HPLC Separation of Geometric Isomers of Carbamyl Peptides

Abstract

Carbamylated peptides that were studied showed much improved resolution on C-18 reversed phase HPLC columns compared to the parent peptides. A number of dipeptides were carbamylated with ethyl-, n-propyl- or isopropyl isocyanates. The three carbamyl derivatives of each dipeptide could easily be resolved. Carbamylated dipeptides with reversed amino acid sequences were also easily separated. Methionine-enkephalin, leucine-enkephalin, Angiotensins I, II and III and substance P were carbamylated with isocyanates derived from certain carcinostatic 2-chloroethyl nitrosoureas. 1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), 1-(2-chloroethyl)-3-(cis-4-hydroxycyclohexyl)-1-nitrosourea (cis-4-hydroxy-CCNU) and 1-(2-chloroethyl)-3-(trans-4-hydroxycyclohexy1)-1-nitroso urea (trans-4-hydroxy-CCNU) gave carbamylated derivatives of each peptide and each mixture of derivatives from a single parent peptide could be resolved. Conditions were found in each case whereby baseline resolution of the corresponding cis-4- and trans-4-hydroxycyclohexylcarbamyl peptides was attained. Cyclohexyl-carbamyl peptides were easily separated from the corresponding peptides from hydroxy-CCNUs. Potential applications are discussed. carbamylation in the expression of antitumor activity or toxicity to normal tissues is still not very clear. A number of DNA polymerases (10, 11), DNA ligase (12), glutathione reductase (13), Serine proteases (14, 15) and tubulin polymerization (16) have been shown to be inhibited by carbamylation by nitrosoureas or isocyanates.

The question of whether carbamylation by CENUs and their metabolites is selective to certain enzymes is an important one because nitrosoureas such as CCNU and Methyl-CCNU are converted to antitumor-active metabolites (8, 17, 18). If it is found that the individual metabolites target different proteins, it could have important applications in chemotherapy and drug design.

In order to determine whether such selectivity of carbamylation exists, it was required that analytical methodology be developed that would permit separation of peptides with the same sequence but with different carbamyl groups. These studies offer hope that if peptides with up to 11 aminoacids are carbamylated by CCNU or the 4-hydroxy-CCNUs, one may be able to separate them by HPLC. This limit may be extended if one need only separate cyclohexyl carbamyl peptides from mixtures of geometric and positional isomers of hydroxycyclohexyl carbamyl peptides as might occur in the cell.