The lysosomal degradability of poly(alpha-amino acids) based on poly(L-glutamic acid) and its derivatives/copolymers was evaluated to gain insight into the subcellular fate of the macromolecules as water soluble polymeric drug carriers. The results indicate that both the incorporation of hydrophobic comonomers and modification of the carboxylic groups of glutamic acid side chains with hydroxyalkylamine increase the lysosomal degradability of the copolymers. Decreased lysosomal degradability of L-glutamic acid copolymers containing tripeptides terminated in p-nitroanilide (drug model) in the side chains confirmed that drug conjugation alters the degradation pattern of the polymeric carriers. The percentages of the enzymatic release of p-nitroaniline from its polymeric complex with time is relatively independent of the contents of the tripeptidyl p-nitroanilides attached to the polymeric conjugates. Determination of the degradation products by electrospray mass spectroscopy showed that no fragments less than 10(3) D were generated by lysosomal enzymes, whereas the main degradation products by papain and chymotrypsin were tripeptides and tetrapeptides. The conclusions derived from these data strongly suggest that these macromolecules, if used as lysosomotropic drug carriers, may accumulate in the lysosomes and limit their usefulness in some applications.

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SynopsisThe random copolymerization of the N-carboxyanhydrides of y-benzyl-L-glutamate and L-valine using triethylamine as the initiator in low dielectric media results in high-molecular-weight copolymers at low conversion. This behavior makes it possible to apply the monomer reactivity ratio theory, which was derived for addition polymerizations, and from the use of the copolymer composition equation, the respective monomer reactivity ratios, the average and incremental copolymer compositions, and the monomer feed ratio at any conversion can be determined. A comparison of the reactivity ratios for the copolymerization of y-benzyl-L-glutamate NCA and L-valine NCA in benzene/methylene chloride ( r~ = 2.1, rv = 0.6) with those obtained using dioxane (rc = 2.7, rv = 0.3) indicates that the interchain compositional heterogeneity is greater for copolymers prepared in the dioxane. For example, a t 1009b conversion of the monomeric NCAs, poly[Glu(OBzl)wVa150] prepared in dioxane has an interchain composition ranging from 74 to 0 mol % y-benzyl-L-glutamate, whereas in benzene/methylene chloride the interchain composition of y-benzyl-i-glutamate ranges from 65 to 0 mol %. Once the reactivity ratios are obtained for any pair of a-amino acid N-carboxyanhydrides, the use of the aforementioned parameters relating to interchain composition can give insight into the compositional heterogeneity between chains as a function of conversion and provide a basis for the preparation of random a-amino acid copolymers that are homogeneous.

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The acid catalyzed O → C migration of the benzyl group in the side chain of tyrosine could be reduced by applying HBr in a mixture of phenol and p‐cresol instead of HBr in trifluoroacetic acid for acidolytic deprotection. This side reaction occurs also during the removal of Boc groups. The loss of O‐benzyl protection and the formation of 3‐benzyltyrosine residues could be suppressed by the application of a 7:3 mixture of trifluoroacetic acid and acetic acid. The acid‐ and base‐catalyzed ring closure of β‐benzylaspartyl residues to aminosuccinyl derivatives was also studied. In this case HBr in trifluoroacetic acid was found to be relatively harmless. Deprotection with HBr in a mixture of trifluoroacetic acid and p‐cresol can be applied for peptides that contain both β‐benzylaspartyl and O‐benzyltyrosyl residues. An attempt to reduce the rate of the base‐catalyzed side reaction by application of hindered tertiary amines was abandoned because the tertiary amines which were effective in this respect led to significant reduction of the rate of the desired reaction, the aminolysis of active esters, as well. A satisfactory solution for the problem was found in the selective catalysis of the active ester reaction with 1‐hydroxybenzotriazole o 4‐dimethylaminopyridine. These catalysts do not enhance the rate of ring closure and in their presence essentially pure β‐benzylaspartyl peptides can be produced in good yield.