Proteases peptide synthesis

Maggiora LL, Smith CW, Zhang ZY (1992) A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis. J Med Chem 35 3727-3730... 

M Baca, TW Muir, M Schnolzer, SBH Kent. Chemical ligation of cysteine-containing peptides synthesis of a 22 kDa tethered dimer of HIV-1 protease. J Am Chem Soc 117, 1881, 1995. 

The pore size of porous titania can be up to 2000 A. Titania is used for the purification of proteins and as a support for bound enzymes. The purification of /1-lactoglobulin from cheese whey, of protease from pineapple, /5-lactamase, and amylase can be achieved with titania. The latter two purifications are impossible on alumina. Titania is also used as a support in peptide synthesis. The separation of plasmid DNA is shown in Figure 3.24. 

W. Kullmann, Kinetics of Chymotrypsin- and Papain-Catalysed Synthesis of (Leu-cine)enkephalin and (Methionine)enkephahn , Biochem. J. 1984, 220, 405 - 416 W. Kullmann, Protease-Catalyzed Peptide Synthesis , Adv. Biosci. 1987, 65, 135- 140. 

Scheme 5.1 The principle of protease-catalysed kinetically controlled peptide synthesis.

Use of Proteases in Peptide Synthesis. Typically peptides are synthesized the standard solid or liquid phase methodologies (56, 57). However, both of these techniques require harsh chemical reactions which are detrimental to certain amino acids. Furthermore, in practical terms most peptide syntheses are limited to the range of 30 to 50 amino acid residues. Hence, peptide synthesis is still somewhat problematic in many cases. In certain situations, the alternative method of peptide synthesis using proteases is an attractive choice. With this form of synthesis, one can avoid the use of the noxious and hazardous chemicals used in solid or liquid phase peptide synthesis. Since the reactions are enzyme catalyzed, racemization of the peptide bond does not occur. This technique has been used with success in the synthesis and semisynthesis of several important peptides including human insulin (55,59). 

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 technique of using proteases in peptide synthesis has been carefully reviewed (60,61) and the technology is advanced to the state that commercially prepared kits for this type of synthesis are available. Nevertheless, due to the well defined methodological routines devised for generjd synthesis of peptides for laboratory use, the standard chemical approaches are likely to remain the methods of choice. 

Fragment condensation of peptides corresponds to a reverse protease reaction -peptide synthesis instead of cleavage - and this is well known in the hterature as well. In fact, proteases have been used extensively for peptide coupling (Jakubke etal, 1985 1996 Jakubke, 1987 1995 Luisi etal, 1977b). This work has shown that even small proteins can be synthesized by block-wise enzymatic couphng (see also Kullmann, 1987, and, for some more recent developments, Celovsky and Bordusa, 2000). 

Celovsky, V. and Bordusa, F. (2000). Protease-catalyzed fragment condensation via substrate mimetic strategy a useful combination of solid-phase peptide synthesis with enzymatic methods. /. Pept. Res., 55, 325-9. 

Formation of an amide bond (peptide bond) will take place if an amine and not an alcohol attacks the acyl enzyme. If an amino acid (acid protected) is used, reactions can be continued to form oligo peptides. If an ester is used the process will be a kinetically controlled aminolysis. If an amino acid (amino protected) is used it will be reversed hydrolysis and if it is a protected amide or peptide it will be transpeptidation. Both of the latter methods are thermodynamically controlled. However, synthesis of peptides using biocatalytic methods (esterase, lipase or protease) is only of limited importance for two reasons. Synthesis by either of the above mentioned biocatalytic methods will take place in low water media and low solubility of peptides with more than 2-3 amino acids limits their value. Secondly, there are well developed non-biocatalytic methods for peptide synthesis. For small quantities the automated Merrifield method works well. 

Proteases can be used for the synthesis of peptides in a way analogous to the ester synthesis catalysed by lipases. The most successful industrial example of enzymatic peptide synthesis is described in section 4.6 aspartame synthesis. In the industrial process in Europe the equilibrium position is shifted towards synthesis because the... 

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. 

Kasche, V. (1989) Proteases in peptide synthesis. In Proteases a Practical Approach, edited by U.Bond and R.Beynon, pp. 125-143. Oxford IRL Press. 

The first applications of solid phase synthesis were to peptides, hence it is no surprise that there have been several reports of HRMAS studies of peptide systems attached to a support. One of the earliest reports of HRMAS in a supported sample was that of Wang-bound lysine, whose structure was determined by TOCSY and HMQC HRMAS NMR.38 More recently, HRMAS NMR has been used to identify several peptidomimetic inhibitors of hepatitis C virus NS3 protease while on the resin.79 However, it is perhaps a bit surprising that more has not been made of HRMAS in attacking problems of relevance to peptide synthesis, although most recent interest is moving that way. Combinatorial chemistry and solid phase organic chemistry has been a much more active area using HRMAS techniques. 

The protease-catalyzed synthesis of peptide bonds is known as the plastein reaction ( ). Plastein itself is defined as the product formed by this reaction which is insoluble in trichloroacetic acid solutions 6). The plastein reaction has been most extensively investigated by researchers in Japan (, ... 

Both alkaline proteases form an intermediate, the acyl-enzyme complex, on the reaction coordinate from the amino acid component to the dipeptide, which is formed by the triad Ser-(or Cys-)-His-Asp (or -Glu) (see Chapter 9, Section 9.5). The acyl-enzyme complex can be formed with the help of an activated amino acid component such as an amino acid ester. The complex can react either with water to the undesired hydrolysis product, the free amino acid, or with the amine of the nucleophile, such as an amino acid ester or amide, to the desired dipeptide. The particular advantage of enzyme-catalyzed peptide synthesis rests in the biocatalyst specificity with respect to particular amino acids in electrophile and nucleophile positions. Figure 7.26 illustrates the principle of kinetically and thermodynamically controlled peptide synthesis while Table 7.3 elucidates the specificity of some common proteases. 

When one is using proteases in a direct reversal of their normal hydrolytic function, the equilibrium position is very important in limiting the attainable yield in equilibrium-controlled enzymatic peptide synthesis. If both reactants and products are largely undissolved in the reaction medium as suspended solids, thermodynamic analysis of such a system shows the reaction will proceed until at least one reactant has dissolved completely, towards either products or reactants ( switchlike behavior). In case of a favorable equilibrium for synthesis, the yield is maximized in the solvent of least solubility for the starting materials (Hailing, 1995). Thermolysin-catalyzed reactions ofX-Phe-OH (X = formyl, Ac, Z) with Leu-NH2 yielded X-Phe-Leu-NH2 with equilibrium yields > 90% over a range of solvents. Some predictions, such as a linear decrease in yield with the reciprocal of the initial reactant concentrations, could be verified (Hailing, 1995). 

Schuster M, Aaviksaar A, Haga M, Ullmann U, Jakubke HD. Protease-catalyzed peptide synthesis in frozen aqueous systems the freeze concentration model. Biomed Biochim Acta 1991 50 S84-S89. 

M. D. Bednarski, and M. R. Callstrom, Carbohydrate protease conjugates Stabilized proteases for peptide synthesis, J. Org. Chem., 60 (1995) 2216-2226. 

Peptide synthesis is an extremely important area of chemistry for the pharmaceutical industry, and like any specialized area of chemistry, has its own set of unique problems associated with it. Racemization and purification of final products are two of the most difficult problems in this area. The use of enzymes has been explored as a possible answer to these problems since 1938 [29]. However, proteases needed to catalyze peptide synthesis are subject to rapid autolysis under the conditions needed to affect peptide coupling, so this has generally not been a practical approach until cross-linked enzyme crystals of proteases became available. The synthetic utility of protease-CLCs was demonstrated by the thermolysin CLC (PeptiCLEC -TR)-catalyzed preparation of the aspartame precursor Z-... 

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