Betides and methods for screening peptides using same

Claim 1

A betide of the formula: X N --X 1 --X 2 --X 3 --X m --X 4 --X 5 --X 6 --X C , where X N is an acyl or other N-terminal group or a peptide up to about 50 amino acids in length having such an N-terminal group; X C is OH, NH 2 or other C-terminal group or a peptide up to about 50 amino acids in length having such a C-terminal group; X m is either des-X or a peptide up to about 50 amino acids in length, and X 1 -X 6 are each independently des-X, a betide amino acid, a natural α-amino acid or an unnatural α-amino acid, provided however that at least one of X 1 to X 6 is a residue of a betide amino acid of the formula: ##STR13## and that at least another of X 1 to X 6 is either a residue of a different betide amino acid of the formula: ##STR14## or an α-amino acid, wherein R o is H, R is H and R 2 is H or methyl, and R 3 is an acyl group with R 2 and R 3 being chosen so that said betide amino acid is selected from the group consisting of b-Ala, b-hAla, b-Val, b-Leu, b-Ile, b-Lys, b-Arg, b-His, b-Pro, b-hSer, b-Thr, b-Phe, b-Tyr, b-Trp, b-Asp, b-hGlu, b-Asn, b-Gln, b-hCys and b-hMet; and provided further however that additional residues of betide amino acids can optionally be included in X n , X m and X c whereby said betide closely resembles a peptide having the corresponding natural amino acid instead of said betide amino acid in the respective position.

Claim 9

A method for screening for a desired property in a peptide having at least one residue with C.sup.β alkylation, which method comprises (A) making a betide by a chain elongation protocol by a) providing a resin or a peptide intermediate having an amino acid residue with a free α-amino group at the N-terminus thereof, b) providing an unnatural α-amino acid having the formula: ##STR18## wherein R o is H or CH 3 , R is H or lower alkyl, R l is a labile α-amino-protecting group, and R 5 is a labile amino-protecting group, R 1 and R 5 being respectively removable under conditions that do not cause the removal of the other; c) coupling said unnatural amino acid either to said resin or to said N-terminus of said intermediate; d) removing R 1 and coupling at least one α-amino protected amino acid or peptide thereto; e) removing R 5 from said intermediate and carrying out an alkylation reaction at said β-site prior to any other reaction at said site; and f) following the completion of said alkylation reaction, providing a reactant selected from the group consisting of carboxylic acids, active esters or anhydrides, acyl halides, isocyanates, isothiocyanates and sulfonyl chlorides and causing an addition reaction to occur at said alkylated site of removal of R 5 to create a bond that cannot be selectively cleaved without cleaving peptide bonds, and (B) testing said betide for said desired property in a biological assay so as to obtain a result which is indicative of the same property of said peptide having such a residue with C.sup.β alkylation.

Claim 10

A method for screening for a desired property in a peptide having at least one residue with C.sup.β alkylation, which method comprises (A) making a betide by a chain elongation protocol by a) providing a resin or a peptide intermediate having an amino acid residue with a free α-amino group at the N-terminus thereof, b) providing an unnatural α-amino acid having the formula: ##STR18## wherein R o is H or CH 3 , R is H or lower alkyl, R l is a labile α-amino-protecting group, and R 5 is a labile amino-protecting group, R 1 and R 5 being respectively removable under conditions that do not cause the removal of the other; c) coupling said unnatural amino acid either to said resin or to said N-terminus of said intermediate; d) removing R 1 and coupling at least one α-amino protected amino acid or peptide thereto; e) removing R 5 from said intermediate and carrying out an alkylation reaction at said β-site prior to any other reaction at said site; and f) following the completion of said alkylation reaction, providing a reactant selected from the group consisting of carboxylic acids, active esters or anhydrides, acyl halides, isocyanates, isothiocyanates and sulfonyl chlorides and causing an addition reaction to occur at said alkylated site of removal of R 5 to create a bond that cannot be selectively cleaved without cleaving peptide bonds, and (B) testing said betide for said desired property in a biological assay so as to obtain a result which is indicative of the same property of said peptide having such a residue with C.sup.β alkylation.

Claim 11

A method for making a desired betide X N --X 1 --X 2 --X 3 --X m --X 4 --X 5 --X 6 --X C , where X N is an acyl or other N-terminal group or a peptide up to about 50 amino acids in length having such an N-terminal group; X C is OH, NH 2 or other C-terminal group or a peptide up to about 50 amino acids in length having such a C-terminal group; X m is either des-X or a peptide up to about 50 amino acids; and X 1 -X 6 are each independently des-X, a betide amino acid, a natural α-amino acid or an unnatural α-amino acid, provided however that at least one of X 1 to X 6 is a residue of a betide amino acid of the formula: ##STR19## and that at least another of X 1 to X 6 is either a residue of an α-amino acid or a different betide amino acid of the formula: ##STR20## wherein R o is H or CH 3 , R and R 2 are independently H or substituted or unsubstituted lower alkyl, and R 3 is an acyl group, an isocyanate group, a thioisocyanate group or a sulfonyl group; and provided further however that additional residues of betide amino acids can optionally be included in X n , X m and X c , which method includes the steps of a) providing a peptide intermediate having an amino acid residue with a free α-amino group at the N-terminus thereof, b) providing an unnatural α-amino acid having the formula: ##STR21## wherein R 1 is a labile α-amino-protecting group, R is hydrogen or lower alkyl and R 5 is a labile amino-protecting group which can be removed under conditions that do not cause the removal of R 1 , c) coupling said unnatural amino acid to the N-terminus of said intermediate to extend the length thereof by one residue; d) removing R 5 without removing R 1 from said extended intermediate; e) alkylating the primary amino group at the site of removal of R 5 with dimethoxybenzhydryl chloride to create a secondary amino group, f) alkylating said secondary amino group by treating with an aldehyde and reducing the reaction product thereof to create a tertiary amino group, g) removing said dimethoxybenzhydryl moiety to recreate a secondary amino group, h) causing an addition reaction to occur at said recreated secondary amino group with a reactant selected from the group consisting of carboxylic acids, active esters or anhydrides, acyl halides, isocyanates, isothiocyanates and sulfonyl chlorides, and i) removing R 1 and optionally alkylating the primary amino group at the site of removal of R 1 and optionally coupling one or more amino acids or peptides thereto, whereby a peptide chain having the length of said desired alkylated betide is created.

Claim 12

A method for making a desired betide X N --X 1 --X 2 --X 3 --X m --X 4 --X 5 --X 6 --X C , where X N is an acyl or other N-terminal group or a peptide up to about 50 amino acids in length having such an N-terminal group; X C is OH, NH 2 or other C-terminal group or a peptide up to about 50 amino acids in length having such a C-terminal group; X m is either des-X or a peptide up to about 50 amino acids; and X 1 -X 6 are each independently des-X, a betide amino acid, a natural α-amino acid or an unnatural α-amino acid, provided however that at least one of X 1 to X 6 is a residue of a betide amino acid of the formula: ##STR19## and that at least another of X 1 to X 6 is either a residue of an α-amino acid or a different betide amino acid of the formula: ##STR20## wherein R o is H or CH 3 , R and R 2 are independently H or substituted or unsubstituted lower alkyl, and R 3 is an acyl group, an isocyanate group, a thioisocyanate group or a sulfonyl group; and provided further however that additional residues of betide amino acids can optionally be included in X n , X m and X c , which method includes the steps of a) providing a peptide intermediate having an amino acid residue with a free α-amino group at the N-terminus thereof, b) providing an unnatural α-amino acid having the formula: ##STR21## wherein R 1 is a labile α-amino-protecting group, R is hydrogen or lower alkyl and R 5 is a labile amino-protecting group which can be removed under conditions that do not cause the removal of R 1 , c) coupling said unnatural amino acid to the N-terminus of said intermediate to extend the length thereof by one residue; d) removing R 5 without removing R 1 from said extended intermediate; e) alkylating the primary amino group at the site of removal of R 5 with dimethoxybenzhydryl chloride to create a secondary amino group, f) alkylating said secondary amino group by treating with an aldehyde and reducing the reaction product thereof to create a tertiary amino group, g) removing said dimethoxybenzhydryl moiety to recreate a secondary amino group, h) causing an addition reaction to occur at said recreated secondary amino group with a reactant selected from the group consisting of carboxylic acids, active esters or anhydrides, acyl halides, isocyanates, isothiocyanates and sulfonyl chlorides, and i) removing R 1 and optionally alkylating the primary amino group at the site of removal of R 1 and optionally coupling one or more amino acids or peptides thereto, whereby a peptide chain having the length of said desired alkylated betide is created.