Dipeptides formation

Fmoc-Cl, NaHC03, aq. dioxane, 88-98% yield. Diisopropylethylamine is reported to suppress dipeptide formation during Fmoc introduction with Fmoc-Cl. ... 

The very first investigations on this topic indicated that racemization can be monitored in micro reactors and that the degree of racemization seems not to be higher than in conventional organic synthesis. For dipeptide formation from the penta-fluorophenyl ester of (Ji)-2-phenylbutyric acid and (S)-a-methylbenzylamine, racemization of 4.2% was found [158]. At higher concentration (0.5 M instead of 0.1 M), a higher degree of racemization was found (7.8%). 

OS 26] [R 4] [P 18] For dipeptide formation from the pentafluorophenyl ester of Boc-D-alanine and (S)-a-methylbenzylamine an extent of racemization of 5.6% was found [86]. This experiment also served to demonstrate monitoring of the racemization of an a-amino acid used in peptide synthesis. 

In the following section, we describe protocols for tests aimed at screening for compounds capable of interfering with some of the main activities of this factor, such as (a) recognition and binding of initiator tRNA (b) codon-dependent ribosomal binding of fMet-tRNA leading to the formation of a 30S or 70S initiation complex (c) ribosome-dependent hydrolysis of GTP and (d) accommodation of fMet-tRNA in the ribosomal P-site and formation of the first peptide bond (initiation dipeptide formation). 

FMF Chen and NL Benoiton. Diisopropylethylamine eliminates dipeptide formation during acylation of amino acids using benzoyl chloride and some alkyl chlorofor-mates. Can J Chem 65, 1224, 1987. 

Exogenous factors affecting the rate of cyclic dipeptide formation 680... 

The rates of formation of various cyclic peptides and DKPs have been documented and shown to be affected by a wide range of physicochemical and structural parameters. Goolcharran and Borchardt examined the effects of exogenous (i.e., pH, temperature, buffer species, and concentration) and endogenous (i.e., primary sequences) factors affecting the rate of cyclic dipeptide formation, using the dipeptide analogues of X-Pro-/)-nitroaniline (X-Pro-/>NA where X represents the amino acid residue of the respective cyclic dipeptide). 

Modification of the amino acid residues located on the N-terminal side of Pro was shown to have a major influence on the rate of cyclic dipeptide formation. For the series of dipeptide analogues of X-Pro-/>NA, the half-lives of cyclic dipeptide formation in 0.5 molG phosphate buffer (pH 7) at 37 °C were reported as follows X = Gly 5.1 days, X = Val 2.5 days, X = Ala 1.1 days, X = /3-cyclohexylalanine 0.8 days, X = Arg 0.7 days, and X = Phe 0.5 days. Increased bulkiness of alkyl and aryl substituents have been previously shown to increase the rate of cyclization due to intramolecular reactions. This however does not seem true for the series studied by Goolcharran and Borchardt as the Ala analogue cyclized twice as fast as the bulkier analogue. From the study it is evident that simple steric bulk of substituents alone cannot be used to effectively explain the effects involved in the formation of cyclic dipeptides from various peptide precursors. 

Sequence inversion and racemization have been associated with uncatalyzed formation of the cyclic dipeptides and has been shown to greatly complicate the kinetics of formation. Cyclic dipeptide formation, by uncatalyzed processes, is rapid enough to pose an apparent threat to the stability of proteins and a possible rationale for the posttranslational N-acetylation of proteins that have been observed in higher organisms. The rate of DKP formation will also depend on the carbonyl ester protecting groups or the structures of the peptide-resin linkage in the solid-phase mode. Furthermore, cyclization is a concentration-independent reaction and demands the use of dilute solutions. ... 

Interestingly, the 3-chloro (3-lactam 66, Scheme 23, upon treatment with (S)-LeuOBn in DMF as solvent, afforded detectable dipeptide formation (15%) in the absence of any promoter. When the same coupling is carried out under identical conditions but with the assistance of 1 equivalent of NaN3, the yield rises to 85%. Finally, by using KCN instead of NaN3, a complete reaction at room temperature is... 

D. N. Reinhoudt, 1998, Large acceleration of alpha-chymotrypsin-catalyzed dipeptide formation by 18-crown-6 in organic solvents, Biotechnol. Bioeng. 59, 553-556. 

S Doekel, MA Marahiel. Dipeptide formation on engineered hybrid peptide synthetases. Chem Biol 7 373-384, 2000. 

The anchoring of an amino acid to the solid support by esterification is often more difficult, and even hazardous, for some residues and can lead to epimer-ization, dipeptide formation, and low substitution. Thus, we recommend the purchase of resins preloaded with the first C-terminal Ai-protected amino acid these are commercially available from various manufacturers. 

Note Do not use this activation procedure for coupling of Arg (conversation of anhydride to the 5-lactam) or Gly (dipeptide formation). 

Fig. 11. Reaction scheme for (I) substrate amino acid activations and dipeptide formation, (II) racemization, and (III) N-methylation. J and E2 are symbols for enzyme activities (from peptide synthetase modules) either on the same or separate proteins. R1 and R2 are amino acid side chains, where R1 is part of the first amino acid activated by the peptide synthetase indicates the linkage between 4 -phosphopantetheinylated enzyme and the substrate or peptide intermediate. AdoMet, S-adenosylmethionine AdoHcy, S-adenosylhomocysteine...

Like modular PKSs, peptide synthetases also epimerize some substrates and/or intermediates. For example, the starter substrate amino acid of cyclosporin A is D-Ala. Racemization of alanine is not catalyzed by an integrated subunit of cyclosporin A synthetase, but by alanine racemase. This is a separate, pyridoxal phosphate-dependent enzyme [ 193]. In contrast, Grsl and Tycl covalently activate L-Phe as a thioester and subsequently epimerize the amino acid [194]. D-Phe is the only epimer accepted as a substrate for dipeptide formation by Grs2 and Tyc2 [195, 196]. No racemization activity is detected in a pantetheine-deficient mutant of Grsl [197]. Deletion mutagenesis pointed to the requirement of the COOH-terminal part of the module for epimerizing L-Phe to D-Phe [180]. In contrast, the biosynthesis of actinomycin D, a bicyclic chromo-pentapeptide lactone (Fig. 10), involves formation of the dipeptide 6-MHA (methylanthranilic acid)-L-Thr-L-Val prior to epimerization of the L-Val exten-... 

Dipeptide formation is also prevented by an intermediate N,0-bis(trimethylsilylation) of the amino acid with chlorotrimethylsilane (TMSCl). The silyl groups are cleaved during aqueous workup leading to Fmoc amino adds in 78-94% yields. Following the suggestion of Yadav et al. that carbamates can be produced from amino acids under neutral conditions by reaction with chloroformates in the presence of zinc dust, A/ -Fmoc amino acids were obtained rapidly (15-20 min) in high yields without detectable dipeptide contaminants. ... 

Figure 23 Differentiation of initiation and elongation epimerization domains by intermodular transfer activity, (a) Intermodular transfer activity assay. Peptidyl or aminoacyl specificity of epimerization domains is monitored by tripeptide formation or dipeptide formation, respectively, (b) Proposed mechanism of aminoacyl epimerization domain and peptidyl epimerization domain in NRPS.42...

Aspartame Dipeptide formation from d,l-phenylalanine with thermolysin kilo-tons Holland Sweetener Company [8]... 

Dioeotide Formation. Figure 2 presents the time course of the dipeptide formation reaction. The limiting reactant, Z-Tyr-OMe, is depleted rapidly, being converted via nucleophilic attack of the glycinamide to the desired dipeptide product, Z-Tyr-Gly-NH2 via trans-esterification to a transient intermediate, Z-Tyr-OHx and via hydrolysis to the undesired side product Z-Tyr-OH. Dipeptide yield, based on the more expensive amino acid substrate, Z-Tyr-OMe, is 75% after 30 minutes using an enzyme concentration of 1 /iM, Both the... 

Scheme 7.21 Comparison of the efficiency of a dipeptide formation using an active ester from a dichlorotriazine anchored to different resins.

Carboxylic acids can be attached to these linkers using methods of ester bond formation such as carbodiimide/DMAP [23] and acid chloride/base. For the loading of N-protected-a-amino acids in particular, an array of different methods has been developed to minimize enantiomerizahon and dipeptide formation during the esterification reaction. These include the use of MSNT/N-methylimidazole [24], mixed anhydrides generated with 2,6-dichlorobenzoyl chloride [25], esters of 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene [26] and acid fluorides [27]. Phenols and N-protected hydroxylamines have been immobilized using the Mitsunobu reaction [28, 29], The latter are particularly useful for the preparation of hydroxamates [29, 30],... 

Coupling procedure for the 3rd amino acid 3rd Dipeptide Formation of DKP (%)... 

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