Peptide, sequencing synthesis

Synthesis of constrained peptide sequences has now been followed by combinations of nonpeptide molecules. As greater constraints are introduced, the numerical productivity falls, but presumably the proportion of leads increases. 

A systematic replacement of any amino acid in the sequence for photoreac-tive analogues allows a photoaffinity scanning of the binding interface. Since solid-phase synthesis is limited in the length of the peptide, Schultz et al. developed a sophisticated method which makes it possible to incorporate unnatural amino acids into large peptide sequences. The photoreactive amino acid was linked to transfer RNA, which inserted the amino acid into the required position by in vivo translation [44]. 

NL Benoiton. Quantitation and the sequence dependence of racemization in peptide synthesis, in E Gross, J Meienhofer, eds. The Peptides Analysis, Synthesis, Biology, Academic, New York, 1981, Vol 5, pp 341-361. 

It is interesting to note that serine peptidases can, under special conditions in vitro, catalyze the reverse reaction, namely the formation of a peptide bond (Fig. 3.4). The overall mechanism of peptide-bond synthesis by peptidases is represented by the reverse sequence f-a in Fig. 3.3. The nucleophilic amino group of an amino acid residue competes with H20 and reacts with the acyl-enzyme intermediate to form a new peptide bond (Steps d-c in Fig. 3.3). This mechanism is not relevant to the in vivo biosynthesis of proteins but has proved useful for preparative peptide synthesis in vitro [17]. An interesting application of the peptidase-catalyzed peptide synthesis is the enzymatic conversion of porcine insulin to human insulin [18][19]. 

Fig. 3.3. Major steps in the hydrolase-catalyzed hydrolysis of peptide bonds, taking chymo-trypsin, a serine hydrolase, as the example. Asp102, His57, and Ser195 represent the catalytic triad the NH groups of Ser195 and Gly193 form the oxyanion hole . Steps a-c acylation Steps d-f deacylation. A possible mechanism for peptide bond synthesis by peptidases is represented by the reverse sequence Steps f-a.

Figure 4 Retro-inversion of host defense peptides. Synthesis of RI peptides is achieved by substituting o-amino acids at all stereocenters within a peptide and reversal of peptide sequence (RI - R3 in the i-peptide and R3 RI in the Rl-peptide). By rotating the Rl-peptide at 180° it can be seen that the three-dimensional space occupied by the amino acid functional (R) groups is retained in comparison to the i-peptide although the peptide backbone has been reversed.

A possibility to overcome the time-consuming solution-phase peptide synthesis, but avoiding specific synthetic problems that cannot be addressed on tbe solid support, is to follow a combined solution-/solid-pbase approach. In such an approach the majority of the peptide sequence is typically assembled on solid support, but critical steps are performed in solution. Below, some exemplary peptides are discussed. 

Mechanism of Action An aminoglycoside that binds directly to the 303 ribosomal subunits causing a faulty peptide sequence to form in the protein chain. Therapeutic Effect Inhibits bacterial protein synthesis. 

For the synthesis of double-stranded symmetrical and unsymmetrical monocystine peptides the formation of an intermolecular disulfide bridge is required. For homodimerization of cysteine peptides all the methods discussed in Section 6.1.1 can be applied taking into account the reactivity of the different oxidative agents toward sensitive amino acid residues present in the peptide sequences. Synthetic approaches based on the direct use of suitable cystine derivatives can be envisaged, at least for small-size peptides since disproportionation would in all cases retain the homodimeric structure 241... 

Due to the S—N acyl shift, the stepwise synthesis using a S-palmitoylated cysteine derivative is not advisable. A more convenient approach is to assemble the peptide sequence and to acylate the cysteine thiol group upon its selective deprotection, retaining, however, the protection of other reactive groups. For this purpose the Cys(StBu) and Cys(Acm) deriv-... 

The last stage of peptide chain synthesis is termination. The genetic code specifies three stop codons, indicating the termination of a coding sequence. When the ribosome encounters one of these stop codons on the mRNA, certain release factors... 

Methods of chemical synthesis of polypeptides and of cloning and mutating genes now allow us to alter peptide sequences at will and to design completely new proteins 355-356b The methods are discussed in Chapters 3, 5, and 26. The following are examples. 

The development of chiral peptide-based metal catalysts has also been studied. The group of Gilbertson has synthesized several phosphine-modified amino adds and incorporated two of them into short peptide sequences.[45J,71 They demonstrated the formation of several metal complexes, in particular Rh complexes, and reported their structure as well as their ability to catalyze enantioselectively certain hydrogenation reactions.[481 While the enantioselectivities observed are modest so far, optimization through combinatorial synthesis will probably lead to useful catalysts. The synthesis of the sulfide protected form of both Fmoc- and Boc-dicyclohexylphosphinoserine 49 and -diphenylphosphinoserine 50 has been reported, in addition to diphenylphosphino-L-proline 51 (Scheme 14).[49 To show their compatibility with solid-phase peptide synthesis, they were incorporated into hydrophobic peptides, such as dodecapeptide 53, using the standard Fmoc protocol (Scheme 15).[451 For better results, the phosphine-modified amino acid 50 was coupled as a Fmoc-protected dipeptide 56, rather than the usual Fmoc derivative 52.[471 As an illustrative example, the synthesis of diphe-nylphosphinoserine 52 is depicted in Scheme 16J45 ... 

The commercial availability of protected /V-methyl amino acids [(Me)Xaa] of many proteinogenic amino acids (as well as other V-alkyl amino acids), the availability of procedures for the synthesis of protected V-alkyl analogues of all the protein amino acids, and the availability of synthetic procedures for site-selective alkylation during SPPS (see Section 10.1.2.1) allows the alkylation of nearly all peptide bonds in a given parent peptide. The synthesis of a series of V-alkyl peptide analogues based on the sequence of a given bioactive peptide (linear or cyclic) in which each peptide bond is successively alkylated and evaluation of the biological activity of this series will be called herein A-alkyl-scan (for example Me-scan, Et-scan, etc.)... 

Synthetic pathways towards the dimethylene isosteres comprise several disconnections, indicated by the essential bond forming reactions (Scheme 2). Several methods yield racemic dipeptide analogues. These are usually incorporated into the peptide sequence and the resulting epimeric peptides are separated. When either R1 or R2 = H, asymmetric syntheses towards the required enantiomer are available. When both R1 and R2 H, only the reduction of the i )[CH=CH] precursor yields homochiral compounds. As many co-amino acids (R1 = R2 = H) are commercially available, their synthesis needs not be discussed here. 

The linker 37 with the first amino acid attached, compound 38, can be applied to stepwise solid-phase peptide synthesis. At the end of the synthesis, when the desired peptide sequence is completed, the thioacid-modified peptide fragment is cleaved from the solid support by HF and further S-alkylated with a N-bromoacetylated peptide to form an endothioester bond. The cleaved thioacid can also be reacted with an alkyl bromide to form the corresponding thioester. 

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