Active residues

On the other hand, potential measurements at the free surface of purified water have shown50 that the value for a flowing surface differs by about 0.3 V from that for a quiescent surface, as a result of adsorption of surface-active residual impurities in the solution (probably also coming from the gas phase). Since emersed electrodes drag off the surface layer of the solution as they come out of the liquid phase, the liquid layer attached to emersed solid surfaces might also be contaminated. 

There are two catalytically active residues in pepsin Asp-32 and Asp-215. Their ionizations are seen in the pH-activity profile, which has an optimum at pH 2 to 3, and which depends upon the acidic form of a group of pKa 4.5 and the basic form of a group of pKa 1.1.160,161 The pKa values have been assigned from the reactions of irreversible inhibitors that are designed to react specifically with ionized or un-ionized carboxyl groups. Diazo compounds—such as A-diazoacetyl-L-phenylalanine methyl ester, which reacts with un-ionized carboxyls—react specifically with Asp-215 up to pH 5 or so (equation 16.28).162-164 Epoxides, which react specifically with ionized carboxyls, modify Asp-32 (equation 16.29). 

HARVEST [Highly Active Residue Vitrification Experimental Studies] A process for immobilizing nuclear waste by incorporation in a borosilicate glass. Developed from FIN-GAL. Piloted by the UK Atomic Energy Authority at Sellafield, in the late 1970s, but abandoned in 1981 in favor of AVM, the French vitrification process. 

When Wa = RC(=0), that is, acyl (Figure 1.11), Wa is not removable without destroying the peptide bond. When Wa = ROC(=0) with the appropriate R, the 0C(=0)-NH bond of the urethane is cleavable. When Wb = NHR, Wb is not removable without destroying the peptide bond. When Wb = OR, the 0=C-0R bond of the ester is cleavable. During activation and coupling, activated residue Xaa may undergo isomerization, and aminolyzing residue Xbb is not susceptible to isomerization. 

More /V-acylurea is generated if tertiary amine is present because the latter removes any protons that might prevent the rearrangement (see Section 2.12). The two intramolecular reactions also occur to a greater extent when interaction between the O-acylisourea and the /V-nucleophile is impeded by the side chain of the activated residue. This means that more 2-alkoxy-5(4//)-oxazolone and /V-acylurea are generated when the activated residues are hindered (see Section 1.4). A corollary of the above is that the best way to prepare an /V-acylurea, should it be needed, is to heat... 

NL Benoiton, YC Lee, R Steinauer, FMF Chen. Studies on the sensitivity to racem-ization of activated residues in couplings of /V-bcnzyloxycarbonyldipeptides. Int J... 

CONSTITUTIONAL FACTORS THAT DEFINE THE EXTENT OF STEREOMUTATION DURING COUPLING THE N-SUBSTITUENT OF THE ACTIVATED RESIDUE OR THE PENULTIMATE RESIDUE... 

FIGURE 4.11 Data showing the effect of the /V -substituent of the activated residue on stereomutation.31 Percentable -d-l-isomer formed in couplings in dimethylformamide at+5°C. Ester.HCl salts neutralized with /V-methyl morpholi ne. MxAn = Mixed anhydride using ClC02iPr with 5-minute activation time at -5°C. DCC = dicyclohexylcarbodiimide, HOBt = 1-hydroxybenzotriazole. 

Enantiomerization of the activated residue is affected by the nature of the aminolyzing residue as well as the nature of its carboxy substituent. The more hindered the incoming nucleophile, the slower the coupling rate, and hence the greater the danger for isomerization. In apparent disaccord with this is the observation that more... 

It has been known for years that the activated residues of acyl- and peptidylamino acids enantiomerize during coupling (1.9). However, the racemization tests available (see section 4.9) did not allow for a valid comparison of the tendency of residues to isomerize because they incorporated a variety of aminolyzing residues and N-substituents. Valid demonstration of the different sensitivities of residues was provided by classical work on the synthesis of insulin. It was found that a 16-residue segment with O-tert-butyltyrosine at the carboxy terminus produced 25% of epimer in HOBt-assisted DCC-mediated coupling in dimethylformamide, and the same segment with leucine at the carboxy terminus produced no epimer. Only when series such as Z-Gly-Xaa-OH coupled with valine benzyl ester became available was it possible to compare many residues with confidence. Unfortunately, it transpires that the issue is extremely complex. 

The danger of epimerization during the coupling of segments exists for all cases, except when me activated residue is Pro and Gly, with a few exceptions (see Section 7.23). The obvious is to design a strategy mat involves activation at these residues only. Options to try to minimize me side reaction for activation at other residues are as follows ... 

During the first decade when solid-phase synthesis was executed using Fmoc/tBu chemistry, the first Fmoc-amino acid was anchored to the support by reaction of the symmetrical anhydride with the hydroxymethylphenyl group of the linker or support. Because this is an esterification reaction that does not occur readily, 4-dimethylaminopyridine was employed as catalyst. The basic catalyst caused up to 6% enantiomerization of the activated residue (see Section 4.19). Diminution of the amount of catalyst to one-tenth of an equivalent (Figure 5.21, A) reduced the isomerization substantially but did not suppress it completely. As a consequence, the products synthesized during that decade were usually contaminated with a small amount of the epimer. In addition, the basic catalyst was responsible for a second side reaction namely, the premature removal of Fmoc protector, which led to loading of some dimer of the first residue. Nothing could be done about the situation,... 

FIGURE 6.10 The side chain of histidine is readily acylated (A) by activated residues. The imidazolide produced is an activated species similar to the intermediate generated by reaction (B) of a carboxylic acid with coupling reagent carbonyldiimidazole. (Staab, 1956). Imida-zolides acylate amino and hydroxyl groups. Isomerization of histidyl during activation results from abstraction (C) of the a-proton by the 7t-nitrogen. 

Peptide bond formation involves activation of the carboxyl group of an amino acid residue, followed by aminolysis of the activated residue by the amino group of a second amino acid residue. Two types of activated molecules are recognized those that are not detectable but are postulated and those that are detectable and can be isolated. Postulated intermediates are necessary to account for the formation of the detectable intermediates. The postulated intermediates are consumed as fast as they are formed, either by aminolysis by an amino group or by nucleophilic attack by an oxygen nucleophile, which produces activated molecules that are also immediate precursors of the peptide. More than one activated compound may be generated by a postulated intermediate. Activated esters, acyl halides and azides, and mixed and symmetrical anhydrides are isolatable activated compounds that are generated from postulated intermediates. Peptides are produced by one of three ways ... 

Pyridine (1) is a weak and good base and good solvent for effecting aminolysis of acyl fluorides (see Section 7.12) and for preparing Boc-amino-acid iV-carboxyanhydrides (see Section 7.14) and activated esters by the carbo-diimide method (see Section 7.7), especially the esters of Boc-amino acids, as it prevents decomposition of the activated residue (see Section 7.15). It is the preferred base for aminolysis of acyl fluorides in dichloromethane. 

Trimethylpyridine (collidine) (2) is more basic than pyridine and is recommended as being superior for minimizing enantiomerization of the activated residue during o/inium salt-mediated reactions of protected segments with an amino group and Fmoc-Cys(R)-OH with the hydroxyl group of a linker-resin (see Section 8.10). 

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