Vol. 20, No. 6, 1965 . BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS OPTICAL RESOLUTION OF D,L AMINO ACIDS BY GAS CHROMATOGRAPHY AND MASS SPECTROMETRY B. Halpern, J. W. Westley, Ingilt von Wredenhagen and Joshua Lederberg Instrumentation Research Laboratory and Kennedy Laboratories for Molecular Medicine Department of Genetics, Stanford University School of Medicine Palo Alto, California Received August 9, 1965 The significance of optical activity for the recognition of life and - hence {ts utility for biochemical exploration needs no elaboration (Lederberg 1965), (Ulbricht 1962). Recently, a number of gas liquid chromatographic (G.L.C.) procedures have been developed, whereby important metabolites, like amino acids, can be scanned for optical activity, with very high sensitivity (Gil-Av., Fischer and Charles 1965), (Halpern and Westley 1965 a,b) (Pollock 1965). The same principle can be generalized for complex mixtures by ratio- detection of D- and L- input reagents when these form resolvable diastereo- isomeric complexes with the target material. We now show the use of mass spectrometry for the ratio-detection, as well as to identify the optically active species. For this purpose we prepared an artificial mixture of D and L enantio- meric resolving agents, in which the L reagent was labelled with 2 deuterium atoms (L*), After coupling with the target material, the product was gas chromatographed and the peaks collected and passed into a mass spectrometer. For each symmetrical molecule (e.g. glycine), the D and L reagents are unre- solved and the label ratio will remain uniform through the peak. However, if an asymmetric molecule is encountered, which gives rise to resolvable diaster- eoisomers, the deuterated reagent will be concentrated in one peak, distorting the ratio. If the target molecule is racemic (D L), two peaks will also be 710 Vol. 20, No. 6, 1965 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS formed (one containing L*D plus D L; the other L*L plus DD); but the label ratio in each peak will remain constant, We chose trifluoracetyl-thiazoli~ dine~4-carboxylic acid chloride as the reagent, because both enantiomers are available (Ratner and Clarke 1937), and deuterium can be incorporated into position 2 with deuteroformaldehyde. Also mass spectrometric fragmentation patterns of its condensates with amino acid esters yielded characteristic peaks which could be used to fdentify both the reagent and the amino acid (Figure 1). Figure 1 MASS SPECTRAL FRAGMENTATION OF TFA~THIAZOLIDINE-4- CARBOXELIC ACID CONDENSATION PRODUCTS uc “a f re * DA, ir CO NH CH C00cH, —p ' H(o) " nor cock, cars, Fragment a m/e 184 $—— clk + val vf —LO—NH—GH—COOGH, ———9 |, — CHC N= CH — Conc, WN 1 (0) Coch, Base Peak, fragment b, M-156 In a typical assay, the amino acid sample was esterified with thionyl chloride— methanol and the excess reagent and solvent removed. An excess of the resolv- ing agent (L* plus D) in an inert solvent was added to the residue and the sus- pension neutralized with triethylamine. After washing with water, the solution was injected into the gas chromatograph and the emerging components collected for introduction into the mass spectrometer. By monitoring the ratio for fragment (a) [184:186] as well as the ratio (b:b+l) for the base peak (M-156] 711 Vol. 20, No. 6, 1965 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS a fast sensitive recording for optical activity was obtained. In addition, the position of the base peak was also used to confirm the identity of the optically active amino acids present (Table), (Figure 2). TABLE: MASS SPECTRAL MONITORING OF G.L.C. FRACTIONS * CORRECTED FOR ISOTOPIC ABUNDANCE. ) Molecular Weight G.L.C. Ratio(a:at2) Fragment(b) Ratio (b+156) and Iden- Optical Identity Fraction m/e 184:186 m/e (b:bt1) tity of Amino Acid of Fraction 1 28:2.5 158 100:8.5 314-alanine L 2 1:24 158 4.5:100 314-alanine L 3 38:41 144 97:100 300-glycine DL 4 55:56 172 100:96 328-aminobutyric acid DL 5 33:32 172 100 :98 328=-aminobutyric acid DL 6 2.5333 200 8.5:100 356-leucine D 7 23:2 200 100:8 356-leucine D 8 100:2 184 100:5 340-proline L 9 12:31 184 12:100 340-proline L *G.L.C. analysia were carried out on a Wilkens 600C Aerograph, fitted with a micro collector and using a 5' X 1/8" $.S. column containing 5% SE 30 on chromosorb W. The separation temperature was 180°C and the Ny flow was 28 ml/min. Mass Spectra were determined on a Bendix-Time-of-Flight Spectrometer and the col- lected sample fractions introduced directly into the ion source. The utility of mass spectrometric detection, thus substantiated, points to a general method for the speedy, facile detection and identification of minute amounts of optically active materials. Hardware for direct coupling of the gas chromatograph to the mass spectrometer (Gohlke 1959, 1962), (Ebert 1961) was not yet available to us for this study. However, the results of other 712 Vol. 20, No. 6, 1965 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 184/186 Abundance m/e 01 1 -Figure 2 ANALYTICAL RESOLUTION OF LABELLED INPUT REAGENT 50 Abundance m/e b/b+1 Fractions Fractiong 1 4 1 ) i ® 1 Lt 1 i i 4567 8 9 123456789 Fractiong 4 5 8 Gas Chromatograph* *G.L.C. Fraction: 1. 2. 3. 4. 5. 6. 7. 8. 9. D L* D D D L* D D L* reagent-L-alanine reagent-L-alanine Yreagent-glycine and L* reagent-glycine reagent-L aminobutyricacid and L* reagent-D aminobutyricacid reagent-D aminobutyricacid and L* reagent-L-aminobutyricacid reagent-D leucine reagent-D leucine reagent-L proline reagent-L proline 713 Vol. 20, No. 6, 1965 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS workers suggests that the technique should thus have a sensitivity in the sub- microgram or nanogram range, rendering it useful for the monitoring of metabol- ic reactions as well as the identification of accumulated asymmetric metabol- ites. References Brenner, M. and Huber, W., Helv. Chim. Acta, 36, 1109 (1953). Ebert, A. A., Anal Chem., 33, 1865 (1961). Gil-Av., E., Charles, R. and Fischer, G., J. Chrom. 17, 408 (1965). Gohlke, R. S., Anal. Chem., 31, 535, (1959) and Anal. Chem., 34, 1332 (1962). Halpern, B. and Westley, J. W., Biochem Biophys Res. Com., 19, 361 (1965). Halpern, B., and Westley, J. W., Chem. Com., 293, (1965). Lederberg, J., Nature, 207, 9 (1965). Pollock, G. Oyama, V., and Johnson, R., J. Gas Chrom., 3, 174 (1965). Ratner, S. and Clarke, H. T., J.A.C.S., 59, 200 (1937). Ulbricht, T.L.V., "The Optical Asymmetry of Metabolites", Comparative Bio- Chemistry, ed. Florken and Mason, Vol. 4, Academic Press (1962). Prepared under the following grants:.- NASA 64-4618-33; AFOSR-886-65; National Institutes of Health-04270 714