PTC derivatives

A major advance was devised by Pehr Edman (University of Lund Sweden) that has become the standard method for N terminal residue analysis The Edman degrada tion IS based on the chemistry shown m Figure 27 12 A peptide reacts with phenyl iso thiocyanate to give a phenylthwcarbamoyl (PTC) denvative as shown m the first step This PTC derivative is then treated with an acid m an anhydrous medium (Edman used mtromethane saturated with hydrogen chloride) to cleave the amide bond between the N terminal ammo acid and the remainder of the peptide No other peptide bonds are cleaved m this step as amide bond hydrolysis requires water When the PTC derivative IS treated with acid m an anhydrous medium the sulfur atom of the C=S unit acts as... 

Step 1 A peptide is treated with phenyl isothiocyanate to give a phenylthiocarbamoyl (PTC) derivative... 

Step 2 On reaction with hydrogen chloride m an anhydrous solvent the thiocarbonyl sulfur of the PTC derivative attacks the carbonyl carbon of the N terminal ammo acid The N terminal ammo acid is cleaved as a thiazolone derivative from the remainder of the peptide... 

In all cases, previously reported polymer-bound PTCs are ones which contain one PTC site/functionalized arm of polymer. Because of the ability to vary the nature of the substrate introduced onto the polymer arm, the preparation of a polymer-bound PTC with more than one PTC site/ functionalized arm of polymer becomes possible. In theory, then, the number of grams of polymer backbone material needed to carry a particular level of required PTC active-site equivalency would be less for a "multi-site substance compared to previously reported polymer-bound "single-site" PTCs. Similar considerations apply as well to nonpolymeric "multi-site" PTCs derived from simple, polyhalo substrates. Therefore, in general, "multi-site" PTCs offer the potential (1) of providing greater PTC activity on a PTC site/g of PTC needed for catalytic activity basis and (ii) of effecting a particular synthetic transformation under milder and/or more efficient conditions. Thus, economy of scale and efficiency are important considerations for both polymeric and nonpolymeric "multi-site" PTCs. 

In the first stage — coupling — a phenylisothiocyanate (PITC) is reacted with the a-amino group of the N-terminal residue to form a phenylthiocarbamyl (PTC) derivative. 

In order to determine the carboxy-terminus of Hez-PBAN,. 200 pmol of purified peptide was digested with carboxypeptidase P. Released amino acids were periodically analyzed as their PTC derivatives. Leu was found to be the C-terminal residue followed by Arg, Pro, Ser, Phe, Tyr, and Lys, respectively, thus confirming the automated Edman degradation data (Table I, run 5). However, none of the data could distinguish between a C-terminal amide or free acid. 

Future perspectives on ptCs derived from Cj (and its experimental detection)... 

In addition to stability issues, cyclization to the ATZ and cleavage occurs considerably more slowly with proUne than with other residues, and conversion of the ATZ of glycine to the PTH is particularly slow. If a sequencer is inadvertently allowed to continue running without the conversion reagent (25% TEA), the problem is often detected by seeing an additional, earlier elution peak at each glycine cycle, while other residues are unaffected. The earlier peak is the more polar (open chain) PTC derivative. All other residues seem to convert quite well without 25% TEA, perhaps the result of tenaciously bound residual TEA. ... 

Figure 4.26 Chemisorbed PTCs derived via surface chlorination...

Applied Biosystems). Recently, a system for the separation of the less stable phenylthiocarbamyl (PTC) derivatives was published (Heinrik-son and Meredith, 1984) and a commercial kit is now available (Waters, Pico-Tag) which greatly enhances not only the speed of analysis, but also increases the sensitivity of detection to the low picomole range. The system is reproducible but requires a skilled operator. 

There is a wide choice of amino acid derivatives that can be used. Phenylisothlocyanate (PITC) reacts with both primary and secondary amino groups to form moderately stable phenylthlocarbamyl (PTC) derivatives that are separated on a reversed-phase column and can be detected in a UV detector at 254 nm wavelength (13). Detection limits are at about the plcomole level. Sample derivatization with PITC takes around 20 minutes and requires close attention to details if consistent results are to be obtained. The presence of salts such as NaCl in the sample interferes with the derivatization of several of the amino acids, and care must be taken in processing samples containing salts. 

Numerous chemical derivatization strategies have been developed also to direct the fragmentation reactions of protonated peptides toward the selective formation of single characteristic sequence-type product ions [85-87]. For example, Sununerfield and coworkers have demonstrated that N-terminal derivatization, with phenyliso-thiocyanate to form the corresponding phenylthiocarbamoyl (PTC) derivative, results in exclusive fragmentation of the amide bond between the first two amino... 

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