FUNCTIONALIZED SEMITELECHELIC POLY[N-(2-HYDROXYPROPYL)METHACRYLAMIDE]FOR PROTEIN MODIFICATION

Semitelechelic poly[N-(2-hydroxypropyl)methacrylamide]s (ST-PHPMA) wit h different functional end groups, namely carboxyl, methyl ester, hydr azide, and amino groups, were prepared by chain transfer free-radical polymerization. 2,2'-Azobisisobutyronitrile (AIBN) was used as an init iator and 3-mercaptopropionic acid, methyl 3-mercaptopropionate, 3-mer captopropionic hydrazide, and 2-mercaptoethylamine were used as chain- transfer agents. The semitelechelic polymers have been characterized b y end-group analysis, size-exclusion chromatography (SEC), and matrix- assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). The effects of the concentrations of the mercaptans a nd the initiator on the molecular weight of the polymers have been inv estigated. The higher the concentration of mercaptan, the lower the mo lecular weight of ST-PHPMA. The concentration of initiator did not hav e a significant effect on the molecular weight of the semitelechelic p olymers. The end groups of the ST polymers can be readily transformed by polymeranalogous reactions. A model protein, a-chymotrypsin, has be en modified with ST-PHPMA-CONHNH2 and ST-PHPMA-COOSu and the conjugate s characterized by MALDI-TOF MS. The activity of modified chymotrypsin s toward a high molecular weight substrate, P-Gly-Leu-Phe-NAp (where P is the HPMA copolymer backbone, and NAp is p-nitroanilide), was sligh tly lower than the activity of the native enzyme. The cleavage of a lo w molecular weight substrate, Z-Gly-Leu-Phe-NAp, by modified chymotryp sins was dependent on their structure. Whereas the activity of the ami no group modified chymotrypsins was higher than that of the native enz yme, the activity of carboxyl-modified chymotrypsins was lower than th at of the native enzyme. In summary, the data seem to indicate that ST -PHPMA is an effective protein-modifying agent.