Alkylation N-terminal

An alternative approach for the analysis of blood samples from the same group of Iranian mustard gas victims has been described (50). As mustard gas alkylates amino acids in hemoglobin, adducts will be formed, which remain in the bloodstream for some time. Selective cleavage of the alkylated N-terminal valine of the a-chain of hemoglobin was carried out by using the modified Edman reagent pentafluorophenyl isothiocyanate. After derivatiza-tion of the adduct-derived pentafluorophenylthio-hydantoin with heptafluorobutyric anhydride, the... 

A number of adducts to amino acid residues have been identified by Noort and colleagues (1996) and Black et al. (1997a, b). Six different histidine residues, three glutamic acid residues, and both of the N-terminal valines were found. Alkylated cysteine, aspartic acid, lysine, and tryptophan were also detected. While the N1 and N3 histidine adducts were found to be most abundant, it was the alkylated N-terminal valine adducts that were most useful for subsequent quantification. See Detection of DNA and protein adducts of vesicants, below, for analytical details. 

Figure 5. Selective cleavage of alkylated N-terminal valine with pentafluorophenyl isothiocyanate...

Peptides typically are prepared for this ligation process using a-alkyl thioesters, because they are simple to make at the time of peptide synthesis. However, due to the relatively slow reaction kinetics of alkyl thioesters, most native chemical ligation processes have been catalyzed through the use of thiol compound additives, such as benzyl mercaptan or thiophenol (Dawson et al., 1997). These compounds react with the initial a-alkyl thioester to form another intermediate, an aryl thioester, which is more reactive toward the N-terminal cysteine on the other peptide to be coupled. A study... 

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. 

The simplest approach to isosteric replacement of one or both sulfur atoms of the cystine disulfide with a methylene or ethylene moiety is given for natural bioactive peptides when one cysteine residue is located in the N-terminal sequence position and the related amino group or peptide extension is not involved in the bioactivity. This allows for direct side chain to backbone (N-terminus) cyclization via amide bonds with suitable 5-carboxyalkyl derivatives of the second cysteine residue, or with the oo-carboxy group of aminodicarboxylic adds containing an alkyl side chain that mimics the Ca to Ca spacer in cystine. Thereby, the length and degree of branching of the sulfide or alkyl spacer can additionally be varied. 

Fig. 1.25. Regulation of alkylation repair in E. coK by methylation of the Ada protein. The effect of methylating agents, such as N-nitroso-N-methyl urea lead to the formation of methyl phospho-triesters (P-Me) of DNA, as well as various base adducts. The Ada protein possesses an N-termi-nal and a C-terminal domain. In one of the first steps of alkylation repair the methyl groups of the phosphotriester is transferred to the Ada protein. The Ada protein is methylated on a Cys residue at its N-terminal domain and thereby transformed into an active transcription activator. In its methylated form the Ada protein binds to the control region of various genes to stimulate their transcription. Among the genes under the control of the Ada protein are its own gene, as well others required for DNA repair (alkB, alkA). After Lindahl et al., 1988.

Use of hydrazine hydrate and alkyl hydrazines results in azapeptides with the corresponding N-terminal unprotected azaamino acid residues. 11 Reaction at the substituted amino group of the hydrazine is favored, affording predominantly the correct (a-sub-stituted) product. 

The assumption is an all-or-none normalization of each of the 3 residues. It is interesting how well this appears to explain the data although there is no a priori reason why that should be so. The residues initially labeled A, B, and C have been tentatively identified as Tyr 25, 92, and 97, respectively (300). The normalization of Tyr 92 (B) causes a decrease in molar absorbance at 287 nm of 700 with little change in either viscosity or rotation. Of the three this is the most accessible residue in the X-ray structure. The normalization of the other two causes changes of 1000 each in absorbance and is accompanied by both viscosity and rotation changes. The second to normalize is Tyr 25 (A) presumably by dissociation of the N-terminal portion of the chain from the body of the molecule, which would expose this residue as in S-protein. The last is Tyr 97 (C) whose exposure requires disruption of the entire structure. This residue is also the most buried of all the tyrosine residues according to the X-ray structure. However, the accessibility of Tyr 97 to chemical modification in S-protein and Met 30 to alkylation in RNase-A indicate that the region around Tyr 97 may be easily deformable. If this is so, why should Tyr 97 be the last to normalize. 

In this method, the cysteine-thioester cyclization generates a cyclic peptide 86a (see Scheme 23) with a Xaa-Cys bond whose thiol moiety is then used for tethering to the core through an S-alkylation reaction.191 The requirement for the cyclization reaction is a linear precursor 84 containing both an N-terminal Cys and a C-terminal thioester. Such a peptide precursor can be conveniently synthesized by a stepwise solid-phase synthesis on a thioester resin 81 using Boc chemistry (Scheme 22). Cleavage by HF after assembly of the peptide sequence will produce the desired precursor with an N-terminal Cys and a C-terminal thioester 84. The crude peptide is then purified by RP-HPLC and the purified unprotected peptide is then circularized in aqueous conditions buffered at pH > 7.0. 

As continuation and extension of the parallel synthesis and libraries from libraries concept, there is the simultaneous synthesis of various libraries (i.e., a resin-bound library is converted in parallel into several new libraries employing different reagents). Figure 12 illustrates the strategy. The initial step is the preparation of the dipeptide library 45. Introduction of the benzyl group was achieved by selective N-alkylation of N-terminally trityl-protected resin-bound amino acids in the presence of lithium f-butox-ide and benzyl bromide. As expected, the alkylation of the amide nitrogen... 

Table 5.8 Stereoselective N-terminal alkylation of dipeptides by chiral phase-transfer catalysis.

Asymmetric phase-transfer catalysis with (S,S)-lg can be successfully extended to the stereoselective N-terminal alkylation of Gly-Ala-Phe derivative 61 (i.e., the asymmetric synthesis of tripeptides), where (S,S)-lg turned out to be a matched catalyst in the benzylation of DL-61, leading to the almost exclusive formation of DDL-62. This tendency for stereochemical communication was consistent in the phase-transfer alkylation of DDL-63, and the corresponding protected tetrapeptide DDDL-64 was obtained in 90% yield with excellent stereochemical control (94% de) (Scheme 5.30) [31]. 

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