬ nucleotides are produced by initial reaction between a nucleoside and a monofunctional nucleosido phosphorus (III) reagent, followed by sequential reaction with additional nucleosidophosphorus (III) monofunctional reagents to build a phosphite chain having the desired sequence of bases. Oxida¬ tion of the phosphite to phosphate produces the desired poly¬ nucleotide. Letsinger and Lunsford, Journal of the American Chemical Society v Vol. 98, pp. 3655 to 3661 (1976) describe a liquid phase synthesis in which a 5' blocked nucleoside is reacted with ethylphosphorodichloridite to produce a nucleo- sido phosphoromonochloridite adduct that is useful as a chain- building reagent. This initial synthesis is carried out at extremely low temperature (approximately -70 β C) in the presence of toluene to produce the active intermediate monoadduct Group) and a very heavy collidine hydrochloride precipitate. Reac¬ tion takes approximately thirty minutes at the extremely low temperature conditions, which are necessary to avoid dimeri- zation of the adduct reagent. Stringent control of the laboratory environment is also necessary in order to eliminate oxidation and moisture problems. After removal of the preci¬ pitate by centrifugation, the reaction mixture is allowed to warm to room temperature for an additional thirty minutes and then repeatedly coevaporated with toluene to remove the un- reacted methylphosphorodichloridite. The purified monoadduct is reacted with a 3' blocked 5' reactive nucleoside to produce a dinucleosido intermediate, after which the terminal 5' site of the second nucleoside is deblocked and chain building car- ried out by repetitive reaction with additional monoadduct. Since there are four different bases in polynucleo- tides, it is necessary to synthesize four different monofunc¬ tional nucleosido phosphorus (III) active intermediates, each in accordance with the laborious process outlined above. Even under the stringent conditions outlined above, this process is susceptible to a side reaction which yields an inert 3' 3' dinucleotide byproduct OMPI (Blocking Moiety) Because of the propensity for dimerization, not only are the reaction conditions extreme, but also very precise control of the reaction is necessitated. Moreover, the collidine hydro- chloride precipitate produced in each coupling step is very heavy and tends to interfere with processing and clog process equipment. Serious problems with corrosion of process equip¬ ment have also been encountered. In an effort to reduce dimerization Fourrey and Shire, Tetrahedron Letters, Vol. 22, pp. 729-732 (1981) searched for new phosphine derivatives which exhibit a better selectivity for primary than secondary alcohols of nucleo- sides. In their synthesis,, the 5*-0-trityl thymidine is reacted with methylphosphoroditetrazolide to produce a mono- adduct which is thereafter reacted with 3*-0-acetyl thymidine to produce a 3' 5' dinucleosido phosphite. The authors also describe deprotecting the 3'OH and reacting with additional methylphosphoroditetrazolide and 3'-0'-acetyl thymidine to produce fully protected TpTpT. The paper expresses an expec- tation that work would show the reaction conditions useful for the synthesis of oligonucleotides. All of Fourrey and Shire's reactions are conducted in a homogeneous liquid phase system. In a modification of the liquid phase synthesis of Letsinger and Lunsford, a heterogeneous reaction system was developed and disclosed by Matteucci and Caruthers, Journal of the American Chemical Society, Vol. 103, pp. 3185-3191 (1981). In this modified process, the initial condensation reaction is between the nucleosido phosphorus (III) active intermediate and a 5* reactive nucleoside which is attached at its 3' position to a resin. Typically, a silica resin is utilized and the initial substrate has the following structure (resin) While the use of a solid phase substrate facilitates recovery of the polynucleotide and simplifies processing, it does not eliminate the need to synthesize four different nucleosido phosphorus (III) intermediates. Nor does it obviate the dimerization problems inevitably attendant on this basic syn¬ thesis scheme. Moreover, the heavy collidine hydrochloride precipitate tends to coat the resin substrate, retard the rate of reaction and reduce yields. In a further modification, Mateucci and Caruthers substituted a tetrazolide group in lieu of the chloride on the monofunctional adduct, thereby eliminating the problem of collidine hydrochloride precipitation during the chain buil¬ ding reaction. However, this modified process requires an additional step and does not avoid the necessity for extremely OMPI low temperature and carefully controlled preparation of four separate monofunctional nucleosido phosphorus (III) active intermediate reagents. Nor does it eliminate the dimeriza¬ tion problems encountered in the preparation of the interme¬ diate reagents. Moreover, the methoxynucleosido monotetra- zolide reagent is quite unstable and must be stored at very low temperature. To avoid the problem of instabilit •ry of the reagent, Beaucage and Caruthers, Tetrahedron Letters, Vol. 22, pp. 1859-1862 (19 ' 81) have developed a methόxy nucleosido phos- phoromono(N-dimethyl)amidite (Blocking (CH-J Moiety) which is storage stable and can be converted to the ono- tetrazolide as needed for the coupling reaction, hile this avoids the problem of the storage stability of the tetra¬ zolide, it does not avoid the necessity of preparing four separate nucleosido phosphorus (III) active intermediate reagents for use in the chain-building reaction. Nor does it avoid the necessity of synthesizing reagents at very low tem¬ peratures under carefully controlled conditions or the problem of dimerization. Thus, the above described methods for polynucleotide synthesis have suffered from a common set of drawbacks relating to processing conditions,- dimerization and the neces¬ sity of preparing four separate nucleosido phosphorus (III) active intermediate chain-building reagents. Summary of the Invention Among the several objects of the present invention, therefore, may be noted the provision of a novel process for preparing polynucleotides; the provision of such a process which avoids the need for preparation of relatively unstable monomeriσ nucleosido phosphorus (III) monofunctional interme¬ diates; the provision of such a process which avoids the problem of deactivation of a reactive resin substrate caused by precipitation of a scavenger amine hydrochloride; the pro- vision of such a process which avoids clogging of processing equipment by amine hydrochloride precipitate; the provision of such a process which minimizes 3'-> 3* dimerization in the course of the preparation of reagent; the provision of such a process which avoids the necessity of repetitive reaction with monomeriσ nucleosido phosphorus (III) intermediates; the provision of such a process in which reaction can be carried out under moderate conditions and in a simple fashion without the necessity of highly precise control; the provision of such a process which can be carried out in process equipment con- structed of conventional alloys without serious corrosion problems; the provision of such a process which produces polynucleotides in high yield; and the provision of improved techniques for detaching a polynucleotide from a substrate resin after the polynucleotide synthesis is complete. Briefly, therefore, the present invention is directed to a novel process for producing a polynucleotide in which a bifunctional phosphorus (III) reagent is reacted with a substrate comprising a nucleoside whose 5" position is reactive and whose 3' position is blocked by attachment to a solid phase resin. The bifunctional reagent corresponds to the formula L - P - I OR, where L is a functional leaving group reactive with a primary or secondary alcohol without forming a hydrogen halide, and -OR, is a nonfunctional substituent. This reaction forms a monofunctional t resin nucleosido phosphorus (III) intermediate corresponding to the formula where R^ is hydrogen or hydroxyl, S is a resin backbone, R 3 is a linking group, B, is a base, and OR, and L are as defined above. The resin intermediate is condensed with a nucleoside whose 5' position is blocked and whose 3' position is reactive, thereby producing a dinucleosido phosphite inter- mediate corresponding to the formula where D-- is a blocking group, OR,, R 2 B, and R, are as defined above, and B 2 is a base which may be the same as or different from B, . The D,- blocking group is thereafter removed by reaction with a Lewis acid to provide a reactive hydroxyl group at the terminal 5' position, and this 5' hydroxyl is reacted with the aforesaid bifunctional reagent to produce a terminal monofunctional - O - P - I OR, moiety at the 5' position, L and OR, being as defined above. The cycle of condensing the terminal monofunctional moiety with 5' blocked 3' reactive nucleoside, removing D 5 and reacting the resultant 5' deblocked substrate with bifunc¬ tional phosphorus (III) reagent is repeated until a chain of desired nucleoside sequence is produced. The nonfunctional R, group is cleaved from each phosphorus moiety with a rea¬ gent which effects substitution of a hydrogen in place there¬ of; and each phosphorus moiety is oxidized. A polynucleotide is thereby produced having the desired nucleotide sequence. The invention is further directed to a novel process for producing a polynucleotide in which bifunctional phospho¬ rus (III) compound is reacted with a substrate comprising a nucleoside whose 5' position is reactive and whose 3* position is blocked by attachment to a solid phase resin. The bifunc¬ tional reagent corresponds to the formula G - P - G I OR where G is a functional leaving group reactive with a primary or secondary alcohol and OR, is a non-functional substi- tuent. The substrate corresponds to the structural formula where R 2 is hydrogen or hydroxyl, S is resin backbone, B, is a base, and R, is a linking group comprising arylene group. The reaction forms a monofunctional resin nucleosido¬ phosphorus (III) intermediate corresponding to the formula where -OR,, R 2 , 3 , B,, S, and G are as defined above. The resin intermediate is condensed with a nucleoside whose 5* position is blocked and whose 3' position is reactive, thereby producing a dinuσleosidophosphite intermediate cor¬ where D 5 is a blocking group, -OR,, R 2 , B,, R 3 , and S are as defined above, and B 2 is a base which may be the same as or different from B, . The D- blocking group is thereafter removed by reaction with a Lewis acid to produce a reactive hydroxyl group at the terminal 5' position, and this 5' hydroxyl is reacted with the aforesaid bifunctional com¬ pound to produce a terminal monofunctional OR η - P I - G moiety at the 5* position, G and OR, being as defined above. The cycle of condensing the terminal monofunctional moiety with 5' blocked 3' reactive nucleoside, removing D 5 , and reacting the resulting 5' deblocked substrate with bifunc¬ tional phosphorus (III) compound is repeated until a chain of desired nucleoside sequence is produced. Each phosphite moiety is oxidized to phosphate and the nonfunctional R, group is cleaved from each phosphorus moiety. A polynucleo¬ tide is thereby produced having the desired nucleotide sequence. The invention is further directed to a resin adapted for use as a substrate in an arm of at least 5 A between the resin backbone and the first nucleoside and preferably with the preparation of a polynucleotide, the resin having a pendent structure comprising H 0 I II 0 II -N-C- (CH 2 ) fc- (NH) m (C) n (CH 2 ) p Ar-NH 2 where Ar is a substituted or unsubstituted arylene group, k is an integer between 0 and 4 inclusive, m is 0 or 1, n is 0 or 1, p is an integer between 0 and 4 inclusive, n is 0 when k + m = 0, and + ra + n + p is stl. Also included in the invention is a resin adapted for use as a precursor in the preparation of a polynucleotide, the resin having a pendent structure comprising H O 0 -N-C-(CH 2 ) k -(NH) m -(C) n -(CH 2 ) p -Ar-N0 2 where Ar, k, m) n, and p are as defined above. -Further included in the invention is a resin adapted for use as a substrate in the preparation of a polynucleotide, the resin having a pendent structure comprising H O O H ■ N-C-(CH 2 ) k (NH) m -(C) n -(CH 2 ) p -Ar-N-R 8 where Ar, k, m, n, and p are as defined above and R„ is either OR, - P - G or OR, - P - G OMP where G is a functional leaving group reactive with the secon¬ dary alcohol and R g is alkyl, aryl, hydrogen, or a salt- forming cation. Other objects and features will be in part apparent and in part pointed out hereinafter. Description of the Preferred Embodiments In accordance with the present invention, an improved process has been discovered in which a polynucleotide chain may be built by repetitive alternate reaction of a 3' blocked, 5' reactive resin nucleosido substrate with a bifunctional phos¬ phorus (III) reagent compound and condensation of the resul¬ tant intermediate with a 5' blocked, 3' reactive nucleoside. This process avoids the necessity for repetitive reaction with monomeric nucleosido phosphorus (III) active intermediate rea- gents and, in particular, avoids the necessity of the prepara¬ tion of four different monomeric nucleosido phosphorus (III) monofunctional reagents for use in providing the desired sequence of bases in the polynucleotide chain. Because the preparation of monomeric nucleosido phosphorus (III) reagents is entirely eliminated, the process of the invention is simplified as compared to the processes previously used in the art. Both the bifunctional reagent and the 5* blocked, 3' reactive nucleoside used in the synthesis are quite stable by comparison with the monofunctional nucleo- sido phosphite of the prior art. Control requirements are much less exacting, the process manipulations are significantly less laborious, and the reactions involved in the process of the invention can be carried out at room temperature. OMPI In a preliminary step of one embodiment of the process of the invention, a bifunctional reagent is prepared corresponding to the formula L - P - L I OR 1 where L is a functional leaving group reactive with a primary or secondary alcohol and OR, is a non-functional substituent. Most preferably, L is a tetrazolide group but other functional groups known in the art to condense with alcohols can also be used. Thus, for example, L may be a heterocyclic base radical such as triazolide, nitrotriazolide, benzotriazolide, imidazo- lide, nitroimidazalide. The non-functional substituent OR, is preferably ψethoxy but, alternatively, R, may be another lower alkyl, substituted lower alkyl, aryl, substituted aryl, or any other non-functional substituent which does not inter- fere with the reaction of the bifunctional. reagent with the 5' hydroxyl groups of a resin nucleosido substrate. Exemplary groups which may constitute R, include ethyl, propyl, tri- chloroethyl, tribromoethyl, benzyl, cyanoethyl, chlorophenyl, and nitrobenzoethyl. To prepare the bi-functional reagent a phosphoro dihalidite precursor corresponding to the formula X - P - X I OR χ where X is halogen is reacted in an organic solvent with tetrazole, triazole, nitrotriazole, or any other source of a leaving group of the type defined above. The reaction is car¬ ried out under an inert atmosphere at a temperature of 0°C or OMPI lower in the presence of an amine scavenger for hydrogen halide. Conveniently, this reaction may be carried out by adding methylphosphorodihalidite to a solution containing the leaving group source and the scavenger. Preferably, the solu- tion is saturated or supersaturated with respect to tetrazole or other leaving group source, and is mixed with the methyl¬ phosphorodihalidite in such relative proportions that there is a molar excess of approximately 2 to 5 moles of leaving group source per mole of methylphosphorodihalidite. Various polar solvents may be used for this reaction, including tetrahydro- furan, dimethylsulfoxide, formamide, dimethylformamide, thio- phene, thiophane, pyridine, piperidine and acetonitrile. After reaction is complete, the amine hydrohalide precipitate is removed by centrifugation or filtration. The supernatant solution of bifunctional phosphorus (III) reagent may be used directly in the reaction with 3' blocked 5' active resin nucleoside. This solution is stable for weeks when stored at -20°C but, since its preparation is simple, it may as conveniently be prepared daily. The initial substrate for the preparation of a poly¬ nucleotide is a 3' blocked, 5' reactive resin nucleoside sub¬ strate corresponding to the formula where S is a resin backbone, R 3 is a linking group, R 2 is hydrogen or hydroxyl, and B, is a base. OMPI The 5' position is deblocked by means of a Lewis acid and the deblocked substrate reacted with the bifunctional phosphorus (III) intermediate L - P - L OR, to produce the resin nucleosido phosphorus (III) monofunctional intermediate In this synthesis step a heterogeneous irreversible reaction occurs between solid resin substrate and reagent contained in a solvent therefor. As noted above, the reagent may be intro- duced in the solution in which it was produced. It is highly preferred that a substantial molar excess of bifunctional phosphorus (III) reagent be utilized in the preparation of the monofunctional nucleosido phosphorus (III) resin intermediate. If only stoichiometric equivalents of substrate and bifunctional reagent are employed, there may be a significant incidence of resin cross-linking via 5'- 5' dimerization. If only a modest excess of the bifunctional reagent is used, the extent of dimerization may actually be exacerbated. But paradoxically, where a high excess, for OMPI example, a molar ratio of 15:1 or greater is used, each reac¬ tive site on the substrate is surrounded by a "swarm" of bi¬ functional reagent molecules which thus form a barrier inhibi¬ ting the kinetic contact between the resin monofunctional nucleosido phosphorus (III) intermediate and unreacted sub¬ strate. Such contact would otherwise result in formation of the dimer. High yields are also promoted by a substantial excess of bifunctional reagent. Thus, the reaction proceeds as follows: Formation of the resin intermediate proceeds at ambient tem¬ perature in an essentially instantaneous, quantitative manner. After reaction is complete, the resin, now comprising the resin nucleosido phosphorus (III) intermediate, is readily separated by filtration or centrifugation and the solution of bifunctional phosphorus (III) reagent is available for use in reaction with further amounts of substrate. After removal of the excess bifunctional reagent, the nucleosido phosphorus (III) intermediate is reacted with a 5' blocked 3 1 reactive nucleoside to produce a dinucleosido phos¬ phite intermediate: To carry out this reaction, a solution of the 5' blocked 3* reactive nucleoside is mixed with the solid resin intermediate in an inert atmosphere, preferably at a molar ratio of at least about 3 moles incoming nucleoside to intermediate. Solvents useful in the synthesis of the bifunctional reagent and the reaction of that reagent with the substrate are also useful as vehicles for the nucleoside in its reaction with the resin interemdiate. Preferably, the nucleoside solution is in the range of 1.0 to 1.5 molar. Heterogeneous reaction again takes place essentially at room temperature, and quantitative yields are realized after a period of approximately five minutes. To build the chain, the blocking group on the ter¬ minal nucleoside is removed by means of a Lewis acid such as zinc bromide, thus providing a reactive hydroxyl group at the terminal 5' position which is again reacted with bifunctional reagent to once more produce a terminal monofunctional - P - L i OR, moiety at the 5' position. This terminal group can be con¬ densed with another 3' reactive 5* blocked nucleoside and this cycle repeated with a sequence of incoming nucleosides that provides the desired base sequence in the product. In order to produce the polynucleotide final product, the phosphite linkages are oxidized to phosphate, preferably by reaction with iodine, and the R, group cleaved from each phosphorus moiety by means of a reagent which effects substi- tution of hydrogen in lieu thereof. This cleavage can be car¬ ried out, for example, by reaction with a mixture of triethyl- amine and thiophenol. The oxidation of the phosphorus (III) to phosphorus (V) is preferably carried out at each nucleoside condensation step. Alternatively, however, it is feasible to carry out the oxidation reaction the entire chain has been generated. In a particularly preferred embodiment of the inven¬ tion, the polynucleotide chain is built on a nucleosido resin substrate in which the linking group, i.e., R 3 in the struc¬ ture comprises an arylene group, more preferably an arylene group combined with other moieties, so that, on a molecular scale, there is substantial bulk in the link between the inorganic resin and the first nucleoside of the nucleotide chain. Use of such a linking group reduces the loading capacity of the resin and is believed to increase the average distance between potential cross-linking functionalities. Whether for these or other reasons, arylene-containing linking groups have been found to inhibit cross-linking between adjacent polynucleotide chains as they are grown on adjoining sites of the resin back- bone. In this embodiment of the invention, it has been found possible to build the chain using either a bifunctional (III) reagent of the type described above, i.e., corresponding to the formula L - P - L I OR, where L is a leaving group which does not form a hydrogen halide, or a reagent corresponding to the formula X - P - X I OR., where X is a halide such as chloride, bromide, or oxygen. Generically, therefore, this embodiment of the invention allows the use of a bifunctional (III) phosphorus compound correspon- ding to the formula G - P - G I OR 1 where G is essentially any functional group reactive with a primary or secondary alcohol and OR, is as defined above. Use of a non-halide is preferred, however, to minimize coating of the substrate or clogging process equipment with an amine- hydrochloride precipitate. It is particularly preferred that R 3 comprise of a length of at least 5 A between the resin backbone and the first nucleoside. More particularly, it is preferred that the pendent structures corresponds to the formula H O O H I -N-C-(CH 2 ) k -(NH) m (C) n (CH 2 ) pj -Ar-N-R 5 - where Ar is the substituted or unsubstituted arylene, k is an integer between 0 and 4 inclusive, m is 0 or 1, n is 0 or 1, p is an integer between 0 and 4 inclusive, n is 0 where k + m = 0, k + m + n + p is at least 1, and R 5 is a phosphite or phosphate linkage. In this structure, Ar is preferably unsub¬ stituted phenylene or phenylene substituted with a nonfunc¬ tional moiety such as alkyl, alkoxy, aryl, or aryloxy. How- ever, if desired, napthylene or other polynuclear arylene groups may be used. Specific structures which may be especially convenient to prepare include that in which Ar is phenylene, k = l, m = 0, n = 0, and p = 0; that in which Ar is phenylene, k = 3, m = 0, n = 0, and p = 0; that in which Ar is phenylene, k = 5, m = l, n = l, and p = 0; and that in which k = 0, m = l, n = l, and p = 0. To prepare structures having a linking group of the type outlined above, a precursor resin having pendent amine groups is condensed with a nitroarylcarboxylic acid to produce the corresponding amide. This condensation is facilitated by initially preparing a linking group intermediate comprising an active ester of the nitroarylcarboxylic acid and an N-hydroxy- imide. Such linking group intermediate corresponds generally to the structure H O 0 l II II -N-C(CH 2 ) k -(NH) m - (C) n (CH 2 ) p -Ar-N0 2 where k, m, n, p, and Ar are as defined above and R_ com¬ prises an imido moiety bonded to the oxygen through an imido nitrogen. Thereafter, the linking group intermediate is reacted with the pendent amine of the resin, resulting in splitting off of the imide and effecting the desired conden¬ sation between the carboxylic acid and pendent amine to form the amide. In preparing the linking group intermediate, it is particularly preferred that the nitroarylcarboxylic acid, which corresponds to the structural formula O O li l| 0 2 N-Ar- (CH 2 ) p (C) n (NH) m - (CH 2 ) k -C-OH where Ar, k, m, n, and p are as defined above, be esterified with an N-hydroxyimide of a dicarboxylic acid, most prefer¬ ably an imide corresponding to the formula where q is 2 or 3, In this instance, active ester has the structure ) n -(CH 2 ) -Ar-N0 2 In the course of the reaction forming the active ester, the nitroarylcarboxylic acid is initially reacted with a compound such as N,N-dicyclohexylcarbodiimide. ' JHE ^ OMPI Preparatory to reaction of the active ester with pendent amine groups of the resin, the amine groups are pre- activitated, for example, by treating the precursor resin with a strong organic base such as pyridine. Any base more nucleo- philic than the pendent amine . is effective as a hydrochloride scavenger and/or for deprotonation of the amine. The precursor resin containing pre-activated pendent amine groups is preferably contacted with a solution of active ester linking intermediate, conveniently the solution in which the active ester is intially formed. Alternatively, the ester can be removed from the reaction soluton and redissolved or reslurried in pyridine or other organic base. In either case, when the pre-activated resin is contacted with active ester, heterogeneous reaction ensues, resulting in splitting of the ester, formation of an activated resin having pendent groups comprising the structure H H 0O o -N-C-(CH 2 ) k -(NH) m -(C) n -(CH 2 ) p -Ar-N0 2 Preferably, the reaction between the active ester linking intermediate and the pendent amino groups of the precursor resin are conducted in the presence of a strong base such as pyridine. The activated resin is recovered by filtration and preferably washed with pyridine , and with other volatile organic solvents such as, for example, alcohols or ethers. Thereafter, the terminal nitro group on the pendent structure of the activated resin (hereinafter sometimes referred to as "the third precursor resin") is reduced and converted to an amine group. Conveniently, the reduction is carried out by heterogeneous reaction between the third pre¬ cursor resin and a noncatalytic chemical reducing agent con- tained in a solvent therefor. Preparatory to the reduction reaction, any remaining pendent amine groups of the original precursor resin (hereinafter sometimes referred to as "the fourth precursor resin") are capped by acylation in the presence of a strong base such as pyridine. Thereafter the reduction reaction is carried out, for example, by contacting the third precursor resin with an aqueous solution of an alkali metal dithionate at a pH of greater than 7.0, prefer¬ ably about 8.5. The desired pH is conveniently established by the presence of an alkali metal bicarbonate as a buffer. Alternatively the reduction may be carried out with a boro- hydride salt. The product second precursor resin having a structure comprising H O O i t* |l -N-C-(CH 2 ) k -(NH) m -(CH 2 ) -(NH) m -(C) n -(CH 2 ) p -Ar-NH 2 is separated from the reaction solution by filtration or cen- trifugation, washed extensively with water to remove excess reducing agent, and then preferably washed with a low molecu¬ lar weight alcohol and/or ether to dry it. The second precursor resin is then condensed with a bifunctional phosphorus (III) compound corresponding to the structure G - P - G I OR, where G and OR, are as defined above. Again, this reaction is conveniently carried out heterogeneously by contacting the second precursor resin with a solution of the phosphorus (III) compound, preferably with alkoxyphosphodihalidite. The second precursor resin is washed with an alcohol, a nitroalkane, and tetrahydrofuran, prior to condensation with the phosphorus compound. The product of the condensation reaction (referred to hereinafter as the "final precursor resin") has a pendent structure comprising H O O H -N-C-(CH 2 ) k -(NH) m -(C) n -(CH 2 ) p -Ar-ά-R 8 where Ro corresponds to the formula OR, I - P - G where R, , and -G are as defined above. The reaction is car¬ ried out in the presence of a strong organic base such as pyridine as a scavenger for the leaving group. The final pre¬ cursor resin is preferably separated from the liquid phase reaction product by conventional solid/liquid separation means, as by centrifugation or filtration. The final precursor resin is then condensed with a 3* reactive 5' blocked nucleoside to produce a nucleosido resin intermediate substrate corresponding to the formula OR.. I P - G where B, , R 2 , D,., Ar, k, m, n, and p are as defined above and R e corresponds to the formula OR, OMPI where -OR g is nonfunctional with respect to the condensation of the terminal leaving group G with the incoming nucleoside, -ORg is also nonfunctional with respect to subsequent reac¬ tions of 5' deblocked substrate and additional bifunctional phosphorus (III) compound. If desired, the phosphite group — P — Nucleoside can be converted to the phosphate linkage T 6 by oxidation in the manner described above. Once the nucleosido resin intermediate has been produced, it may be used in the manner described above for the preparation of the polynucleotide chain. The bifunctional phosphorus compound used in the chain-building reactions may comprise leaving groups L, which are reactive with a primary or secondary alcohol but do not form a hydrogen halide in condensing with an alcohol. However, in the embodiment where R 3 comprises an arylene group, the alternative use of an alkoxyphosphorodihalidite reagent is both acceptable and preferred. Further in accordance with the invention, improved methods and reagents have been discovered for detaching the polynucleotide chain from the solid state resin after the chain-building reactions have been completed. Previously, detachment of the chain from the solid state resin had been accomplished by use of a mixture of acetic acid, pyridine, and isoamyl nitrite. However, it has now been discovered that effective detachment may be achieved using a mixture of pyr¬ idine and isoamyl nitrite alone, omitting acetic acid, any other low molecular weight alkanoic acid, or any salt of an organic or inorganic base and an alkanoic acid, from the cleaving reagent. Where the detachment of the polynucleotide is carried out in the substantial absence of any low molecular weight alkanoic acid or salt thereof, it has been found that nearly 100% yields can be obtained by carrying out the reac¬ tion at moderately elevated temperatures, preferably at least about 45°C, typically in the range of between 50°C to 70°C, over a period of typically 24 hours. According to prior practice, in which acetic acid had been used, by-product for- mation was incurred in the chain detachment reaction, and overall yields tended to be only 30% to 35%. Although the proportions of components of the clea¬ ving reagent are not believed to be critical, it has been found that a volumetric ratio of between about 5 parts pyri- dine and about 20 parts pyridine per part isoamyl nitrite provides effective results. Other lower alkyl esters of nitrous acid may be used in place of isoamyl nitrite, while other strong organic bases, particularly other tertiary amines, may be used in place of pyridine. Optionally, the cleaving reagent can further comprise a solvent for the base and nitrous acid ester, such as, for example, tetrahydrofuran. After detachment from the inorganic resin, the polynucleotide may be purified by thin layer chromatography, preferably using a two-solvent system such as, for example, methanol/chloroform (1:9) and ammonium hydroxide, water, and n-propanol (35:5:60). OMPI_ V/IPO In polynucleotide synthesis, the yield of each step dictates how long a given chain may be propagated. Because the process of the invention provides very high yields, in the range of 99.4% at each step, it can be exploited in the syn- thesis of a very long chain nucleic acids. For example, the final yield in the synthesis of a 52 residue polynucleotide material is 78% of the starting material. Such yields are not attainable by the conventional phosphite chemistry procedure. Moreover, the manipulations required in the process of the invention are so simple and straightforward that a semi¬ skilled technician with no extensive chemical knowledge can accomplish the synthesis. By contrast, in the phosphoramidite procedure of the Beaucage and Caruthers, successful synthesis is dependent on the exercise of a very high level of skill. As noted, the method of the process of the invention avoids the production of any dinucleotide inert side product, and amine hydrochloride precipitate is generated only in the preliminary preparation of the bifunctional phosphorus (III) intermediate and not in the generation of the polyphosphite chain. The cost of the materials utilized in each step is less than half, probably no more than about a quarter of that for the conventional process. As further noted above, in the conventional phos¬ phite process 5' blocked 3' monofunctional phosphite substi- tuted nucleoside intermediates are repetitively reacted with a 5' deblocked nucleosido resin substrate. By contrast, the process of the invention may be referred to as a "transverse phosphite" process since it proceeds by alternate reaction of the substrate with bifunctional reagent and condensation of the resultant terminal functional group on the resin with a 5' blocked, 3' reactive nucleoside. The following examples illustrate the invention. Example 1 Methylphosphorodichlorodite (0.1 ml) was added under an inert atmosphere to a well stirred solution of collidine (0.7 ml) and 1H tetrazole (2.5 mM) in anhydrous tetrahydro- furan (3.0 ml) at room temperature. Instantaneous reaction produced methylphosphoroditetrazolide and a collidine hydro¬ chloride precipitate. The precipitate was removed by centri- fugation. The solution of methylphosphoroditetrazolide produced was sufficient for two condensations with 150 mg portions of 5' deblocked deoxynucleoside resin. Example 2 A 1.0 ml aliquot of the solution of methylphosphoro- ditetrazolide, prepared as described above, was added to a solid resin having a silica backbone and a pendent 5' reactive deoxynucleoside attached to the backbone at the 3' position through a succinamide linkage (Vydac resin-150 mg; alterna¬ tively 75 mg. Fractosyl resin may be used). These proportions represent a molar ratio of approximately 20 moles methylphos¬ phoroditetrazolide per mole of deoxynucleoside-bearing resin. The mixture was gently swirled for five minutes, thereby producing a terminal monofunctional tetrazolide substituted phosphite at the 5' position on the deoxynucleoside pendent moiety. The resin was thereafter separated by filtration from the supernatant solution containing excess methylphos- phoroditetrazolide. The recovered resin was flushed with argon and a solution of 5' dimethoxytrityl deoxynucleoside (0.050 mM) in anhydrous tetrahydrofuran (0.5 ml) was mixed with the resin. These proportions represent a molar ratio of 5 M of the 5' blocked, 3' reactive nucleoside per mole of the terminal monofunctional tetrazolide substituted resin nucleo¬ side phosphite. The mixture was swirled for five minutes, during which time the 5' blocked, 3* reactive deoxynucleoside condensed with the monofunctional phosphite to produce a resin dinucleosido phosphite intermediate, the terminal nucleoside of which was blocked at the 5* position. The cycle was then repeated by deblocking the ter- minal 5' site with zinc bromide and reacting with additional methylphosphoroditetrazolide under the conditions described above, to once more produce the terminal monofunctional tetrazolide substituted resin nucleosido phosphite. This monofunctional intermediate was reacted with a 5' blocked, 3' reactive nucleoside containing the next base for a polynucleo¬ tide of desired base sequence. As measured by release of dimethoxytritanol after each cycle, condensations employing blocked derivatives of dA, dC, dG and dT proceeded reprodu- cibly at greater than 98% yield. The above cycles were repeated in a thirteen step synthesis to produce the polynucleotide 3' TAGGAATATAGCGG 5'. At the conclusion of this thirteen step synthesis, trityl alcohol was recovered at 94% of the initial resin deoxynu¬ cleoside, thereby reflecting an average yield of 99.4% for each step. As necessary, the polynucleotide may be detached from the resin by conventional deesterification reactions known to the art. EXAMPLE 3 N-hydroxysuccinamide was condensed with p-nitro- . phenylacetic acid in dioxane solution, to produce a total of 552.4 mg of N-succinamidyl-p-nitrophenylacetate. The dioxane. was removed and the N-succinamidyl-p-nitrophenylacetate mixed with and partially dissolved in anhydrous pyridine (12 mL) . A slight pink color appeared as the active ester was mixed with the pyridine. Since dissolution was not complete, a slurry was produced, and this slurry used in the next step of the synthesis. Silated silica resin (1 g) sold under the trade designation "Fractosyl" was mixed with the partial solution/ slurry of N-succinamidyl-p-nitrophenylacetate in pyridine, and the resulting mixture shaken overnight at room tempera- ture. On the next day, the solid phase activated resin was separated from the supernatant organic liquid by filtration.. The resin was thereafter washed four times with pyridine, three times with methyl alcohol, and three times With ethyl ether. A partially dried orange/rust colored activated resin was obtained. Drying of the resin was completed in an oven. Dried, activated resin was reacted with acetic an¬ hydride in pyridine to acylate any pendent amine groups which had not been condensed with N-succinamidyl-p-nitrophenyl- acetate. This procedure prevented any free amine from compe- ting with subsequent reactions. The nitro group on the pendent structure of the activated resin was reduced with sodium dithionate. Prepara¬ tory to the reduction reaction, the activated resin was washed twice with pyridine, twice with methanol, and twice with a 0.5 M solution of sodium bicarbonate. The resin was then suspended in a sodium bicarbonate buffer solution (pH 8.5) and sodium dithionate (1.004 g) added to the suspension at 40°C. The reduction reaction mixture was held at 40°C for one hour, after which the resin was separated by filtration, washed again with pyridine, methanol, and sodium bicarbonate solution, and resuspended in a fresh aliquot of buffer solu¬ tion (20 mL) . Sodium dithionate was again added and the reduction reaction sequence repeated. Thereafter, the resin was separated from the supernatant aqueous phases by filtra¬ tion and washed extensively with 0.5 M sodium bicarbonate solution. The resin obtained was suitable as a precursor for condensation with bifunctional phosphorus (III) compound in the production of a resin substrate for a polynucleotide synthesis. » EXAMPLE 4 P-nitrophenylacetic acid (0.45 g) , N-hydroxysuccin- amide (0.3453 g) , and dicyclohexylcarbodiimide (0.5674 g) were mixed in dixsoane solution, and reaction carried out for the formation of N-succinamidyl-p-nitrophenylacetate. This reaction was repeated using, in lieu of p-nitrophenylacetate, other nitroarylcarboxylic acids, inclu¬ ding specifically N-p-nitrobenzoyl-6-aminocaproic acid (0.78 g reacted with 0.3453 g N-hydroxysuccinamide) , and 4-(p-nitrophenyl)butyric acid (0.523 g reacted with 0.3453 g N-hydroxysuccinamide) . In each case, esterification between the nitroaryl¬ carboxylic acid and N-hydroxysuccinamide proceeded, with for¬ mation of a by-product dicyclohexylurea precipitate which was separated by filtration. OMPI VItO EXAMPLE 5 Silated silica resin containing pendent amine groups (0.5 g) sold under the trade designation "Fractosyl" was prepared for condensation with an active ester by contac- ting it with pyridine (2 mL) and triethylamine (0.5 mL) . The resultant mixture of resin, pyridine and triethylamine was shaken for 45 minutes, and the bases evaporated under vacuum, after which the pre-activated resin was mixed with an addi¬ tional portion of pyridine (5. mL) . To the mixture of silica resin and pyridine was added a substantially equivalent portion of N-succinamidyl-p- nitrophenylacetic acid, and the resultant amidation reaction mixture shaken for 20 hours. Thereafter, the solid resin was separated by filtration, washed with three 5-mL portions of pyridine, three 5-mL portions of methanol, and one 5-mL por¬ tion of ethyl ether, and then dried under vacuum for one-half hour. The dried activated resin was contacted with pyri¬ dine (3 mL) and acetic anhydride (300 mL) and the resultant mixture shaken for one hour to affect acylation of any free amine groups on the resin. Thereafter, the resin was washed with a 0.5 M solution of sodium carbonate (100 mL) and a 0.5 M solution of sodium bicarbonate (100 mL) . The washed acti¬ vated resin was then contacted with a solution of 0.2 M sodium dithionate in 0.5 M sodium bicarbonate (5 L; pH 8.76) for reduction of the nitro group. EXAMPLE 6 A precursor resin produced in the manner described in Example 5 was washed with methyl alcohol, three times with nitromethane, and three times with tetrahydrofuran. There¬ after, the resin was contacted with a solution of methylphos- phorodichloridite (100 mmL) , pyridine (0.75 mL) , and tetra¬ hydrofuran (3 mL) , thereby effecting condensation of the methylphosphorodicloridite with the amine group and forming a pendent moiety corresponding to the structure Alternate condensation with 5' blocked 3* reactive nucleoside, deblocking of the 5* position, and further condensation with methylphosphorodichloridite was carried out until the nucleo- tide sequence TCTAGCTTCCTTCT had been obtained. At each step of the polynucleotide synthesis the phosphite linkage was oxidized to a phosphate with iodine, and the resin recovered by filtration and washed with tetrahydro¬ furan. Condensations with the methylphosphorodichloridite were carried out in the presence of pyridine as a hydrochlo- ride scavenger. Deblocking of the 5* hydroxyl was carried out using a zinc bromide solution. EXAMPLE 7 A polynucleotide resin prepared on a silica resin substrate, as described in Example 6, was detached using a mixture of pyridine (2 mL) and isoamyl nitrite (0.3 mL) at 40°C. After five hours, 54% recovery of the free polynucleo¬ tide had been achieved, and at 24 hours an 89% recovery had been achieved. This procedure was repeated at 60°C, at which temperature an 84% yield was obtained in five hours and a 100% yield in 24 hours. By comparison, carrying out the polynucleotide detachment reaction at room temperature using a reagent com¬ prising pyridine, isoamyl nitrite, and acetic acid produced a yield of only 32% in five hours, and 33% after 24 hours. * Thus, the process of the invention provides a poly- nucleotide preparation that is simple, rapid and commercially feasible in terms of labor and material costs. It is also adapted to implementation in state of the art processing equipment utilized in genetic engineering processes. In par¬ ticular, it may be adapted for automation in the commercially available "gene machine". Moreover, the process of the invention provides a commercially practicable and cost effec¬ tive route to the synthesis of genes which specify small pep- tide hormones and which can be engineered into recombinant DNA plasmids for the manufacture of such hormones by bacteria. In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained. As various changes could be made in the above methods without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. OMPI

Claims: Claims WHAT IS CLAIMED IS: 1. A process for producing a polynucleotide, com¬ prising the steps of: reacting a bifunctional phosphorus (III) reagent with a sub¬ strate comprising a nucleoside whose 5' position is reactive and whose 3' position is blocked by attachment to a solid phase resin, said reagent corresponding to the formula L - P - I OR, where L is a functional leaving group reactive with a primary or secondary alcohol without forming a hydrogen halide, and OR, is a nonfunctional substituent, thereby producing a monofunctional resin nucleosido phosphorus (III) intermediate corresponding to the formula where R 2 is hydrogen or hydroxyl, S comprises a resin back- bone, R 3 is a linking group, B, is a base and OR, and L are as defined above; condensing said resin intermediate with a nucleoside whose 5' position is blocked and whose 3' position is reactive, thereby producing a resin dinucleosidophosphite intermediate corre- sponding to the formula where D 5 is a blocking group, -OR,, R 2 , R_, B, and S are as defined above and B_ is a base which may be the same as or different from B, ; removing the D 5 blocking group by reaction with a Lewis acid to provide a reactive hydroxyl group at the terminal 5* position; reacting said 5* hydroxyl with said bifunctional reagent to produce a terminal monofunctional OMPI moiety at said 5* position, L and -OR, being as defined above; repeating the cycle of condensing the terminal monofunctional moiety with 5' blocked, 3* reactive nucleoside, removing D_, and reacting the resultant 5* deblocked substrate with bi¬ functional phosphorus (III) reagent until a chain of a desired nucleoside sequence is produced; and cleaving the nonfunctional R, group from each phosphorus moiety with a reagent which effects substitution of hydrogen in place thereof, and oxidizing each said phosphorus moiety, thereby producing a polynucleotide having the desired nuσleo- tide sequence. 2. A process as set forth in claim 1 wherein said bifunctional phosphorus (III) reagent and said 5* reactive 3' blocked resin nucleoside substrate are mixed in a molar ratio of at least about 15 moles of said reagent per mole of said 5' reactive substrate for carrying out the reaction which produces said monofunctional resin nucleosidophosphorus (III) intermediate. 3. A process as set forth in claim 2 wherein said monofunctional resin nucleosidophosphorus (III) intermediate is produced by a heterogeneous reaction between. said reagent, contained in a solvent therefor, and said resin substrate. 4. A process as set forth in claim 2 wherein said 3' reactive 5' blocked nucleoside and said monofunctional resin nucleosido phosphorus (III) intermediate are mixed in a molar ratio of at least about 3 moles of said 3' reactive nucleoside per mole of said intermediate for carrying out said condensation. OMPI 5. A process as set forth in claim 4 wherein said resin dinucleosidophosphite intermediate is produced by a heterogeneous reaction between said 3' reactive 5' blocked nucleoside, contained in a solvent therefor, and said substrate. 6. A process as set forth in claim 1 wherein R, has a length of at least 5 angstroms and comprises the struc¬ ture H O O H I II II I -N-C-(CH 2 ) k -(NH) m -(C) n -(CH 2 ) p -Ar-N-R 5 - where Ar is a substituted or unsubstituted arylene group, k is an integer between 0 and 4 inclusive, m is 0 or 1, n is 0 or 1, p is an integer between 0 and 4 inclusive, n is zero » where k + m = 0, k + m + n + p is at least 1, and R-. is selected from the group consisting of OR c I 6 and PR, P II 0 where -OR g is nonfunctional both with respect to the afore¬ said condensation of said terminal monofunctional moiety with 5* blocked, 3' reactive nucleoside, and with respect to the reaction of 5' deblocked substrate with said bifunctional phosphorus (III) reagent. 7. A process as set forth in claim 6 wherein Ar comprises phenylene. 8. A process as set forth in claim 6 wherein the preparation of said substrate comprises reacting a nucleoside, whose 5' position is blocked and whose 3' position is reac¬ tive, with a final precursor resin having a pendent structure comprising H O O II ' -NI-CII- (CH 2 ) k -(NH) m -(CIf) n -(CH 2 ) p -Ar-NI-R 8 where Ar, k, m, n, and p are as defined above and R„ corre¬ sponds to the formula G - P - G I OR 1 where G is a functional leaving group reactive with a secon¬ dary alcohol, and R, is as defined above. 9. A process as set forth in claim 8 further comprising the preparation of said final precursor resin by reacting a second precursor resin with a bifunctional phos¬ phorus (III) compound, said second precursor resin having a pendent structure comprising H O O -N I-CI-(CH 2 )|ς-(NH) m -(CI!) n (CH 2 ) p -Ar-NH 2 where Ar, k, m, n, and p are as defined above, and said bi- - - i, 42 functional phosphorus (III) compound corresponds to the formula G - P - G 1 OR, where G and R, are as defined above. 10. A process as set forth in claim 9 further comprising the preparation of said second precursor resin by reduction of a third precursor resin having a pendent struc¬ ture comprising (CH 2 ) k -(NH) m -(C) n -(CH 2 ) p -Ar-N0 2 where Ar, k, m, n, and p are as defined above. 11. A process as set forth in claim 10 further comprising the preparation of said third precursor resin by reacting a fourth precursor resin with a linking group inter¬ mediate, said fourth precursor resin having a pendent struc¬ ture comprising -NH 2 said linking group intermediate comprising the structural formula O O R7-O- 1C-(CH 2 ) k -(NH) m -(C li) n -(CH 2 ) p -Ar-N0 2 where k, m, n, p, and Ar are as defined above and R_ com¬ prises an imido moiety bonded to the oxygen through an imido nitrogen. OMPI 12. A process as set forth in claim 11 wherein said linking group intermediate is prepared by reacting a nitro¬ arylcarboxylic acid with an N-hydroxyimide of a dicarboxylic acid, while removing by-product water from the reaction zone, said nitroarylcarboxylic acid corresponding to the formula p o 0 2 N-Ar-(CH 2 )p(C II) n (NH) m -(CH 2 ) k -CII-OH where Ar, k, m, n, and p are as defined above, and said N-hydroxyimide corresponding to the formula where q is 2 or 3. 13. A process as set forth in claim 1 wherein R 3 comprises an arylene group and wherein the polynucleotide is detached from the resin backbone by contacting the resin and attached nucleoside with a cleaving reagent comprising a tertiary amine and an alkyl ester of nitrous acid, said cleaving reagent being substantially free of any low molecu¬ lar weight alkanoic acids and of any salts of such acids. 14. A process for producing a polynucleotide com¬ prising the steps of: reacting a bifunctional phosphorus (III) compound with a sub¬ strate comprising a nucleoside whose 5' position is reactive and whose 3' position is blocked by attachment to a solid phase resin, said compound corresponding to the formula where G is a functional leaving group reactive with a primary or secondary alcohol and -OR, is a nonfunctional substi- tuent, and said substrate corresponds to the formula where R 2 is hydrogen or hydroxyl, B, is a base, R, is a linking group comprising an arylene group, and S comprises a resin backbone, thereby producing a monofunctional resin nucleosidophosphorus (III) intermediate corresponding to the formula where -O - j R 2 , B,, G, R 3 , and S are as defined above; condensing said resin intermediate with a nucleoside whose 5' position is blocked and whose 3' position is reactive, thereby producing a resin dinucleosidophosphite intermediate corre¬ sponding to the formula where D 5 is a blocking group, -OR, , R 2 , R 3 , B, , and S are as defined above, and B 2 is a base which may be the same as or different from B,; removing the D,. blocking group by reaction with a Lewis acid to provide a reactive hydroxyl group at the terminal 5' position; reacting said 5' hydroxyl with said bifunctional compound to produce a terminal monofunctional OR, - P i 1 - G moiety at said 5' position, G and -OR, being defined as above; repeating the cycle of condensing the terminal monofunctional moiety with 5' blocked 3* reactive nucleoside, removing D 5 , and reacting the resultant 5* deblocked substrate with bifunctional phosphorus (III) compound until a chain of the desired nucleoside sequence is produced; and oxidizing each said phosphorus moiety, and cleaving the non¬ functional R, group from each phosphorus moiety with a rea¬ gent which effects substitution of hydrogen in place thereof, thereby producing a polynucleotide having the desired nucleo- tide sequence. 15. A process as set forth in claim 14 wherein R 3 has a length of at least 5 angstroms and comprises the struc¬ ture » H O O H -Nl-C«-(CH 2 ) k -(NH) m -- (CII) n - (CH 2 ) p -Ar-NI-R 5 - where Ar is the substituted or unsubstitued arylene group, k is an integer between 0 and 4 inclusive, m is 0 or 1, n is 0 or 1, p is an integer between 0 and 4 inclusive, n is 0 where k + m = 0, k + m + n + is at least 1, and R_ is selected from the group consisting of OR c I 6 - P - and OR.. I 6 P - II o where -OR g s nonfunctional both with respect to the afore¬ said condensation of said terminal monofunctional moiety with 5' blocked 3' reactive ^ nucleoside, and with respect to the reaction of 5' deblocked substrate with said bifunctional phosphorus (III) compound. » 16. A process as set forth in claim 15 wherein the preparation of said substrate comprises reacting a nucleoside, whose 5' position is blocked and whose 3* position is reac¬ tive, with a final precursor resin having a pendent structure comprising H O O H -N I-CII-(CH 2 ) k -(NH) m -(CII) n -(CH 2 ) p -Ar-NI-R 8 where R,, Ar, k> m, n, and p are as defined above and Rg corresponds to the formula - P - G I OR, where G is a functional leaving group reactive with a secon¬ dary alcohol, and R. is as defined above. 17. A process as set forth in claim 16 further comprising the preparation of said final precursor resin by reacting a second precursor resin with a bifunctional phos¬ phorus (III) compound, said second precursor resin having a pendent structure comprising H O O I H II -N-C-(CH 2 ) k -(NH) m -(C) n (CH 2 ) p -Ar-NH 2 -^JsCT where Ar, k, , n, and p are as defined above and said bifunctional phosphorus (III) compound corresponds to the formula - where G and R, are as defined above. 18. A process as set forth in claim 17 further comprising the preparation of said second precursor resin by reduction of a third precursor resin having a pendent struc¬ ture comprising ) n -(CH 2 ) p -Ar-N0 2 where Ar, k, m, n, and p are as defined above. 19. A process as set forth in claim 18 further comprising the preparation of said third precursor resin by reacting a fourth precursor resin with a linking group inter¬ mediate, said fourth precursor resin having a pendent struc¬ ture comprising -NH 2 said linking group intermediate comprising the structure R7-O-C f- (CH 2 ) k -(NH) m -(C ?) n -(CH 2 ) p -Ar-N0 2 where k, m, n, p, and Ar are as defined above and R_ com- prises an imido moiety bonded to the oxygen through an imido nitrogen. 20. A process as set forth in claim 19 wherein said linking group intermediate is prepared by reacting a nitro¬ arylcarboxylic acid with an N-hydroxyimide of a dicarboxylic acid while removing by-product water from the reaction zone, said nitroarylcarboxylic acid corresponding to the formula where Ar, k, m, n, and p are as defined above, and said N-hydroxyimide corresponding to the formula where q is 2 or 3. 21. A process as set forth in claim 14 wherein the polynucleotide is detached from the resin backbone by contac¬ ting the resin and attached nucleoside with a cleaving reagent comprising a tertiary amine and an alkyl ester of nitrous acid, said cleaving reagent being substantially free of any low molecular weight alkanoic acids and of any salts of such acids. OMPI 22. A resin adapted for use as a substrate in the preparation of a polynucleotide, said resin having a pendent structure comprising H O -U- (CH 2 ) k -(NH) m -(C) n -(CH 2 ) p -Ar-NH 2 where Ar is a substituted or unsubstituted arylene group, k is an integer between 0 and 4 inclusive, m is 0 or 1, n is 0 or 1, p is an integer between 0 and 4 inclusive, n is zero when k + m = 0, and k + m + n + ^ 1. 23. A resin adapted for use as a precursor in the preparation of a polynucleotide, said resin having a pendent structure comprising n -(CH 2 ) p -Ar-N0 2 where Ar is a substituted or unsubstituted arylene group, k is an integer between 0 and 4 inclusive, m is 0 or 1, n is 0 or 1, p is an integer between 0 and 4 inclusive, n is 0 when k + m = 0, and k + m + n + $s- 1. 24. A resin adapted for use as a substrate in the preparation of a polynucleotide, said resin having a pendent structure comprising HO O H -N M-Cl-(CH 2 ) k -(NH) m -(CII) n -(CH 2 ) p -Ar-N1-R 8 * &__&___. . where Ar is a substituted or unsubstituted arylene group, k is an integer between 0 and 4 inclusive, is 0 or 1, n is 0 or 1, p is an integer between 0 and 4 inclusive, n is zero where k + m = 0, k + m + n + p l, and Rg is selected from the group consistin of where G is a functional leaving group reactive with a secon¬ dary alcohol, and R g is alkyl, aryl, hydrogen, or a salt- forming cation.

Inventors: BINGHAM SCOTT E, CAO TIN M, SUNG MICHAEL T, BINGHAM, SCOTT, E., CAO, TIN, M., SUNG, MICHAEL, T.
Applicants: UNIV SOUTHERN ILLINOIS, SOUTHERN ILLINOIS UNIVERSITY FOUNDATION
Application year: 1983
Main classification: C07H15
IPC codes: C07H21, C08G79