Stepwise chain assembly peptide synthesis

In a general sense exopeptidases should be the enzymes of choice for stepwise chain assembly since once formed the internal peptide bonds of the growing chain can no longer be proteolytically cleaved from this type of peptidase. Carbox-ypeptidase exhibit superior properties for the stepwise synthesis, especially, carbox-ypeptidase Y (CPD-Y)12101 or other serine peptidases of this type. In principle, aminopeptidases can also be used starting from the C-terminus. Because under these conditions not only the carboxyl component but also the amine component has a free a-amino function, product isolation is more difficult, particularly, if one component is used in excess. Otherwise, stepwise synthesis from the C-terminus is not a problem in chemical peptide synthesis. 

HBTU activation has been adapted for automated stepwise solid-phase peptide synthesis for both f-butoxycarbonyl (Boc) and fluorenylmethoxycarbonyl (Fmoc) strategies. For the former, a simple, effective protocol has been developed, which involves simultaneous in situ neutralization with coupling (extra equivalents of base are required). This protocol is particularly suitable for assembling complex peptides, arising from sequence-dependent peptide chain aggregation. Since aggregation occurs when protonated a-ammonium peptide-resin intermediates are neutralized, simultaneous neutralization and acylation can help to overcome this phenomenon. For Fmoc chemistry, efficient protocols are also available for both batch and continuous-flow systems. ... 

The polymer-bound p-nitrobenzophenone oxime (71d) has been found to be a suitable support for stepwise peptide synthesis. Protected peptides can be assembled on 70d by coupling and deprotection steps similar to those employed in the usual Merrifield solid-phase procedures (Scheme 39). Cleavage of peptides from 71d can be accomplished with hydrazine and amino acid esters under mild conditions, which do not affect benzyl ester side-chain protecting groups. 

The basic success of an automated biopolmyer synthesis depends on the chemistry used for chain assembly. A well-understood and well-characterized chemical process has the best chance for success on an automated instrument. Fortunately, many of the reactions used in peptide and oligonucleotide synthesis are well characterized and approach 100% stepwise yields with minimal side reactions. Advances in this field are continually resulting in higher quality products. 

It was left to the ingenuity of R. Bruce Merrifield, an organic chemist at the Rockefeller University, to develop an innovative solution to this problem that totally revolutionized the field of peptide synthesis. His plan was to assemble the peptide chain in a stepwise manner by adding new amino acids at the A terminus while the Oterminal end was attached to a solid polymeric support of chloromethylated polystyrene, which is now referred to as the Merrifield. In this fashion, all of the excess reagents, impurities, and by-products could be easily removed by washing the resin after each operation, and the pure polypeptide could be cleaved from the solid support as the last step in the synthesis. Merrifield was awarded the Nobel Prize in Chemistry in 1984 in recognition of his contributions to the invention and development of the solid-phase method for the synthesis of peptides. 

The peptide was then separated from the by-products and unreacted starting material and the process was repeated. Merrifield succeeded in assembling a peptide chain in a stepwise manner while it was attached at one end to a solid support that could easily be removed by the proper solvents. It soon became apparent to Merrifield that the solid phase technique should be applicable to units other than amino acids. He extended it to the synthesis of... 

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