Synthesis of cooligopeptides and functionalized aliphatic polyesters by chemo-enzymatic synthesis

摘要

Introduction: There is a constant need for new biomaterials within the field of tissue engineering. Aliphatic polyesters have over a long time been of interest for biomedical applications because they are biodegradable and biocompatible. However, the lack of bioactivity can cause problems when it is sought for regenerative and medical applications. We have synthesized block and random cooligopeptides of lysine and alanine by chemo-enzymatic synthesis using Papain (Caracia papaya) as catalyst. The same method was used to design a bioactive copolymer, where an aliphatic polyester was functionalized with oligopeptides through chemo-enzymatic synthesis. This is a promising synthesis technique which has been used to prepare many different types of peptides. It is also cost effective, environmentally friendly and gives high purity peptides. The synthesis method and synthesized polyester shows interesting results in terms of being an effective synthesis route for bioactive materials and for being used in for example tissue engineering. Method: Random and block cooligopeptides of alanine and lysine ethyl ester were prepared by chemo-enzymatic synthesis and papain was used as catalyst. Furthermore the synthesis method was used to functionalize an aliphatic polyester by grafting on oligopeptides to the chain. The method has been described in our previous work. Briefly, amino acid ethyl esters were dissolved together with the catalyst in a phosphate buffer solution (PBS). The reaction was heated to 40°C and allowed to react for 30 min. The reaction was stopped by removing the enzyme from the solution, using centrifugation filters, and the product was purified by dialysis (MWCO of 500-100 g/mol). The aliphatic polyester was synthesized through ring opening polymerization using a yttrium salan complex (salanY(OiPr)) as catalyst. The cooligopeptides and functionalized polyester were analyzed by proton and carbon-13 nuclear magnetic resonance (1H-NMR, 13C-NMR) and by Size exclusion chromatography (SEC). The cytotoxicity was analyzed by a standard cell viability test. Results: Random and block cooligopeptides of alanine and lysine were successfully prepared by chemo-enzymatic synthesis. The NMR results showed full monomer conversion of alanine in both types of cooligopeptides and 47% and 68% conversion of lysine in the block and random cooligopeptide, respectively. The average molecular weight was approximately 600 g/mol for both structures. The cell viability test showed that the cooligopeptides exhibited low cell cytotoxicity. The block cooligopeptides have slightly lower cell viability compared to the random cooligopeptides which is thought to be because of the longer positively charged chains of lysine. The same synthesis method was used to prepare aliphatic polyesters functionalized with oligopeptides. They were successfully prepared and characterized by 1H-NMR, 13C-NMR and SEC. These results show that chemo-enzymatic synthesis can be used not only to prepare different types of oligopeptides but also for grafting peptides onto aliphatic polyesters. Cell viability test of the biomaterial show no cell cytotoxicity. Conclusions: Chemo-enzymatic synthesis can be used for synthesis of block and random cooligopeptides as well as peptide functionalized aliphatic polyester. The results confirms that chemo-enzymatic synthesis is a promising synthesis method not only for designing different peptides and but also for functionalization and bioactivation of polyesters. The cell viability test show no cell cytotoxicity which implies that the synthesized materials could be interesting for biomedical applications such as implants for tissue engineering.