Derivatization of amino acids

Pentafluorobenzyl bromide has been used in the derivatization of mercaptans [55] and phenols [36], m the analysis of prostaglandins [37], and in quantitative GC-MS [5S] 1,3 Dichlorotetrafluoroacetone is used for the derivatization of amino acids to the corresponding cyclic oxazolidinones and allows the rapid analysis of all 20 protein ammo acids [d] Pentafluorophenyldialkylchlorosilane derivatives have facilitated the gas chromatographic analysis of a wide range of functionally substituted organic compounds, including steroids, alcohols, phenols, amines, carboxylic acids, and chlorohydrms [4]... 

Fig. 7-8. Derivatization of amino acids with OPA and a thiol compound.

Derivatization of a racemic compound with an achiral group may play an important role in the analysis of a chiral compound (Fig. 7-15). In the case of substances with low or no UV-activity, the compounds can be rendered detectable by introducing an UV-absorbing or fluorescent group. If the racemate itself shows selectivity on a chiral stationary phase (CSP), this method can be applied to reduce the limit of detection. Examples have been reported in the literature, especially for the derivatization of amino acids which are difficult to detect using UV detection. Different derivatization strategies can be applied (Fig. 7-16). 

Liu Z, Minkler PE, Lin D, Sayre LM. 2004. Derivatization of amino acids with N,N-dimethyl-2,4-dinitro-5-fluorobenzy-lamine for liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 18 1059. 

CE has been applied extensively for the separation of chiral compounds in chemical and pharmaceutical analysis.First chiral separations were reported by Gozel et al. who separated the enantiomers of some dansylated amino acids by using diastereomeric complex formation with Cu " -aspartame. Later, Tran et al. demonstrated that such a separation was also possible by derivatization of amino acids with L-Marfey s reagent. Nishi et al. were able to separate some chiral pharmaceutical compounds by using bile salts as chiral selectors and as micellar surfactants. However, it was not until Fanali first showed the utilization of cyclodextrins as chiral selectors that a boom in the number of applications was noted. Cyclodextrins are added to the buffer electrolyte and a chiral recognition may... 

EHJM Jansen, RH van den Berg, R Both-Miedema, L Doom. Advantages and limitations of precolumn derivatization of amino acids with dabsyl chloride. J Chromatogr 553 123-133, 1991. 

The post-column derivatization of amino acids by the ninhydrin technique is a well known method for routine analysis of amino acids [7-9]. The amino acids are usually separated by ion-exchange chromatography and then converted into UV-absorbing derivatives for quantitation. The ninhydrin reaction is often used for TLC detection of amino acids and proteins. 

Derivatization of amino acids with [14C]DNS-C1 is very useful for protein sequencing [203,204]. The reactions involved are the same as described in the Fluorimetric Derivatization section (Section 4.2.1.1.1). The plates are observed under UV light for qualitative identification, but are quantitated by removing the spot from the plate for scintillation counting. Alternatively, the plates may be scanned with a radiochromatogram scanner. 

This section describes some derivatives of amino acids prepared by the condensation of a reagent with the amino group only or with both functional groups with the formation of a cyclic derivative. A number of reactions of this type have been tested for the derivatization of amino acids [191 ], mostly for a limited number. Some of the main reactions only will be mentioned here. 

Knapp, D. (1979). Derivatization of amino acids and peptides. Handbook of Analytical Derivatiza-tion. Wiley-Interscience, New York. pp. 243—302. 

Only for derivatization of amino acids with monofunctional amines nonvolatile products can be formed. "Bifunctional reagents the by-products are MeOH (EtOH) and DMFA (749 16). 

The typical example of one-stage derivatization of amino acids is their treatment by isopropyl bromide in presence LiH with the formation of N-isopropy-lated isopropyl esters. Unfortunately, this reaction can take place only in high-boiling aprotic bipolar solvents like dimethyl sulfoxide (DMSO, = 189°C, RInonpoiar 90 18) tWs is a significant restriction for its application, in practice. A more important method is based on the reaction of amino acids with methyl or phenyl isothiocyanates with the formation of 3-methyl (phenyl) hydantoins ... 

Pre-column derivatization—RPC analysis of phenylisothiocyanate (PITC), o-phthaldehyde (OPA), 9-fluorenylmethyl chloroformate (FMOC), or other derivatives of amino acids with UV or fluorescence detection is most common. This is the preferred methodology for life science research because of its higher sensitivity. More examples of precolumn derivatization of amino acids are shown in the life science section of this chapter. 

Precolumn derivatization. Derivatization of amino acids by NDA was carried out as follows a 250 pL sample solution in a 2 mL glass bottle was mixed sequentially with 750 pL 0.1 mol/L borate buffer (pH 9.25), 250 pL 2 mmol/L NDA methanol solution and 250 pL 20mmol/L KCN aqueous solution. The mixture was vortexed for 1 min and allowed to stand for 30 min at room temperature. The resulting mixture was subjected to analysis. 

Cyanuric chloride on monosubstitution with nucleophiles such as methanol or 4-amino-azobenzene followed by displacement of a second chlorine with alanine amide gave compounds which are used for precolumn derivatization of amino acids. The diastereoisomers formed are resolved by reverse-phase HPLC <92MI 6l2-0i>. Enantiomeric amino acids are separated by HPLC on bis[carbamoyl(alkyl)methylamino]-6-chloro-l,3,5-triazine derived stationary phases <93JC277>. 

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