CA1312991C - Solid supports for peptide synthesis - Google Patents

Abstract

SOLID SUPPORTS FOR PEPTIDE SYNTHESIS
Abstract of the Disclosure Solid supports of the formula P-C6H4-CH(R1)-NHCOCH2Q-C6H4-COCH(R2)-X

wherein P and the 1,4-phenylene (C6H4) to which it is attached is a copoly(styrene-divinylbenzene) matrix, Q is oxo or thio, R1 is methyl, phenyl, 4-methylphenyl or 4-methoxyphenyl, R2 is hydro-gen, lower alkyl, phenylalkylene or phenyl and X is chloro, bromo or iodo are disclosed. They can be prepared by coupling an amine-containing copoly-(styrene-divinylbenzene) resin, for example, benz-hydrylamine resin, with a 4-(.alpha.-haloalkanoyl)phenoxy-acetic acid or a 4-(.alpha.-haloalkanoyl)phenylthioacetic acid. The solid supports are useful for solid phase peptide synthesis.

Description

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Field of the Invention This invention relates to solid supports useful for solid phase peptide synthesis (SPPS), to the use o~ phenoxyacetic acid and phenylthioacetic acid derivatives to provide a "spacer" or "linking group"
function to the solid supports and to the use of the solid supports in SPPS.

Ba~ckground of the Invention During the last 25 years, SPPS has been developed into an invaluable tool for the prepara-tion of peptides. One aspect of the technology which can create a problem, however, is the cleavage of the peptide from the solid phase. Most often, such cleavages are done by acids with strongly pro-tonating properties such as hydrofluoric acid and hydrobromic acid. The use of these strong acids, however, does not always give an acceptable or desired result. They can give rise to undesirable side reactions such as side chain alkylation by carbocations generated from the alcohol component of ~. .

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protective groups, or dehydration of the protonated side chains of aspartyl and glutaryl residues fol-lowed by acylation reactions of the resulting acylium ions. Moreover, the use of the strong acids can be contraindicated because the chemist employing a combination of solid phase and fragment condensa-tion techniques for peptide synthesis may wish to cleave only the peptide from the solid phase and retain protective groups on the peptide fragment.
The quest for alternative methods for cleaving peptide-resins has resulted in the development of several photolabile solid supports for SPPS. D.H.
Rich and S.K. Gurwara, J. Amer. Chem. Soc., 97, 1575 (1975) report the use of 4-(bromomethyl)-3^
nitrobenzoic acid to form a photolabile linking group between a polystyrene-base resin and the pep-tide being generated. Other photolabile linking groups for resins reported include an c~-methylphen-acyl ester linkage, S.S. Wang9 J. Org. Chem., 41, 3258(1976), and a 4-(2-oxypropionyl)phenylacetamido-methyl linkage, F.S. Tjoeng and G.A. Heavner, J.
Org. Chem., 48, 355 (1983). In Canadian patent 1,108,348, issued September 1, 1981, Rich and Gurwara have extended their investigations to in-clude the use of 4-((t- butyloxycarbonyl)amino-methyl)-3-nitrobenzoic acid to form a photolabile linking group.

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The present application discloses new photo-labile solid supports for preparing peptides of high quality. Their stability under the conditions used to elaborate the peptide by stepwise coupling of 5aminoacids or fragments, and the facility with which the completed peptide ^an be cleaved from the resin, render the solid supports as useful and advantageous support systems for SPPS.

Summary_of the Invention 15The photolabile solid supports of this inven-tion are represented by formula 1 wherein Pg together with the 1,4-phenylene (C6-H4) to which it is attaohed9 represents a copoly-(styrene-divinylbenzene) matrix, Q is oxo or thio, R1 is methyl, phenyl, 4-methylphenyl or 4-methoxy-: : ~
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phenyl, R2 is hydrogen, lower alkyl, phenylalkyle-ne or phenyl, and X is chloro, bromo or iodo.

A preferred group of photolabile solid supports is represented by formula 1 wherein P, Q and R1 are as defined hereinabove, R2 is lower alkyl and X is chloro or bromo.

A more preferred group of solid supports is represented by formula 1 wherein P is as defined hereinabove, Q is oxo, R1 is phenyl 3 R2 iS lower alkyl and X is chloro or bromo.

A most preferred solid support is the compound of formula 1 wherein P is as defined hereinabove, R1 is phenyl, R2 is methyl and X is chloro or bromo.

Also included within the scope o~ this inven-tion is the use of organic acids of formula 2 H00CCH2Q-C6H4-cOcH(R2)-x 2 ;, . . . . .

wherein Q, R2 and X are as defined hereinabove for use in the production of a linking unit, i.e.
-COCH2Q-C6H4-COCH(R2)-, which is an integral portion of the photolabile solid supports of formula 1.

The photolabile solid support of formula 1 can be obtained by a process comprising coupling an organic acid of formula 2 wherein Q, R2 and X are as defined hereinabove with a resin of formula 3 P-c6H4-cH(Rl)-NH2 3 wherein P and R1 are as defined hereinabove.

Details of~ the Invention For convenience, the "photolabile solid 9Up-ports" of this application are hereinafter desig-nated simply as "solid supports".

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The symbol "C6H~ represents a divalent 1,4-phenylene radical.

The symbol P, together with the phenylene to which it is attached, represents the radical derived by removal of a proton from the para position of a phenyl group of a phenyl group-containing solid polymeric support of the type used in SPPS. In keeping with the practice of the art, the symbol is of a general form and with the phenylene represents the matrix or polymeric backbone of the phenyl-containing resin support in which at least a portion of the phenyl groups, usually rang.ng from 1 to 20%, are covalently bound to the remaining portion shown for the general formula 1, namely the portion CH-(Rl)-NHcocH2Q-c6H4-cocH(R2)-x. In this connection, a preferred polymeric support, from which the radical can be derived, is a polystyrene-divinylbenzene copolymer in which the divinylbenzene is cross-linked to the extent of 0.5 to 2% by weight of the copolymer, particularly the commercially available copoly(styrene-1% divinylbenzene) resin and copoly(styrene-2% divinylbenzene) resin.

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- The term "lower alkyl" as used herein means alkyl radicals containing one to three carbon atoms and includes methyl, ethyl, propyl and l-methyl-ethyl.

The term "phenylalkylene" as used herein means radicals having an alkylene radical containing one to three carbon atoms joined to a phenyl group and includes benzyl, 2-phenylethyl, 3-phenylpropyl, 1-methyl-2-phenylethyl and 2-phenylpropyl.

The term "residue" with reference to an amino acid means a radical derived from the corresponding ~-amino acid by eliminating the hydroxyl of the carboxyl group and one hydrogen of the ~-amino group.

In general, the abbreviations used herein ~or designating the amino acids and the protective groups are based on recommendations o~ the IUPAC-IUB
Commission on Biochemical Nomenclature, see Bio-chemistry, 11, 1726-1732 (1972). For instance, Pro, Gln, Ala, Gly, Ile, Arg, Asp, Phe, Serj Leu, Cys and Tyr represent the "residues" of L-proline; L-glu-tamine, L-alanine, glycine, L-isoleucine, L-arg-inine, L-aspartic acid, L-phenylalanine, L-serine, L-leucine, L-cysteine and L-tyroslne, respectively.

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The starting materials for the process, namely the organic acids of formula 2 and the resins of formula 3, are known. For example, the organic acids, phenoxyacetic acid derivatives and phenyl-thioacetic acid derivatives, are described by C.R.
Hughes et al., Canadian patent 1,068,695, issued December 25, 1979. The resins o~ formula 3 wherein R1 is methyl, phenyl, 4-methylphenyl or 4-methoxy-phenyl have been described often; for example, see J.M. Stewart and G.R. Matsueda, U.S. patent 3,954,709 issued May 4, 1976, and G.R. Matsueda and J.M. Stewart, Peptides, 2, 45 (1981).

With reference to the process, an organic acid of formula 2 is coupled with a resin of formula 3 to give the corresponding solid support of ~ormula 1.
Coupling can be achieved readily by activating the earboxy group of the organic aeid. Examples of the aetivated form o~ the carboxy group are the aeid ehloride~ anhydride, azide, aetivated ester, or 0-aeyIurea of a dialkylearbodiimide. Deseription of these aetivated forms can be found in general text-books on peptide chemistry. Thereafter, the organic aeid with its aetivated earboxy group is reaeted with a resin of formula 3 to yield the eorresponding solid support of formula 1.

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A practical and convenient manner for achieving the coupling comprises firstly reacting the organic acid of formula 2 with one to two molar equivalents of N,N'-dicyclohexylcarbodiimide in an inert sol-vent, for example, methylene dichloride or dimethyl formamide, at O to 50 C for 30 minutes to two hours; secondly, isolating the resulting activated form of the organic acid (i.e. the O-acylurea acti-vated intermediate); and finally, reacting the lat-ter with about an equimolar amount (with respect to the amine content) of the resin of formula 3 in an inert solvent, ~or instance methylene dichloride, dimethylformamide, or a mixture thereof, at O to 50 C, usually from about three hours to one or two days. Thereafter, the desired solid support is collected from the reaction mixture by filtration, washed and dried.

Other details The solid supports of formula 1 have been used successfully for the preparation of a variety of peptides; for example, see Examples 2 and 3 herein.

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When the solid supports are employed for this purpose, the standard methods of solid phase peptide synthesis are used. A leading reference on these methods is G. Barany and R.B. Merrifield in "The Peptides: Analysis, Synthesis, Biology", E. Gross and J. Meienhofer, Eds., Academic Press, New York, NY, 1980, Vol. 2, Chapter 1. Accordingly, the solid support is coupled with the initial synthetic unit which is a N~ (and side chain, if appropriate) protected amino acid corresponding to the C-terminal amino acid of the desired peptide. This latter coupling can be done according to the potassium fluoride method, K. Horiki et al., Chem. Lett., 165 (1978), or by the cesium salt method, B.F.
Gisin, Helv. Chim. Acta, 56, 1476 tl973). There-after, the appropriate N~ (and side chain, if appropriate) protected amino acids are coupled step-wise to form the peptide-solid support wherein the peptide portion thereof has the desired sequence of amino acid residues.

Subsequent cleavage of the peptide or protected peptide from the solid support can be effectively and efficiently done by photolysis. The photolytic cleavage of the peptide-solid support of this invention is accomplished readily by dissolving or suspending the peptide-solid support in a solvent : . .. . .
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which provides a suitable media for photolysis reactions. Examples of such photolytically stable solvents include dioxane, dimethylformide, the lower alkanols such as methanol, ethanol or isopropanol, dimethylacetamide and N-methylpyrrolidone. A pre-ferred solvent is a mixture of dimethylformamide and ethanol (4:1, V/V).

The solution or suspension of the peptide-solid support is purged with an inert gas, for example, argon or nitrogen, to remove any dissolved oxygen.
Thereafter, the solution or suspension is irradiat-ed, preferably at a wavelength of 350 nm at a tem-perature ranging from -10 C to 25 C for 4 to 24 hours. In this manner, the peptide is cleaved from the solid support. If required, any side chain protective groups present on the cleaved peptide are removed thereafter by standard methods designed for that purpose.

Alternatively, the preceding cleavage can be accomplished by using an appropriate nucleophile;
for example, sodium hydroxide in aqueous dioxane or benzyltrimethylammonium hydroxide in methanol.

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~12-The following examples illustrate further this invention. Abbreviations used in the examples in-clude BOC: t-butyloxycarbonyl; TFA: trifluoroacetic acid; CH2C12: methylene chloride; DIEA: diiso-propylethylamine; DMF: dimethylformamide; EtOH:
ethanol; DCC: N,N'-dicyclohexylcarbodiimide. Solu-tion percentages are calculated on a volume/volume basis unless sta~ed otherwise. The following terms are trademarks: Pyrex and Rayonet.

Preparation of (4-(2-Chloropropionyl)phenoxy)-acetamidomethyl-copoly(styrene-1% divinybenzene) resin (Formula 1: P together with the 1,4-phenylene to which it is attached is a copoly(styrene-l~ divi-nylbenzene) matrix, Q is oxo, R1 is phenyl, R2 is methyl and X is chloro).
A suspension of benzhydrylamine resin (5.0 g, amine content = 0~42 mM/g) in CH2C12 (100 ml) was stirred at room temperature (20-22 C) for 30 minutes. The resin was collected on a ~ilter, wash-ed with 5% DIEA in CH2C12 and then CH2C12.
Thereafter, the washed resin was dried under vacuum.

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A solution of (4-(2-chloropropionyl)phenoxy)-acetic acid (2.28 g) and DCC (0.99 g) in CH2C12 (20 ml) was stirred at 0 C for one hour. The suspension was filtered and the filtrate was added a stirred suspension of the previously washed ben-zyhydrylamine resin in 20 ml of CH2C12. The total mixture was stirred at room temperature for 48 hours. Thereafter, the solid material in the mixture was collected on a filter, washed with C-H2C12 and dried under vacuum to give the title solid support (4.48 g, substitution: 0.568 mM/g);
negative Kaiser test, E. Kaiser et al., Anal. Bio-chem., 34, 595 (1970); ir (KBr): carbonyl absorption at 1650-1680 cm~l.
By following the procedure of example 1 but replacing the benzhydrylamine resin with 4-methyl-benzhydrylamine resin, the solid support of formula 1 in which P together with the 1,4-phenylene to which it is attached is a copoly(styrene-1% divinyl-benzene) matrix, Q is oxo, R1 is 4-methylphenyl, R2 is methyl and X is chloro is obtained.

Similarly, by~following the procedure of exam-ple 1 but replacing (4-(2-chloropropionyl)phenoxy)-acetic acid with (4-(2-bromopropropionyl)phenyl-thio)acetic acid, the solid support of formula 1 in ::

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which P together with the 1,4-phenylene to which it is attached is a copoly(styrene-1% divinylbenzene) matrix, Q is thio, R1 is phenyl, R2 is methyl and X is bromo is obtained.

Example 2 Preparation of(Leu5)enkephaline of Formula H-Tyr^Gly-Gly-Phe^Leu^OH

N-(t-Butyloxycarbonyl)-leucine was linked to the title solid support of Example 1 (substitution =
0.568 mM/g) by the potassium fluoride method of K. Horiki et al., Chem. Lett., 165 (1978), using 9 molar equivalents of KF and 3.6 molar equivalents of N~-BOC-leucine in DMF at 7G C for 24 hours, to give (4-(2-(N-(t-butyloxycarboxyl)^leucine3propio-nyl)phenoxy)acetamido-copoly(styrene-1% divinylben-zene) resin. The dried amino acid-solid support showed a leucine content of 0.399 mM/g for the pro^
duct, as determined by deprotection of an aliquot, followed by picric acid titration, B.F. Gisin, Anal. Chim. Acta, 58, 248 (1972). The latter amino acid^solid support was used to build up the amino acid sequence of the desired peptide by the solid ~ .

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phase technique of R.B. Merrifield, J. Amer. Chem.
Soc., 85, 2149 (1963). N-BOC amino acids were used; the hydroxyl of the side chain of the tyrosine unit being protected with a benzyl protective group (Bzl). BOC-deprotection was effected with TFA.
Coupling was effected with DCC (2 molar equi-valents). Cleavage of the completed protected pep-tide from the solid support was done by photolysis in the following manner:
A suspension of the protected peptide-solid support (5~25 g) in DMF: EtOH (4:1), contained in a Pyrex vessel, was purged with argon for one hour at room temperature. Utilizing a Rayonet RPR-206 pho-tochemical chamber (Southern New England Ultraviolet Co., Hamden, Connecticut, U.S.A.), the suspension was irradiated at 350 nm for 24 hours. The reaction mixture was filtered. The filtrate was concentrated to dryness under vacuum. The residual oil was dis-solved in a minimum of methanol. The solution was added dropwise to stirred diethyl ether (225 ml).
The flocculent precipitate was collected and washed with cold diethyl ether to yield BOC-Tyr(Bzl)-Gly-Gly-Phe-Leu-OH (1.26 g). Serial treatment of the latter product with TFA to remove the BOC group and hydrogenation (Pd/C) to remove the Bzl group gave the known title compound, J. Hughes et al., Nature, 258, 577 (1975).

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Example 3 Preparation of ~-Casomorphin of Formula H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH

N-(t-Butyloxycarbonyl)-isoleucine was linked to the title solid support of Example 1 in the same manner as described for N-(t-butyloxycarbonyl)-leucine in Example 2 to give (4-(2-(N-(t-butyloxy-carbonyl)-isoleucyl)propionyl)phenoxy)acetamido-copoly(styrene-1% divinylbenzene) resin (isoleucine content = 0.734 mM/g)~ The dried amino acid-solid support was used, according to the same method of Example 2, to generate the desired amino acid sequence on the solid support in a quantitative yield. The hydroxyl of the tyrosyl residue was pro-tected with a Bzl. In a similar manner to that described in Example 2, the peptide was cleaved quantitatively from the solid support by photolysis to yield BOC-Tyr(Bzl)-Pro-Phe-Pro-Gly-Pro-Ile-OH.
Serial treatment of the latter product with TF'A to remove the BOC group and hydrogenation (Pd/C) to remove the Bzl group gave the known title compound, V. Brantl et al., Hoppe-Seyler's Z. Physiol. Chem., 360, 1211 (1979), in a 70% yield.

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Claims (10)

1. A solid support of formula 1 P-C6H4-CH(R1)-NHCOCH2Q-C6H4-COCH(R2)-X 1 wherein P, together with the 1,4-phenylene to which it is attached, is a copoly(styrene-divinylbenzene) matrix, Q is oxo or thio, R1 is methyl, phenyl, 4-methylphenyl or 4-methoxyphenyl, R2 is hydrogen, lower alkyl, phenylalkylene or phenyl, and X is chloro, bromo or iodo.

2. A solid support of claim 1 wherein R2 is lower alkyl and X is chloro or bromo.

3. A solid support of claim 1 wherein Q is oxo, R1 is phenyl, R2 is lower alkyl and X is chloro or bromo.

4. A solid support of claim 3 wherein R2 is methyl and X is chloro or bromo.

5. A solid support of claim 3 wherein P, together with the 1,4-phenylene to which it is attached, is copoly(styrene-1% divinylbenzene) matrix.

6. A process for preparing a solid support of claim 1 which comprises coupling an organic acid of formula 2 HOOCCH2Q-C6H4-COCH(R2)-X 2 wherein Q, R2 and X are as defined in claim 1 with a resin of formula 3 P-C6H4-CH(R1)-NH2 3 wherein P and R1 are as defined in claim 1 to obtain the corresponding solid support.

7. A process of claim 6 wherein the coupling is achieved by activating the carboxy group of the organic acid, and reacting the organic acid with its activated carboxy group with the resin of formula 3.

8. A process of claim 6 wherein the activated carboxy group is an activated ester or O-acylurea of a dialkylcarbodiimide.

9. The use of an organic acid of formula HOOC-CH2Q-C6H4-COCH(R2)-X wherein Q, R2 and X
are as defined in claim 1 for the manufacture of a solid support of claim 1 for application in SPPS.

10. In a process for preparing a peptide by SPPS
wherein the solid phase is a solid support of claim 1, the step which comprises cleaving the peptide or protected peptide from the solid support by photo-lysis.