Peptides: dipeptide synthesis

Prolyl aminopeptidases (PAP) are exopeptidases that hydrolytically cleave off an N-terminal Pro from peptides. The enzymes belong to the a/p hydrolase fold proteins. A PAP from Streptomyces thermoluteus carrying the active site nucleophile mutation S144C was used as a catalyst for the synthesis of proline-containing peptides. Dipeptide synthesis was obtained with an amino acid methyl or benzyl ester as the acyl donor and prolyl-OBz as the nucleophile [21]. Under alkaline conditions, cycli-zation and polymerization of prolyl-OBz was observed. 

The dipeptide synthesis with free thermolysin (2 mg/ml) was carried out in water 10 mM of CLCa at pH 6,8 and 20 °C. The yield of peptide was below 10%, due to some reaction byproducts coming from the hydrolysis of methyl ester group of the peptide and consequent product hydrolysis as is shown in the following reactions ... 

Following peptide bond synthesis, the ribosome Is translocated along the mRNA a distance equal to one codon. This translocation step is promoted by hydrolysis of the GTP in eukaryotic EF2-GTP. As a result of translocation, tRNAj , now without its activated methionine, is moved to the E (exit) site on the ribosome concurrently, the second tRNA, now covalently bound to a dIpeptIde (a peptIdyl-tRNA), Is moved to the P site (Figure 4-26, step U). Translocation thus returns the ribosome conformation to a state in which the A site Is open and able to accept another amlnoacylated tRNA complexed with EFlct-GTP, beginning another cycle of chain elongation. 

In order to introduce reactive azides/alkynes in peptides, the Rutjes group utilized alcalase mediated dipeptide synthesis with amino add amides and either acetylenic or azido amino... 

The synthetic potential of papain in low water systems has been explored with acyl donors bearing the carbamoylmethyl (Cam) leaving group. Dipeptide synthesis proceeded in >80% yield, and yields up to 60% were obtain for the synthesis of bioactive peptides like dermorphin-(l-4) (Boc-Tyr-D-Ala-Phe-Gly-NH2). Papain is also used as a versatile protease in polymer chemistry for the synthesis of amino acid oligomers and cooligomers of a-hydroxy acids and amino acids [22]. 

Many related so-called thermolysin-like proteinases (TLPs) from various Grampositive strains have been described [47], including neutral proteases from Bacillus subtilis, and some of these variants are applied in peptide synthesis. Several metal-loenzymes acting as carboxy- or aminopeptidase have also been characterized, but these variants have not been extensively used in peptide synthesis. A bovine carboxy-peptidase A [39] and orange carboxypeptidase C [68] have been applied for dipeptide synthesis in water-organic solvent mixtures, both under thermodynamic and xmder kinetic control. 

Gaertner et al. [48] investigated peptide bond formation catalyzed by PEG-proteases in benzene. The substrate specificity of PEG-chymotrypsin for ester hydrolytic activity in aqueous solution was almost the same as that of unmodified enzyme [51] both PEG-modified and unmodified chymotrypsins catalyzed the hydrolysis of aromatic amino acid esters but these enzymes did not hydrolyze the basic amino acid ester. In the case of dipeptide synthesis in benzene, however, not only Bz-iyr-Phe-NHa but also Bz-Lys-Phe-NH2 was synthesized from corresponding amino acid derivatives by PEG-chymotrypsin. The change in the substrate specificity, which has been reported in a few cases, might be due to a surrounding environment on the enzyme molecule. 

Higher peptides are prepared m an analogous way by a direct extension of the logic just outlined for the synthesis of dipeptides... 

Describe the synthesis of the dipeptide Lys-Ala by Merrifield s solid phase chemical method of peptide synthesis. What pitfalls might be encountered if yon attempted to add a leucine residue to Lys-Ala to make a tripeptide ... 

Substitution of a dipeptide unit by a cychc dipeptide derivative within a peptide chain can induce certain conformational restraints that may alter the biological response via changing receptor selectivity. A facile procedure for synthesis of pyrazinone ring-containing opioid mimetics [21] has been elaborated, based on the cycHzation of readily available dipep-tidyl chloromethyl ketones [22] (Scheme 6). This method affords 2(IH)-pyrazinone derivatives containing substituents with desired functional groups at positions 3 and 6 in high yield. 

The same reagents can be used to form amides from carboxylic acids and amines, a method which is applicable to peptide synthesis. Condensation of A-benzyloxycarbonyl-L-phenylalanine and ethyl glycinate hydrochloride gave an 85% yield of purified dipeptide. 

OS 25] [R 4] [P 17] For dipeptide formation from the pentafluorophenyl ester of (J )-2-phenylbutyric acid and (S)-a-methylbenzylamine an extent of racemization of 4.2% was found [86]. At higher concentration (0.5 instead of 0.1 M), a higher degree of racemization was found (7.8%). This experiment also served to demonstrate monitoring of the racemization of a simple carboxylic acid used in peptide synthesis. 

Racemization studies in the synthesis of the tripeptide Z-Gly-Phe-Gly from Z-Gly-Phe and Gly-OC2H5 revealed that in THF at room temperature such racemization occurred to the extent of about 5%, in DMF at -10 °C, however, less than 0.5%. 53 103 In the synthesis of Boc-Val-Tyr-OC2H5 (50%) from Boc-Val and Tyr-OC2H5 with CDI, a small amount of 0-acylation of tyrosine (4%) also occurred in the dipeptide. 11] A V -Carbonyldibenzimidazole was found inferior to CDI in the synthesis of peptides because of poorer yields and more rigorous reaction conditions needed. 53... 

Application of amino acid silyl esters or A-silyl amino acid silyl esters as amino components is very convenient in peptide synthesis with CDI, because the resulting peptide silyl esters are easily hydrolyzed to dipeptides during the usual work up. They need not be saponified in a separate step, as would be the case with the corresponding alkyl esters. Furthermore, no racemization occurs with this method.tl8],tl9]... 

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... 

Another example of the ability of proteinogenic amino acids, small peptides, and amines to catalyse the formation of new C-C bonds has been demonstrated by Weber and Pizzarello they were able to carry out model reactions for the stereospecific synthesis of sugars (tetroses) using homochiral L-dipeptides. The authors achieved a D-enantiomeric excess (ee) of more than 80% using L-Val-L-Val as the peptide catalyst in sugar synthesis (in particular D-erythrose) via self-condensation of glycol aldehyde. 

If prebiotic peptides and/or proteins were in fact initially formed in aqueous solution (the hypothesis of biogenesis in the primeval ocean ), the energy problems referred to above would have needed to be solved in order for peptide synthesis to occur. As discussed in Sect. 5.3, there is some initial experimental evidence indicating that the formation of peptide bonds in aqueous media is possible. An important criterion for the evolutionary development of biomolecules is their stability in the aqueous phase. The half-life of a peptide bond in pure water at room temperature is about seven years. The stability of the peptide bond towards cleavage by aggressive compounds was studied by Synge (1945). The following relative hydrolysis rates were determined experimentally, with the relative rate of hydrolysis for the dipeptide Gly-Gly set equal to unity ... 

Fig. 5.2 Peptide synthesis using Leuchs anhydrides amino acid 1 (with residue Ri) is reacted with phosgene to give the Leuchs anhydride. This reacts with amino acid 2 (residue R2) to give the peptide carbamate. The dipeptide is obtained after cleavage of C02...

The amino acid, activated by uptake of SO3, reacts with a second molecule of amino acid to form the dipeptide, which can in turn react further to form a tripeptide (and so on). This peptide synthesis model, which is supported by experimental evidence, appeals because of its simplicity it may well correspond much more closely to conditions on the primeval Earth than do some other models (Chen and Yang, 2007). 

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