Protein solid-phase synthesis

Beausoleil, E., Truong, K.T., Kirshenbaum, K., and Zuckermann, R.N. Influence of monomer structural elements in hydrophilic peptoids. In Innovations and Perspectives in Solid Phase Synthesis and Combinatorial Libraries Peptides, Proteins, and Nucleic Acids, R. Epton (Ed.), 2001, Mayflower Scientific Press Kingswin-ford, UK, pp. 239-242. 

Atherton E, Logan CJ, Sheppard RC, Peptide synthesis, part 2 Procedures for solid phase synthesis using W-lluorcnylmcthoxycarbon-ylamino acids on polyamide supports Synthesis of substance P and of acyl carrier protein 64—74 decapeptide. J Chem Soc Perkin Trans 1 1981 ... 

Hammer RP, Albericio F, Gera L, Barany G. Practical approach to solid-phase synthesis of C-terminal peptide amides under mild conditions based on photolysable anchoring linkage. Int J Peptide Protein Res 1990 ... 

The third approach (iii), the solid-phase synthesis, has not been yet implemented for the preparation of protein-like HP-copolymers with heterogeneous blockiness, although the possibility—inherent in this method—to form the macromolecular chains with a well-defined chemical sequence of monomeric units is of great interest for the problem. That is why, these possibilities will also be discussed briefly in Sect. 2.4. 

Keywords. Short monolithic columns, Monoliths, Chromatography, Separation, Purification, Proteins, DNA, Bioconversion, Solid-phase synthesis... 

Moreover, if the average yield per step decreases only slightly, let us say down to 85%, the overall yield of the convergent synthesis is still quite acceptable -37%-, but now the overall yield of the linear synthesis would be only 0.004%. It is clear, therefore, that for polypeptides of any complexity and for proteins, linear syntheses in solution are not practicable even if the yields of each step are kept high. However, solid-phase peptide synthesis can be quite efficient. This is because solid-phase synthesis represents an improvement in linear methodology which has, as yet, not found an equivalent in convergent methods [17]. 

For such an integrated research activity, differently modified peptides and proteins that carry modifications whose structure can be changed at will through synthesis are invaluable tools. Therefore, the synthesis of the lipidated peptides is an important theme. Lipidated peptides can typically not be accessed via standardized peptide synthesis methods. However, employing the synthetic tools developed and presented here, most types of lipidated peptides can now be synthesized and obtained in pure form. Even though solution-phase approaches still play a significant role in the synthesis of lipidated peptides, the recently developed solid-phase synthesis methods delineate the preferred strategy to access the majority of the required lipidated peptides. 

Ribonuclease A (RNase A) was selected as the target enzyme for solid-phase synthesis because its sequence was known (Scheme S), 22 25 and an X-ray structure had been deduced. 24 Importantly, it had been shown that this 124-residue protein could be reduced and unfolded and then reoxidized to re-form the four disulfide bonds with recovery of full enzymatic activity. 25 ... 

To illustrate the diversity of strategies available for solid-phase synthesis, several fairly recent protein syntheses can be cited. SRY, an 80-residue DNA binding protein, was syn-thesized[62] on a Pepsyn support (polyacrylamide gel beads) in a continuous flow machine using Fmoc/tBu protection and TBTU/HOBt activation. HPLC gave a homogeneous product with molecular weight 10051 Da (calculated 10033 Da). It bound to DNA, as expected. 

An alternative to the synthesis of proteins by classical fragment synthesis in solution or by solid-phase synthesis on a support is the use of enzyme-catalyzed condensation of amino acids or peptides. This possibility was first demonstrated in 1938 91 with the synthesis of poorly soluble benzoyl-leucyl-leucine anilide by papain catalysis. After many years, this approach was extended to the preparation of peptide hormones such as Leu-enkephalin 92 and dynorphin(l -8).[93 This was made possible by the use of highly purified enzymes and by careful control of reaction conditions. The basic principles of protease-catalyzed peptide bond formation have been discussed.194 ... 

Scheme 6 Strategy for Solid-Phase Synthesis of vMIP I Protein Using a Safety-Catch Linked32 ...

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