This invention relates to reagents useful to derivatize C-terminal proline residues of peptides. More particularly, the invention relates to reagents which react with the mixed anhydride reaction product of a peptide having a C-terminal proline residue and acetic anhydride.

Background of the Invention The C-terminal sequencing of prolyl containing peptides has been difficult to achieve. For example, Hawke U.S. Patent 4,837,165 describes a C-terminal peptide sequencing procedure utilizing a trimethylsilyl reagent. The Hawke reagent requires the formation of an oxazolinone which has not been demonstrated to occur when a proline residue is at the C-terminus.

Summary of the Invention This invention provides reagents useful for the derivatization of prolyl containing peptides. The reagents of the invention comprise a class of compounds having a 3-amino-2-halo pyridine structure. Such compounds function as nucleophilic reagents with respect to acetic acid anhydride activated proline residues. Under appropriate conditions the amide nitrogen of the proline residue may displace the halogen of the reagent to provide a cleavable cyclic derivative. The reagents of the invention thus accommodate C-terminal sequencing of prolyl containing peptides.

Detailed Description of the Invention The reagents useful in the invention have the structural formula I

in which x is a halogen, preferably chlorine or fluorine and y, if present, is an alkyl group, preferably having from about one to about six carbon atoms or an oxide (O-) .

A peptide having a terminal proline residue is reacted with acetic anhydride in known manner (see, e.g. , co-pending U.S. Patent Application Serial No. 07/311,966) to provide a linear mixed anhydride which has one component of the generic formula II

O 0 0

I! II II

CH ₃ -C-NH-Z-pro-C-0-C-CH ₃ in which Z is one or more amino acid residues and pro is a proline residue.

The linear mixed anhydride is then reacted with a reagent of the invention to yield a derivative of the formula II component as illustrated by the following equation:

(conditions) N V

Cyclization by displacement of the halo (X) substituent of the reagent by the amide nitrogen of proline yields a cleavable cyclic derivative having the

which hydroly.zes to a shortened peptide

0 O

// II CH3-C-NH-Z-C-OH

and a proline derivative

Cyclization may be facilitated by the use of 3 amino-2-fluoropyridine to provide a fluorine atom as a leaving group. Alternatively, the ring nitrogen of the pyridine moiety of the peptide addition product may be methylated, e.g., by reaction with methyl iodide to enhance the ability of the halo substituent of the reagent to function as a leaving group.

Exemplification of the Invention The peptide Ala-phe-pro was reacted with acetic anhydride to produce a linear mixed anhydride under the conditions indicated by the following equation II

NH ₂ -Ala-Phe-pro-C-OH Acetic Anhydride

Temperature 50° Solvent - CH3CN 30'

O 0 0

II II II

CH ₃ C - ala - phe - pro-C-O-C-CHβ

The reaction mixture components illustrated by equation II was reacted with 3-amino-2 chloro pyridine as indicated by Equation IIIA and IIIB

0 0

II II

IIIA. CH3 - C - NH - Ala - Phe - Pho - C - OH

No reaction

0 0 0

// 11 11

IIIB. CH ₃ - C - NH - ala-phe-pro-C-o-C-CH ₃

0 0

1/ II

CH ₃ C - NH Ala - Phe - Pro - C - NH

- f)

Cyclization did not occur in the reaction specifically described by Equation IIIB. Displacement of the chlorine by nucleophilic attack through the proline amide nitrogen on the pyridyl N-methylated reagent yielded species consistent with the proposed structures based on FAB mass spectrometry.

Cleavage, for example, by reaction with aqueous base 5% triethylamine (aq) yields the shortened peptide

O O li II

CH ₃ - C - NH - ala - phe - C - OH

and a proline derivative

which may further hydrolyze to yield additional products: