Reagents phenylisothiocyanate

FIGURE 5.19 N-Tertninal analysis using Edman s reagent, phenylisothiocyanate. Phenylisothiocyanate combines with the N-terminus of a peptide under mildly alkaline conditions to form a phenylthiocarbamoyl substitution. Upon treatment with TFA (trifluo-roacetic acid), this cyclizes to release the N-terminal amino acid residue as a thiazolinone derivative, but the other peptide bonds are not hydrolyzed. Organic extraction and treatment with aqueous acid yield the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative. 

The primary structure (i.e., the amino acid sequence) of a protein can be determined by stepwise chemical degradation of the purified protein. By far the most powerful and commonly used technique for doing this is the automated Edman degradation. The amino terminal amino acid residue of the polypeptide is reacted with Edman s reagent (phenylisothiocyanate) to form the phenylthiocar-bamyl derivative, which is removed without hydrolysis of the other peptide bonds by cyclization in anhydrous acid. The amino acid derivative is converted to the more stable phenylthiohydantoin and identified by HPLC. The process can be repeated many times, removing the amino acids from the amino terminus of the polypeptide one residue at a time and identifying them until the entire sequence... 

The precolumn technique that is most frequently employed today was developed during the early 1980s [32,33]. For this method, The classical Edman reagent phenylisothiocyanate (PITC) is used for amino acid derivatization after hydrolysis. Separation of the PTC amino acids i then accomplished by HPLG, with detection at 254 nm. Although standard Cig columns available Irom a variety of vendors are suitable for separation of the PTC-derivatized amino acids, there are specific columns that have bqen optimized for this purpose (e.g.. Waters). Approximately 0.5 /ug of peptMe should be hydrolyzed for analyses using precolumn derivatization. ... 

The new N-terminal residue can then be removed as a phenylhydantoin in a second cycle of phenylcarbamoylation and cyclization. This method of stepwise degradation, while discovered several decades earlier (Bergmann et al. 1927), reached practicality only in 1950 when Edman modified the reagent and applied chromatographic procedures for identification of the cyclic products. The improved reagent, phenylisothiocyanate, smoothly converts the peptide to the phenylthiocarbamoyl derivative which is cyclized and cleaved by the action of hydrochloric acid (dissolved in an organic solvent such as dioxane) ... 

A drawback to conventional amino analysis by chromatography is the need for pre- or post-column derivatization to improve sensitivity. Ninhy-drin, the reagent originally applied for detection, has been increasingly displaced by other reagents such as phenylisothiocyanate,71 9-fluorenylethyl chloroformate,72 and o-phthaldialdehyde (OPA). OPA allows fluorimetric detection, which offers the potential for greater sensitivity.73 A limitation of OPA is that it doesn t derivatize secondary amines, so an additional reaction must be added for proline detection. And, as noted for amine analysis in section A5.4.2, such derivatization adds to the analysis time and may yield unstable products. 

Two other reagents used in HPLC are 9-fluorenyl methoxycarbonyl chloride (FMOC) and phenylisothiocyanate (PITC). 9-fluorenyl methoxycarbonyl chloride is becoming increasingly popular in protein chemistry research because it reacts with secondary amines and also offers rapid analysis of protein hydrolysates. 

Sequencing is a stepwise process of identifying the specific amino acids at each position in the peptide chain, beginning at the N-terminal end. Phenylisothiocyanate, known as Edman s reagent, is... 

An indirect method makes possible the general use of lithium as well as magnesium organometallic reagents for the synthesis of dithioesters their addition to phenylisothiocyanate followed by alkylation gives A-phenylimidothioesters (themselves valuable intermediates), which are readily sulfhydrolysed [151,152],... 

Synthesis of saturated and unsaturated dithioesters through reaction of Grignard reagents with phenylisothiocyanate, alkylation and sulfhydrolysis... 

With allylic Grignard reagents, the carbophilic addition on the thiocarbonyl of phenylisothiocyanate occurred as expected exclusively with inversion of the allylic chain. From crotyl and prenyl magnesium halides the compounds (4f) and (4g) were obtained in yields of 77 and 72%, respectively. 

PITC (phenylisothiocyanate) Aabs = 254 nm. Phenylthiocarbamyl amino acid derivatives are moderately stable at room temperature (1 day). PITC reacts well with both primary and secondary amino acids. Reaction time is approximately 5 minutes at room temperature. Excess reagent must subsequently be removed under vacuum. Also, for hydrolyzed samples, hydrochloric acid must be completely removed prior to derivatization. As a result, even though the actual reaction time is reasonably fast, the total time for various sample manipulations can add up to 2 hours. This is partially compensated by the extremely fast separation possible (12 minutes). Detection is by UV absorption only. Detection limits are typically in the high picomole range. Short column life can result due to unreacted PITC getting into the column. Unlike some of the other reagents, PITC quantifies tyrosine and histidine very well. PITC analysis is available as a commercially prepackaged system dubbed Pico-Tag by Waters Corporation. Representative references include 184-188. See Fig. 11 for a typical separation. 

Dansyl chloride and phenylisothiocyanate (PITC) are the derivatizating agents most used in UV detection. Dansyl chloride reacts with the primary and secondary amino groups of peptides in a basic medium (pH 9.5), forming dansylated derivatives that are very stable to hydrolysis but are photosensitive. The derivatives are detectable in UV at 254 nm and by fluorescence. Dansyl sulfonic acid is formed as a by-product of the reaction, and excess reagent reacts with the dansyl derivatives to form dansyl amide the conditions of derivatization must therefore be optimized in order to avoid the formation of such by-products to the extent possible. The conditions of the reaction with dansyl chloride and of the separation of the derivatives thus formed have been thoroughly studied (83,84). Martin et al. (85) carried out derivatization using an excess concentration of dansyl chloride of 5 -10-fold in a basic medium (lithium carbonate, pH 9.5) in darkness for 1 h. 

The Edman degradation method for polypeptide sequence determination. The sequence is determined one amino acid at a time, starting from the amino-terminal end of the polypeptide. First the polypeptide is reacted with phenylisothiocyanate to form a polypeptidyl phenylthiocarbamyl derivative. Gentle hydrolysis releases the amino-terminal amino acid as a phenylthiohydantoin (PTH), which can be separated and detected spectrophoto-metrically. The remaining intact polypeptide, shortened by one amino acid, is then ready for further cycles of this procedure. A more sensitive reagent, dimethylaminoazobenzene isothiocyanate, can be used in place of phenylisothiocyanate. The chemistry is the same. 

PTC coupling reagent—Mix 21 ml of absolute ethanol, 3 ml of triethylamine, 3 ml of phenylisothiocyanate (Sigma catalog P-1034), and 3 ml of water. Store tightly covered at 4°C. 

---