Combinatorial libraries have become an important tool in drug discovery. The compounds which are members of a combinatorial library are typically prepared in a parallel array synthesis, whereby reactions of the same type are performed, either simultaneously or in close temporal proximity, with different reagents.

Reagents bonded to solid supports are often used to facilitate the removal of contaminates such as reaction by- products and excess starting materials from the reaction mixtures in a parallel array synthesis. The contaminant is converted by the solid support bound reagent to a form which can be more easily removed from the reaction mixture than the original contaminant, e. g., by filtration, extraction, absorption onto ion exchange resin or other means. The solid support bound reagent residue can be then be separated from the reaction mixtures by simple filtration.

Examples of solid support bound reagents include N- (2- aminoethyl) aminomethyl polystyrene, aminomethylated polystyrene and piperidine-4-carboxylic acid polyamine resin. Others are described in the 1999 Calbiochem- Novabiochem Corporation Catalog, pages 214-216. However, very few support bound reagents are presently available, and the solid support bound reagents which are currently known react with only a limited number of contaminate types. For example, solid support bound reagents which react with oxidizing agents are presently unavailable. Solid support bound reagents of this type would facilitate removal of

oxidizing agents from reaction mixtures and the preparation of combinatorial libraries by parallel array synthesis.

It has now been found that ascorbic acid bonded to insoluble polymers reacts with oxidizing agents in reaction mixtures (Example 3). Sensitive functional groups such as disulfides are not affected by this reaction (Example 3).

Based on this discovery, novel polymers with ascorbic acid bonded thereto, methods of preparing said polymers, methods of removing oxidizing agents from a solution and methods of removing oxidizing agents from the reaction mixtures of parallel array syntheses are disclosed.

One embodiment of the present invention is an insoluble polymer comprising ascorbic acid bonded thereto.

Another embodiment of the present invention is a method of removing an oxidizing agent from a solution, e. g., a reaction mixture. The method comprises the step of reacting the oxidizing agent with an insoluble polymer comprising ascorbic acid bound thereto. Preferably, the polymer is then removed from the solution by, for example, filtration.

Another embodiment of the present invention is a method of preparing an insoluble polymer with ascorbic acid bound thereto. The method comprises the step of reacting ascorbic acid with an insoluble polymer comprising pendent groups-X- R.-X-is a covalent bond or an inert linking group and R is a heteroatom functional group which can react with a primary alcohol or alkoxide to form a covalent bond with the oxygen atom of the alcohol or alkoxide.

Yet another embodiment of the present invention is a method of removing an oxidizing agent (s) from the reactions of a parallel array of reactions. A polymer of the present invention is reacted with the oxidizing agent (s) in the

reactions of the parallel array, thereby forming a product from the oxidizing agent. The product and the polymer are then separated from the reaction mixtures. The parallel array of reactions can be used in a process for preparing a combinatorial library.

Yet another embodiment of the present invention is a method of preventing oxidation of a compound (s). The method comprises the step of storing the compound (s) in the presence of a polymer of the present invention. In one example, the compound (s) are members of the same combinatorial library.

The ascorbic acid resins of the present invention can be used to remove oxidizing agents from reaction mixtures with organic, protic or aprotic polar solvents without modifying sensitive functional groups such as disulfides.

Because they can be separated from reaction mixtures by simple filtration, these polymers are suitable for use in small scale reactions and in automated procedures.

Therefore, the polymers of the present invention are particularly useful in parallel array syntheses by which combinatorial libraries are prepared.

The present invention is directed to novel insoluble polymers having ascorbic acid bound thereto or immobilized thereon. Preferably, at least 0.1 millimoles of ascorbic acid are covalently bonded to or immobilized on each gram of polymer, more preferably between about 1.0 millimoles of ascorbic acid and even more preferably at least 5.0 millimoles. Ascorbic acid is preferably bonded to the polymer at its primary alcohol. The polymers of the present invention are also referred to herein as"ascorbic acid resins".

Ascorbic acid resins can be prepared from polymers having pendent groups-X-R. The pendent groups on a polymer can be the same or different, but are preferably the same.

R is a heteroatom-containing functional group which can react with ascorbic acid to form a covalent linkage with ascorbic acid. In one example, R comprises a leaving group covalently bonded to a carbon atom and can react with a primary alcohol or alkoxide to form an ether. In another example, R is a group which can react with a primary alcohol or alkoxide to form an ester.

The term"leaving group"has the meaning commonly afforded the term in the field of organic chemistry. A "leaving group"is covalently bonded to a carbon atom and can be displaced by a nucleophilic reagent such that a new covalent bond is formed between the carbon atom and the residue of the nucleophilic reagent. Suitable leaving groups are those which can be displaced by the primary alcohol or corresponding alkoxide of ascorbic acid to form an ether linkage between the pendent group and the ascorbic acid residue. Examples include a primary, secondary or tertiary chlorides (e. g., trityl chloride), bromides, iodides or sulfonate esters. Procedures for forming an ether linkage between a primary alcohol and a carbon bound to a leaving group and are disclosed, for example, in Fréchet and Haque, Tetrahedron Letters 1975: 3055 (1975), the entire teachings of which are incorporated herein by reference.

Groups which can react with primary alcohols to form ester linkages include anhydrides, acid halides and carboxylic acids. Thus, the primary alcohol of ascorbic acid can react with these groups, resulting in the formation of an ester linkage between the pendent group and the

ascorbic acid residue. Procedures for carrying out these transformations are well known in the art. A specific example of immobilizing ascorbic acid onto a polymer by forming an ester linkage between pendent groups of a polymer and ascorbic acid is provided in Examples 1 and 2.

-X-is a covalent bond or an inert linking group. As used herein, an"inert linking group"is a moiety which is substantially unreactive towards ascorbic acid and towards the reagents in a reaction mixture to be treated with the ascorbic acid resin. The choice of a suitable linking group depends upon the use of the ascorbic acid resin, for example, the type of reaction mixture to which the ascorbic acid residue is to be added. Examples of linking groups include alkylene [- (CH2) n--NHCO- (CH2)-I-0- (CH2)-I <BR> <BR> <BR> <BR> (-alkylene-O-) n-CO- (CH2) 2-/ (-alkylene-O-) n-CH2CH2NHCO- (CH2) m~ n -CH2NH (-alkylene-O) n-CO- (CH2) m-and alkylene-NHCO- (CH2) m-. n is an integer from 1 to about 100 and m is an integer from 1 to about 20. The skilled artisan will be able to select, using no more than routine experimentation, linking groups which are suitable for a particular application.

The polymers of the present invention are required to be insoluble. As used herein, the term"insoluble"means that the polymer does not dissolve in solvents used to carry out organic reactions, for example, in protic solvents such as water and alcohols, in organic solvents such as ethers (e. g., diethyl ether, THF, 1,4-dioxane, glyme and diglyme), ketones (e. g., acetone and methyl ethyl ketone), aromatic solvents (e. g., benzene, toluene and xylene), hydrocarbons (e. g., alkanes, cycloalkanes and petroleum ethers), nitriles (e. g., acetonitrile) and nitroalkanes (e. g., nitromethane) and in polar aprotic solvents such as HMPA, DMF and DMSO.

Consequently, they are crosslinked polymers having from 0

to about 20% crosslinking, preferably 0.5% to about 2.0% crosslinking. Examples of suitable polymers include crosslinked polystyrenes, polyacrylamides or polyacrylates.

Polystyrenes can be crosslinked with, for example, divinylbenzene ("DVB"); polyacrylamides and polyacrylates can be crosslinked with, for example, bis 2-acrylamidoprop- 1-yl polyethylene glycol.

Specific examples of polystyrenes which can be used to form the ascorbic acid resins of the present invention include DVB crosslinked polystyrenes having-(ethylene-O-) n- ethylene-NH-CO-CH2CH2COOH,-CH2NH-CO-CH20- (CH2CH20) n- COCH2CH2COOH or-COOH attached to phenyl groups in the polymer backbone. Polystyrenes with linking groups comprising polyethylene glycol are referred to herein as a "polystyrene-polyethylene glycol copolymer"or"PS-PEG".

Polystyrenes with-COOH attached to phenyl groups in the polymer backbone are referred to herein as"carboxylated polystyrenes". PS-PEG and carboxylated polystyrene are commercially available. The preparation of an ascorbic acid resin from carboxylated polystyrene is described in Examples 1 and 2.

Specific examples of polyacrylamides which can be used to form the ascorbic acid resins of the present invention include bis 2-acrylamidoprop-1-yl polyethylene glycol crosslinked polyacrylamides having-CH (CH3)-CH2O-(CH2CH2O) n- CH2CH2NHCOCH2CH2-COOH (also referred to as"PEGA"resins") or -(CH2) 3NHCO-CH2CH2COOH(CH2) 3NHCO-CH2CH2COOH attached to the nitrogen atom of amides in the polymer backbone. These polymers are also commercially available from Calbiochem-Novabiochem.

The ascorbic acid resins of the present invention can be used to remove oxidizing agents from solutions such as reaction mixtures by adding resin to the solution and

allowing the oxidizing agent to react with the ascorbic acid immobilized on the resin. In this manner, oxidizing agents can be removed from reaction mixtures carried out in protic solvents, polar aprotic solvents and organic solvents.

Examples of oxidizing agents which can be removed from solutions include I2, Br2, Cl2, CrO3 CrO3/pyridine, SeO2, SO3/pyridine or peroxides such as H2Ozor benzoyl perodide.

The immobilized ascorbic acid generally reacts with oxidizing agents at room temperature within minutes to about two to three hours. However, the reaction can be carried out at other temperatures, for examples between about 0° C and about 50° C. Whether additional reaction time or resin is required so that all the oxidizing agent reacts can be determined by any suitable means known to the skilled artisan for determining the presence or concentration of the oxidizing agent. For example, the concentration of Cl2, Br2 or I2 in a solution can be determined by monitoring the characteristic color of the oxidizing agent visually or spectrophotometrically. The presence of peroxides can be detected by the potassium iodide method disclosed on page 238 of the"CRC Handbook of Laboratory Safety, 3rd Edition, A. Keith Furr editor, CRC Press.

The ascorbic acid resin"removes"oxidizing agents from a solution or reaction mixture oxidizing agents by converting them into a product, referred to as the "oxidizing agent residue". The oxidizing agent residue can be readily separated from the solution reaction mixture, e. g., by filtration, extraction, absorption onto ion exchange resin or other suitable means.

C12, Br2 and 12 are converted by the ascorbic acid resin into Cl, Br and I, which generally precipitate from organic solvents. The precipitate can be removed by

filtration or aqueous extraction. Cl-, Br-and I-can be removed from aqueous solution by absorption onto ion exchange resin, followed by filtration.

Peroxides are converted by ascorbic acid resin into carboxylic acids, which can be removed from organic solvents by extraction with aqueous base. Carboxylic acids which are water soluble can be removed from aqueous solvents by absorption onto ion exchange resins followed by filtration.

After reaction with ascorbic acid resin, oxidizing agents such as CrO3 CrO3/pyridine and Set, are converted into forms which are insoluble in most organic solvents. These reaction products can therefore be separated from most organic solvents by filtration or extraction with water.

They can be separated from water by absorption onto ion exchange resin followed by filtration.

"Combinatorial library"has the meaning commonly afforded the term in the art. For example, a combinatorial library is a collection of at least three, preferably ten and more preferably at least twenty-five structurally different compounds. Typically, the compounds of a combinatorial library have an invariant portion, referred to as the"scaffolding"and a variable portion which differs in each member of the combinatorial library.

The compounds of a combinatorial library are prepared by carrying out a plurality of reactions of the same type using different reagents. Typically, these reactions are performed in parallel, i. e., simultaneously in or close temporal proximity. Reactions that are performed in parallel to prepare a combinatorial library are referred to as a"parallel array synthesis". Because they are typically insoluble in reaction solvents, ascorbic acid resins can be easily separated from the small scale reactions generally

used in parallel array syntheses. Thus, ascorbic acid resins are particularly suited for removing oxidizing agents from the reaction in a parallel array syntheses.

Ascorbic acid resins can also be used to prevent oxidation of compounds by storing the compounds in the presence of ascorbic acid resins. For example, ascorbic acid resins can be added to a solution being used to store a compound. The resin prevents oxidation of the compound by reacting with oxidizing agents in the solvent. The resin can be filtered and oxidizing agent residues removed, as described above, when the compound is ready for use.

Alternatively, the compound and ascorbic acid resin can be added to the same vessel or vial when the compound is being stored neat. The resin prevents oxidation of the compound by reacting with oxidizing agents present in the vessel or vial. When the compound is ready for use, it can be dissolved in a suitable solvent. The resin can then be filtered and the oxidizing agent residues removed, as described above.

The invention is illustrated by the following examples, which are not intended to be limiting in any way.

Example 1-Preparation of Polystyrene Carbonyl Chloride Under an N2 atmosphere, a 500 mL three-necked round bottomed flask equipped with an overhead stirrer and addition funnel was charged with carboxylated polystyrene resin (10 grams, 2.77 mmol CO2H/gram resin), anhydrous CH2Cl2 (120 mL), and anhydrous dimethylaminoformamide (DMF) (2 mL).

Next, oxalyl chloride (10 mL, 114 mmol) was added via a slow dropwise addition from an addition funnel. After stirring overnight under N2, the solvent was removed under vacuum using a gas dispersion tube. The resin was subsequently washed with anhydrous CH2Cl2 (3 x 100 mL). After the last wash, the resin was dried under vacuum for 2-3 hours. This resin was carried on directly into subsequent reactions; chloride determination on an aliquot showed a loading of 2.5 mmol/gram, which corresponded to 100% conversion to the acid chloride.

Example 2-Preparation of L-Ascorbic Acid Immobilized Polystyrene Polystyrene carbonyl chloride resin (10 grams, 2.5 mmol/gram), prepared as described in Example 1, was suspended in anhydrous CH2Cl2 and DMF (3: 2,100 mL). L- ascorbic acid (14 grams, 80 mmol), dimethylaminopyridine (DMAP) (1.46 grams, 12 mmol) and pyridine (9.7 mL, 120 mmol) were added to the suspension. The reaction was allowed to proceed overnight and the resin was washed with anhydrous CH2Cl2 (3 x 100 mL), DMF (3 x 100 mL) and finally methanol (2 x 100 mL). The resin was dried under vacuum. The loading was determined by the weight gain (2.2 mmol/gram).

Example 3-Removal of I, From Reaction Used to Prepare Oxytocin BisAcm-oxytocin, with the sequence H-Cys (Acm)-Tyr-Ile- Gln-Asn-Cys (Acm)-Pro-Leu-Gly-NH2 (SEQ ID NO.: 1) was prepared with a Rainin Synthesizer on RINK-amide resin using Fmoc-chemistry. The two cysteine thiols were protected with acetoamidomethyl groups (Acm). The linear peptide was removed from the resin in a solution containing 94% trifluoroacetic acid, 2% anisole, 2% triisopropylsilane and 2% water (four hours, room temperature).

The linear polypeptide was precipitated with diethyl ether and purified on a preparative C18 reversed phase HPLC column (250 x 22 mm) with a linear gradient of H20 containing 0.045% trifluoroacetic acid, and 60% acetonitrile in H2O containing 0.039% trifluoroacetic acid at a flow rate of 10 mL/minute, followed by lyophilization of the major fractions. The purified product was analyzed by reversed phase HPLC on a Hewlett Packard 1000 Series Instrument.

Analytical HPLC was performed with a 6%/minute linear gradient of buffer B in buffer A (A = 0.045% trifluoroacetic acid in water; B = 60% acetonitrile/40% water/0.039% trifluoroacetic acid) over fifteen minutes at a flow rate of 1 mL/minute using a Vydac C18 5pm, (0.46 cm x 25 cm) column.

Electron spray mass spectrometry (LC ESMS) was acquired on a Hewlett Packard 1100 MSD Instrument. Found: [M + Ho+ = 1151; calc. [M + H] + = 1151.

The linear polypeptide was converted to a cyclic polypeptide by Acm deprotection from the two protected cysteines of the purified linear peptide and intermolecular disulfide bond formation from the resulting liberated thiols. Acm deprotection and disulfide bond formation was accomplished by reacting the purified linear peptide with an

excess of iodine in 10% acetic acid in H20 at room temperature for two hours. To remove excess I2 from the reaction mixture, ascorbic acid resin, prepared as described in Examples 1 and 2, was added directly to the reaction mixture. After 2 hours, the resin was removed by filtration.

The formation of the cyclic product was confirmed by LC ESMS. Found: [M + H] 1007; calc [M + H] + = 1007.

EOUIVALENTS Those skilled in the art will be able to recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.