The Enzymatic Resolution of Aromatic Amino Acids Richard Sheardyl, L. Llotta, E. Steinhalt, R. Champion, J. Rinkw, M. Planutis, J. Sallnkas, T. Boyer, and D. Carcanague The Pennsylvania State University, Hazleton, PA 18201 T h e significance of the concepts of stereoisomerism is often not well understood by thetypical undergraduate organic chemistry student. T h e notions of chirality, optical activitv. and resolution donot lend themselves to e&v&nla* . nations of their principles. Certainly, the use of models enables one to demonstrate chiralitv. hut the conceots of ontical activity and resolution presenya more difficuit prohiem. T h e best approach is throueh the use of a laboratorv exoeriment. his kxperiment should he designed to demonstrate as many principles of stereoisomerism as oossihle h u t should he effiiiint in terms of time and preparation. The use of biological molecules affords an excellent avenue to this end. For example, the activity of an enzyme is dictated by its geometrical structure, and this structure is ultimately a function of the sequence and chiralities of its constant amino acids. Since most naturally occurring amino acids are of the L-configuration, most enzymes whiih catalyze reactions involving amino acids are active only in the presence of L-amino acids. Thus.. a iudiciouslv chosen eniyme can he employed to separate L-amino acids from Damino acids (i.e., a resolution) due to its specificity. One enzyme that has been successfully used in the resolution of a number of amino acids is paoain ( I . 2). T h e vrocedure, however, has two disadvantayes. 1) he e n ~ y n ereaction is complete only after a couple of days. 2) Furthermore. the harsh hydrolysis of the gioups could lead some racemization and ultimate loss of optical purity. On the other hand, m-chymotrypsin, which hydrolyzes the peptide bonds of aromatic amino acids as well as their esters and amides, has proven to he an excellent agent in the resolution of hoth naturally occurring aromatic amino acids (i.e., phenylalanine, tryptophan, and tyrosine) and derivatized amino acids. For example, hoth L-p-fluoro- and L-p-chlorophenylalanines have heen shown to he substrates for the enzyme (3). We wish to report that L-o-fluoro- and L-mfluorophenylalaninesare also substrates for this enzvme. Initially, we were interested in obtaining opticafly pure fluorophenylalanines from the commerciallv available D.L mixtures. The ease and speed of the a-chymbtrypsin prockdure, relative to the papain procedure, suggested that it could easily he incorporated into the regular organic laboratory course. T h e phenylalanines are converted to their ethyl or methyl esters via standard techniques and then treated with enzyme. Commercially available esters may also be used to preclude the esterification step. The enzyme reaction is complete within 45 min. Workup of the reaction mixture vields the L-ohenvlalanine as the acid and the Dphenylalanine as its e;ter.~uantities sufficient for meaning1'111polarimetry data and of hirh ~ u r i.t vare obtained - ootical . . for i l l species.-
-
td
'
The authors wish to acknowledge the support of a PSU-FSSF grant for this project. This paper was presented at the 18th Middle Atlantic Regional Meeting. American Chemical Society. May 21-23, 1984. For example, esterificationmay be achieved by adding the amino acid (2.0 g) to 150 mL of ice cold alcohol containing 40 mL of SOCI, followed by warming to 40 OC and maintaining that temperature for 2 h (6). Esterification may also be achieved by refluxing an alcohol solution of the amino acid containing 1-2 mL of concentrated H,SO, for 2 h. Note: Normally, 2.0 g of ester hydrochloride can be obtained from the esterification of 2.0 g of amino acid.
646
Journal of Chemical Education
Other experiments using a-chymotrypsin have been re(4,5), but they offer disadvantages ported in THIS JOURNAL in that they require additional synthetic steps or are nonpreparative in nature. The experiment reported here is flexible in that any aromatic amino acid mav he used. includine unnaturally occurring derivatives. The esterification step may lw excluded since all of the naturaliv occurrine arumatic amino acids are commercially available as their ethyl or methyl esters. In addition, a-chvmotrv~sin. .. . all amino acids. and amino acid derivatives areinexpensive and stable a t 4 "C for many months. T h e experiment is divided into three parts: 1)the esterification (which, again, may be excluded); 2) the resolution; and 3) the determination of ootical nuritv. ~* Anv - -, ~ suitnhle - ~ ~ - ~ - ~ - - esterification procedure may he'used to obtain the ethyl or methvl ester of the ~ h e n v l a l a n i n eHowever. .~ it is imoerative that the resultant D,L-ester he free of any "nreactkd acid. For the resolution and polarimetry steps, we found it best that students work in pairs in order to obtain sufficient quantities of desired products. Each pair of students needs a pH meter to monitor the progress of the reaction. T h e resolution, as well as the polarimetry, can be completed in two and one-half 3-h laboratory sessions. n
.
~
~
Experimental Procedures Resolution with aGhymotrypsin The aromatic amino acid esters (either commercially available or synthesized by us) were treated with the enzyme in amanner similar toTong, et al. (3).We used bovine pancreatic ol-chymotrypain Type I1 (E.C. 3.4.21.1) purchased from Sigma Chemical Co., St. Louis, MO. Two grams of the D,L-phenylalanylester (either ethyl or methyl) was dissolved in 30 mL of H20 and the pH of the solution adjusted to 5.0 with 0.2 M LiOH. Two hundred forty milligrams of the enzyme was dissolved in 5 mL of H20 and added to the amino acid ester solution. The pH of the solution began to drop rapidly. Dropwise addition of 0.2 MLiOH from a buret maintained the pH at 5.0. The pH stabilized within 10-20 min and the reaction was allowed tosit for an additional 10-20min. The reaction was swirled or stirred frequently throughout the addition of the LiOH. The solution was concentrated at 50 OC with aspiration until it was very cloudy. To prevent foamingduring this process, a little Sigma AntiFoam was added to the solution. The flask containing the white crvstals was allowed to sit in an ice bath for 45 min or -in- ~the ~--. refrigerator overnight. These crystals may remain in the refrigerator for up to a week with no serious problem. This is a convenient ~
~
~
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~~
.. ~ ~ ~ - .
stonninr noint-~
l ' h s cr)*tuls of I.-phenylalaninc acid were collected by suction filtmtiun, washed with a iew miliilirerp o f r u l d ethanol and dried in
anwennt 50°C ThefiltrstewnsadiustrdtuvH9 withO2M l.iUH and the D-esterwas extracted with three washings of ethyl acetate. The pooled organic washings were dried over NalS01 and filtered. In order to obtain the ester as its hydrochloride, the ethyl acetate solution was treated with a solution of 1.0 mL SOCll in 10 mL of ice cold ethanol. Care should be exercised when preparing the SOClz solution. The best method is to add the SOClz very slowly to ice cold ethanol to avoid splattering of the ethanol. Furthermore, all procedures using the SOCla should he performed in the hood. Once the SOClz solution had been added to the ethyl acetate, the resultant solution was purged with dry filtered air for about 10 min. The organic solvents were then gently distilled off,with aspiration,in the hood. The resultant residue was dissolved in absolute ethanol (25 mL), and the ethanol was again distilled off. This procedure is necessary to remove residual SOC12. The resultant crystals were thendissolved in 1-2 mL ofwarm ethanol and precipitated with 150
Table 1. Results of the Enzymatb Resolution MP ('C)
Compound
Lit.
Table 2. Base Comparlson for Enzyme Resolvtlon
bb20
(MolesD.L-Ester)/2 Moles Base Used
Base Used
Found
Lit.
Found
280-284 155-157
-33.4 T 3.9
-33 4 35
224-228
-23
-228
1.159 X 1.159 X 1.159 X
LiOH
284 DPhenylalanineMelhylEsterV59-161 .-OF-Pheny alan ner 250-255 L-PF-Phenylalanine Ethyl Ester" 0-PF-PhenylalanineEthyl Esterd L-mF-Phenylalminec 239-243 L-mF-PhenyialanineEthyl Ester@ DmF-Phenylalanine Ethyl Esterd L-OF-PhenylalanineS 226-231 L-OF-Phenylalanine Ethyl Estd C-OF-Phenyialanine Ethyl Esterd L-Fheny alan ned
+
Pvrltyandaulhentlclly of all compounds wereverified by NMR, elemental analyds, and TLC. Melting points repated here are uncorrected. Valves lw all esters are for thelr hydmchlaides. 'From cammerclallyavallabls. 0.L-~henylalanlns methyl ester. c = 1.3%In H a 1 L
mL of ether. The D-phenylalanyl ester thus obtained was collected by suction and allowed to dry thoroughly. Deiermlnatlon of Optlcal Purity The compounds obtained above were dissolved in the solvent and to the concentration as reoorted in Table 1. It is imperative that the polarimetry solutions be ahsolutely clear. The specific rotations were then determined from the observed rotation (as obtained from the polarimeter) by the following equation: (aJDZ0 = a,blle
(1)
where soh, is the observed rotation. 1 is the cell ~ a t leneth h in decimeters, and c is the concentration of the c&npouniin g/ mL of solution. Experimental rotations were determined on a Perkin-Elmer 241 polarimeter. Any polarimeter capable of determining rotations of i0.50° or more should be suitable for the determinations. Comparison of the specific rotations of these compounds to literature values (when available) confirmed optical purity (see Tahle 1). Since literature values for the fluorophenylalanvl ethvl esters were not available.. o ~ t i c a louritv for thesecomp&nds was determined in the following manner. Since the L-fluoro~henvlalanines obtained from the enzvme . . preparation were optically pure, conversion of these back to their ethyl esters via a procedure that would lead to no racemization would give rise to optically pure L-esters. Di-
.
NaOH KOH
%
Differences
1.156 X lo-= 1.159 X lo-3 1.152 X I O P
0.26 000 0.60
rect comparison of the specific rotations of these L-esters thus obtained to those of the D-esters isolated from the enzyme preparation indicated optical purity for all fluorophenylalanyl ethyl esters. I t should be noted that the values for the specific rotations of these esters are being reported here for the first time. As further proof of optical purity, another set of experiments was performed. The actual amount of D,L-ester reacting with the enzyme was determined quantitatively by careful titration of the enzyme reaction with standardized LiOH s ~ l u t i o nThe . ~ results in Table 2 indicate tbat exactly 0.50 equivalents of the D,L-ester actually reacted. The results in Tahle 2 also indicate tbat high optical purity was obtained when using NaOH or KOH as the base. The enzymatic resolution 01' aromatic aminnacid esters hy ,I-chymotrypiin is an excellent experiment to demonstrate many aspects of stereoisomerism, organic synthesis, and hiochemistry. I t illustrates: 1) the differences in the physical and biological properties of enantiomers; 2) the speed and efficiency of enzyme-mediated reactions; 3) the concept of optical activity; 4) methods of resolution; and 5) techniques to determine optical purity. Literature Clted ~
~
(11 Mahrig, J. R.;Shapiro.S.M. J Cham.Educ. 1976.53,586. (21 D0herty.D. G.;Popenae, E.A. J.Biol. Chem. 1951.189,417.
(8) T0ng.J.H.; Peiitc1erc.C.: D'Lorio,A.:Bonoitan,N.L. Con. J.Biocharn. 1911,189,877. (4) Clement, G. E.: Potter. R. J. J. Chem. Educ. 1971.8.695. (5) Bender,M.L.:Kezdy, F. J.; Wed1er.F.C.J. Chsrn.Educ. 1967,44,84. (6)Green8tein.L. P.; Winitz.M. "Chemiatryofthe AminoAeids". Vol2; Wiley: New York. 1961.
. .
a The drop in pH of the enzyme reaction is due to the production of H,O+. Since the D,L-esteris 50% Lester, and since 1.0 equivalent of H30+ is produced per equivalent of Lester, 0.50 equivalents of H,Of
is produced per equivalent of D.L-ester.Therefore, quantitative titrations of the enzyme reaction indicate the extent of reaction.
Volume 63 Number 7 July 1966
647
