Peptides derivatizing agents

Most HPLC instruments monitor sample elution via ultraviolet (UV) light absorption, so the technique is most useful for molecules that absorb UV. Pure amino acids generally do not absorb UV therefore, they normally must be chemically derivatized (structurally altered) before HPLC analysis is possible. The need to derivatize increases the complexity of the methods. Examples of derivatizing agents include o-phthaldehyde, dansyl chloride, and phenylisothiocyanate. Peptides, proteins, amino acids cleaved from polypeptide chains, nucleotides, and nucleic acid fragments all absorb UV, so derivatization is not required for these molecules. 

Chemical derivatization of an analyte is performed to improve the selectivity and sensitivity of that analyte. There are two approaches to the use of derivatizing agents in HPLC. Precolumn derivatization, which is performed before the analytes are separated, is most commonly used for small molecules such as amino acids, whereas postcolumn derivatization, which is performed after the separation but before the analytes reach the detector, is often used for larger molecules such as peptides. 

Peptides are commonly detected by absorbance at 200-220 nm. However, most of the compounds present in wine may interfere in the ultraviolet detection of peptides when low wavelengths are used. Thus, for the analysis of these compounds it is useful to apply sensitive and selective detection methods. To this end, it is possible to form derivates of the peptides that can be detected at higher and more specific wavelengths. Detection by fluorescence can also be used to detect peptides containing fluorescence amino acids (tyrosine and tryptophan). For peptides without this property, the formation of derivates with derivatizing agents have been proved to be very useful (Moreno-Arribas et al. 1998a). 

The amino acid composition of peptides is generally assayed by carrying out acid hydrolysis with 6 M hydrochloric acid, followed by determination of the individual amino acids by HPLC. Various types of HPLC have been employed to separate amino acids, but reversed-phase chromatography on Cigcoiumns is the most commonly system used. Detection of amino acids normally involves derivatization, since their maximum absorbance is at 214 nm in which many other compounds also can absorb. The most frequently used derivatizing agents are dansyl chloride, phenyl isothiocyanate and o-phthaldialdehyde. 

For those without native fluorescence, two common approaches have been employed, namely, derivatization and indirect fluorescence. Derivatizing agents should be pure, low fluorescent, and stable, as well as react quickly and uniquely with the analytes and, thus, formed compounds should be strongly fluorescent and stable. These include dansyl chloride, fluorescamine, 4-clair-7-nitro-benz-2-oxa-l,3-diazole (NBD), o-phthaldialdehyde (OPA), fluorescein isothiocyanate (FITC), and naphthalene-2,3-dicarboxaldehyde (NDA), which have been used for the analyses of amino acids, peptides, proteins, thiols, and sugars with LOD in 1-100 nM range. Compared to LINF, approaches based on derivatization provide the advantages of relatively low cost and versatility in instrumentation, but they may suffer from contamination and loss of temporal information. 

Amino acid ester isocyanates are useful synthetic building blocks, precursors to peptides and azapeptides, 7 8 chiral derivatizing agents,9 13 and reagents for the preparation of chiral chromatographic media.11 12 (S)-2-lsocyanato-3-phenylpropanoate (phenylalanine methyl ester isocyanate) has been used as a... 

USE Derivatizing agent for primary, secondary amines, la sequencing of peptides by Ed man degradation. In amino add analyses by HPLC (Pico-Tag). 

The identification of N-terminal amino acids in peptides and proteins is of considerable practical importance because it constitutes an essential step in the process of sequential analysis of peptide structures. Many N-amino acid derivatives have been proposed for this purpose and the ones most commonly studied by TLC are 2,4-dinitrophenyl (DNP)-and 5-dimethylaminonaphthalene-l-sulfonyl (dansyl, Dns)-amino acids, and 3-phenyl-2-thiohydantoins (PTH-amino acids). Recently, 4-(dimethylamino)azobenzaie-4/-isothio-cyanate (DABITC) and phenyl-isothiocyanate (PITC) have also been investigated as derivatizing agents of amino acids. 

Peptides may also be delected nsing derivatizing agents to impart electrochemical, UV, or fluorescence properties on the analyte of interest. Thongh a vast number of possible derivatizing reagents exist, discussion here will be limited to those that have been used with electrochemical detection. 

Amino Acid Composition and Peptide Structure. Similar to some of the previously mentioned derivatization agents, peptides containing electroactive amino acids exhibit increased anodic sensitivities over nonelectroactive peptides in the presence of Cu(II), resulting from the additive effect of the amino acid and Cu(II) oxidation. At the cathode, sensitivities of electroactive peptides are equal to those for nonelectroactive peptides except for shorter tripeptides [157] where, in some cases, cathode signals are repressed by the presence of tyrosine. 

There is little doubt that of the various methods available for the indirect HPLC resolution of enantiomers of amino acids, use of MR has been most successful. The reagent meets satisfactorily the characteristic features, as enumerated above, as a derivatizing agent in different situations. Marfey s method has been widely used for structural characterization of peptides, confirmation of racemization in peptide synthesis, and detection of small quantities of D-amino acids. Many other reagents used for prederivatization of free amino acids suffer inherent problems, for example, inability to react with all proteinogenic amino acids, unstable derivatives, poor detectability of certain amino acid derivatives, or lack of quantitative yield of the reaction. 

Amines are another important group of analytes. Mellbin and Smith [72] compared three different fluorescent reagents, dansyl chloride, 4-chloro-7-nitrobenzo-1,2,5-oxadiazole, and o-phthaldialdehyde, for derivatization of alkylamines. The dansyl tag was found to be the most effective. Hamachi et al. [73] described the application of an HPLC-POCL method for determination of a fluorescent derivative of the synthetic peptide ebiratide. Another comparative study was done by Kwakman et al. [74], where naphthalene-2,3-dialdehyde and anthracene-2,3-dial-dehyde were evaluated as precolumn labeling agents for primary amines. The anthracene-2,3-dialdehyde derivatives were not stable, especially in the presence of hydrogen peroxide, and the POCL detection of these derivatives was therefore... 

Three approaches can be employed to separate peptide stereoisomers and amino acid enantiomers separations on chiral columns, separations on achiral stationary phases with mobile phases containing chiral selectors, and precolumn derivatization with chiral agents [111]. Cyclodextrins are most often used for the preparation of chiral columns and as chiral selectors in mobile phases. Macrocyclic antibiotics have also been used as chiral selectors [126]. Very recently, Ilsz et al. [127] reviewed HPLC separation of small peptides and amino acids on macrocyclic antibiotic-based chiral stationary phases. 

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