Enantiomeric Purities of Amino Acids Using Carbohydrate-Based Isothiocyanates

By: Manfred P.Schneider, FB C – Bergische Universität Wuppertal, D-42097 Wuppertal, Germany schneid@uni-wuppertal.de, AnalytiX Volume 10 Article 2

Introduction

In the first paper of this series (Analytix Nr. 1/2010), an efficient enantiomeric analysis of a series of alkyl oxiranes was described, using inexpensive reversed-phase columns as an alternative to high-cost so-called “chiral columns”. The analysis utilised isothiocyanate derivatisation reagents based on monosaccharide such as BGITC (Figure 1). The oxiranes were first converted by reaction with isopropyl amine into the corresponding ß-amino alcohols. In a second step, these alcohols were derivatised with BGITC into the corresponding diastereomeric thioureas. Baseline separations were observed in all cases, thus establishing a highly efficient and general method for the analysis of this class of molecule.

 

Figure 1 Structure formulas of isothiocyanate derivatisation reagents based on monosaccharide

Figure 1 Structure formulas of isothiocyanate derivatisation reagents based on monosaccharide (90245) (T5783) (335622) (44891) (88102) (04669)

 

Amino acids

The above reagents (Figure 1) have been shown to be also highly suitable for the enantiomeric analysis of amino acids of widely varying structures such as proteinogenic-, nonproteinogenic (non-coded)-, and non-natural- (1,2) amino acids as well as α,α’-disubstituted (3) and ß-amino acids (4).To their advantage, native (underivatised) amino acids (“straight out of the bottle” or the reaction medium) react under mild conditions and at a rapid rate (at room temperature) with these reagents, leading to the corresponding diastereomeric thioureas (Figure 2). These can then be injected directly into the HPLC without the need for further purifi cation. In the majority of cases, baseline separations are achieved.

 

Figure 2 Amino acids: formation of diastereomeric thioureas (schematic) [* denotes centre of chirality]

Figure 2 Amino acids: formation of diastereomeric thioureas (schematic) [* denotes centre of chirality]

 

As derivatives of natural monosaccharides, all of the above reagents are enantiomerically pure by defi nition, and the ratios of the subsequent diastereomers directly refl ect the enantiomeric composition of the amino acid in question. This requires, of course, that both enantiomers react rapidly and quantitatively and with the same rate in order to avoid a diastereoselectivity during the derivatisation process. For new target molecules, this must be ascertained in every case by calibration with the corresponding racemate. The approach described in this article frequently has distinct advantages over the so-called direct method employing chiral stationary phases in that a) the separation of diastereomers is usually simpler to perform and often provides better resolutions, b) the choice of chromatographic conditions is much greater and these can be more easily optimised, and c) the reagents already contain chromophores (fl uorophores) for convenient UV-detection and no pre-column derivatisations (OPA, PITC) are required as is frequently the case with chiral columns. In view of the range of novel derivatisation reagents which recently became available (PGITC, PGalITC, NGalITC) (5), the method is an interesting alternative and can also substitute for the frequently employed Marfay’s reagent (6,7).

In principle, all of the above reagents can be employed for the analysis of amino acids. Thus, Kinoshita et al. [1] achieved baseline separations in the analysis of various α-amino acids employing GITC (Aldrich T8783) and AITC (Sigma-Aldrich 90245). The introduction of benzoyl groups (BGITC Aldrich 335622) [2] and naphthoyl groups (NGaIITC Sigma-Aldrich 04669) [5] considerably enhanced the UV- and fl uorescence detectability of the corresponding thioureas by factors ranging from 6 (BIGTC) to 40 (NGalITC). Furthermore, the introduction of these residues often provides considerable improvement to the separation of these diastereomers, as did the incorporation of extremely bulky pivaloyl groups, such as in PGITC (Sigma-Aldrich 44891) and PGaIITC (Sigma-Aldrich 88102).

While simple RP-18 columns are usually employed, the separation conditions can be varied widely in order to achieve the best separating conditions. Various mobile phases have been used in order to optimise the separation conditions. These include MeOH : phosphate buff er (pH 2.8) [1] over MeOH : H2O : phosphate buff er (pH 7) to acetonitrile : water/0.1% trifl uoroacetic acid (3,5). In certain cases, the reagent may interfere with the separation if its retention time is very near that of the analyte. The addition of small amounts of ethanolamine is suffi cient to destroy excess reagent by formation of the corresponding thiourea which elutes at widely diff erent retention times. The method – after further optimisation of the separation conditions – allows the separation of all racemic, proteinogenic amino acids in one go [1].

In Tables 1 and 2 the results obtained with the above reagents in the analysis of a variety of structurally different amino acids are summarised by listing capacity factors k, separation factors α and resolutions R, or simply ΔtR1/2. The listed data is representative; however, the absolute values may change, depending on the actual separation conditions (type of column, mobile phase, fl ow rate etc.).

 

Table 1 Separations of proteinogenic amino acids

Table 1 Separations of proteinogenic amino acids

 

Table 2 Separations of non-proteinogenic amino acids

Table 2 Separations of non-proteinogenic amino acids

 

The detection limits are already very low in the case of GITC (ca. 5 ng) and can be further reduced by the introduction of aromatic chromophores or fl uorophoric groups. This is particularly important in cases where body fl uids are analysed directly.

 

Summary

The method described above allows the rapid, efficient and inexpensive determination of enantiomeric purities in a wide variety of structurally varied amino acids. By using the suitable derivatisation reagent, baseline separations are observed in nearly all cases. The method is quite general and applicable to a) detection of trace amounts of amino acids in biological samples; b) check for racemisations, and c) monitoring asymmetric syntheses of amino acids. The method is clearly adaptable to automation using reaction batteries and auto-samplers. The method is thus applicable both on a laboratory scale and in online quality control. It is thus highly suitable for monitoring asymmetric syntheses of amino acids including enzyme-catalysed transformations.

 

Experimental

5 mg of the corresponding amino acid is dissolved in 50% (v/v) aqueous acetonitrile containing 0.55%(v/v) triethyl amine to give a fi nal volume of 10 mL. To 50 μL of this stock solution 50 μL of 0.66% (w/v) BGITC in acetonitrile is added. The resulting solution is shaken on a laboratory shaker for 30 min, after which 10 μl of 0.26% (v/v) ethanolamine in acetonitrile is added and shaking is continued for another 10 min. Ethanolamine reacts with any excess of BGITC and the resulting thiourea derivative is eluated well behind any of the amino acid derivatives. The mixture is then diluted to a fi nal volume of 1 mL and a 10 μL aliquot is injected into the HPLC. (RP-18, mobile phase MeOH :H2O [67 mM phosphate buff er ( pH 7) = 65 : 27 : 8 up to 70:25:5 and 80:15:5], depending on the case, fl ow rate 0.5 mL/min, compare tables).

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Materials

     

References

  1. Nimura, N., Ogura, H., Kinoshita,T. Reversed-phase liquid chromatographic resolution of amino acid enantiomers by derivatization with 2,3,4,6-Tetra-O-acetyl-ß-D-glucopyranosyl isothiocyanate. J. Chromatogr. 202 (1980), 375–9; and other papers in this series: Kinoshita, T., Kasahara, Y., Nimura, N. ibid. 210 (1981) 77–81; Nimura, N., Kasahara,Y., Kinoshita, T. ibid.213 (1981), 327–30; Nimura, N., Toyoma, A., Kinoshita, T. ibid.213 (1984), 547–52.
  2. Lobell, M., Schneider, M. 2,3,4,6-Tetra-O-benzoyl-ß-Dglucopyranosyl isothiocyanante: an efficient reagent for the determination of enantiomeric purities of amino acids, ß-adrenergic blockers and alkyloxiranes by high performance liquid chromatography using standard reversed phase columns. J. Chromatogr. 633 (1993), 287–94.
  3. Peter, A., Olajos, E., Casimir, R., Tourwe, D., Broxterman, Q.B., Kaptein, B., Armstrong, D.W. High performance liquid chromatographic separation of the enantiomers of unusual α-amino acid. J. Chromatogr. 871 (2000) 105–13; for an industrial application, see: German Patent Application: DE 198 33853 A1, Degussa-Hüls AG, 28 July1998.
  4. Peter, A., Lazar, L., Fulop, F., Armstrong, D.W. High-performance liquid chromatographic separation of ß-amino acids. J. Chromatogr. A 926 (2001), 229–38.
  5. Schneider, M., unpublished.
  6. Review: Görög, S., Gazdag, M. Enantiomeric derivatization for biomedical chromatography. J. Chromatogr. B 659 (1994), 51–84.
  7. Bushan, R., Bruckner, H. Marfey’s reagent for chiral amino acid analysis: A review; Amino Acids 27 (2004), 231–47. Bushan, R., Kumar, V., Tanwar, S. Chromatographic separation of enantiomers of non-protein α-amino acids after derivatization with Marfey’s reagent and its four variants, Amino Acids 36 (2009), 571–9.

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