ANALYTICAL

BIOCHEMISTRY

Gas Chromatographic of Amino Acids MAMORU Tokyo

63, 308-320 (1975)

Determination of the Optical Using N-Trifluoroacetyl Menthyl HASEGAWA

Research Laboratory 8-6-6 Asahimachi,

AND

of Kyowa Machidashi,

Purities Esters

ISA0 MATSUBARA Hakko Tokyo,

Kogyo Japan

Co.,

Ltd.

Received May 18, 1973; accepted May 1, 1974 Nineteen a-amino acids and one p-amino acid were resolved as their NTFA menthyl esters by gas-liquid chromatography. Neutral and basic amino acids were well resolved on some conventional packed columns. For the complete resolution of acidic amino acids, a capillary column was used. The results of the applications to some biochemical substances showed that this method is of value for practical usage. The mechanism of the resolution was also investigated and showed that the difference between the derivatives of the enantiomeric pairs in the ability to form intermolecular hydrogen bond contributes essentially to the resolution.

The simple and rapid determination of the optical purities of amino acids has been desired in many biochemical fields and peptide chemistry. Since the pioneering work by Casanova and Corey (1) who resolved racemic camphor by gas-liquid chromatography (glc), numerous approaches have been made, and enantiomeric alkancs (2)) alcohols (3-5)) hydroxy acids (6-g), amines (9-12), amino acids and diastereomeric peptides (13) have been resolved in various degrees. To resolve racemic amino acids, two different methods have been developed. In one of which some optically active stationary phases have been employed (14-17) I and in the other the diastereomeric derivatives of amino acids have been used (18-24). Although it has many advantages, the former method is not suited to routine or semiroutine analysis, because it needs specially prepared stationary phases such as N-trifluoroacctyl-n-phenylalanyl-nleucine cyclohexyl ester (141, and also in most cases a capillary column equipped instrument. In the latter method, 2-alkanols, 3-alkanols, and N-trifluoroacetyl-S-prolyl chloride have been used as the resolving agents. These agents should be optically pure especially when traces of one amino acid isomer in much larger quantities of the other isomer is to be determined. This difficulty has led investigators to troublesome 308 Copyright @ 1975 by Academic Prrss, Inc. All rights of reproduct.ion in any form rcsrrvcd.

GLC

RESOLUTION

OF

RACEMIC

AMINO

ACIDS

309

works, and in some cases clear cut results have not obtained. Thus the practical usage of this method has been limited. In this study, a naturally occurring optically pure alcohol, that is l-menthol, was used and the diastereomeric method was investigated. Vitt et al. reported that several aliphatic amino acids were resolved as the menthyl esters (24). But we could not synthesize these derivatives successfully by their conditions. By the method described below, 19 a-amino acids and 1 p-amino acid were resolved well as the N-trifluoroacetyl menthyl esters on packed or capillary columns. We also describe the results of the applications to some biochemical substances. Moreover, the resolution mechanism was investigated and is discussed here. MATERIALS

Preparation

AND

METHODS

of Derivatives

All the compounds studied were obtained from commercial sources with exception of amidases used for the resolution of racemic phenylglycine. Aliphatic and aromatic amino acids. 250 mg of l-menthol was added to 1.0 mg of amino acid and esterified at 105°C for 1-3 hr in an oil bath by Fisher’s method. Then the excess HCl and menthol were completely removed bubbling with dry N, gas, followed by addition of 0.5 ml of trifluoroacetic anhydride (TFAA) The reaction mixture was vibrated for 5 min at room temperature and the excess TFAA was removed by the gentle stream of dry N, gas. 0.5 ml of dry ethylacetate was added and a 1 ~1 aiiquot was applied to glc. In order to examine the difference in the reaction velocity between the enantiomeric pairs, menthylation and N-trifluoroacetylation were stopped at each interval. Hydroxy amino acids. To the trifluoroacetylated menthyl esters obtained as described above, 0.5 ml of TMS-HT (hexamethyldisilazane and trimethylchlorosilane in anhydrous pyridine, Tokyo Kasei Co,) was added and stirred for 5 min at room temperature. After rembval of the excess TMS reagent by the gentle stream of N, gas, the residual products were dissolved in 0.5 ml of ethylacetate. Acidic and basic amino acids. 5.0 mg of amino acids were added into 3 ml of dry HCl-methanol and esterified at 80°C for 1 hr. After removal of the excess reagent under vacua, 2.0 g of l-menthol was added and interesterified at 105°C for 3 hr bubbling with dry HCl gas. The menthyl esters obtained thus were then N-trifluoroacetylated. Preparation of L- and D-alanine derivatives. Following the method for t.he aliphatic. amino acids, L- and n-alanine were added to 3.0 g of

310

HASEGAWA

AND

MATSUBARA

l-menthol and esterified agitating by a magnetic stirrer for 5 hr. After cooling, 50 ml of acetone was added and the resulted precipitates were gathered by filtration, washed with diet,hylether and then with a small volume of acetone for several times, and crystallized from acetone to form white fine platy crystal for the n-isomer and needle-like crystal for the n-isomer. These esters were then IV-trifluoroacetylated. The derivatives thus obtained were identified by infrared and NMR spectrometry. Applications Analysis of alanine in fermentation broths. 0.1 ml of the broth of a racemase defective mutant of Corynebacterium glutamicum (25) was freeze dried, to which 0.5 g of l-menthol was added and heated at 105°C for 3 hr bubbling with dry HCI gas. After removal of HCI and menthol, N-trifluoroacetylation was carried out for 30 min. After evaporation by a stream of N, gas, the residue was dissolved in 0.5 ml of ethylacetate and a 1 ~1 aliquot was applied t.o glc. Determination of the efficiency of enzymatic resolution of racemic phenylglycine. Racemic phenylglycine was treated with various amidases

in some appropriate buffers. Two examples of these enzymes were “Tasinase,” a proteinase from Streptomyces 1033 (Kyowa Hakko Kogyo Co. Ltd.) and the acetone dried cells of Pseudomonas cruciviae. 0.5 ml of the reaction mixtures were freeze dried and treated as described above except the esterification temperature. This reaction was carried out at 65°C in order to prevent the racemization. Analysis of the component amino acids of peptides. r-Glutamylvaline was synthesized from L- or n-glutamic acid and L-valine by the partially purified y-glutamylpeptidase from Micrococcus glutamicus (26). About 5 mg of the peptide was hydrolyzed in 3 N HCl at 100°C for 4 hr and the reaction mixture was freeze dried. The residual materials were then treated according to the method for acidic amino acids. The final derivatives were dissolved in 0.1 ml of ethylacetate and 1 ~1 of the solution was applied to glc. Gas Chromatography

A Yanagimoto model 550F dual flame ionization detector gas chromatograph was used for all amino acids. The stainless steel columns (0.754 m X 3 mm i.d.) were packed with various commercial 5% coated stationary phases on 80/100 mesh Chromosorb W.AW. A Perkin Elmer model 900 gas chromatograph equipped with a stainless steel capillary column (300 ft X 0.01 in. i.d.) coated with Apiezon L was used for acidic and basic amino acids. Chromatographic conditions arc described in each figure.

GLC

Infrared

RESOLUTION

Spectrometry

OF

of Alanine

RACEMIC

AMINO

311

ACIDS

Derivatives

A Shimazu model 27G infrared spectrometer attached with a NaCl cell fixed at various thickness (Nihon Bunko Co.) was used for the determination of the nature of the hydrogen bond formed by the derivat.ives. This hydrogen bond was measured at various concentrations in Ccl, (0.05-1.0 M) . RESULTS

Resolution

AND

DISCUSSION

of Standard Racemic Amino Acids

Vitt et al., esterified amino acids in toluene and resolved some amino acids, we could not obAliphatic

and aromatic amino acids. Although

MET ZOOOL 0

PRO lEOaL ,,

J.JL.;,

, LO

0

20

PHENYLGLYCINE 180"

40

60

0

20

40

,

L P

60 min

FIG. 1. Resolution of aliphatic and aromatic amino acids on PEG-adipate column. Conditions: 5% PEG-adipate column (4 m X 3 mm i.d.), carrier flow = He 10 ml/min, sample size = 1 pl, injection = 250°C.

312

HASEGAWA

0

AND

20

10

MATSUBARA

30

40

50 min

2. Simultaneous analysis of aliphatic and aromatic amino acids. Conditions: 5% PEG-adipate column (4 m X 3 mm i.d.), 14@-200°C at 2°C min, carrier flow = He 10 ml/min, sample size = 1 ~1. FIG.

tain derivatives successfully by their conditions. However, as described in Methods, we directly suspended amino acids in menthol and could synthesize their esters. The thin-layer chromatographic analysis of these reaction mixtures showed that the reactions were almost. complete. By this method, the derivatives of alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, phenylglycine, and tyrosine were obtained. The most. available stationary phases at hand were PEG-adipate, PEG2OM, Silicone DC-550, and EGSS-X. Among them PEG-adipate gave the complete resolution of enantiomeric pairs (Fig. 1) , and the good separation of each amino acid except alanine and valine (Fig. 2). DC-550 also showed the high resolving efficiency, but could not separate L-isoleucine and n-leucine. It is noteworthy that these amino acids can be resolved completely by these conventional packed columns.

I 0

10

20

30

40

50

1 60

min

FIG. 3. Resolution of hydroxy amino acids on PEG-adipate column. Conditions: 5% PEG-adipate column (4 m x 3 mm i.d.), isothermal at 19O”C, carrier flow = HE 10 ml/min, sample size = 1 ~1.

GLC

RESOLUTION

OF

RACEMIC

AMINO

313

ACIDS

Hydroxy amino acids. The trifluoroacetylated esters of hydroxy amino acids such as serine, threonine, homoserine, hydroxyproline, and tyrosine gave only small peaks or unclear results. This may be due to the instability of their 0-TFA groups. The relatively more stable derivatives, i.e., N-TFA-0-TMS menthyl esters were thus examined. As shown in Fig. 3, these derivatives gave good resolution and separation on a PEGadipate column. Basic amino acids. As the solubilities of these amino acids in HClmenthol were extremely low, the esterification was not accomplished by the method for aliphatic amino acids. Then the interesterification from methyl to menthyl ester was examined and gave good results. These amino acids were resolved well on nonpolar stationary phases such as Apiezon-L (Fig. 4a, 4b). The other basic amino acids, arginine and histidine, gave no peaks on any columns tested. This may be caused by the particular instabilities on columns or the low volatilities of their derivatives (18). Acidic amino acids. These amino acids could not be esterified because of their low solubilities in HCI-menthol. But like basic amino acids, the reaction was accomplished successfully by the interesterification. As these derivatives contain two menthyl groups, their boiling points may be relatively high. From this viewpoint, we used nonpolar stationary phases at high column temperature. Glutamic acid gave only partial resolution, and aspartic acid practically no resolution on packed columns, but both amino acids were resolved perfectly on a capillary column (Fig. 412, 4d). There were no evidences that the reaction velocities of menthylation

0

20

40

0

20

0

20

0

20 min

FIG. 4. Resolution of basic and acidic amino acids. Conditions: (a) 5% Apieeon L column (0.75 m X 3 mm i.d.), isothermal at lSO”C, carrier flow = He 15 ml/min. (b) Capillary column (Apiezon L, 300 ft X 0.01 in. i.d.), isothermal at 245°C. (C) Capillary column (Apiezon L, 300 ft x 0.01 in. i.d.1, isothermal at 240°C. (d) Capillary column (Apiezon L, 300 ft X 0.01 in. id.), isothermal at 265°C.

314

HASEGAWA AND MATSUBARA TABLE 1 Separation Factors Measured on Various Stationary

Phases

Separation factor ~ya Amino acid

Structure of side chain R

Alanine

PEG20M

PEG-A

Apiezon-L

DC-550

1.147

1.114

1.111

1.073

1.156

1.141

1.117

1.083

1,160

1.144

1.139

1.077

1.177

1.161

1.171

1.081

4

1.045

1.070

-

-

1.167

1.113

1.081

1.149 1.154 1.0’ 1.050

1.128 1.135 1.0 1.052

1.119 1.119 1.0 1.0

1.077 1.080 1.0 1.0

-

CH* Leucine

-CH&H

/ \

CHa CHs Valine

-CH

/ \

Isoleucine

-CH

CH, CH,

/

-\0 / -CHz-\ 0, \

CH&H3

Phenylglycine Phenylalanine a-ABAd cu-AVAe &ABA/ B-AIBAr

-CH&H, -CH&H&Hs (see Fig. 7b) (see Fig. 7c)

a Mean values obtained from three times chromatography; 4 m columns packed with 5y0 coated stationary phases were used at the column temperature 18O”C, and the carrier flow (He) was 10 ml/min. * Not determined. E No resolution. The pair have just the same retention times. d cu-Amino-n-butyric acid. e cu-Amino-n-valeric acid. f fi-Amino-n-butyric acid. 0 &Aminoisobutyric acid.

and trifluoroacetylation were different between the enantiomeric pairs caused by their diastereomeric relationships. In this way, the protein amino acids except cysteine, arginine, histidine, and tryptophane were well resolved by this method. The unifying method of the derivation is now under investigation for the purpose of the simultaneous analysis of all the protein amino acids.

GLC

Application

RESOLUTION

to Biochemical

Determination

OF

RACEMIC

AMINO

315

ACIDS

Substances

of the optical

purity

of alanine in fermentation

broths.

This method was used for the screening of the racemase defective mutants of Corynebacterium glutamicum. The lyophilized broth of a mutant expected to be racemase defective was directly menthylated and then N-trifluoroacetylated. It was made clear that alanine produced by this mutant was L-isomer only (Fig. 5a). A small amount of the n-isomer which was previously added to the medium as a growth factor was also detected. Thus this mutant was clearly characterized as a racemase defective. Determination of the efficiency of the enzymatic resolution of racemic phenylglycine. To obtain n-phenylglycine, which is one of the two components of aminobenzylpenicillin, racemic phenylglycine amide was treated with a number of amidases. The resolving efficiencies of these enzymes could be determined by this method. Partial resolution occurred by an acetone dried cell of Pseudomonas cruciviae, but no resolution by “Tasinase” (Fig. 5b). Analysis of the component amino acids of peptides. y-Glutamylpeptides can be synthesized by the reverse reaction of hydrolysis catalyzed by the y-glutamylpeptidase from various bacteria (26). To determine the substrate specificity of this enzyme from Micrococcus glutamicw, this 3

b

Ll

1

I 0

I 10

I 20

I min

I 0

I

I 20

I

I 40

I

I 60 mi

FIG. 5. Analysis of the optical purities of amino acids in biological fluids. (a) Alanine fermentation broth of a racemase defective mutant of Corynebacterium glutamicum. Freeze dried broth was directly derived and applied to glc. Conditions: 5% PEG-adipate (4 m X 3 mm id.), isothermal at 160°C. (b) Reaction mixtures of enzymatic resolution of racemic phenylglycine. The enzymes were “Tasinase,” a protease from Streptococcus (b-l), and acetone powdered cells of Pseudomonas cruciviae (b-2). Conditions: 5% PEG-adipate (4 m X 3 mm), isothermal at 180”.

GLC

RESOLUTION

OF

RACEMIC

AMINO

ACIDS

317

These values are the indicators of the standard free energy differences between the diastereomeric pairs with respect to their gas-liquid partition equilibria. As shown in Table 1, the larger the bulkiness of the side chains (especially that around the ,&carbons), the greater the a-values in all the columns tested. Since this bulkiness may be one of the factors that limit the free rotation around the bond-a (Fig. 7a), these mean that the diastereomeric difference in physicochemical properties such as vapor pressures and solubilities in the stationary phases may depend at least partially on the strength of this limitation. No resolution of p-amino-nbutyric acid (P-ABA), the asymmetric center of which is at t.he P-carbon (Fig. 7b), may be due to the relatively lesser restriction of the free rotation around the a-carbon. On the contrary, /3-aminoisobutyric acid (/3AIBA), a structural isomer of P-ABA, whose a-carbon is asymmetric (Fig. 7c), could be resolved on the polar stationary phases. Effects of the polarities of the stationary phases. Table 1 also shows that the (Yvalues of all the amino acids tested increase with the increment of the polarity of the stationary phase. This means that the solutesolvent polar interaction contributes to the resolution and that this interaction is stronger for the derivatives of the n-isomers. No resolution of /3-AIBA on the nonpolar stationary phases suggest that the vapor pressures of these pairs are almost the same as studied by Brennan and Coates (27). Thus the resolution of this amino acid on the polar stationary phases means that the solute-solvent polar interaction takes part certainly in the resolution mechanism. Order of the emergence of the enantiomeric pairs. The L-isomers emerged faster than the n-isomers among all the amino acids tested. The study by the Dreiding Stereomodels (Btichi) and the ir spectra of the derivative revealed a significant explanation for these phenomena. The menthyl group is most stable in one of the two chain conformations (28), and the carbonyl group of the ester bond lie skewed to the C, carbon and the C, carbon attached hydrogen atom of the menthyl group (4). The stereomodel shows that the steric repulsion between bulky C,, isopropyl group of the menthyl moiety, and the amino acid side chain R fixes this R group at the skewed position to the ester carbonyl group (Fig. 8). In this conformation, the polar groups of the OH Men-O-t&R Ial

fltlv Men-0-C-F-,-F’CH. ~~~HCOCF. H NHCOCF.

lb1

OHH (.&n-o-&&&.( GHCOCF~

(Cl

FIG. 7. Amino acid derivatives employed to investigate the resolution (a) a-Amino acid. (b) /3-Amino-n-butyric acid. (c) P-Aminoisobutyric Men represents menthyl group, and * an asymmetric carbon.

mechanism. acid. Here

318

HASEGAWA

I

COCF3

AND

MATSUBABA

F3COC

I

FIG. 8. Proposed preferable conformations of N-TFA menthyl esters of L- and n-amino acid. The menthyl group (painted out in black) is seen over a-carbon of amino acid moiety.

n-isomer derivative, i.e., the ester carbonyl and the amide group lie at t.he same side where the steric hindrance of the isopropyl group is weak. Otherwise the amide group of the n-isomer derivative is fixed at the same side of this isopropyl group. In other words, the amide group of the latter is somewhat shielded by this bulky group from environment. This shielding effect may give n-isomers stronger polar interactions with surrounding substances. The ir spectrum observed at NH stretching band of the derivative supported this idea. Both enantiomer derivatives form a hydrogen bond, but the concentration effects show that this bond is an intermolecular type and is more stable for the n-isomer (Fig. 9). 1.0 H

0.2 M

0.1 N

I

I I I

~ 349

3350 v

+

4 I

I

I

I I clll-

FIQ. 9. Concentration effect on the hydrogen bond formation observed at N-H stretching band of ir spectrum in CCL. The thickness of the cell were 0.1, 0.2, 0.5, 1.0 mm, respectively. () n-alanine derivative ; (- - - -) o-alanine derivative.

GLC

RESOLUTION

OF

RACEMIC

AMINO

ACIDS

319

Figure 10 shows that the intermolecular hydrogen bond (3220 and 3215 cm-l) formed between the derivatives and the polar solvents, i.e., tetrahydrofran and 1,4-dioxane is apparently stronger for the D-isomer. Thus D-isomer derivatives have certainly stronger intermolecular hydrogen bonds between not only themselves but also polar solvents (polar stationary phases). Resolution mechanism. These results obtained above give support to one another. As the result of their higher ability to form the intermolecular hydrogen bond, the D-isomer derivatives may have lower vapor pressures and stronger solute-solvent interactions. This leads to the lesser mobilities of the D-isomers on nonpolar and especially on polar stationary phases. Thus the (Yvalues increase in proportion to the increment of the polarity of the stationary phases. The larger the bulkiness around the p-carbon of the side chain R, the stronger the steric repulsion between R and the isopropyl group of the menthyl moiety; and the time average when the preferable conformation exists grew longer. That is, as the bulkiness increases, the difference between the D- and L-isomer derivatives in the ability to form intermolecular hydrogen bond increases. Thus the (Yvalues also increase in proportion to the increment of the bulkiness of R. The essential contribution of the bulkiness around the p-carbon in R group should be noted. Much smaller (Yvalue of ,&AIBA may be caused by the position of its amide group. This group of the compound can lie somewhat freely in space, and may possess lesser difference in hydrogen bond formation between enantiomeric pairs than a-amino acids. These good agreements of each result suggest that this explanation is reasonable.

a

FIG. 10. Difference in the hydrogen bond formation with polar solvents observed at N-H stretching band, in tetrahydrofran (a), and in 1,4-dioxane (b). C----J Lalanine derivative ; (- - - -) n-alanine derivative (0.2 M).

320

HASEGAWA

AND

MATSUBABA

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

CASANOVA, J., AND COREY, E. J. (1961) Chem. Znd., 1664. STERN, R., ATKINSON, E. R., AND JENNING, F. C. (1962) Chem. Ind., 1758. GAULT, Y., AND FELKIN, J. (1965) Bull. Sot. Chem. Frcmce, 742. ROSE, H. C., STERN, R. L., AND KARGER, B. L. (1966) Anal. Chem. 38, 469. CROSS, J. M., PUTNEY, B. F., AND BERNSTEIN, J. (1970) J. Chromatogr. Sci. 8, 679. GIL-Av, E., AND NUROK, D. (1962) Proc. Chem. Sot., 146. WESTLEY, J. W., AND HALPERN, B. (1968) J. Org. Chem. 33, 3978. KARGER, B. L., STERN, R. L., ROSE, H. C., AND KEANE, W. (1967) in Gas Chromatography 1966, the Institute of Petroleum, London. GORDIS, E. (1966) Biochem. Pharmacol. 15, 2124. FEIBUSH, B., AND GIL-Av, E. (1967) J. Gas Chromatogr. 5, 257. GUNNE, L. M. (1967) Biochem. Pharmacol. 16, 863. CORBIN, J. A., AND ROGERS, L. B. (1970) Anal. Chem. 42, 974. WEICAND, F., PROX, L., SCHNIDHAMMER, L., AND KONIG, W. (1963) Angew. Chem. Intern. 2, 183. KOENIG, W. A., PARR, W., LICHTENSTEIN, H. A., BAYER, E., AND ORO, J. (1970) J. Chromatogr. Sci. 8, 183. FEIBUSH, B., AND GIL-Av, E. (1970) Tetrahedron Lett. 26, 1361. PARR, W., YANG, C., AND BAYER, E. (1970) J. Chromatgr. Sci. 8, 591. PARR, W., AND HOWARD, R. (1972) J. Chromatogr. 66, 141. HALPERN, B., AND WESTLEY, J. W. (1966) Tetrahedron Lett. 21, 2283. GIL-Av, E., CHARLES-SIGLER, R., FISHER, G., AND NUROK, D. (1966) J. Gas Chromatogr. 4, 51. POLLOCK, G. E., AND OYAMA, V. I. (1966) J. Gas Chromatogr. 4, 126. WESTLEY, J. W., HALPERN, B., AND KARGER, B. L. (1968) Anal. Chem. 40, 2046. AYERS, G. S., MONROE, R. E., AND HOSSHOLDER, J. H. (1971) J. Chromatogr. 63, 259. DABROWIAK, J. C., AND COOKE, D. W. (1971) Anal. Chem. 43, 791. Vrrr, S. V., SAPOROWSKAYA, M. B., GUDKOVA, I. P., AND BERIKOV, V. M. (1965) Tetrahedron Lett., 2575. HAGINO, H., AND NAKAYAMA, K., unpublished result in our laboratory. HASEGAWA, M., AND MATSUBARA, I. (1973) Agr. i3iol. Chem. 37, 1985. BRENNAN, N., AND COATES, V. J. (1958) Nature (London) 181, 1401. ELIEL, E. L. (1962) in Stereochemistry of Carbon Compounds, p. 221, McGrawHill Book Company Inc., New York and London.