WO2000075154A2 - Protected nucleosides - Google Patents

Abstract

Processes for the synthesis and purification of protected nucleosides using a solvent system without the use of pyridine as a solvent, and separation processes without the use of chromatography.

Description

PROTECTED NUCLEOSIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U S Provisional Application No 60/137,639 filed June 4, 1999

FIELD OF THE INVENTION

The invention I elates to the synthesis and puiification of protected nucleosides, and more particularly to methods foi the synthesis and purification of protected nucleosides without the use of pyndme as a solvent

BACKGROUND OF THE INVENTION

Nucleosides aie compounds of importance in physiological and medical reseaich, obtained dunng partial decomposition, l e , hydiolysis, of nucleic acids, and containing a puπne or pyπmidme base linked to eithei D-πbose (forming πbonucleosides) or D-deoxyπbose (forming deoxynbonucleosides) They aie nucleotides minus the phosphate group Well-known nucleosides include adenosine (A), cytidme (C), undine (U), and guanosme (G), as well as the deoxynucleosides deoxyadenosme (dA), deoxycytidme (dC), deoxyguanosme (dG), and deoxythymidme (dT) It should be noted that thymidine is actually a deoxynucleoside, and may be refeπed to in the literature as eithei thymidine (T) oi deoxythymidme (dT) Structural 1 epresentations of the foui deoxynucleosides dA, dC, dG and dT are shown in Figuies 1A, 2A, 3A and 4A, lespectively The "deoxy" site in each compound is at the 2' position of the furan πng The active hydioxy sites aie at the 3' and 5' positions In each of dA, dC and dG theie is an exocychc NH, gioup which is protected, piefeiably by acylation, as discussed below Nucleosides are multi-functional compounds, having both ammo and hydroxy functional groups They may be used in automatic synthesizers to produce ohgonucleotides as well as synthetic genes Ohgonucleotides and genes are formed by stringing together nucleosides m a predetermined sequence through phosphate ester linkages between the 3'-hydroxyl group of one nucleoside and the 5'-hydroxyl group of the next

In order to conduct syntheses selectively and efficiently, it is necessary to block or "protect" specific functional groups in order to achieve reaction at the desired sites The "protecting" groups are designed to be removed under specific carefully controlled conditions, usually under relatively mild and typically acidic conditions To be useful as precursors m the synthesis of high value pharmaceuticals, it is necessary that protected nucleosides be of very high purity (I e , greater than about 99%, preferably greater than about 99.5%) Unless otherwise indicated, purity percentages herein are expressed as percent area as measured by HPLC, but may be expressed as percent by weight, where indicated

Typically, the protection of nucleosides involves the denvatization of both ammo and hydroxy functional groups, except foi thymidine, which requnes only the protection of hydroxyl groups Vanous schemes are employed to achieve these protected nucleosides, but usually the N-protected derivatives (most often N-acylated) are isolated and purified before protecting the hydroxyl gioups. For example, the 2- ammo group of dC, and the 6-ammo group of dA are protected by benzoyl groups, while the 2-amιno group of dG is protected by an isobutyryl group The hydroxyl group, typically a 5'-hydroxyl group in all of the nucleosides, is generally protected by a 4,4'-dιmethoxytπtyl (DMT) group When phosphorylated, a protected nucleoside will react at the 3'-hydιoxyl group A phosphorylated protected nucleoside will then react with a second nucleoside at the 5' position after the 5' position has been deblocked by removal of the DMT gioup The phosphorylation thus occurs between the 3'-hydιoxyl group of the first nucleoside and the 5'-hydιoxyl group of the second nucleoside to form a dmucleotide By repeating the phosphorylation procedure, the synthesizer can produce ohgonucleotides containing a predetermined sequence of nucleosides

Discussions of the synthesis and protection of nucleosides by deπvatization may be found m many references, including the following, all of which are incorporated herein by reference One method of protecting nucleosides is described in Ti, et al , "Transient Protection Efficient One-flask Syntheses of Protected Deoxynucleosides", J Am Chem Soc , Vol 104, 1316-1319 (1982), which is discussed in more detail below in regard to the examples Other methods of synthesizing protected nucleosides are set forth m Charubala, et al , "Nucleotides XXIII Synthesis of Piotected 2'-Deoxynbonucleosιde-3'-phosphotrιesters Containing the p-Nitiophenylethyl Phosphate Blocking Group", Synthesis. 965 (1984) Still other methods for synthesizing such protected nucleosides are set forth m Kierzek, "The Synthesis of 5'-O-dιmethoxytπtyl-N-acyl-2'-deoxynucleosιdes, Improved 'Transient Protection' Approach", Nucleosides & Nucleotides, 4(5), 641-649 (1985) In all of these references, protection by N-acylation is effected with benzoyl chlonde on adenosme and cytidme derivatives, and with lsobutyπc anhydnde on guanosme denvatives, as is well-known m the art The compounds aie then further piotected by the introduction of methoxytπtyl or dimethoxytπtyl groups, also as is well-known m the art An earlier article on the protection of such nucleosides may be found m Schaller. et al . J Amer Chem Soc , Vol 85, 3821-3827 (1963) Another article on piotected nucleosides is McGee, et al , "A Simple High Yield Synthesis of N²-(2- Methylpropanoyl)-2'-deoxyguanosme", Synthesis, 540 (1983) In all of the reported syntheses, the protected nucleosides must be subjected to purification prior to then use in pharmaceutical syntheses A detailed description of the preparation of protected deoxynucleosides is provided in Jones, "Pieparation of Protected Deoxynbonucleosides", Ohgonucleotide Synthesis A Practical Approach, IRL Press, 23-34 (1984), mcorpoiated herein by reference This reference describes various processes for protecting such nucleosides, protecting the 5'-hydιoxyl group as a tπtyl ethei and the 3'-hydroxyl gioup with benzoyl chloride or a levuhmc anhydnde Foi nbonucleosides, piotection of the 2'- hydroxyl group is also needed, most commonly provided by tert-butyldimethylsilyl (TBDMS) group. The exocyclic amino groups are protected by acylation with a benzoyl moiety (Bz) or an isobutyryl group (iB), as discussed above. Acyl protection is also discussed in Kδster, et al., "N-Acyl Protecting Groups for Deoxynucleosides", Tetrahedron. 37(2). 363-369 (1981). TBDMS is discussed for protection of any hydroxyl group, using 4-(dimethylamino)pyridine (DMAP) as a catalyst, in Chaudhary, et al., "4-Dimethylaminopyridine: An Efficient and Selective Catalyst for the Silylation of Alcohols", Tetrahedron Letters. No. 2, 99-102 (1979).

All of these reported preparation procedures use pyridine as a solvent in the synthesis processes. Pyridine has been found to be chemically compatible with the nucleosides, the protected nucleosides, and with the various reactants used to form such products. However, pyridine is highly toxic and its ability to dissolve nucleosides is limited. Large amounts of pyridine have to be used in the reaction to maintain the nucleosides in solution. These large amounts of pyridine are difficult to remove from the protected nucleoside products. Thus, the pyridine solvent processes have very low productivity, and the processing costs are high. It would, therefore, be desirable to have a process for preparing protected nucleosides that does not require the use of pyridine as a solvent.

As discussed in Chaudhary, et al, cited above, 4-(dimethylamino)pyridine (DMAP) is often used as a catalyst in processes in which pyridine is the solvent. In fact, DMAP has become a standard catalyst for use in such processes. However. DMAP is also considered highly toxic, is very expensive, and is difficult to separate from the desired product. It is, therefore, also desirable to provide a process which does not require the use of DMAP as a catalyst. Another reason for the use of pyridine is that it is a mild base, and can neutralize acids formed in the reaction mixture. In this capacity it acts as an "acid scavenger". Many of the chemical reactions in the protection process produce acid byproducts, such as HC1. For example, benzoyl chloride or isobutyryl chloride are used to acylate the amino group. In the acylation reaction, these acid chloride compounds react with the amino group to produce HC1 as a byproduct. Similarly, in the tritylation reaction, dimefhoxytrityl chloride (DMT-C1) or other trityl chlorides react with hydroxy groups to generate HC1 as a byproduct of this reaction. It is necessary to neutralize the acid produced by these reactions to prevent the acid from undesirably reacting with the nucleosides, and therefore an acid scavenger is needed in such processes.

The automatic synthesis of ohgonucleotides as well as synthetic genes has previously been carried out on a milligram scale for research purposes. More recently, ohgonucleotides are being used in larger quantities in the formation of commercial products such as anti-sense drugs that prevent the synthesis of disease- causing proteins in the human body. The very sensitive nature of the protecting groups together with the variety of polar and non-polar impurities generated during the syntheses of these derivatives makes their purifications complicated, expensive, and difficult to scale-up to industrial-scale production. Therefore, procedures are now needed to manufacture nucleosides in relatively large industrial quantities. Column chromatography, especially flash silica gel chromatography, has been used extensively to purify protected nucleosides on a small to medium scale. This method requires the use of large volumes of high purity solvents in proportion to the amount of material purified. The method is also labor-intensive, requiring precise monitoring to make the fraction cuts at the appropriate times to maximize yield of desired product. For these reasons, large-scale use of this method of purification can be very costly.

The equipment required to conduct flash silica gel chromatography on a multi- kilogram scale is expensive to purchase and operate. For example, one commercially available production scale chromatography unit is capable of separating up to about 4 kg of material per run. Run times can vary from 18 to 36 minutes, at an elution rate of 7 liters per minute. The basic unit investment is very expensive, coupled with the cost (and subsequent disposal cost) of 125 to 250 liters of expensive high purity solvent per run. In addition, some products need to be purified multiple times by cliiomatogiaphy to reach the desired purity, each time at a significant yield loss The high costs associated with chromatographic purification make it unattractive to industrial-scale operations

For the above reasons, it is desirable to provide a process for protecting nucleosides which does not required the use of pyridine as a solvent, which does not require the use of DMAP as a catalyst, and which does not require the use of separative chromatography to purify the product The present invention meets these needs and offers additional advantages as discussed in the following detailed description The process of acylation for protection of exocychc ammo groups is well known in the art and is descnbed, for example, m Jones, "Preparation of Protected Deoxynbonucleosides," op cit As discussed in that reference, a key pioblem m the preparation of protected deoxynbonucleosides is chemically differentiating between hydroxyl and ammo groups Only m the case of dC has it been possible to selectively acylate the ammo group (N-acylation) without acylatmg the hydioxyl gioup The dA and dG ammo groups have been found to be too weakly basic for such a selective reaction However, it is possible to selectively de-acylate, that is, to differentiate by making use of the more rapid hydrolysis, at pH greatei than 10, of the esters versus the amides Thus the chemical procedure which has been used is to per-acylate the nucleoside, acylatmg both the hydroxyl groups and the ammo group, and then to selectively hydrolyze the esters to leave the desned N-acylated nucleoside Such a per-acylation process, however, has been found to be difficult to use, because it requires isolation of the per-acylated intermediate An alternative piocedure is discussed m the Jones reference for selective N-acylation based on tempoiaiy protection of the hydroxyl groups as trimethylsilyl etheis, and is lefeired to as "TMS- transient protection " Unlike ester groups, the trimethylsilyl etheis can be hydrolyzed in solution without the need for isolation For these leasons, the TMS-transient protection method of N-acylation is the preferred method for use with dA and dG However, the acylation procedures described in the Jones ieference lequire the use of pyridine as a solvent BRIEF DESCRIPTION OF THE DRAWINGS

FIGURES 1A, IB, 1C and ID present a schematic representation of the preparation of protected N⁶-benzoyl-5'-O-(4,4-dimethoxytrityl)-2'-deoxyadenosine from deoxyadenosine free-base. FIGURES 2A, 2B and 2C present a schematic representation of the preparation of protected N⁴-benzoyl-5'-(4,4-dimethoxytrityl) deoxycytidine from deoxycytidine free-base.

FIGURES 3A, 3B, 3C and 3D present a schematic representation of the preparation of protected 5'-O-dimethoxytrityl N²-isobutyryl-2'-deoxyguanosine from deoxyguanosine free-base.

FIGURES 4A and 4B present a schematic representation of the preparation of protected 5'-O-dimethoxytritylthymidine from thymidine free-base.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention protected nucleosides are synthesized in polar organic solvents, purified by liquid-liquid extraction and/or selective adsorption, and then solidified by precipitation or crystallization by adding a solvent in which the product is insoluble. The solidification process is considered precipitation in the case of a product which is amorphous in solid form, and crystallization in the case of a product which is crystalline in solid form. By the use of this process, and repeating the process as needed, essentially pure protected nucleosides are obtained without the use of chromatography and without the use of pyridine as a solvent or DMAP as a catalyst. In addition, the process can be scaled up readily to any desirable scale. For purposes of this application, an essentially pure nucleoside is one of greater than about 98% purity, and preferably greater than about 99%o purity, and most preferably greater than about 99.5% pure, all by weight. The process of the present invention is applicable to the preparation of protected deoxyribonucleosides. As discussed above, the deoxynucleosides dA, dC and dG, each of which have an exocyclic amino (NH₂), are first protected by acylation of that amino group. This step is necessary, because the highly reactive amino groups would otherwise react with the compounds being used to protect the hydroxyl group. As discussed above, the amino groups of dA and dC preferably are protected by benzoyl groups, while the amino group of dG preferably is protected by an isobutyryl group. These acylated compounds will be referred to herein as Bz-dA, Bz-dC and iB-dG, respectively. This step is not used for dT, because it does not have an exocyclic amino group.

In accordance with the present invention, there is provided also a process for selectively protecting the exocyclic amino group of a starting deoxyribonucleoside which has an exocyclic amino group which is to be protected by acylation and at least one hydroxyl group which is to be left unprotected, the process comprising: a) dispersing the starting deoxyribonucleoside in a polar solvent which is substantially free of pyridine and which is a solvent for the protected product formed in step b); and b) selectively acylating said exocyclic amino group of said starting deoxyribonucleoside to form a protected product in which said hydroxyl group(s) are unprotected.

This method for the N-acylation of deoxyribonucleosides does not use pyridine as a solvent or DMAP as a catalyst. This eliminates the need for the difficult step of separating product from pyridine solvent and/or DMAP catalyst, as generally required in prior processes. This also provides a pyridine- free amino-protected product for subsequent protection of hydroxyl groups, as discussed below.

The polar solvent should be one in which the N-acylated deoxynucleoside produced in step b) is soluble. The starting deoxynucleoside of step a) need not be fully soluble in the solvent provided it can be dispersed to carry on the acylation process of step b). The preferred polar solvent to use depends on the particular deoxynucleoside being acylated, as discussed further below. The preferred acylation piocedures for protecting exocyclic ammo groups in accordance with the present invention vary for each of the three deoxynbonucleosides which require acylation, dA, dC and dG As indicated above, dC can be acylated directly by selective acylation, while dA and dG preferably are acylated indirectly via TMS-transient protection

The direct acylation of dC will be discussed first In this acylation procedure, a starting material of dC free-base preferably is used as the starting deoxynucleoside The dC free-base may be derived from commercially available dC salts, such as dC- HC1 salt A preferred process for producing dC free-base from dC-HCl salt is to fust dissolve the dC-HCl m a suitable polai solvent, prefeiably a protic polai solvent such as a C, to C₃ alcohol, with methanol particularly preferred A non-aqueous oiganic base, such as the acid-scavengers discussed below, is then added to neutralize the dC- HC1 A pieferred organic base is a tertiary amine such as tnethylamme (TEA) The mixture should be refluxed until the dC-HCl converts to dC free-base Then the dC free-base is precipitated out of solution by adding a sufficient amount of a second solvent which is miscible with the first polar solvent, but m which the fiee-base is insoluble Good results were obtained with CH₂C1₂ as the second solvent

The dC free-base is dispersed in a polar solvent which is free of p\ πdme The solvent is preferably as anhydrous as possible, because any water present must be removed or otherwise tied up pnor to acylation Preferred polar solvents for use in the direct acylation of dC are protic solvents such as straight chain or blanched C, to C₅ alcohols, particularly methanol, ethanol, propanol or butanol Relatively low boiling point secondary alcohols, particularly isopropanol, aie prefened Ethanol is also suitable, but is difficult to use commei cially because of regulatoiy pioblems Methanol is less desirable because it may cause side reactions undei certain conditions Furthermore, isopropanol was found to provide high selectiv ity of acylation, and the product was found to crystallize cleanly out of the isopiopanol An acylatmg leagent, prefeiably a benzoylating leagent, is then added to the dC solution Benzoic anhydride is a suitable reagent for acylation of dC by benzoylation Benzoic acid, which requires a base catalyst, and benzoyl chloride aie not as suitable foi use in this process A preferred molar ratio of benzoic anhydride to dC was found to be about 1 2 to 1 3 An excess of benzoic anhydride results m the formation of the undesirable bisacylation by-product, and too little results in incomplete conversion of the dC to benzoylated dC (Bz-dC) The reaction temperature should be close to, preferably withm about 10°C of, or at the boiling point of the polar solvent The reaction is slower at lower temperatures, which may result in an incomplete reaction which may contaminate the product with unreacted starting materials For isopropanol, which has a boiling point of 82 4° C, the reaction temperature preferably is from about 70°C to about 82 4°C, more preferably from about 75 °C to about 80°C As discussed above, the acylatmg agent which is added to the starting deoxyribonucleoside solution is often m the form of a chlonde or other matenal which pioduces acid bypioduct upon leactmg with the nucleosides In such cases, the acid byproduct must be neutralized, or it may cause decomposition of the nucleoside Therefore, an acid scavenger preferably is included in the reaction mixture A preferred acid scavenger is a tertiary amine, which fonns a salt with the acid, with a particularly preferred tertiaiy amine being tπethylamme (TEA) Other suitable tertiary amines include any tn-alkyl amine of the geneial formula N(R)₃, wheiem R represents the same or different C, to C₆ alkyl groups In addition to TEA, such tπ- alkyl ammes include tπmefhylarmne, tπpropylamme and di-isopropyl-monoethyl amine (DIPEA) Other suitable tertiary ammes include tπethanolamme

As indicated above, the acylation of dA preferably is achieved by transient TMS protection of both the ammo and hydroxyl sites, followed by benzoylation, and then selective deprotection of the hydroxyl sites to leave just the ammo sites benzoylated, thus forming benzoylated dA (Bz-dA) The polar solvent in which the starting dA is dispersed is preferably an aprotic polar solvent, pieferably as anhydious as possible Suitable aprotic polar solvents include tetrahydrofuran (THF), acetonitrile, amides, such as dimethylfoπnamide (DMF), dimethylacetamide and N-methylpyrrohdone, as well as dimethylsulfoxide (DMSO), dimethylsulfone and hexamethylphosphate (HMPA) In a piefeπed embodiment of this piocess, dA monohydrate (dAΗ₂O) is suspended in a polai solvent such as tetiahydiofuian (THF) with about 10 equivalents of tnethylamme (TEA) THF is a preferred solvent foi the TMS protection and subsequent benzoylation steps because it provides solubility foi the intermediates and is inert to the reagents Of the 10 eq of TEA, 7 eq is to neutralize HC1 produced from the chemical reactions and 3 eq to buffei the reaction mixture to ensure a slightly basic environment About 5 equivalents of tπmethylchlorosilane (TMSC1) is added slowly while maintaining the reaction at a relatively low temperature of about 0 to 10°C, preferably about 2 to 5 °C The TMSC1 reacts at active OH and NH₂ sites, and also quenches the water of hydration from the dA monohydrate The reaction should be monitored, as by TLC and/oi HPLC, until all of the dA is consumed About 2 equivalents of benzoyl chloride

(BzCl) are added and the reaction is continued until no intermediate from the first step is observable At this point, the exocyclic ammo groups and the hydroxyl groups are all benzoylated

The benzoylated hydroxyl groups are then restored to their ongmal hydroxyl form by hydiolysis The hydrolysis leaction selectively depiotects the

1 sites, while leaving the ammo sites benzoylated A pieferred hydiolysis method is a t\\ o- phase step-wise hydrolysis with NaOH After the hydiolysis proceeds half wa\ the aqueous phase is separated and neutralized immediately with HC1 solution to reduce the pH to about 8 to 9 The remaining organic phase is hydrolyzed \\ ith another portion of NaOH until the hydrolysis is complete Then the aqueous phase is separated, combined with the first portion of aqueous phase, and again neutralized with HC1 solution to a pH of about 8 to 9 A miscible organic solvent m which the Bz-dA product is insoluble is then added to cause the product to precipitate out of solution The precipitated product may then readily be collected, as by filtration The strategy of this process is to utilize the phase sepaiation between THF and H₂0 to provide a means for isolating the product Two-step hydiolysis, lather than one-step, provides better control of the reaction to avoid extensive over-hydrolysis which was encountered using a conventional one-step method The acylation of dG pieferably is achieved also via TMS-transient protection In this case the acylation is by isobutyryl protection of the exocyclic ammo group to form isobutyryl dG (lB-dG) A preferred acylation agent for this reaction is isobutyryl chlonde (lBCl) As with the benzoylation of dA, m a preferred embodiment of this process dG monohydrate (dGΗ^O) is suspended in a polar solvent such as tetrahydrofuran (THF) with about 10 equivalents of tnethylamme (TEA) THF was found to be the best solvent for the TMS protection dG and the subsequent acylation steps because it provides solubility for the intermediates and is inert to the reagents As with the acylation of dA, of the 10 eq of TEA, 7 eq is to neutiahze HCl pioduced from the chemical reactions, and 3 eq to buffer the reaction mixture to ensure a slightly basic environment About 5 equivalents of tπmethylchloiosilane (TMSC1) is added slowly while maintaining the reaction at a lelatively low tempeiature of about 0 to 10°C, preferably about 2 to 5 °C The reaction should be monitored, as by TLC and/or HPLC, until all of the dG is consumed About 2 equivalents of lBCl is added and the reaction is continued until no intermediate from the first step is observable At this point, the exocyclic ammo group and the hydroxyl groups have all been acylated by the lBCl

The acylated hydroxyl groups are now restored to their original hvdroxyl form by hydrolysis The hydrolysis reaction selectively deprotects the hydroxyl sites, while leaving the ammo sites isobutyryl protected A preferred deprotection method is by hydrolysis with NaOH, which can be a simple one-step hydrolysis After the hydrolysis is complete, the reaction mixture is neutralized with an acidic material, such as solid NH₄C1, to reduce the pH to about 8 to 9 The organic solvents are then removed by vacuum distillation A miscible organic solvent in which the lB-dG product is insoluble is then added to cause the product to precipitate out of solution The precipitated product may then readily be collected, as by filtration

The pyndine-fiee deoxynbonucleosides which have had their exocyclic amino groups protected by acylation may now be used to foim 5'-protected deoxyribonucleoside products In the following piocess, the 5'-hydroxyl gioup of the deoxynbonucleoside is protected, and the protected pioduct is punfied and separated out of solution as a solid Purification and solidification comprise liquid-liquid extraction steps which take advantage of the fact that the protected nucleoside products are insoluble m watei, while soluble m selected polar and non-polar solvents

In accordance with the present invention, there is provided a piocess for prepanng an essentially pure 5 ^-protected deoxynbonucleoside compnsing a) dissolving a starting deoxyribonucleoside in a polar, aprotic solvent which is inert to the starting deoxynbonucleoside, to the 5'-protected deoxynbonucleoside, and to the other reactants, wherem any exocyclic ammo groups of the starting deoxynbonucleoside are protected, preferably by acyl protection, b) reacting the starting deoxynbonucleoside in said solution with a protecting reagent to form a 5'-protected deoxynbonucleoside product, c) removing polar impunties by one or more liquid-liquid extractions usmg immiscible polar and non-polar solvent systems m which the product preferentially partitions into the non-polar phase, and the impurities preferentially partition into the polar phase, and d) removing non-polai impurities by solidifying the product out of solution while leaving the non-polar impunties m solution

In step (a), the starting deoxynbonucleoside may be one that already has its exocyclic ammo group protected, or dT which does not have an exocyclic ammo group Protection of the exocyclic ammo group may be made by the above-described acylation protection process of the present invention, or by any other suitable protection process The nucleoside to be protected is dissolved m a polar, aprotic organic solvent Examples of polar, aprotic solvents suitable for use m this step include amides, such as dimethylformamide (DMF), dimethylacetamide and N-methylpyrrohdone, as well as dimethylsulfoxide (DMSO), dimethylsulfone and hexamethylphosphate (HMPA) DMF is particularly preferred For the reasons discussed above, pyridine is not used as a solvent in the processes of the piesent invention In step (b), a protecting reagent is added to the starting deoxyribonucleoside solution which selectively protects the 5'-hydroxyl group of the deoxyribonucleoside. The protecting reagent must react with the 5' hydroxyl group preferentially over the 3' hydroxyl group to form a removable protecting group at the 5' position. A preferred method of protecting this hydroxyl group is to form a trityl derivative compound, a process referred to as "tritylation". Preferred protecting groups are trityl, methoxytrityl and dimethoxytrityl groups. Preferred protecting reagents are trityl chloride and substituted trityl chlorides. For example, dimethoxytrityl chloride (DMT-Cl) is used in the examples set forth below. During the tritylation reaction, the 3 '-hydroxyl group should be left unreacted. This is necessary to permit selective bonding of the 5' and 3' positions during subsequent ohgonucleotide synthesis. The tritylation reaction is preferably conducted at a temperature ranging from about -10°C to about 40 °C, more preferably from about 10°C to about 25 °C. Protecting reagents such as trityl chloride or substituted trityl chlorides produce an acid byproduct upon reacting with nucleosides. In such cases, the acid byproduct must be neutralized, or it may cause decomposition of the nucleoside. Therefore, an acid scavenger is preferably included in the reaction mixture. Preferably the acid scavenger is present in a mole ratio to the tritylation agent of about 1 : 1 to about 3:1. A preferred acid scavenger is a tertiary amine, which forms a salt with the acid. A particularly preferred tertiary amine is triethylamine (TEA). Other suitable tertiary amines include any tri-alkyl amine of the general formula N(R)₃, wherein R represents the same or different C, to C₆ alkyl groups. In addition to TEA, such tri-alkyl amines include trimethylamine, tripropylamine and di-isopropyl- monoethyl amine (DIPEA). Other suitable tertiary amines include triethanolamine. Although pyridine is not used as a solvent in the process of the present invention, a small amount of pyridine can be used as an acid scavenger. When used in small amounts, the pyridine can be purified out of the product with the other impurities. The polar solvents and acid scavengers must be chemically compatible with the starting nucleosides, the reactants, and the nucleoside products to which they are exposed They must also not interfere adversely with the reaction of the reactants with the nucleosides Because the DMT-Cl used in tritylation is highly reactive towards water, all materials involved m the tntylation must be anhydrous In the preferred acylation processes of the present invention, Bz-dA may pick up water from the TMS-transient protection acylation reactions, and therefore need to be dehydrated It was found that ordinary thermal and vacuum drying techniques were unsatisfactory for lemovmg the water contained in this material, because the water may exist in hydrate form In accordance with another aspect of the present invention, an azeotropic dehydration process has been developed which is effective to remove water in the Bz-dA

The azeotropic dehydration process of the present invention is applied to the solution of the starting deoxynbonucleoside dissolved m the polar aprotic solvent pnor to the addition of the protecting reagent The dehydration process comprises adding a dehydrating solvent to the solution of step (a) that forms an azeotiope with water and the polar aprotic solvent, and distilling the azeotrope from the mixtuie Foi a starting solution of deoxynbonucleoside in the polar aprotic solvent dimethylformamide (DMF) suitable dehydrating solvents include one or moie C, to C₁₀ hydrocarbons, which may be linear, branched or cyclic, and may be substituted or unsubstituted Preferred dehydrating solvents include pentane, hexane and heptane, particularly hexane It was also found desirable to include a small amount of a tertiary amine, such as TEA, to stabilize the acylated nucleosides dunng the azeotropic dehydration step The protecting reagent should be added to the solution of the starting deoxynbonucleoside under controlled conditions of tempeiature and addition rate The progress of the reaction should be monitored, as by HPLC analysis The objective of the momtoπng is to control and optimize the conversion without generating too much of over-tntylated impurities The reaction is quenched by the addition of water when the optimal point is reached An additional advantage of tritylating the starting deoxyribonucleoside in the polar aprotic solvent rather than in pyridine is that the reaction does not require the use of a catalyst. As discussed above, tritylation in pyridine generally requires the use of a catalyst, such as 4-(dimethylamino)pyridine (DMAP) which is a toxic material that is very expensive and difficult to separate from the final product.

Step (c) is a purification step that removes polar impurities from the product. The solution of the protected nucleoside inevitably contains polar and non-polar impurities which must be removed from the final nucleoside product. As discussed above, the final product should be at least about 98% pure, preferably at least about 99%o pure, and more preferably at least about 99.5% pure, by weight. Among the polar impurities which need to be removed are the amino-protected nucleosides which either did not react with the hydroxyl protecting agent, or which may have two acyl protecting groups (identified as bis-acylated products). Among the non-polar impurities which need to be removed are the bis-tritylated materials, which contain two trityl protecting groups, and impurities derived from DMT-Cl. These tend to be the most difficult impurities to remove because their polarity is relatively close to that of the desired protected nucleosides.

Polar impurities are removed from the solution of protected nucleoside product by a liquid-liquid extraction process which takes advantage of the difference in solubility between protected nucleosides and polar impurities in polar solvent system. The protected nucleosides are generally insoluble in water and basic aqueous salt solutions, while many of the polar impurities are soluble in water or such solutions. Therefore water, or preferably an aqueous solution of water and a basic salt such as a soluble carbonate or bicarbonate salt, can be used to extract polar impurities from the protected nucleoside product. By adding water or a basic aqueous solution to the initial product solution in polar, aprotic solvent, followed by extracting with an immiscible non-polar solvent, such as methylene chloride, the protected nucleoside is transferred to the non-polar phase, while the majority of polar impurities are left in the polar phase. Preferred basic salts include sodium and potassium carbonate and bicarbonate, particularly sodium bicarbonate. Preferably, the basic aqueous salt solution contains about 1% to about 10%, preferably about 2% to about 5%, by weight salt. Other halogenated solvents, such as chloroform and 1,2-dichloroethane may also be employed as the non-polar solvent. The product solution in the non-polar solvent can be repeatedly extracted in this manner with water or a basic aqueous solution to achieve higher purity level. It has been found that an aqueous solution comprising about 1 to about 10% by weight NaHCO₃, preferably about 2% to about 5%, and about 0 to about 40% DMF, is suitable for the removal of polar impurities. After the polar solvent system has been mixed with the non-polar phase, the mixture is allowed to settle, and is separated. The desired protected product remains in the non-polar phase, while the undesired polar impurities are carried away in the polar phase. The process may be repeated as many times as necessary to remove the polar impurities and obtain the desired product purity.

This polar solvent system selectively extracts out all or most of the polar impurities. To remove additional polar impurities, the resulting product solution optionally may be treated further with a suitable adsorbent, such as activated carbon. Other suitable adsorbents, such as silica, alumina, and molecular sieves, are well- known to-those skilled in the art. When polar impurities are below desirable levels, the adsorbent with adsorbed impurities may be readily removed, as by filtration. This has been found to be a desirable step in the purification of the protected dA and dC products, but unnecessary in the purification of dG and dT.

In step (d), the product is solidified out of solution by a process which further purifies the product. The product may be solidified out of solution by crystallization, in the case of a crystalline product, by precipitation in the case of an amorphous product, or by a combination of crystallization and precipitation in the case of products which form both crystalline and amorphous solids. Thus the teπn solidification is intended to encompass the processes of crystallization, precipitation and combinations thereof by which a solid product comes out of solution.

Solidification may be effected by the use of either non-polar solvents or polar solvents. In non-polar solvent solidification, a non-polar phase containing the dissolved product is combined with a miscible solvent in which the product is insoluble in an amount effective to crystallize a ciystallme pioduct, 01 to piecipitate an amorphous product from the non-polar phase For crystallization, preferably, the miscible solvent in which the product is insoluble is added slowly to the product solution For precipitation, preferably, the product solution is added slowly to the miscible solvent in which the product is insoluble

When the solidification is taking place in a polar solvent, the product is transferred or re-dissolved into a polar phase which comprises a polar solvent that is miscible with water Because the present protected nucleosides are all insoluble m water, water can be used to solidify the product from the polar phase To solidify the product from the polar phase, the polar phase containing the dissolved product is combined with a suitable amount of water effective to solidify the product out of solution The water may be added to the polar phase, or the polai phase may be added to the water When this process was used to solidify dC product out of solution, the polar phase was added slowly to water The product was found to be a mixture of crystal and amorphous solids

Many polar solvents are suitable for use m this solidification process In addition to being miscible with water and a solvent foi the product, the polai solvent must also be inert to the product Preferably, the polar solvent also has a boiling point of less than 100°C, the boiling point of water, to facilitate subsequent removal by evaporation Suitable solvents include acetonitrile, acetone, and lower (C, to C₃) alcohols, particularly methanol

When the product is solidified in non-polar solvents, crystallization effectively removes most of the non-polar impurities The non-polar impurities remain dissolved in the non-polar solvents as the pioduct crystallizes out of solution Such a crystallization process is preferably used with DMT-dT and Bz-DMT-dA Howevei, when the product is solidified by precipitation to an amorphous form, it was found that additional purification may be needed to remove non-polai impunties In such a case, an additional step of liquid-liquid extraction is used to remove non-polar impurities Such a process is preferably used with protected nucleosides which form amorphous solids, as was found to be the case with Bz-DMT-dC and iB-DMT-dG In liquid-liquid extraction to remove non-polar impunties, the crude pioduct is isolated m a polar phase in which it is soluble A mixture of DMF and watei is prefened for use in the liquid-liquid extraction punfication of these products The impurities are then extracted with a non-polar solvent, or a non-polar solvent system The non-polar solvent should be a solvent for the non-polar impurities, but not for the nucleoside product In addition, the polar phase and non-polar solvent system must be immiscible with each other This will cause the non-polar impurities to partition into the non-polar solvent phase, while keeping the majority of the product in the polar solvent phase The non-polar impurities can then be removed by phase separation Mixtures of aromatic hydrocarbons and aliphatic hydrocarbons are suitable non-polar solvent systems, particularly ones containing about 6 to 12 carbon atoms, with or without hetero atoms A mixtuie of cum en e and hexane is a prefened composition Preferably, the mixture of cumene and hexane compnses about 10 to about 90 parts cumene to about 90 to about 10 parts hexane, by volume, moie preferably about 1 to about 3 parts cumene to 1 part hexane, with a mixtuie of about 2 parts cumene to 1 part hexane being particularly pieferred

The liquid-liquid extraction to lemove non-polai impunties may be pei formed either before or after the liquid-liquid extraction to lemove polar impurities However, preferably the non-polar extraction is peifomied before the polar extraction This is because the same non-polar solvent which is used to dissolve the product in the polar extraction step can be used m the final crystallization or precipitation step Furthermore, the non-polar extraction can be performed with the pioduct dissolved in a polar phase comprising the polar aprotic solvent m which the nucleoside is initially provided Thus, it is simpler and moie efficient to peifoim the non-polar exti action first

To solidify the product out of solution by piecipitation or ciystalhzation, the non-polar phase in which the product has been isolated, and fiom which the polar impurities have been removed is mixed with an immiscible non-polar solvent in which the product is insoluble A wide lange of solvents can be used for this purpose, so long as the non-polar phase and the non-polai solvent are miscible with each othei For crystalline pioducts, the non-polar solvent is prefeiably added slowly to the non- polar phase comprising the product This causes the product to crystallize, while keeping non-polar impurities m the mother liquor For amorphous products, the non- polar phase comprising the product is preferably added slowly to the non-polar solvent This causes the pioduct to precipitate, while keeping the non-polar impurities in solution

The process of the present invention offers many advantages over prior processes in which pyridme is used as the process solvent Because the polar aprotic solvent used m the present piocess are bettei solvents for the nucleosides than pyridme, the reactions can be run at much higher nucleoside concentiations Thus, higher volumetric efficiency is obtained As discussed above, the present piocess eliminates, or at least gieatly reduces the use of pyridme and DMAP, which are considered highly toxic substances The purification process steps of the present invention, including extraction, adsorption, and pi ecipitati on/cry stalhzation are common operations, and aie moie efficient and less costly opeiations than cinematography Because of the simplicity and othei improvements of the piesent process, the overall pioduct yield is also improved Further, the combination of these improvements results m a significant lowering of the processing costs foi protected nucleosides Finally, the new processes aie scalable to any desirable scale to meet industrial demand

Figures 1A to ID present a schematic representation of a preferred method of preparing protected N⁶-Benzoyl-5'-O-(4,4-dιmethoxytπtyl)-2'-deoxyadenosme from deoxyadenosine free-base, with the acylation being via TMS-transient protection The deoxyadenosine fiee-base of Figuie 1A is combined with TMSC1 to foim TMS protected dA As shown m Figuie IB, two TMS groups attach to the exocyclic NH-, group, as well as to the 3' and 5' OH gioups The TMS-protected dA is then combined with benzoyl chloride (BzCl or PhCOCl) to foim benzoylated TMS-dA, which is then hydrolyzed to restoie the OH gioups, resulting in the acyl-protected Bz-dA of Figure 1C This material is then tπtylated by the addition of DMT-Cl to form the final protected product N⁶-Benzoyl-5'-0-(4,4-dιmefhoxytπtyl)-2'- deoxyadenosme as shown in Figure ID

Figures 2A, 2B and 2C present a schematic representation of a preferred method for the preparation ofprotected N⁴-benzoyl-5'-(4,4-dιmethoxytπtyl) deoxycytidine from deoxycytidine free-base In this case direct acylation is used, without the need for a TMS protected intermediate The deoxycytidine free-base of Figure 2A is reacted with benzoic anhydride (Bz₂O) to form the acyl protected Bz-dA of Figure 2B This material is then tntylated by the addition of DMT-Cl to form the final protected product N⁴-benzoyl-5'-(4,4-dιmethoxytπtyl) deoxycytidine as shown m Figure 2C

Figures 3A, 3B, 3C and 3D present a schematic representation of a prefened method for the preparation ofprotected 5'-O-dιmethoxytntyl N²-ιsobutyryl-2'- deoxyguanosine from deoxyguanosme free-base, with the acylation being via TMS- tiansient protection The deoxyguanosme free-base of Figuie 3A is combined with TMSC1 to form TMS-piotected dG As shown m Figuie 3B, two TMS gioups attach to the exocyclic NH₂ group, as well as to the 3' and 5' OH gioups The TMS- piotected dG is then combined with isobutyryl chloride to form acylated TMS-dG, which is then hydrolyzed to restore the OH gioups, resulting in the acyl-piotected lB-dG of Figure 3C This material is then tntylated by the addition of DMT-Cl to form the final protected product 5'-O-dιmefhoxytrιtyl N²-ιsobutyryl-2'- deoxyguanosme as shown in Figure 3D

Figures 4A and 4B present a schematic representation of a preferred method for the preparation ofprotected 5'-O-dιmethoxytπtylthymιdme fiom thymidine fiee- base Because thymidine does not have an exocyclic NH₂ group, there is no acylation step The thymidine free-base of Figure 4A is reacted with DMT-Cl to fonn the final protected product 5'-O-dιmethoxytπtylthymιdme as shown in Figure 4B EXAMPLES

In the following examples protected nucleosides are made in accordance with the methods of the present invention. The examples set forth processes for making protected- forms of the nucleosides deoxyadenosine (dA), deoxycytidine (dC), deoxyguanosme (dG) and deoxythymidme (dT). Each of these nucleosides has special properties which affect the manner in which the protection process is performed.

The amino groups of deoxyadenosine and deoxyguanosme are difficult to protect by acylation because the acylating agents tend to react with both the free amino groups and with hydroxyl groups. Therefore, the acylation process is conducted with a transient TMS protection step, which provides the necessary amino selectivity. This is discussed in the Ti, et al. reference, as cited above. The process for the acylation of dA was found to result in water being present in the acylation product. Therefore, a dehydration step is included in the process prior to protection by tritylation. Deoxycytidine does not need to be acylated via TMS protection, because the acylation process is naturally selective between the desired amino sites and the undesired hydroxyl sites.

In the following examples, the solid protected forms of dA and dT were crystalline in structure, and therefore they were solidified out of solution by crystallization. On the other hand, dG was found to be amorphous, and was therefore solidified by precipitation. When dC was solidified out of solution, it was found to be a combination of crystalline and amorphous. The solidification of dC is therefore a combination of crystallization and precipitation. Generally, the crystallization process successfully reduced the level of non-polar impurities to the desired level. On the other hand, it was found that a step of liquid-liquid extraction to remove non-polar impurities was desirable before the precipitation of dC and dG in order to improve the purity of the final product. Other particular steps in the processing of the different nucleosides will be apparent from the following detailed descriptions of examples. EXAMPLE 1 Preparation of N⁶-Benzoyl-2'-deoxyadenosine λ ia TMS-transient Protection

Example 1A

N⁶-benzoyl-2'-deoxyadenosme was prepared on a 500 g scale by the following process which makes use of TMS-transient protection To a 12-L 4-neck flask equipped with an overhead stirrer, thermometer, mtiogen mlet nd addition funnel was added 500 g (1 86 mol) 2'-deoxyadenosme monohydrate (dAΗ₂0), 5 0 L anhydrous tetrahydrofuran (THF) and 2 69 L anhydrous tnethylamme (heremaftei "TEA") (18 6 mol, 10 0 eq) The mixture was stirred under nitrogen in an ice bath (inner temperature about 2°C) to give a turbid dA solution The addition funnel was charged with 1 18 L tnmethylchlorosilane (TMSCl) (9 3 mol, 5 eq), which was added dropwise to the dA solution over one hour, with the temperature maintained at about 2-5 °C, to form TMS-protected dA The mixture was stirred at this temperature for another hour, and 430 mL benzoyl chloride (BzCl, 3 72 mol, 2 0 eq) was then added slowly over one hour, while maintaining the temperature at about 2-5 °C The mixture was continuously stirred at room temperature for another hour, forming benzoylated TMS-dA The reaction mixture was then cooled, and 2 L of water was added slowly with stirring, maintaining the temperature at less than 10°C The mixture was then allowed to settle for phase separation, and the aqueous phase was set aside While maintaining the organic phase at about 2-5 °C, 1 0 L of 4 N NaOH was added slowly over one hour, and stirred for an additional hour The mixture was allowed to settle, and the aqueous phase removed An additional 600 mL 4 N NaOH was added to the organic phase, which was then stmed for another houi The mixtuie was allowed to settle and the aqueous phase removed The separated aqueous phases were then combined, and neutralized by the addition of 4 N HCl at about 2-5 °C until reaching a pH of about 8, dunng which solids were formed To the mixture was added 5 L 1 :1 ethyl acetate/t-butyl methyl ether (TBME), and the mixture stirred for about 30 minutes at room temperature. The solids were filtered and washed twice with 0.5 L 1 :1 ethyl acetate/TBME. The solids were then vacuum dried at about 40°C for about 24 hours. A typical yield was about 70%.

Example IB

An alternative process for making N⁶-Benzoyl-2'-deoxyadenosine used NH₄OH instead of NaOH in the hydrolysis step. This preparation was on a 200 g scale, and also used TMS transient protection. 200 g (0.743 mol) dA-H₂0, 3.0 L anhydrous THF and 1.04 L anhydrous Et₃N (7.4 mol, 10.0 eq) were combined, and the mixture stirred under nitrogen in an ice bath (inner temperature 2°C) to give a turbid dA solution. 592 mL TMSCl (3.715 mol, 5 eq) was added dropwise to the dA solution over one hour, with the temperature maintained at about 2-5 °C. The mixture was stirred at this temperature for another hour to convert dA to TMS-protected dA. 173 mL BzCl (1.486 mol, 2.0 eq) was then added slowly over one hour. The mixture was continuously stirred at room temperature for another hour to convert the TMS- protected dA to benzoylated TMS-dA.

The mixture was then cooled to about 2-5 °C, and to this 25 mL of H₂0 was slowly added to quench any unreacted TMSCl. The mixture was then vacuum filtered to remove the solid TEAΗC1 salt, which was further washed with anhydrous 3 L THF, and the filtrate and washes were combined.

While maintaining this mixture at about 2-5 °C, 750 mL of water was added slowly over one hour. Then 1.5 L 29% NH₄OH was gradually added over 30 minutes. To make the mixture homogeneous, 1.5 L of methanol was then added. Stirring was continued for 1-3 hours at room temperature until the reaction was about 75% complete.

Volatile solvents were removed by vacuum distillation to leave a gummy residue. To the residue was added 500 mL water and 3 L 1:1 ethyl acetate/TBME, and the mixture stirred for about 30 minutes at room temperature. White solids were observed to precipitate out. The solids were filtered and washed with 250 mL water, and vacuum dried at room temperature for about 12 hours. A typical yield was about 70%.

EXAMPLE 2 Preparation of N⁶-Benzoyl-5'-O-(4,4-dimethoxytritvι)-2'-deoxyadenosine

This example demonstrates a method for preparing N⁶-benzoyl-5'-(4,4- dimethoxytrityl)-2'-deoxyadenosine from Bz-dA. 142 g of Bz-dA prepared in accordance with Example IB (containing an unknown amount of H₂0) was combined with 500 mL of dimethylformamide (DMF), 40 mL of TEA, and 500 mL of hexane. The mixture was heated to reflux and stirred under nitrogen in a flask equipped with a Dean-Stark trap. About 17 g of a heavy layer containing water and DMF was collected over 5 hours. The heavy layer was analyzed and found to contain about 12 g of water. The amount of Bz-dA was recalculated to be 130 g, or 0.366 mol, based on the amount of water removed. This step removed the residual water without having to subject the material to a drying step.

The mixture was cooled to ambient temperature and transferred into a separatory funnel to separate the DMF layer containing TEA and Bz-dA from the remaining hexane. The bottom DMF layer was separated and transfened back into the flask, together with an additional 100 g of TEA (0.99 mol, ~3.0 eq based on Bz-dA). A solution of DMT-Cl (164 g, 98%, 0.476 mol, 1.30 eq based on Bz-dA) in 500 mL of CH₂C1₂ was added dropwise to the above mixture over 1.5 hours. During the addition, the temperature of the mixture was controlled so that it did not exceed 25 °C. Upon the completion of the addition, the mixture was stiιτed at ambient temperature for another 30 minutes. The mixture was then transferced into a jacketed flask, which was equipped with a mechanical stirrer, an addition funnel, and a bottom take-off valve. The mixture was extracted with 2% aqueous NaHC0₃ solution ( 1000 mL) and 4 times with a mixture of DMF and 2% NaHCO₃ solution (1000 mL each time). The mixture was then extracted again with 2% NaHCO₃ solution (1000 mL) to remove residual DMF. The resulting CH₂C1₂ solution was concentrated to about 500 mL and transferred into an addition funnel.

To the solution was added 4 L of hexane-TBME (t-butyl methyl ether) (2:1) mixture over one hour with vigorous stirring. A homogeneous solution was obtained first, but a white precipitate product started to appear in about 30 minutes. The mixture was cooled to 15 °C, stirred for 3 hours, and filtered. The product filter cake was dried under vacuum at 40 °C until reaching constant weight, and was found to be over 99%. pure, with a yield of about 80%.

EXAMPLE 3

Preparation of N²-Benzoyl-2'-deoxycvtidine

The following method was used to make N²-benzoyl-2'-deoxycytidine from a starting material of dC-HCl salt. As a preliminary step, the dC-HCl salt was neutralized to dC free-base as follows. 300 g dC-HCl (1.13 moles) and 144 g TEA (1.42 moles) were charged to 300 mL CH₃OH under stinϊng. The suspension was heated to reflux, forming a homogenous light brown solution, and heating was then discontinued. 1.5 L CH₂C1₂ was then gradually added to this solution under stirring, whereby a white solid was formed. Stirring at room temperature was continued for one about hour, and then the solid was filtered out. The solid was then washed with an additional 500 mL CH₂C1₂, and dried under vacuum for 30 minutes. 260 g of dC free-base product having a purity of about 98% was obtained, which was a yield of about 100%).

The dC free-base (2'-deoxycytidine) may then be used to prepare N²-benzoyl- 2'-deoxycytidine (Bz-dC) as follows. 250 g dC was mixed with 3.75 L isopropanol (I-POH) and heated to 60°C. 250 g benzoic anhydride (Bz₂O) (1.1 mole). predissolved in 1.5 L I-POH was added into the above mixture over 5 minutes, and another 500 mL I-POH was used to rinse the inlet. This mixture was continuously heated to 75 °C and aged at 75 °C for 30 minutes. Another 25 g Bz,0 (0.1 1 mole) predissolved in 500 mL I-POH was added to the above mixture over 5 minutes, and the mixture was heated at 75 °C for another 30 minutes. Then, an additional 25 g Bz₂O (0.11 mole) predissolved in 500 mL I-POH was added to the above mixture over 5 minutes, and the mixture was heated at 75 °C for another 30 minutes. In all, a total 300 g Bz₂O (1.32 moles) was used, for a molar ratio of Bz₂O to dC of about 1.2. The mixture was then cooled to about 2-5 °C over one hour, stirred for an additional 30 minutes at about 5 °C, and filtered. In one test run, after vacuum drying at 37°C for 12 hours, 285 g of Bz-dC as a white solid was obtained, which was a yield of about 80%. NMR showed good purity, with only 0.55% I-POH identified in the sample.

EXAMPLE 4

Preparation of N⁴-BenzoyI-5'-(4,4-dimethoxytrityl) deoxycytidine

The following procedure was used to prepare N⁴-benzoyl-5'-(4,4- dimethoxytrityl) deoxycytidine from Bz-dC. 300 g of Bz-dC (0.904mol) was dissolved in 1.6 L of DMF at 60 °C, and the solution cooled to room temperature. This solution was mixed with 238 mL of TEA (1.71 mol, 1.90 eq. based on the Bz-dC), and the mixture stirred under nitrogen until a homogeneous solution was obtained. 413 g of 98% DMT-Cl (1.22mol, 1.35 eq. based on the Bz-dC) was added gradually over 30 minutes, and the charging port rinsed with 800 mL of DMF. The temperature of the reaction mixture was maintained between 25 and 30 °C during this period. Upon the completion of the addition, the mixture was stirred at 25 °C for an additional 30 minutes.

Then, 1.0 L of DI water was charged to the reaction mixture gradually over 15 minutes. The mixture was stin^ted until a homogeneous solution was obtained. This solution was then extracted with a mixture of cumene and hexane (3 x 3.0 L, cumene/hexane=2:l) at 40 °C to remove non-polar impurities by liquid-liquid extraction. In some cases, a third phase may be generated during these extractions. If that happens, the bottom and the middle phases should be taken together as the product layer. Once these extractions were complete, the DMF-water phase was then extracted with 3.0 L of hexane to remove residual cumene.

The DMF phase was then separated and diluted with 2.0 L of CH₂C1₂. The resulting solution was extracted with a mixture of DMF and 2% aqueous NaHCO₃ solution (1 :3, 2 x 4 L) to remove polar impurities (including Bz-dC and bis-Bz-dC), together with TEA and TEA-HC1. The CH₂C1₂ phase was then washed with the 2% NaHC0₃ solution (2 x 4 L) to remove residual DMF in the CH₂C1₂ solution. The CH₂C1₂ solution was then separated and stirred with activated charcoal (75 g, Darco G-60 powder) for about 1 hour. Then, the charcoal was filtered off and the charcoal cake rinsed with 200 mL of CH₂C1₂.

The combined filtrate and rinse was stripped to dryness under vacuum. The residue was re-dissolved in 500 mL of acetonitrile. The solution was added into 9 L of water with vigorous stirring over 30 minutes. The product precipitated out as fine powder during this period. Upon the completion of the addition, the resulting mixture was stirred further for one hour and then was filtered. The filter cake was dried in a vacuum at 50°C until the weight loss was less than 0.2% in 24 hours. A typical yield was about 75%, with a purity of over 99%.

EXAMPLE 5 Preparation of N²-Isobutyryl-2'-Deoxyguanosine via TMS-transient Protection

N²-isobutyryl-2'-deoxyguanosine (iB-dG) was made by charging 40 g (0.14 mol) of 2'-deoxyguanosine monohydrate (dG), 0.4 L anhydrous THF, and 194 mL anhydrous triethylamine (1.4 mol, 10.0 eq) to a flask, and stirring the mixture under nitrogen in ice bath (imier temperature 2°C) to give a turbid solution. To this mixture was added 89 mL chlorotrimethylsilane (TMSCl, 0.7 mol, 5 eq) by dropwise addition over about one hour, while maintaining the mixture at about 2-5 °C. The mixture was then stin^ted at this temperature for another hour, during which isobutyryl chloride (iB-Cl, 0.28 mol, 2.0 eq) was added slowly. The mixture was stirred continuously at room temperature until the reaction was complete, about 4-5 hours.

Then, 200 mL H₂O was added to the reaction mixture, which was stirred for 15 minutes, and allowed to settle. The two phases were then separated. The bottom aqueous phase containing the polar impurities was drained out. Then, 400 mL of 2N NaOH was added slowly to the remaining organic phase, while maintaining a temperature of about 2-5 °C. This reaction was mildly exothermic, and the pH of the resultant mixture was about 12. Stiffing was continued for another 2 hours at about 2-5 °C. Then, 40 g of NH₄C1 (s) was added to neutralize the solution to a pH of about 8-9.

Solvents were then removed by vacuum until the solution was cloudy. 600 mL 1 : 1 ethyl acetate/t-butyl methyl ether was added to the residue, and the mixture stirred for about 30 minutes. The solids were then filtered and washed with 100 mL water and 1 : 1 ethyl acetate/t-butyl methyl ether, and dried by vacuum at room temperature for 12 hours. In one test, the solids, which still retained some solvent, weighed 38.8 g, a yield of 76.8%.

EXAMPLE 6 Preparation of 5'-O-Dimefhoxytrityl N²-Isobtιtyryl-2'-Deoxyguanosine

5'-O-dimethoxytrityl N²-isobutyryl-2'-deoxyguanosine was made by charging

12.00 g of N²-isobutyryl-2'-deoxyguanosine (iB-dG, 35.6 mmol), 14.0 mL of triethylamine, and 60 mL of DMF to a flask. The mixture was stirred at ambient temperature until the iB-dG was completely dissolved. Then, a solution of 15.0 g of 98 % DMT-Cl (44.1 mmol, 1.24 eq based on iB-dG) in 60 mL of CH₂C1₂ was added slowly to the mixture over 45 minutes. The temperature was controlled to 27 °C using a water bath. Upon the completion of the addition, the mixture was stirred at ambient temperature for another 2 hours. The reaction mixture was transferred to a separatory funnel and washed with 3% NaHCO₃ solution (2 x 200 mL). The organic layer was dried with Na^O^ The dried solution was added dropwise to 250 mL of toluene with vigorous stirring. Upon completion of the addition, the mixture was stiffed for 2 hours and then filtered. The solid was washed with toluene-and hexane. After drying, 16.1 g of material was obtained. HPLC analysis showed the purity of 5'-0-dimethoxytrityl N²-isobutyryl-2'- deoxyguanosine was 99.05%, with a yield of about 70%.

EXAMPLE 7 Preparation of 5'-O-DimethoxytrityIthymidine

5'-0-dimethoxytritylthymidine (DMT-T) may be made by the following procedure. A solution containing 568 g of 2'-deoxythymidine (dT) (99%, 2.32 mol) and 2.0 L of DMF was prepared at a temperature below about 55 °C. The solution was charged to a jacketed 12-L flask equipped with a bottom take-off valve, a circulating bath, and an addition funnel. 1.0 L of triethylamine (7.19 mol, 3.10 eq based on dT) was added, and the mixture cooled to below 10°C.

962 g of 98% DMT-Cl (2.78 mol, 1.20 eq based on dT) was mixed with 2.5 L of CH₂C1₂ to form a slurry, and the slurry was added slowly to the above flask through the addition funnel over about one hour. During the addition period, the temperature of the mixture should be maintained below 20°C. Upon the completion of the addition, the temperature was raised to about 25 °C, and the mixture was stiffed for an additional hour.

3.0 L of 2% aqueous NaHCO₃ solution was then added to the flask, and the resulting mixture stirred vigorously for about 10 minutes. The mixture was allowed to settle, and the bottom CH₂C1₂ layer was drained through the take-off valve and collected. After the top aqueous layer was drained, the CH₂C1₂ layer was transferred back into the flask. This NaHCO₃ solution wash was repeated twice, and the CH₂C1₂ layer sampled for analysis after each wash. The CH₂C1₂ layer was then transferred back to the 12-L flask, and 6.0 L of cyclohexane was added over 30 minutes at ambient temperature with vigorous stirring. The resulting mass was cooled to 5°C with slow stirring, and filtered. The circulating bath then was switched to its heating cycle with a setpoint of 45 °C. The filter cake-was washed with cyclohexane (2 x 3.0 L) and dried under vacuum suction for 15 minutes.

The solid material was then mixed with 3.0 L of CH₂C1₂ and transferred back to the 12-L flask. The mixture was heated to refluxing to allow the solid to dissolve. When a homogeneous solution was obtained, the circulating bath was switched to its cooling cycle (setpoint: -5 °C). Then about 6 L of hexane was added to the solution over 30 minutes with vigorous stirring. The mixture was then cooled to about 5 °C with slow stiffing, during which the product crystallized out of solution. The solid was filtered off and washed with hexane (2 x 3.0 L). The wet cake was dried under vacuum suction for 15 minutes. A typical yield was about 85%, at a purity of over 99%.

Claims

We claim

1 A process for preparing an essentially pure 5 '-protected deoxynbonucleoside product comprising the steps of a) dissolving a starting deoxynbonucleoside m a polar aprotic solvent which is inert to the starting deoxynbonucleoside, to the 5'-protected deoxynbonucleoside, and to the other leactants, wherem any exocyclic ammo groups of the deoxynbonucleoside aie protected, b) reacting said starting deoxynbonucleoside with a protecting reagent to form a 5'-protected deoxynbonucleoside product, c) removing polar impunties by one or more liquid- liquid exti actions using immiscible polar and non-polar solvents which form polai and non-polai phases m which the pioduct prefeientially partitions into the non-polar phase and the impurities prefeientially partition into the polai phase, and sepaiatmg the non-polai phase from the polar phase, and d) removing non-polar impurities by solidifying the pioduct out of solution while leaving the non-polar impurities m solution

2 The process of claim 1 further comprising the step of lecovermg said 5'-protected deoxynbonucleoside product

3 The process of claim 1 wherem the exocyclic ammo groups are protected by acyl protection

4 The process of claim 1 furthei compnsing a step of lemovmg non- polar impurities by one or more hquid-hquid extractions using immiscible polar and non-polai solvents which form polar and non-polar phases in which the pioduct prefeientially partitions into the polar phase and the impunties piefeientially partition into the non-polar phase, and sepaiatmg the polar phase fiom the non polai phase

5. The process of claim 1 further comprising the step of dehydrating the deoxyribonucleoside of step (a) prior to adding the reagent of step (b).

6. The process of claim 5 wherein said dehydrating step comprises adding a dehydrating solvent which forms an azeotrope with water to the solution of step (a), and then distilling the azeotrope out of the solution.

7. The process of claim 6 wherein said dehydrating solvent is one or more C₅ to C₁₀ hydrocarbons, which may be linear, branched or cyclic, and may be substituted or unsubstituted.

8. The process of claim 7 wherein said dehydrating solvent is selected from the group consisting of pentane, hexane, heptane and combinations thereof.

9. The process of claim 1 wherein said reagent is a tritylation agent and said protected deoxyribonucleoside is a 5'-tritylated deoxyribonucleoside.

10. The process of claim 9 wherein said tritylation agent is dimethoxytrityl chloride (DMT-Cl), methoxytrityl chloride or trityl chloride.

11. The process of claim 1 , wherein an acid scavenger is added to said solution with said reagent.

12. The process of claim 11 wherein said acid scavenger is one or more tertiary amines.

13. The process of claim 12 wherein said acid scavenger is triethylamine (TEA).

14 The process of claim 1 wherem step (c) further comprises selectively adsorbing polar impurities with a solid adsorbent

15 The process of claim 1 wherem said deoxynbonucleoside is selected from the group consisting of deoxyadenosine (dA), deoxycytidine (dC), deoxyguanosme (dG) and deoxythymidme (dT)

16 The process of claim 15 wherem the starting deoxynbonucleoside of step (a) is selected from the group consisting of N⁶-benzoyl-2'-deoxyadenosme (Bz- dA), N²-benzoyl-2'-deoxycytιdme (Bz-dC), N²-ιsobutyryl-2'-deoxyguanosme (iB-dG), and deoxythymidme (dT)

17 The process of claim 1 wherem said polai aprotic solvent is selected fiom the group consisting of dimethylformamide (DMF), dimethylacetamide, N-methylpyrrohdone, dimethylsulfoxide (DMSO), dimethylsulfone, hexamethylphosphate (HMPA) and combinations theieof

18 The process of claim 1 wherem said step (d) compπses solidifying the product out of solution m a non-polar solvent by combining the solution \\ ith a miscible solvent in which the product is insoluble in an amount effective to ciystalhze a crystalline product, or to precipitate an amorphous product from the non-polar phase

19 The process of claim 1 wherem said step (d) compnses transfening the pioduct to a solution in a polar solvent that is miscible with watei, and solidifying the pioduct out of the solution by combining the solution with an amount of \\ atei effective to solidify the product out of solution

20. The process of claim 1 wherein said protected deoxyribonucleoside is N⁶-benzoyl-5'-(4,4-dimethoxytrityl)-2'-deoxyadenosine, said starting deoxyribonucleoside is N -benzoyl-2'-deoxyadenosine (Bz-dA), said polar aprotic solvent is dimethylformamide (DMF), said reagent is dimethoxytrityl chloride (DMT- Cl), and said non-polar solvent of step (c) is CH₂C1₂.

21. The process of claim 21 wherein said step (d) comprises adding a mixture of hexane and t-butyl methyl ether (TBME) to a solution of product in CH₂C1₂ to crystallize the product out of solution.

22. The process of claim 21 wherein the hexane-TBME mixture is about 2 parts hexane to 1 part TBME, by volume.

23. The process of claim 20 further comprising the step of dehydrating the Bz-dA prior to adding the reagent of step (b)

24. The process of claim 22 wherein said dehydrating step comprises adding a dehydrating solvent which forms an azeotrope with water to the solution of step (a), and then distilling the azeotrope out of the solution.

25. The process of claim 1 wherein said protected deoxyribonucleoside is N⁴-benzoyl-5'-(4,4-dimethoxytrityl) deoxycytidine, said starting deoxyribonucleoside is N²-benzoyl-2'-deoxycytidine (Bz-dC), said polar aprotic solvent is dimethylformamide (DMF), said reagent is dimethoxytrityl chloride (DMT-Cl), and said non-polar solvent of step (c) is CH₂C1₂.

26 The process of claim 25 further compnsing, after step (b) and before step(c), a step of removing non-polar impunties by fust adding water to the solution of product m DMF, and then performing one or more hquid-hquid extractions by adding an immiscible non-polar solvent to the solution, allowing polar and non-polai phases to form in which the product preferentially partitions into the polai phase and the impurities preferentially partition into the non-polar phase, and sepaiatmg the polar phase containing the product from the non-polar phase

27 The process of claim 26 wherem immiscible non-polai solvent is a mixture of cumene and hexane

28 The piocess of claim 27 wherem the cumene-hexane mixture comprises about 10 to about 90 parts cumene to about 90 to about 10 parts hexane, by volume

29 The process of claim 28 wheiem the cumene-hexane mixtuie compnses about 2 parts cumene to 1 part hexane, by volume

30 The process of claim 26 wheiem step (d) compnses transferring the product to a polar solvent that is miscible with watei, and solidifying the product out of the solution by combining the solution with an amount of water effective to solidify the product out of solution

31 The piocess of claim 30 wherem the polai solvent of said step (d) is acetonitrile

32 The process of claim 1 wherem said protected deoxynbonucleoside is 5'-0-dιmethoxytπtyl N²-ιsobutyryl-2'-deoxyguanosme, said starting deoxynbonucleoside is N²-ιsobutyryl-2'-deoxyguanosιne (iB-dG), said polar aprotic solvent is dimethylformamide (DMF), said reagent is dimethoxytrityl chloride (DMT- Cl), and said non-polar solvent of step (c) is CH₂C1₂

33 The process of claim 32 wherem step (d) compnses adding a solution of the product m CH₂C1₂ to hexane to precipitate the product out of solution

34 The piocess of claim 1 wherem said protected deoxynbonucleoside is 5'-0-dιmethoxytntylthymιdme, said starting deoxynbonucleoside is 2'- deoxyfhymidine (dT), said polai apiotic solvent is dimethylformamide (DMF), said leagent is dimethoxytrityl chloride (DMT-Cl), said non-polar solvent of step (c) is CH₂C1₂

35 The process of claim 34 wheiem said step (d) compnses adding hexane to a solution of product in CH₂C1₂ to crystallize the product out of solution

36 A process foi selectively protecting the exocyclic ammo group of a starting deoxynbonucleoside which has an exocyclic ammo group which is to be protected by acylation and at least one hydroxyl gioup which is to be left unprotected, the process comprising a) dispersing the starting deoxynbonucleoside a polai solvent which is substantially free of pyπdine and which is a solvent for the piotected product fonned in step b) , and b) selectively acylatmg said exocyclic ammo gioup of said starting deoxynbonucleoside to form a piotected product in which said hydioxyl gιoup(s) are unprotected

37. The process of claim 36 wherem said polar solvent is a straight chain or branched C_l to C₅ alcohol.

38. The process of claim 37 wherem said polar solvent is isopropanol.

39. The process of claim 36 wherein said deoxyribonucleoside is selected from the group consisting of deoxyadenosine (dA), deoxycytidine (dC) and deoxyguanosine (dG).

40. The process of claim 36 further comprising adding an acid scavenger to said polar solvent solution of deoxynbonucleoside.

41. The process of claim 40 wherein said acid scavenger is one or more tertiary amines.

42. The process of claim 41 wherem said acid scavenger is tnethylamme (TEA) or di-isopropyl-monoethyl amine (DIPEA)

43. The process of claim 36 wherem said deoxyribonucleoside is deoxycytidine, which is acylated with benzoic anhydnde.

44. The process of claim 43 wherem said starting deoxynbonucleoside is deoxycytidine free-base.

45. The process of claim 44 wherem said deoxycytidine free-base is made by the neutralization of deoxycytidme-HCl.

46. The process of claim 45wherein the deoxycytidine free-base is made by dissolving deoxycytidine-HCl in a protic polar solvent, adding enough non- aqueous organic base to neutralize the deoxycytidine-HCl, refluxing the mixture until the deoxycytidine-HCl converts to deoxycytidine free-base, and then adding a second solvent which is miscible with the polar protic solvent, but in which deoxycytidine free-base is insoluble, to the mixture until the deoxycytidine free-base precipitates out of solution.

47. The process of claim 46 wherein said protic polar solvent is methanol, said non-aqueous organic base is triethylamine and said second solvent is CH₂C1₂.

48. The process of claim 43 wherein the molar ratio of benzoic anhydride to deoxycytidine is about 1.2 to about 1.3.

49. The process of claim 36 wherein step (c) comprises:

(i) reacting the starting deoxyribonucleoside with a transient protection agent which reacts with both the exocyclic amine group(s) and the hydroxyl group(s); (ii) reacting the transiently protected deoxyribonucleoside with a selective acylation agent which acylates the transiently protected exocyclic amino group(s) but leaves the transiently protected hydroxyl group(s) essentially unreacted; and

(iii) reacting the deoxyribonucleoside with a hydrolyzing agent to return said transiently protected hydroxyl group(s) to hydroxyl form.

50. The process of claim 49 wherein said transient protection agent is a trimethylsilane salt.

51. The process of claim 50 wherein said transient protection agent is trimethylchlorosilane (TMSCl).

52. The process of claim 49 further comprising adding an acid scavenger to the solution.

53. The process of claim 52 wherein said acid scavenger is triethylamine.

54. The process of claim 53 wherein enough triethylamine is added to the solution to neutralize any acid produced during the process.

55. The process of claim 49 wherein said deoxyribonucleoside is deoxyadenosine or deoxyguanosine.

56. The process of claim 49 wherein said deoxyribonucleoside is deoxyadenosine, said transient protection agent is trimethylchlorosilane (TMSCl), said acylation agent is benzoyl chloride, and enough triethylamine is added to the solution to neutralize any acid produced during the process.

57. The process of claim 56 wherein said hydrolyzing agent is NaOH or

NH₄OH.

58. The process of claim 49 wherein said deoxyribonucleoside is deoxyguanosine, said transient protection agent is trimethylchlorosilane (TMSCl), said acylation agent is isobutyryl chloride, and enough triethylamine is added to the solution to neutralize any acid produced during the process.

59. The process of claim 58 wherein said hydrolyzing agent is NaOH or

NH₄OH.