Resolution of Racemic Aspartic Acid, Tryptophan, Hydroxy and Sulfhydryl Amino Acids by Gas Chromatography Glenn E. Pollock and A. H. Kawauchi Exobiology Dicision, Ames Research Center, NASA, Moffett Field, Calif. 94035
THERESOLUTION of diastereomers of amino acids by gas chromatography has been reported by several investigators (1-11) in the past few years. While many protein amino acids have been well resolved using the 2-butyl-N-trifluoroacetyl derivatives, the O,N or S-di-TFA derivatives of serine, hydroxyproline, tyrosine, and cysteine have not been weli resolved. This communication reports the successful resolution of these amino acids as well as aspartic acid and tryptophan. Methods for the synthesis of the derivatives are given. EXPERIMENTAL
Apparatus. A Perkin-Elmer gas chromatograph, Model 881, modified for capillaries was used. Capillary columns, 0.02 inch X 150 feet, coated with Carbowax 20M and DEGS, were used as liquid phases. Reagents. Anhydrous (&)-2-butanol, 3-4N in anhydrous HC1 was used for preparing racemic esters. Optically active (+)-2-butanol and (+)-2,2-dimethyl-3-butanol were used for preparing the L-amino acid-(+)-alcohol (L+) diastereomer for peak assignment. The method of Halpern and Westley (12) was used for the preparation of these optically-active alcohols. Racemic and optically-active L-amino acids (Mann Research Laboratories) of threonine, serine, hydroxyproline, tyrosine, aspartic acid, tryptophan, and cysteine were used for synthesis of the esters. Pentafluorobenzoyl chloride (Peninsular Chem. Res., Inc.), trifluoroacetic anhydride, anhydrous methanol, methylene chloride, (+))-2,2-dimethyl-3-butanol, p-toluenesulfonic acid hydrate, toluene, benzene, pyridine, diethyl ether, and acetyl chloride freshly distilled from N,N-dimethylaniline were the other reagents used in the preparation of these derivatives. Procedure, Preparation of 0- and S-Acetyl-N-TFA-2Butyl Esters of Hydroxyl and Sulfhydryl Amino Acids. Five milligrams of amino acid were placed in a dry, screwcapped vial containing a micro, Teflon-coated stirring bar. To the vial was added 3 ml of the HCI-2-butanol reagent. The vial was capped with a Teflon-lined cap and placed in an oil bath at 100 "C for 3 hours to effect esterification. After (1) F. Weygand, A. Prox, L. Schmidhammer, and W. Konig, Angew. Cliem. bitermt. Ed., 2, 183 (1963). (2) R. Charles, G. Fischer, and E. Gil-Av, Israel J . Cliem., 1,
234 (1963). (3) E. Gil-Av, R. Charles, and G. Fischer, J . Chromatog., 17, 408 (1965). (4) B. Halpern and J. W. Westley, Biochem. Biophys. Res. Commun., 19, 361 (1965). ( 5 ) G. E. Pollock, V. I. Oyama, and R. D. Johnson, J . Gas Chromatog., 3, 174 (1965). (6) B. Halpern and J . W. Westley, Cliem. Commun., 12,246 (1965). (7) S. V. Vitt, M. B. Saporovskaya, I. P. Gudkova, and V. M. Belikov, Tetra/iedro/zLetters, 30, 2575 (1965). (8) G. E. Pollock and V. I. Oyama, J. Gas Chromatog., 4, 126 (1966). (9) B. Halpern and J. W. Westley, Tetrahedron Letters, 21, 2283 (1966). (10) E. Gil-Av, R. Charles-Sigler, G. Fischer, and D. Nurok, J . Gas Cliromatog., 4, 51 (1966). (11) E. Gil-Av and B. Feibush, Tetrahedron Letters, 35, 3345 (1967). (12) B. Halpern and J. Westley, Australian Journal of Chem., 19, 1533 (1966). 1356
0
ANALYTICAL CHEMISTRY
esterification, the HC1-2-butanol was removed in vacuo on a rotary evaporator. To the tube were added 1.0 ml of methylene chloride and 0.2 ml of trifluoroacetic anhydride. The tube was capped, heated at 100 "C for 5 minutes, cooled, and the solvent and excess reagent were removed in vacuo. Two milliliters of anhydrous methanol were added, and the tube was capped and allowed to stand for 20 hours at room temperature. The alcohol treatment quantitatively removed the trifluoroacetyl group from the oxygen and sulfur atoms. The evaporation process was repeated to dryness. To the tube was added a small, dry magnetic stirring bar and 1 ml of anhydrous methylene chloride. The capped tube was cooled in an ice bath to 0-5 "C while stirring. The cap was removed and 0.05 ml of anhydrous pyridine was added with stirring. A pledget of dry cotton was used to stopper the tube. The addition of 0.05 ml of anhydrous, freshly-distilled acetyl chloride was added over a period of 1-2 minutes to the stirred tube from a micropipet. The cotton was removed, the tube capped, removed from the ice bath, and allowed to remain at room temperature for 30 minutes. The solvents and reagents were then removed in vacuo. One milliliter of anhydrous, alcohol-free diethyl ether was then added t o the tube to dissolve the derivative. After centrifugation or filtration, the insoluble precipitate of pyridine hydrochloride was washed with 2.0 ml of anhydrous ether. The combined ether extracts were evaporated to dryness. The derivative was dissolved in ethanol and chromatographed. Preparation of N-Pentafluorobenzoyl-AsparticAcid 2-Butyl Ester. The esterification of aspartic acid with 2-butanol was as described for the hydroxy derivatives. After removal of the alcohol, 1.0 ml of methylene chloride, 0.05 ml of pyridine, and 0.1 ml of pentafluorobenzoyl chloride were added to the aspartic acid 2-butyl ester hydrochloride. The tube was capped and heated at 100 "C for 5 minutes. Reagents were removed in vacuo at 60 "C (starting at room temperature to avoid bumping). One milliliter of anhydrous, alcohol-free diethyl ether was added, dissolving the aspartic acid derivative, but not the pyridine hydrochloride. The tube was centrifuged to remove the precipitate and the liquid was ready for chromatography. Preparation of N-TFA-Aspartic Acid 2,2-Dimethyl-3Butyl Ester. Into a 50-ml round-bottomed flask was placed 0.0025 mole of aspartic acid. To this were added 0.0033 mole of p-toluenesulfonic acid hydrate, 0.01 mole of 2,2dimethyl-3-butano1, and 25 ml of a mixture of toluene and benzene (15:35). The flask was then attached to a DeanStark tube and the tube was filled with the toluene-benzene mixture. This was a critical point because the loss of alcohol in an empty side arm was sufficient to give incomplete esterification in the reaction flask. The flask was refluxed for 32 hours with a condenser protected from atmospheric moisture by a Drierite tube. The liquids were removed in vacuo and the aspartic acid ester p-toluenesulfonic acid salt was acylated with trifluoroacetic anhydride as previously described. The acylated sample was dissolved in dry methylene chloride after removal of the acylation reagents. After several minutes, the free p-toluenesulfonic acid crystallized and was removed by filtration. The filtrate contained the aspartic acid derivative ready for chromatography. Preparation of N,N-di-TFA-Tryptophan 2-Butyl Ester. Tryptophan was esterified and trifluoroacylated as described
Table I.
Gas Chromatographic Data Relevant to the Resolution of Racemic Hydroxy, Sulfhydryl Amino Acids, Aspartic Acid, and Tryptophan Temperature,
Amino acid Threoninec Serinec Hydroxyprolinec Cysteinec Aspartic Aci&
Derivative 0-acetyl-N-TFA-2-butyl ester 0-acetyl-N-TFA-2-butyl ester O,N-diacetyl-2-butyl ester 0-acetyl-N-TFA-2-butyl ester 0-acetyl-N-TFA-2-butyl ester O,N-diacetyl-2-butyl ester 0-acetyl-N-TFA-2-butyl ester O,N-diacetyl-2-butyl ester S-acetyl-N-TFA-2-butyl ester N-TFA-2,2-dimethyl-3-butyl ester
"C
150 140 180 150 140 180 150 180 150 150
He flow rate, ml/min 10 10 10 10
Retention time, minutes 5.63 7.99 11.85 9.88 16.14 17.42 29.97 45.46 24.32 20.86
10
10 10 10 10 10
Resolution, %" 76.4 86.2 77.5 84.1 85.7 58.2 74.0 50.9 86.0 90.0
D( +)
L(+)
5.84 8.32 12.34 10.33 16.92 18.05 30.80 46.74 25.56 22.24
L+/D+~
1.037 1.041 1.041 1.045 1.048 1.036 1.027 1.028 1.050 1.066
N-pentafluorobenzoyl-2-butyl
ester 190 10 42.04 43,55 81.0 1.035 Tyrosined 0-acetyl-N-TFA-2-butyl ester 180 7.50 43.39 45.56 84.2 1.050 Tryptophand N,N-di-TFA-Zbutyl ester 180 7.50 36.11 37.98 73.9 1.051 a Kaiser's ( I S ) graphical method was used for obtaining a realistic and meaningful resolution value. * L + / D + values are reported to indicate degree of resolution, but we have found them misleading depending on the column and for this reason prefer Kaiser's method. These derivatives chromatographed on a 0.02-inch X 150-foot Carbowax 20M column, These derivatives chromatographed on a 0.02-inch X 150-foot DEGS column.
for the hydroxy derivatives. After trifluoroacylhtion, the reagents were removed in vacuo, and the trifluoroacylation step was repeated a second time to assure completion of the reaction. After removal of the solvents, the derivative was ready for chromatography. All of the compounds reported in this section have had their structures verified by mass spectrometry. The overall yields of the hydroxy derivatives range from 85-95 of theory while the aspartic acid and tryptophan derivatives are virtually quantitative (95-100x). Only in the case of cysteine is the The pure S-acetyl-N-TFA-2yield unsatisfactory (22 butyl ester of cysteine was obtained by sublimation in vacuo and chromatographed; its structure was proven by mass spectrometry. Repetition of the method reported here, but in a nitrogen atmosphere, did not significantly improve the yield of the cysteine derivative. The quantitative synthesis of this derivative is still under study. It should be mentioned that while the yields of the hydroxy and sulfhydryl derivatives fall short of loo%, they give equal peaks for a racemic acid, indicating that the side reactions which occur d o not discriminate between the D- or L- forms of the amino acid. This is true even of cysteine which gives such a low yield. Side reactions of a degradative nature occur with the hydroxy amino acids as a result of esterification. Other side reactions of a different type occur as a result of 0-acetylation with acetic anhydride. These reactions are also being studied further by gas chromatographic-mass spectrometric methods. When acetyl chloride-pyridine is used for 0-acetylation, side reactions of the type found with acetic anhydride are either miniscule or nonexistent,
z
z).
RESULTS AND DISCUSSION
Previous results have shown that the hydroxy and sulfhydryl amino acid diastereomers were very poorly resolved as the 0,N-di-TFA 2-butyl esters, but threonine N-TFA free hydroxyl was well resolved (8). However, the free hydroxy derivatives of the other amino acids did not resolve well, so other derivatives were tried. Initially, the 0,N-diacetyl derivatives of serine and threonine were prepared and found t o be resolved fairly well on Carbowax 20M, but higher tem(13) R. Kaiser, "Gas Phase Chromatography," Vol. 1, Butterworths, Washington, D. C., 1963, p 39.
0,N- DIACETYLTHREONINE, 0.3 r
20
2-BUTYLESTER
c
1.0 r
r
0.9 0.8 -
5
25
1
n
0.5
15
0.4
I
H-TFA-TYROSINE, 2-EUTYLESTER
r
:':iA, ,
0.I
0
50
45
40
TIME, min
I5
20
IO
0-ACETYL N - TFA-SERINE, 2-BUTYLESTER
N-T~A-HYDROXYPROLINE, 2-BUTYIESTER r
5
IO
0
L-35!!L 30
40
N,
0-ACETYL 0.5
2-EUTYLESTER
,
J I", 15 10 0-ACEIYL N-TFA-THREONINE, 2-BUIYLESTER
O,N -DlACETYtH~DROXYPROLlHf,
0 , N - DIACEIYLSERINE, 9-EUTYLESTER
35
2
H-TFA-ASPARTIC ACID, 2, 2-Di-METHYL
N-01-TTATRYPTOPHAN, 2-BUTILESTER
c
3-BUIYLESIER
1 j\ 1 ,Al t
h,
,
45
40 TIME,
35
mln
30
30
25 TIME,
20
15
min
Figure 1. Resolution of subject amino acids under conditions given in Table I peratures were necessary, and hydroxyproline was not well resolved. The retention values are shown in Table I. With this clue, however, we attempted t o synthesize the 0-acetyl-N-TFA and S-acetyl-N-TFA derivatives. Initially we tried acylation with methyltrifluoroacetate in methanol, This method acylates only the amino groups. Poor results were always obtained, as some 0,N-diacetyl derivative was formed, indicating incomplete amino acylation. Acylation with trifluoroacetic anhydride followed by alcoholysis of the 0 - T F A provided relatively clean derivatives. We followed the alcoholysis of threonine, serine, and tyrosine using a I/*inch X 5-foot packed column of SE-30 on 60-80 mesh Chromosorb W. This enabled us to see the di-TFA derivative
5z
VOL. 40, NO. 8, JULY 1968
1357
Also shown in Table I and Figure 1 is the pertinent information concerning tryptophan and aspartic acid. The aspartic acid may be resolved as either of the derivatives given with about equal facility although the 2,2-dimethyl-3-butyl ester N-TFA derivative can be chromatographed at a lower temperature than the N-pentafluorobenzoyl derivative, and resolution is slightly better. With the successful resolution of these racemic amino acid diastereomers, only three protein amino acids have not been resolved by gas chromatography: histidine, cystine, and arginine. Temperatures for chromatography of these derivatives may need to be over 200 “C. Most of the phases used thus far in this study have had temperature limits of 200 “C or below for capillary columns. Of course, we may not have tried the best phase with derivatives of these amino acids. For instance, most of the amino acids are resolved on a single column using a polar phase such as Carbowax 20M or BDS; however, tyrosine and tryptophan require the use of the less polar phase, DEGS.
and t o follow the formation of the N-TFA-free OH derivative. Only a few minutes were necessary for the analysis. We found, a s Darbre and Blau (14) reported for somewhat different conditions, that the tyrosine 0 - T F A dissociated very rapidly while serine and threonine derivatives were more stable. Serine existed completely as the free hydroxyl derivative after about 7 hours, while threonine still had a trace of di-TFA after 16 hours, and existed completely as the free hydroxyl after about 20 hours. Tyrosine existed wholly as the free hydroxyl in about 2 hours. Using this solvolysis procedure for all of the subject multifunctional amino acids, we prepared relatively clean derivatives. Some side reactions did occur with sulfhydryl and hydroxy amino acids, as earlier noted by Halasz and Buennig (15). Initially, it was thought that some decomposition of the N-TFA t o form N-acetyl might occur, but the 0-acetyl-N-TFA derivatives showed no trace of the diacetyl derivative; and derivatizing N-TFA valine ester in the presence of acetyl chloride-pyridine showed no N-acetyl valine ester. This method could be used for studying hydroxy derivatives in the presence of the monoamino, monocarboxylic acids without the possibility of side reactions. Table I gives the gas chromatographic information concerning the amino acids reported. The degree of resolution which can be expected from these derivatives under the conditions given is shown in Figure 1.
We acknowledge the help of John Hayes and Michael Romiez in determining the mass spectra of these derivatives and verifying their structures.
(14) A. Darbre and K. Blau, Biochem. Biophys. Acta, 100, 298 (1965). (15) I. Halasz and K. Buennig, 2. A m / . Ckem., 211, 1 (1965).
RECEIVED for review October 23, 1967. Accepted April 23, 1968.
ACKNOWLEDGMENT
Gas Chromatographic Analysis of Tall Oil Fatty Alkyds for Monomer and Dimer Acid Content R. A. L. Paylor, Raymond Feinland,’ and N. H. Conroy American Cyanamid Co. and Arizona Chemical Co., Stamford, Conn.
INCONNECTION with a study of the importance of thermal dimerization during alkyd cooking ( I ) , a method was needed to determine the amount of monomer and dimer acid present in fatty acid alkyd resins after completion of the alkyd cook. AS the first step of the analysis, the acids can be liberated quantitatively from the alkyd by alkaline hydrolysis (2). The problem is then one of analysis of a mixture of monomer and dimer acids. Various methods for the determination of monomer and dimer fatty acids, based on molecular distillation (3-5) and liquid partition chromatography (6) have been reported in the literature. These methods are unsuitable because they require Present address, Clairol Corp., Stamford, Conn. (1) J. T. Geoghegan, H. G. Arlt, Jr., and C. 0. Myatt, “Abstracts,” ACS Meeting, Miami Beach, Fla., April 1967, D 6. (2) ASTM Standards, 20, 650 (1967). (3) T. F. Bradley and W. B. Johnston, Znd. Eng. Chem., 33, 86 (1941). (4) S. A. Harrison and D. H. Wheeler, J . Am. Chem. Soc., 76, 2379 (1945). (5) R. F. Paschke, J. R. Kearns, and D. H. Wheeler, J . Am. Oil
excessive time, special apparatus and skills, and in some cases, they lack sufficient precision. Gas chromatography was therefore investigated as a n alternative technique, and a method has been developed for the analysis of monomer and dimer fatty acids as their methyl esters, This method was found to be rapid and convenient. Results obtained for weighed mixtures of monomer and dimer acids are in satisfactory agreement with the known compositions. EXPERIMENTAL
Hydrolysis. The hydrolysis of the alkyl resin t o liberate the acids was performed according to ASTM Method D 1398-58 (2) without modification. Preparation of Methyl Esters. The preparation of the methyl esters was carried out using a methanol-boron trifluoride procedure based on that reported by Metcalf and Schmitz (7). The boron trifluoride-methanol esterification reagent was prepared by dissolving 27 grams of BFs-etherate (Eastman No. 4272) in 100 ml of methyl alcohol. One gram of sample and 0.2 gram of squalane, the internal standard, were weighed to the nearest 0.1 mg into a small flask. Twenty-six milliliters of the esterification reagent
Chemists’ SOC.,31, 5 (1954).
(6) E. N. Frankel, C. D. Evans, H. A. Moser, D. G. McConnell, and J. C. Cowan, ibid., 38, 130 (1961). 1358
ANALYTICAL CHEMISTRY
(7) L. D. Metcalf and A. A. Schmitz, ANAL,CHEM., 33, 363 (1961).
