Merrifield SPPS gave a new dimension to the peptide synthesis and now the protein synthesis is presented to be a routine work, but the facts are always against these exaggerated claims. There comes various obstacles in the way of protein synthesis, which renders the product sufficiently below the expectations. The work presented here contains the synthesis of two proteins, human insulin and sperm whale myoglobin. The first aim of this work was the synthesis of A chain of human insulin and stabilizing the system for its synthesis with better results. The synthesis of this difficult sequence (which produces secondary structures and breaking of the chain during the reaction) was successfully obtained using the legendary “magic mixture” as carrier solvent. Another improvement in the synthesis was obtained using N,N Fmoc – Tmob-Gly-OH on the N-terminal of the peptide. The introduction of Tmob group has improved purity for the crude peptide. Due to these improvements in the chain this peptide was synthesized again to proceed further work of disulfide bond formation, using different protecting groups combination for the cystine. The synthesis of the disulfide bond occurs in a basic reaction media which as a consequence can lead to numerous side products, the improved solubility has also solved the problem of interference from these undesired products.
The B chain of human insulin was also synthesized using ‘magic mixture’. After synthesis of the chain, purified the peptide with preparative HPLC for the next steps of introducing the disulfide bonds between the two chains. For the synthesis of the inter chain bond the free sulphur of cystine group was first activated with the 2,2’-Dithiodipyridine as activating agent, and then the activated chain was reacted with the A-chain to obtain the required hetrodimer. This product can be identified and characterized using Mass spectrometry and HPLC.
The sperm whale myoglobin was synthesized once with a regular interval of capping and once without capping and then was tried to purify upto best possible purity. Though it was indicated from the mass spectrum that there is any synthesis of the required product, to the crude product was added hemin group and the UV spectrum was recorded, the shift of the UV spectrum (407 nm as against 406 for natural apomyoglobin) with the addition of hemin to crude apomyoglobin indicates a successful synthesis of the myoglobin.
The next purpose of the work obtained was the observation of recemization in different conditions and then to find ways to control or minimize these recemization. As earlier discussed the basic environment is more responsible of the recemizaiton, specially in the activation process, the use of basic catalyst like TBTU increase the racemization, nearly by 20%. To overcome this negative factor specially in the case of very much racemization sensitive Cystine, Cys-o-Pfp active ester was used and the TBTU was totally removed in this specific cycle of activation.
Another effect over racemization is the solvent effect, like the activation reagent the basicity of the solvent also affect the degree of recemization. In the ‘magic mixture’ the presence of basic solvent NMP was also inviting the racemization , to minimize its effect the ratio of NMP was reduced (DMF/DCM/NMP) from 1:1:1 to 3:3:1.
In this work the synthesis of the Insulin A,B and sperm whale myoglobin was successfully performed. The hetrodimer disulfide bond was synthesized and the effect of various reagents on the final product with special reference to recemization were studied and the conditions set to remove or minimize these drawbacks.