Dipeptide buffers buffering

FIGURE 2.17 Anserine (AA/3-alanyl-3-methyl-L-hisddine) is an important dipeptide buffer in the maintenance of intracellular pH in some tissues. The structure shown is the predominant ionic species at pH 7. p. i (COOH)... 

The P-alanyl dipeptides carnosine and anserine (A -methylcarnosine) (Figure 31-2) activate myosin ATPase, chelate copper, and enhance copper uptake. P-Alanyl-imidazole buffers the pH of anaerobically contracting skeletal muscle. Biosynthesis of carnosine is catalyzed by carnosine synthetase in a two-stage reaction that involves initial formation of an enzyme-bound acyl-adenylate of P-alanine and subsequent transfer of the P-alanyl moiety to L-histidine. 

When the log /J/pH measurement of a peptide is performed by the shake-flask or the partition chromatography method (using hydrophilic buffers to control pH), usually the shape of the curve is that of a parabola (see Ref. 371 and Fig. 1 in Ref. 282), where the maximum log I) value corresponds to the pH at the isoelectric point (near pH 5-6). Surprisingly, when the potentiometric method is used to characterize the same peptide [275], the curve produced is a step function, as indicated by the thick line in Fig. 4.5 for dipeptide Trp-Phe. 

FIGURE 1.28 Chromatograms of the HPLC enantiomer separation of Z-protected a-aminophosphinic acids (a,b) and phosphinic acid-ilr-dipeptide (c) on (a and b) a 0-9-(tert-butylcarbamoyl)quinidine CSP and (c) corresponding 0-9-(fcrr-butylcarbamoyl)qninine-CSP, respectively. Experimental conditions Column dimensions, 150 mm x 4 mm ID mobile phase, methanol-50 mM sodium phosphate buffer (80 20 v/v) (pHa 5.6) temperature, 40°C flow rate, 1 mLmin detection, UV at 250 nm and optical rotation detection (ORD). (Reproduced from M. Lammerhofer et ah, Tetrahedron Asymmetry, 14 2557 (2003). With permission.)... 

FIGURE 1.30 Micro-HPLC separation of all 4 stereoisomers of the dipeptide alanyl-alanine as FMOC derivatives (a) and DNP-derivatives (b), respectively, on a 0-9-(tert-butylcarbamoyl)quinine-based CSP. Experimental conditions Column dimension, 150 X 0.5 mm ID mobile phase (a) acetonitrile-methanol (80 20 v/v) containing 400 mM acetic acid and 4 mM triethylamine, and (b) methanol-0.5 M ammonium acetate buffer (80 20 v/v) (pHa 6.0) flow rate, 10 ixLmin temperature, 25 C injection volume, 250 nL detection, UV at 250 nm. (Reproduced fromC. Czerwenka et al., J. Pharm. Biomed. Anal., 30 1789 (2003). With permission.)... 

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. 

Harris, R. C., Marlin, D. J., Dunnett, M., Snow, D. H., and Hultman, E. (1990). Muscle buffering capacity and dipeptide content in the thoroughbred horse, greyhound dog and man. Comp. Biochem. Physiol. 97A, 249-251. 

Conversely, when 6 was extended N-terminally by a small portion of the prosequence, that is by the Lys-Arg dipeptide to give KR-ET-1 (7), and subjected to oxidation in 0.1 M NH4OAC buffer (pH 9.5) at 25 °C for 24 hours the ratio of native to nonnative-type disulfide isomer increases remarkably (88 12 vs 75 25 for 6), whilst isomer 3 is not detectable. In the presence of GSH/GSSG an additional increase to almost quantitative formation of the native isomer was observed (Table 2). 58 This improvement was completely abolished by substituting Asn for Asp at position 8 (D8N-KR-ET-1), whereas most of the increase was maintained with similar carboxamide analogues in positions 10 and 18 (Table 2). 

All of the a-amino-substituted carboxylic acid 4-hydroxyphenacyl phototriggers have proved to be unstable in aqueous environments, hydrolyzing to the free amino acid and the a-hydroxy phototrigger. Nevertheless, the photocaged dipeptide, 4-hydroxyphenacyl alanyl-alanylate is stable to aqueous and buffered media 253 ... 

Piperazine-2,5-dione formation can occur not only in the case of dipeptides esters or amides containing a free amino group, but also during the activation of N-blocked or protected IV-alkyl amino acids containing dipeptides or even tripeptides. Thus, the activated dipeptide Z-Gly-Pro-ONp (76) afforded the Z-protected piperazine-2,5-dione 77 when subjected to buffered dioxane solution at pH 8 (Scheme 29)J159 ... 

The key operational parameter in free-solution capillary electrophoresis is the pH of the running buffer, as the electroosmotic flow and ionization of the analyte can be regulated by this variable. The role of buffers in capillary electrophoresis has been discussed in detail, with emphasis on buffer concentration, buffer type, and pH effects [10]. The effect of organic solvents on separation and migration behavior has been studied for dipeptides [11] and somatostatin analog peptides [12]. The order of migration as well as the selectivity may be manipulated by organic modifiers in... 

Figure 5.4 shows the separation of histidine-containing dipeptides by CZE using a buffer containing zinc ions.7 Copper ions or zinc ions in acetate or phosphate buffers (pH 2.5) were used, with good results. In the absence of metal ions, little resolution was evident, but as the metal ion concentra-... 

Method A A soln of the iV-(P-D-glucopyranosyloxycarbonyl)dipeptide ester (0.2 mmol) in MeOH (10 mL) was added to a soln of P-gJucosidase (40 units) in 0.25 M acetate buffer (pH 5.0, 190 mL). The reaction mixture was shaken for 12-36 h at 37 °C, the pH was adjusted to 10-11 and the soln was extracted with EtOH (4xl00mL). The combined organic phases were dried (MgS04), filtered, and the solvent removed to give the N-terminal deprotected dipeptide esters as colorless oils. 

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