Sensitizer residues

Zarbiv, R., Grunewald, M Kavanaugh, M. P., and Kanner, B. I. (1998) Cysteine scanning of the surroundings of an alkali-ion binding site of the glutamate transporter GLT-1 reveals a conformationally sensitive residue. J. Biol. Chem. 273,14231-14237. 

Androutsellis-Theotokis, A., Ghassemi, F and Rudnick, G. (2001) A conformationally sensitive residue on the cytoplasmic surface of serotonin transporter. J. Biol. Chem. 9,9. 

The general conclusion to be drawn from the experience reported with these various oxidative methods is that with increasing reactivity of the oxidizing agents more side reactions are to be expected at sensitive amino acid residues, in particular at Met, Trp, and Tyr. In this context, the azodicarboxylic acid derivatives could represent a valid alternative 54,55 since these reagents are devoid of any side reaction at these sensitive residues 56 The reaction of di-ferf-butyl azodicarboxylate (1) with a peptide cysteine thiol leads to the intermediate formation of a sulfenohydrazide adduct 2 that reacts with the second thiol to generate the... 

Kuwahara M, Gu Y, Ishibashi K, Marumo F, Sasahi S. 1997. Mercury sensitive residues and pore site in AQP3 water channel. Biochemistry 36 13973-13978. 

Dimethyl Tellurium Difluoride1 3 The apparatus shown in the Figure is set up. 4.7 g (30 mmol) of dimethyl tellurium and 175 ml of fluorotrichloromethane are placed in the 500 ml reaction flask which is cooled to — 78° in an ethanol/dry ice bath. The rotameter and pressure controls are set to deliver a fluorine/nitrogen (or argon) mixture (1/10, v/v). The gas mixture is passed at a flow rate not exceeding 100 m//min through a metal spiral immersed in a — 78° cooling bath and then into the stirred solution of dimethyl tellurium. The reaction is complete after 75 mmol of fluorine have been passed into the flask. Dimethyl tellurium difluoride precipitates as a white solid. The solvent is removed by vacuum distillation under an inert atmosphere and the moisture-sensitive residue is pure dimethyl tellurium difluoride yield 1.3 g (22%) m.p. 82° sublimes at 40D/0.001 torr. 

Cleavage of the 5-(acetamidomethyl) group is achieved with metal ions such as mercury(II) in aqueous acetic acid or silver(I) in TFA.b In the latter case, the trifluoromethane-sulfonate salt was found to be superior to the trifluoroacetate, and silver tetrafluoroborate is an efficient alternative. While sensitive residues such as Met, Trp, and Tyr are largely unaffected by cleavage with silver(I) salts, mercury(II)-mediated cleavage has been reported to give side reactions at Trp residues, P which can be suppressed by operating in the... 

Excellent Sensitivity Residual error plots Appearance of trending indicates a need to improve the model or to remove data that are inconsistent with the Kramers-Kronig relations. 

Several groups have adapted the method to analyze residues in a variety of matrices. Acetic acid (HQAc, 1%) and sodium acetate have been widely used to adjust and maintain pH and promote stability and recovery of base-sensitive residues. HOAc was used to adjust pH by Stubbings and Bigwood to determine residues [sulfonamides, quinolones, (fluoro)quinolones, ionophores, and nitroimidazoles] in chicken muscle. Buffering to acidic conditions improved the extraction efficiency of quinolones. Aeetonitrile extracts were subsequently purified by dSPE (see also Section 4.4.6.1) over Bondesil NH2 sorbent. An aliquot of the extracts was evaporated to dryness and re-dissolved in acetonitrile water (90 10, v/v) before LC-MS/MS analysis. Validation was performed on chicken muscle samples, and matrix-matched standards were used because suppression of the MS response was observed for many of the target analytes. 

Suitable oxidative reagents include iodine (38), Tl(tfa)3 (23), and alkyltri-chlorosilanes-sulphoxide (31). The deprotection/oxidation step can be carried out either in solution, once the free peptide has been cleaved from the resin, or while the protected peptide is still on the resin. In a variation on the theme, silver-mediated deprotection can be followed immediately by a mild oxidizing environment, e.g. addic DMSO (40,41, 95). As specified in the subsections that follow, caution is required when peptide substrates contain sensitive residues, especially Met and Trp (38,96). 

In case the signature matrix is not diagonal, Samantaray and Ghoshal use parameter estimation for isolation of simultaneous faults [11]. Parameters are estimated by least squares optimisation of residuals. In that approach, values for sensitivities of residuals with respect to parameters are needed. Beyond this optimisation problem, knowledge of how sensitive residuals are with respect to certain parameters helps assessing the information in a fault signature matrix. 

Solid-phase ssmthesis is based on the covalent attachment (anchoring) of the growing peptide chain to an insoluble polymeric support or resin carrier, so that unreacted soluble reagents can be removed by filtration and washing without loss of the peptide-resin. Subsequently, the insoluble peptide-resin is extended by a series of addition cycles, which are required to proceed with high yields and fidelities. Excess soluble reagents drive the reactions to completion. Because of the speed and simplicity of the repetitive steps, the solid-phase procedure is amenable to automation. After the chain has been established, it is necessary to release (cleave) the crude peptide from the support imder conditions that do not affect sensitive residues in the sequence. Purification and characterization follow to ensure and verify that the desired structure has been obtained. 

Preston GM, Jung JS, Guggino WB, Agre P (1993) The mercury-sensitive residue at cysteine 189 in the CHIP28 water channel. J Biol Chem 268 17-20... 

Disulfide bridges play a fundamental role in maintaining biologically active conformations of several natural and synthetic peptides. There are several methods by which intramolecular multiple disulfide bonds are formed in synthetic peptides and proteins. The disulfide bond formation is accomplished using either an oxidizing agent or mild basic conditions. All methods have several limitations, including side reactions of oxidation sensitive residues, use of hazardous oxidants and difficulties in their post oxidation removal. 

Table 1. Average surface accessibilities (x) and the differences of the surface accessibility areas of CIDNP-sensitive residues derived from molecular dynamics calculations between the wild-type of EcorL (x) and the three single-site mutants [7]. All area values are given in A. Dot density 1 test sphere radius 1.5 A. denotes the site of mutation substituting the respective tyrosine residue with CIDNP-inert alanine.

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