Simple, Rapid, Inexpensive Determinations of Oxirane Enantiomeric Purities

By: Manfred P.Schneider FB C, Bergische Universität Wuppertal,D-42097 Wuppertal, Germany, AnalytiX Volume 10 Article 1


Analytical methods for the determination of enantiomeric purities should go beyond high-quality standards to include important additional features: simple, rapid and inexpensive. Attractive alternatives to high-cost, so-called chiral columns (chiral stationary phases) are inexpensive reversed-phase (RP) columns in combinationwith suitable derivatisation reagents. In this series of articles, we describe applications of such systems involving suitably substituted monosaccharide-based isothiocyanates for the determination of enantiomeric purities of the title compounds (Figure 1).

All derivatisations are based on the simple transformations of these reagents into the corresponding diastereomeric thioureas by reaction with (primary and secondary) amino groups (Figure 2). These transformations can either be created by reaction of the oxiranes under consideration with amines (leading to the corresponding ß-amino alcohols, see Oxiranes below) or are already part of the molecule to be analysed (amino acids, pharmaceuticals such as ß-blockers etc.).

As derivatives of natural mono-saccharides all of these reagents are optically pure, and the ratios of diastereoisomers thus produced directly reflect the enantiomeric composition of the amino compound in question. The first examples for such applications [using the tetra acetate, GITC Aldrich T5783 and the triacetate of α-D-arabinose AITC Sigma-Aldrich 90245] were introduced between 1980–1984 by Kinoshita et al.1; in the meantime both performance and UV-detection were considerably improved by the introduction of benzoyl groups (BGITC Aldrich 335622)2 and naphthoyl groups (NGaIITC Sigma-Aldrich 04669)3. Also, the introduction of pivaloyl groups (PGaIITC, Sigma-Aldrich 88102) led in many cases to improved separations2.

This series of articles consists of three parts, each addressing one group of the title compounds: oxiranes, amino acids (proteinogenic, non-proteinogenic, ß-amino- and α.α´- disubstituted-), and pharmaceuticals carrying amino functions.

Figure 1 Structure formulas of monosaccharide-based isothiocyanates

Figure 1 Structure formulas of monosaccharide-based isothiocyanates (T5783) (90245) (335622) (04669) (88102) (44891)

Figure 2 Formation of diastereomeric thioureas

Figure 2 Formation of diastereomeric thioureas (schematic) [* denotes centre of chirality]

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Enantiomerically pure, mono-substituted alkyloxiranes (compare Figure 3) are important chiral building blocks4 for the syntheses of numerous natural products, including (inter alia) pheromones5, γ- and δ-Lactones6, various natural products7, and ferroelectric liquid crystals8. The enantiomeric purity levels of these materials can be determined by numerous methods. For low molecular weight alkyl oxiranes, the analyst can utilise complexation gas-liquid chromatography employing chiral metal complexes (Ni, Mn, Co, Zn) as stationary phases9. However, this method may not be applicable to molecules of higher molecular weight, in which case decompositions are occasionally observed. Also, low-separation factors are frequently observed. Modified cyclodextrins can also be employed as chiral stationary phases for this purpose10. Yet, none of these methods seems to be generally applicable for enantiomeric purity determinations irrespective of structure and molecular weight. Since so-called chiral columns may not be commercially available for specific cases or are expensive, reversed phase columns can provide an alternative approach when employed in combination with suitable derivatisation reagents. With this methodology, the best and most generally applicable separations are achieved using a two-step strategy involving a) regioselective ring opening of the oxirane moiety using a simple sterically demanding amine leading to the corresponding ß-amino alcohols, followed by b) derivatisations of the resulting ß-amino alcohols with the above reagents. Based on earlier results by Gal11, this procedure was further optimised by employing 2-propylamine for step a), thereby considerably shortening the time required for the sample preparation, and for step b), employing BGITC for improved UV detection2 (Figure 3).

Figure 3 Conversion of alkyl oxiranes into diastereomeric thioureas using a two-step strategy

Figure 3 Conversion of alkyl oxiranes into diastereomeric thioureas using a two-step strategy [* denotes centre of chirality]

In using PGalITC (Sigma-Aldrich 88102) and GITC (Aldrich T5783) only partial or no separations are achieved. Clearly the very best results were obtained using BGITC (Aldrich 335622). Base-line separations are achieved in all cases (α=1.15–1.23; RS = 2.14 –2.29) and all retention times are within a reasonable time-range (Table 1).

Table 1 Separation of diastereomeric BGITC – derivatives obtained from oxirane

Table 1 Separation of diastereomeric BGITC – derivatives obtained from oxirane – derived ß-amino alcohols. Column: Li Chrospher 100 RP-18 (5μm); flow rate= 0.5ml min-1 ; t0 = 3.1 min; wavelength of detection = 231 nm; R= substituents as shown in Figure 3. k´ = capacity factors; α = separation factor; RS = resolution.

In view of the mild separation conditions (quite in contrast to the high temperatures required in GC separations), no decompositions are observed. Both enantiomers were converted completely and with the same rate. The analytically determined ratios (integrations) are thus a true reflection of the enantiomeric compositions. In Table 1 the separation parameters for 13 different alkyl oxiranes are summarised together with the separation conditions. Owing to their different retention times, it was also possible to separate a mixture of 10 oxirane-derived ß-amino alcohols (derived from 5 racemic oxiranes of different substitution patterns) in one single experiment (Figure 4).

Figure 4 Separation of ten oxirane–derived ß-amino alcohols as diastereomeric thiourea derivatives in a single experiment.

Figure 4 Separation of ten oxirane–derived ß-amino alcohols as diastereomeric thiourea derivatives in a single experiment. Mobile phase, methanol-water (90:10); flow rate, 0.5 ml min-1; 0.7 nmol of each derivative are injected. Components were eluted in the following order: always (R) - before (S) for R = C2, C4, C6, C8, C10.

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The method described above allows the rapid and inexpensive determination of enantiomeric purities in a wide variety of structurally varied alkyl oxiranes. Base-line separations are observed in all cases and the determined ratios are a true reflection of the enantiomeric purities. Themethod is clearly adaptable to automation using reaction batteries and auto-samplers. The method is thus applicable both on a laboratory scale and in on-line quality control. It is thus highly suitable for monitoring asymmetric syntheses of oxiranes4 as well as enzyme-catalysed transformations

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50 μL of the corresponding alkyl oxirane ismixed with 200 μL of 2-propylamine in a 1 mL vial (having a tightly sealed, Teflon-lined cap) and heated at 100 °C for 2 hours. Excess 2-propylamine is evaporated in a slow stream of N2 after which 950 μL of acetonitrile are added. 50 μL aliquots of this solution are mixed with 0.66% (w/v) BGITC in acetonitrile and the mixtures are allowed to react for 30 min at room temperature. After dilution with CH3CN to a final volume of 1mL, 7 μL aliquots are injected into the HPLC (RP-18,mobile phase MeOH: H2O = 85:15 up to 95:5, depending on the case, flow rate 0.5 mL/min, compare table).

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  1. Nimura, N., Ogura, H., Kinoshita, T., Reversed-phase liquid chromatographic resolution of amino acid enantiomers by derivatisation with 2,3,4,6-Tetra-O-acetyl-ß-D-glucopyranosyl isothiocyanate J.Chromatogr. 202 (1980) 375–379; 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) 327–330; Nimura, N., Toyoma, A., Kinoshita, T., ibid. 213 (1984) 547–552.
  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) 287–294.
  3. Schneider, M., unpublished.
  4. Schaus, S.E., Brandes, B.D., Larrow, J.F., Tokunaga, M., Hansen, K.B., Gould, A.E., Furrow, M.E., Jacobsen, E.N., Highly selective hydrolytic kinetic resolution of terminal epoxides catalyzed by chiral (salen) CoIII complexes. Practical synthesis of enantioenriched terminal epoxides and 1,2- diols; J.Am. Chem. Soc., 124 (2002) 1307–1315 and a wealth of literature cited therein.
  5. Mori, K., Sasaki, M., Tamada, S., Suguro, T., Masuda, S., Heterocycles 10 (1978) 111-; loc cit. synthesis of optically active 2-ethyl-1,6-dioxaspiro [4.4] nonane(chalcogran), principle aggregation pheromone of Pityogenes chalcographus (L.) Tetrahedron 35 (1979) 1601–1605; Johnston,B.D., Oehlschläger, A.C., Synthesis of the Aggregation Pheromone of the Squarenecked Grain Beetle Cathartus quadricollis J. Org. Chem., 51 (1986) 760–763.
  6. Haase, B., Schneider, M., Enzyme assisted synthesis of enantiomerically pure δ-lactones, Tetrahedron: Asymmetry 48 (1993) 1017–1026; Habel,A., Boland,W., Efficient and flexible synthesis of chiral γ- and δ-Lactones, Org. Biomol. Chem. 6 (2008) 1601–1604.
  7. Masaoka, Y., Sakkibara,M., Mori, K., Synthesis and Absolute Configuration of (+)-8-Hydroxyhexadecanoic Acid, an Endogenous Inhibitor for Spore Germination in Lygodium japonicumAgric. Biol. Chem. 46 (1982) 2319–2324.
  8. Sagakuchi, K., Kitamura, T., Shiomi, Y., Koden, M., Kuratate, T., Chem. Lett. 1991, 1383; Kusumoto, T., Sato, K., Hiyama, T., Takehara, S., Osawa, M.,Nakamura, K., Fujisawa, T., ibid. 1991, 1623.
  9. Schurig, V., Practice and theory of enantioselective complexation gas chromatography, J. Chromatogr. A 965 (2002) 315–356.
  10. König, W.A. Gas Chromatographic Enantiomer Separation with modified Cyclodextrins, Hüthig, Heidelberg 1991.
  11. Gal, J., Determination of the enantiomeric composition of chiral epoxides using chiral derivatisation and liquid chromatography, J. Chromatogr. 331 (1985) 349 –357.


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