Serine proteases peptide synthesis

A different approach toward cyclic peptides has been presented by Leatherbarrow and coworkers, employing ring-dosing metathesis (RCM) on a solid support [50], The authors reported on the synthesis of conformationally strained cyclic peptides of the Bowman-Birk inhibitor type, which are naturally occurring serine protease... 

A practical enzymatic procedure using alcalase as biocatalyst has been developed for the synthesis of hydrophilic peptides.Alcalase is an industrial alkaline protease from Bacillus licheniformis produced by Novozymes that has been used as a detergent and for silk degumming. The major enzyme component of alcalase is the serine protease subtilisin Carlsberg, which is one of the fully characterized bacterial proteases. Alcalase has better stability and activity in polar organic solvents, such as alcohols, acetonitrile, dimethylformamide, etc., than other proteases. In addition, alcalase has wide specificity and both l- and o-amino acids that are accepted as nucleophiles at the p-1 subsite. Therefore, alcalase is a suitable biocatalyst to catalyse peptide bond formation in organic solvents under kinetic control without any racemization of the amino acids (Scheme 5.1). 

As discussed above, proteases are peptide bond hydrolases and act as catalysts in this reaction. Consequently, as catalysts they also have the potential to catalyze the reverse reaction, the formation of a peptide bond. Peptide synthesis with proteases can occur via one of two routes either in an equilibrium controlled or a kinetically controlled manner 60). In the kinetically controlled process, the enzyme acts as a transferase. The protease catalyzes the transfer of an acyl group to a nucleophile. This requires an activated substrate preferably in the form of an ester and a protected P carboxyl group. This process occurs through an acyl covalent intermediate. Hence, for kineticmly controlled reactions the eii me must go through an acyl intermediate in its mechanism and thus only serine and cysteine proteases are of use. In equilibrium controlled synthesis, the enzyme serves omy to expedite the rate at which the equilibrium is reached, however, the position of the equilibrium is unaffected by the protease. 

The optimum yield of a condensation product is obtained at the pH where Ka has a maximum. For peptide synthesis with serine proteases this coincides with the pH where the enzyme kinetic properties have their maxima. For the synthesis of penicillins with penicillin amidase, or esters with serine proteases or esterases, the pH of maximum product yield is much lower than the pH optimum of the enzymes. For penicillin amidase the pH stability is also markedly reduced at pH 4-5. Thus, in these cases, thermodynamically controlled processes for the synthesis of the condensation products are not favorable. When these enzymes are used as catalysts in thermodynamically controlled hydrolysis reactions an increase in pH increases the product yield. Penicilhn hydrolysis is generally carried out at pH about 8.0, where the enzyme has its optimum. At this pH the equiUbrium yield of hydrolysis product is about 97%. It could be further increased by increasing the pH. Due to the limited stability of the enzyme and the product 6-aminopenicillanic acid at pH>8, a higher pH is not used in the biotechnological process. 

Peptides with C-terminal phosphonates, initially reported to have antibacterial properties, have also been found to possess inhibitory properties toward serine proteases)28 The synthesis of peptide phosphonates (Section 15.1.8) usually requires protection of the phos-phonic moiety as a diester, followed by selective deprotection in the final stage. The importance of peptide thiols (Section 15.1.9) is exemplified by captopril, an orally active angiotensin converting enzyme inhibitor used as a treatment for hypertension)29 These peptide thiols are prepared by the reaction of sulfanylalkanoyl amino acids with a-amino esters followed by deprotection of carboxy and sulfanyl groups. Other peptide thiols have been reported to be inhibitors of zinc metalloproteases, collagenases, and aminopeptidases. 

The synthesis of peptides with C-terminal phosphonate moieties results in a mixture of diastereomers when racemic 1-aminoalkylphosphonates are used. In most biological applications, e.g. inhibition of serine protease by peptidyl diphenyl 1-aminoalkylphosphonates, only one diastereomer reacts with the enzyme.14 Pure diastereomers can be obtained by separation of the mixture and simple crystallization frequently works well. Several excellent commercially available HPLC columns are potentially useful for the separation of diastereomers. Alternatively, optically active 1-aminoalkylphosphonic adds can be used as starting materials. 

This strategy was employed by Semple and co-workers [29] in the synthesis of the N(10)-C(17) fragment of cyclotheonamides, a family of 19-membered cyclic penta-peptides isolated from the sponge Theonella swinhoei, which are serine protease inhibitors. 

Bikunin (Bik), a peptide excreted in the urine, is one of the primary inhibitors of the trypsin family of serine proteases. This peptide plays a key role in inflammation and innate immunity because of its two Kunitz-type binding domains [1, 2], Bik suppresses proteolytic activity in a variety of tissues and can also exert localized anti-inflammatory effect [3-5], Inflammation is an important indicator of infection, cancer, and tissue injury in acute and chronic states. In acute inflammation, fluids and plasma components accumulate in the affected tissues due to vascular dilation. Subsequent activation of platelets and increased presence of immune cells occur during repair. Long-standing inflammation may be present before the disorder is identified. Due to its inhibitory role and potential use as an early marker of inflammation, we will review the synthesis, structure, pathophysiology of Bik as well as the various approaches for its measurement in this chapter. 

The constmction of synthetic selenocysteine-containing proteins or selenium-containing proteins attracts considerable interest at present, mainly for the reason that it can be used to solve the phase problem in X-ray crystallography. Selenomethionine incorporation has been used mostly uutil now for this purpose. There are also two reports ou uew synthetic selenocysteine-containing proteins. In one case, the active site serine of subtUisin has been converted into a selenocysteine residue by chemical means, with the result that the enzyme gains a predominant esterase instead of protease activity. In the second case, automated peptide synthesis was carried out to produce a peptide in which all seven-cysteine residues of the Neurospora crassa metallothioueiu (Cu) were replaced by selenocysteine. The replacement resulted iu au alteration of both the stoichiometry and the affinity of copper binding. ... 

A type I thioesterase domain is present at the NHj-terminal of the animal FAS and is responsible for catalyzing hydrolysis of the completed fatty acyl chain from the enzyme. The active site contains both conserved serine and histidine residues [87] and is thought to function via a mechanism similar to that of the serine proteases [50] however, no conserved acidic residue is present to complete the charge relay/transfer. A second variety of thioesterase (type II) is encoded as a separate protein and interacts with the multifunctional FAS to release medium chain fatty acids [88, 89]. This enzyme has a weak sequence similarity to the type I thioesterase, which includes the conserved active site serine and histidine residues. These enzymes are also homologous to proteins encoded by genes involved in the synthesis of peptide antibiotics [90,91] (see below). 

A step-by-step peptide synthesis from the N- to the C-terminus is not possible with chemical methods as it risks partial epimerization due to the repeated carboxy activation procedures, In constrast, the stereo- and regiospecificity of serine and cysteine proteases ensures integrity of the stereogenic center and allows ecological reaction conditions without side-chain protection. Scheme 4 shows the synthesis scheme using clostripain and chymo-trypsin as catalysts.The second coupling reaction was carried out by enzyme catalysis in a frozen aqueous system (see Section 4.2.3.1). 

The protease family of enzymes has long generated considerable interest due to their role in peptide degradation and possibly peptide synthesis. Although many distinct families of serine proteases seem to exist, the two best studied ones are chymotrypsin and subtilisin. Several MD studies have been reported on serine proteases in aqueous media " but only little is known about the dynamics of serine proteases in non-aqueous solvents. ... 

Directed evolution has also been very effective for increasing enzyme activity in organic solvents 14> For example, the serine protease subtilisin can catalyze specific peptide syntheses and transesterification reactions, but organic solvents are required to drive the reaction towards synthesis. Sequential rounds of error-prone PCR and visual screening yielded a subtilisin variant with twelve amino acid substitutions that was 471 times more active than wild-type in 60% dimethylforma-mide (DMF)[145- 22° this enzyme is much more effective for peptide and polymer synthesis. 

The mechanism of peptide bond synthesis is thought to resemble the reverse of the acylation step in the serine protease, with the base of A2486 (A2451 in E. coli) playing the same general base role as histidine-57 in chymotrypsin. This A is universally conserved within the central loop of domain V [28]. 

During hydrolysis catalyzed by serine proteases an acyl-enzyme complex transfers the acyl group to water. However, in enzymatic synthesis, the acyl group is not transferred to water but to a nucleophile, that is, to an amino group or amino acids/peptides. Thus a transpeptidation reaction takes place during the enzymatic modification [46,57]. 

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