Peptide kinetically controlled

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). 

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

Chen, S.-T., Chen, S.-Y. and Wang, K.-T., Kinetically controlled peptide bond formation in anhydrous alcohol catalyzed by the industrial protease alcalase. J. Org. Chem., 1992, 57, 6960. 

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. 

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. 

Kasche, V. (1986) Mechanism and yields in enzyme catalysed equilibrium and kinetically controlled synthesis of -lactam antibiotics, peptides and other condensation products. Enzyme Microb. Technol., 8, 4-16. 

Kinetically controlled synthesis N-Xaa-esters Xaa-amides Corresponding peptides 90 14... 

To avoid the problems with kinetic control of a reaction during peptide bond formation, it is recommended that longer peptide phosphonates be synthesized stepwise by extension in the N-terminal direction, starting with diphenyl l-(aminoalkyl)phosphonates. The longer coupling time will provide peptide products that are usually an equivalent (or close to equivalent) mixture of diastereomers. The synthesis of longer peptides is demonstrated by the synthesis of Boc-Val-Pro-Valp(OPh)2, which is an excellent inhibitor of human neutrophil elastase (kobs/[I] = 27,000 M 1s 1)J38 ... 

There are two basic strategies for enzyme-catalyzed peptide synthesis equilibrium- and kinetically controlled synthesis. The former is the direct reversal of proteolysis and involves the condensation of an amino component with unactivated carboxyl component. The latter proceeds by the aminolysis of an activated peptide ester. 

Schellenberger V, Jakubke H-D. Protease-catalysed kinetically controlled peptide synthesis. Angew. Chem. Int. Ed. Engl. 1991 30 1437-1449. 

Trp, Leu, Met), and it was selected for making the Tyr-Gly, Phe-Leu, and Phe-Met peptide bonds. Papain was selected for Gly-Gly and Gly-Phe peptide bonds. Bromelain is very similar to papain as far as its specificity. All these proteases are serine or cysteine type, and the peptide bond formation can be done under kinetically controlled conditions. Thermolysin is an asparyl protease, and it was selected mainly for Phe-Leu and Gly-Phe bonds. MeCN, EtOAc, and methyl caproate were used as solvents with a controlled amount of buffer or at fixed water amount. 

In contrast to the equilibrium-controlled approach which ends with a true equUibrium, in the protease-catalyzed kinetically controlled synthesisf l the product appearing with the highest rate and disappearing with the lowest velocity would accumulate. This approach requires the use of acyl donor esters as carboxy components (Ac-X) and is limited to proteases which rapidly form an acyl-enzyme intermediate (Ac-E). Serine and cysteine proteases are known to catalyze acyl transfer from specific substrates to various nucleophihc amino components via an acyl-enzyme intermediate. In reactions of this type, the protease reacts rapidly with an amino acid or peptide ester, Ac-X, to form a covalent acyl-enzyme intermediate, Ac-E, that reacts, in competition with water, with the amino acid or peptide-derived nucleophile HN to form a new peptide bond (Scheme 3). The partitioning of the acyl-enzyme intermediate between water and the added nucleophile is the rate-limiting step. Under kinetic control, and if k4[HN] k3[H20], the peptide product Ac-N should accumulate. However, the soluble peptide product will be degraded if the reaction is not terminated after the acyl donor ester is consumed. 

The solubilizing capacity of the choline residue is so pronounced that even substrates combining two hydrophobic amino acids are homogeneously soluble in aqueous buffer without any additional cosolvent. These favorable physical properties were also used in the enzymatic formation of peptide bonds. The amino acid choline ester 38 acts as the carboxyl component in kinetically controlled peptide syntheses with the amino acid amides 39 and 40 [52] (Fig. 11). The fully protected peptides 41 and 42 were built up by means of chymotrypsin in good yields. Other proteases like papain accept choline esters as substrates also, and even butyrylcholine esterase itself is able to generate peptides from these electrophiles. 

Kinetically controlled syntheses, which are more often studied, can only be carried out by enzymes forming a reactive acyl-enzyme intermediate (serine or cysteine protease. Scheme 5). The reaction starts with weakly activated amino acids (e.g. esters), and the rapidly formed reactive intermediate RCOE is attacked by nucleophiles like amines and water. If k-i and A 4[H2NR ] > < 3[H20], the desired peptide accumulates. Short reaction times, low enzyme concentrations and the danger of secondary hydrolysis of the peptide product are characteristics of these reactions. The optimal pH usually lies above pH 8. 

Besides being used for the production of the precursors 6-APA and 7-ADCA, penicillin amidases are also able to couple 6-APA and 7-ADCA with D-phenyl-glycine ester or amide in a kinetically controlled enzymatic peptide synthesis forming ampicillin or cephalexin (38, 39). These reactions (Scheme 21) have a great potential for being commercialized in the near future [95]. 

The involvement of milk protein-derived cytomodulatory peptides to determine the viability of cancer cells is a field of great interest. Commercial yoghurt starter cultures hydrolyse casein to produce bioactive peptides that control colon cell kinetics in vitro. Bioactive sequences of casein modulate cell viability in different human cell cultures. Peptides from an extract of Gouda cheese inhibited growth of leukemia cells even at 1 pmol/L [223]. They were able to induce apoptosis in the tumor cells. Cancer cells are more reactive to peptide-induced apoptosis than non-malignant cells [224]. Casein-derived peptides could have a role in the prevention of colon cancer by blocking proliferation of the epithelium and by... 

Glycosidase-catalyzed glycoside synthesis is quite analogous to protease-catalyzed peptide synthesis. As with proteases, glycosidases may be used under either equilibrium or kinetically controlled conditions for synthetic purposes (Fig. 11.3-... 

Important for the nucleophile efficiency in kinetically controlled peptide bond formation... 

In contrast to the equilibrium-controlled approach the peptidase-catalyzed kinetically controlled peptide synthesis (for a review see reference1851) needs much less enzyme, the reaction time to reach maximal product yield is significantly shorter, and the product yield depends both on the properties of the enzyme used and the substrate specificity. Kinetic control means that the product appearing with the highest rate and disappearing with the lowest velocity would accumulate. Whereas the equilibrium-controlled approach ends with a true equilibrium, in the kinetic approach the concentration of the product formed goes through a maximum before the slower hydrolysis of the product becomes important. The product will be hydrolyzed if the reaction is not stopped after the acyl donor ester is consumed and true equilibrium is allowed to be reached. 

Figure 12.5-9. Comparison of the equilibrium (a) and the kinetically controlled approach (b) of peptidase-catalyzed peptide synthesis.

Serine and cysteine peptidases are not perfect acyltransferases. Therefore, it is useful to have a method for the prediction of the outcome of the kinetically controlled peptide synthesis. In order to get a simple efficiency parameter we decided to introduce the partition value p11061 analogous with the definition of the Michaelis constant according to Eq. (3), where P2 = Ac-OH, P3 = Ac-N, and N = HN. 

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