Acyl peptide synthesis with

The acylated peptides (Myr)GCX-Bimane 31 a-e (X = G, L, R, T, V), which are found in certain nonreceptor tyrosine kinases and ct-subunits of several heterotrimeric G-proteins, were synthesized in solution using common solution-phase peptide synthesis with X-myristoylglycine as a building block. These model peptides were used for acylation studies with palmitoyl-CoA in phospholipid vesicles at physiological pH. For such uncatalyzed spontaneous reactions only a modest molar excess of acyl donor species (2.5 1) was necessary. Unprotected side chains of threonine or serine are not interfering with this S-acylation (Scheme 14). 

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. 

Acyl chlorides. Acyl chlorides are formed rapidly by reaction of carboxylic acids with SOCl2 and pyridine in CH2C12 at 25°. The dicyclohexylammonium salts of carboxylic acids react particularly rapidly (ca. 1 minute). The acid chlorides prepared in situ in this way react with amines in the presence of DMAP or DBU to form amides in >85% yield. This SOCl2-Py method is also useful for peptide synthesis with slight racemization. 

Similarly, carbonylimidazolinm salts have been introduced. For example, CBMIT 15 is described as an efficient amino acylating reagent for peptide synthesis with sterically hindered amino acids (36). 

A new peptide synthesis with adducts of phosphorous compounds and tetrahalomethanes has been reported N-(Chlorophosphoryl)pyri-dinium betaines are remarkably reactive acylating agents for the preparation of esters, amides, and peptides. They can easily be prepared from phosphorous acid or its esters, pyridine, and mercuric chloride Peptides have also been prepared through polymeric N-acoxydicar-boxylic acid imides with markedly reduced coupling times at elevated temperature (70°). The solid-phase method, first used in the synthesis of peptides, gains wider application. Recently a solid-phase Wit-tig synthesis of olefins has been published . 

Peptide synthesis with active acyl compds. CON <... 

However, like other methods of test to be discussed in the following section, the salt formation with pyridine hydrohalides in the control of completed amino acylation of amino groups unfortunately interferes in the peptide synthesis with masking functions labile to acid [133] and — as already mentioned — with other basic centres on polymer like guanidyl, indolyl, and imidazolyl residues. 

Acylation of peptides by aliphatic or aromatic carboxylic acids is followed by different routes. The respective carboxylic acids are supplied by linked ptolyke-tide synthases, either of the fatty acid or various polyketide synthase types, and introduced as either CoA esters or activated directly as adenylates. Several such activating systems have been characterized (see Section III.A.5). To initiate peptide synthesis with CoA esters, respective transferases arc required ... 

Selenophenyl N-carbobenzoxy-dl-alaninate and methyl glycinate in acetonitrile allowed to stand 12 hrs. at room temp. -> methyl N-carbobenzoxy-dl-alanyl-glycinate. Y 78.5%. F. e., also with the Na-salts of the free acids by refluxing 4 hrs. in tetrahydrofuran, s. H.-D. Jakubke, Z. Ghem. 3, 65 (1963) B. 97, 2816 (1964) peptide synthesis with other active acyl compounds, such as acyloximes or 3-methyl-1-phenylpyrazolyl esters, s. G. Losse, A. Barth, and K. Schatz, A. 677, 185 (1964). 

With the dicyclohexylcarbodiimide (DCQ reagent racemization is more pronounced in polar solvents such as DMF than in CHjCl2, for example. An efficient method for reduction of racemization in coupling with DCC is to use additives such as N-hydroxysuccinimide or l-hydroxybenzotriazole. A possible explanation for this effect of nucleophilic additives is that they compete with the amino component for the acyl group to form active esters, which in turn reaa without racemization. There are some other condensation agents (e.g. 2-ethyl-7-hydroxybenz[d]isoxazolium and l-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline) that have been found not to lead to significant racemization. They have, however, not been widely tested in peptide synthesis. 

HONZL RUDINGER Peptide Synthesis Peptide synthesis by coupling ot acyl azides with amino esters... 

In the second major method of peptide synthesis the carboxyl group is activated by converting it to an active ester, usually a p-nitrophenyl ester. Recall from Section 20.12 that esters react with ammonia and amines to give fflnides. p-Nitrophenyl esters are much more reactive than methyl and ethyl esters in these reactions because p-nitrophenoxide is a better (less basic) leaving group than methoxide and ethoxide. Simply allowing the active ester and a C-protected amino acid to stand in a suitable solvent is sufficient to bring about peptide bond formation by nucleophilic acyl substitution. 

Much more important than these reactions, however, are the reactions of CDI and its analogues with carboxylic acids, leading to AAacylazoles, from which (by acyl transfer) esters, amides, peptides, hydrazides, hydroxamic acids, as well as anhydrides and various C-acylation products may be obtained. The potential of these and other reactions will be shown in the following chapters. In most of these reactions it is not necessary to isolate the intermediate AAacylazoles. Instead, in the normal procedure the appropriate nucleophile reactant (an alcohol in the ester synthesis, or an amino acid in the peptide synthesis) is added to a solution of an AAacylimidazole, formed by reaction of a carboxylic acid with CDI. Thus, CDI and its analogues offer an especially convenient vehicle for activation of... 

Another -activation of amino acids for peptide synthesis is achieved by preparing sulfenamides from sulfenylimidazoles. A sulfenylimidazole is formed in situ from the sulfenyl chloride (prepared from the disulfide and chlorine) and imidazole, which reacts further with an amino acid ester to give a sulfenamide in high yield. Conversion of such sulfenamides with IV-acyl amino acids by means of triphenylphosphine affords dipeptides with racemization of less than 0.5%.[481... 

With the C-terminal residue introduced as part of the BAL anchor and the penultimate residue incorporated successfully by the optimized acylation conditions just described, further stepwise chain elongation by addition of Fmoc-amino acids generally proceeded normally by any of a variety of peptide synthesis protocols. 

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]. 

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