Anal. Chem. 1998, 70, 1059-1065
Articles
Characterization and Evaluation of d-(+)-Tubocurarine Chloride as a Chiral Selector for Capillary Electrophoretic Enantioseparations Usha B. Nair,†,§ Daniel W. Armstrong,† and Willie L. Hinze*,‡
Departments of Chemistry, University of MissourisRolla, Rolla, Missouri 65401, and Wake Forest University, P.O. Box 7486, Winston-Salem, North Carolina 27109
A new macrocyclic of the bis(benzylisoquinoline) alkaloid family, d-(+)-tubocurarine chloride (DTC), has been evaluated as a chiral selector for the separation of optical isomers of organic carboxylates using capillary electrophoresis (CE). The pertinent physicochemical properties, such as absorption spectrum, isoionic point, and solution conformation, of DTC were determined. The effects of varying such experimental parameters as DTC concentration, pH, and methanol content in the running buffer were assessed. CE separation of the enantiomers of 18 different compounds was achieved using DTC as the chiral selector under optimized background electrolytic conditions. A vast amount of the recent capillary electrophoretic literature focuses on the utilization of chiral selectors in the running buffer to affect the resolution of optical isomers.1-11 Among the many recently described chiral selectors, perhaps the macrocyclic antibiotics have had the most immediate and dramatic impact.4 In particular, the oligophenolic glycopeptide antibiotics (vancomycin, teicoplanin, ristocetin A) and ansamycins (rifamycins) are enjoying widespread applications in capillary electrophoresis (CE) for the separation of enantiomers.4,12-17
In addition to such chiral antibiotics, there are, of course, many other different types of macrocyclic compounds. For example, the bis(benzylisoquinoline) alkaloids represent an important group of macrocyclic compounds, in which two benzyltetrahydroisoquinoline residues are linked together asymmetrically.18 The more important members of this class include curine, chondrocurine, and tubocurarine, which differ with respect to the position and number of the hydroxyl and methoxy groups present on the two aromatic rings. These alkaloids all have two stereogenic centers. Herein, the pertinent physicochemical properties and the use of one member of this family of macrocyclics, specifically 7′,12′dihydroxy-6,6′-dimethoxy-2,2′,2′-trimethyltubocuraranium chloride hydrochloride, or simply d-(+)-tubocurarine chloride (DTC), of South American arrow poison fame, as a chiral selector in CE for enantiomeric separations are reported. Results indicate that tubocurarine can function as an effective chiral additive for the CE separation of the optical isomers of compounds containing a carboxylate moiety in the vicinity of the stereogenic center. The selection of enantiomeric organic carboxylates as the general analyte class was made on the basis of a previous conductivity study, which indicated that tubocurarine selectively interacted with organic anions, including enantiospecific binding of some N-acetylR-amino acids.19
†
University of MissourisRolla. Wake Forest University. § Current address: Pharmaceutical R&D Department, Forest Laboratories, Inc., Farmingdale, NY 11735. (1) Ward, T. J. Anal. Chem. 1994, 66, 633A-640A. (2) Novotny, M.; Soini, H.; Stefansson, M. Anal. Chem. 1994, 66, 646A-655A. (3) Lin, B.; Zhu, X.; Koppenhoefer, B.; Epperlein, U. LC-GC 1997, 15, 40-46. (4) Ward, T. J. LC-GC 1996, 14, 886-894. (5) Camilleri, P. Chem. Commun. 1996, 1851-1858. (6) Nishi, H.; Izumoto, S.; Nakamura, K.; Nakai, H.; Sato, T. Chromatographia 1996, 42, 617-630. (7) Chankvetadze, B.; Endresz, G.; Blaschke, G. Chem. Soc. Rev. 1996, 141153. (8) Stalcup, A. M.; Gahm, K. H. Anal. Chem. 1996, 68, 1360-1368. (9) Wang, J.; Warner, I. M. Anal. Chem. 1994, 66, 3773-3776. (10) Stalcup, A. M.; Agyei, N. M. Anal. Chem. 1994, 66, 3054-3059. (11) Shelton, C. M.; Seaver, K. E.; Wheeler, J. F.; Kane-Maguire, N. A. P. Inorg. Chem. 1977, 36, 1532-1533. (12) Armstrong, D. W.; Rundlett, K. L.; Chen, J. R. Chirality 1994, 6, 496-509. (13) Gasper, M. P.; Berthod, A.; Nair, U. B.; Armstrong, D. W. Anal. Chem. 1996, 68, 2501-2514. ‡
S0003-2700(97)00860-3 CCC: $15.00 Published on Web 02/07/1998
© 1998 American Chemical Society
EXPERIMENTAL SECTION Materials. Amethopterin, potassium hydroxide, sodium dihydrogen phosphate, and amino acid derivatives were purchased from Sigma Chemical Co. (St. Louis, MO). (+)-Tubocurarine chloride pentahydrate and all other chiral compounds containing carboxylic acid functional groups were obtained from Aldrich Chemical Co. (Milwaukee, WI). HPLC grade methanol, acetone, sodium hydroxide, and reagent grade hydrochloric acid were obtained from Fisher Scientific Co. (St. Louis, MO). (14) Rundlett, K. L.; Gasper, M. P.; Zhou, E. Y.; Armstrong, D. W. Chirality 1996, 8, 88-107. (15) Fanali, S.; Desiderio, C. J. High Resolut. Chromatogr. 1996, 19, 322-326. (16) Sharp, V. S.; Risley, D. S.; McCarthy, S.; Huff, B. E.; Strege, M. A. J. Liq. Chromatogr. Relat. Technol. 1997, 20, 887-898. (17) Ward, T. J.; Dann, C.; Brown, A. P. Chirality 1996, 8, 77-83. (18) Shamma, M. The Isoquinoline Alkaloids; Academic Press: New York, 1972.
Analytical Chemistry, Vol. 70, No. 6, March 15, 1998 1059
Methods. All capillary electrophoretic separations were performed on a Beckman P/ACE 2000 CE unit (Beckman, Palo Alto, CA) equipped with a 50-µm × 47-cm fused-silica capillary (40 cm to the detection window). Data acquisition was done using System Gold software. The CE was used in the reversed polarity mode, i.e., with the anode at the detector end. Direct UV detection was employed using a detector wavelength of 254 nm. The temperature was maintained at 25 °C. The run voltage for all separations was -15 kV. Methanol or acetone was used as the electroosmotic flow marker. As reported for other quaternary ammonium CE additives, tubocurarine (DTC) electrostatically interacts with the capillary wall. Hence, a rinse program to condition the capillary was used before each sample run. This consisted of four rinses, each of 2.0-min duration, using (i) doubly distilled water, (ii) 0.10 N HCl, (iii) doubly distilled water, and (iv) 0.50 N KOH. An additional methanol rinse was done after every 2-3 h of capillary use. This capillary conditioning regimen ensures the reproducibility of the EOF velocities and migration times. Samples were introduced into the capillary by the pressure injection mode at 0.50 psi for 1-3 s. All samples (analyte concentration, 1.0 mg/mL) were dissolved in methanol or methanol/phosphate buffer solution (1:1, v/v) and filtered with 0.45-µm nylon syringe filters (Alltech, Deerfield, IL) prior to analysis. Phosphate buffer solutions were prepared by adjusting the pH of 0.05 M sodium dihydrogen phosphate to the desired value using aqueous hydrochloric acid or sodium hydroxide. The tubocurarine solutions were prepared by dissolving appropriate amounts of the solid in the buffer or buffer/methanol mixture. For most of the CE separations, the running buffer solution consisted of 15 mM tubocurarine dissolved in 20:80 (v/ v) methanol/aqueous 0.05 M phosphate, buffered to a pH of 5.0, 6.0 or 7.0. The tubocurarine solutions appear to decompose when exposed to air; such solutions turned brownish-yellow within 12 h at the voltages employed in the CE runs. Hence, all tubocurarine running buffer solutions were stored in the dark and refrigerated when not in use. Caution: It should be noted that tubocurarine is a potent neuromuscular blocker20,21 and toxic, targeting nerves and skeleton muscles. Hence, all safety precautions recommended by the commercial supplier should be followed, particularly when handling the solid material. Specifically, the material safety data sheet recommends that the user “wear appropriate NIOSH/MSHAapproved respirator, chemical-resistant gloves and safety goggles” and “use [the solid] only in a chemical fume hood”.22 The LD50 values for mice/rats are reported to be in the range 28-33 (or 0.5-1.2) mg/kg for oral (or intraperitoneal injection) administration.23,24 (19) Alcantar, C. G.; Eliseev, A. V.; Yatsimirsky, A. K. Bioorg. Med. Chem. Lett. 1995, 5, 2993-2998. (20) Miller, R. D.; Savarese, J. J. In Anesthesia; Miller, R. D., Ed.; ChurchillLivingstone: New York, 1986; pp 889-943. (21) Punnen, S.; Gonzalez, E. R.; Krieger, A. J.; Sapru, H. N. Pharmacol. Biochem. Behavior 1984, 20, 85-89. (22) Material Safety Data Sheet (for D-Tubocurarine chloride, CAS Registry No. 57-94-3; MF C37H42CL2N206); Sigma-Aldrich Chemical Co.: St. Louis, MO, 1997; 4 pp. (23) The Merck Index; Budavari, S., Ed.; Merck & Co., Inc.: Rahway, NJ, 1989; p 1542. (24) Lamanna, C.; Hart, E. R. Toxicol. Appl. Pharmacol. 1968, 13, 307-315.
1060 Analytical Chemistry, Vol. 70, No. 6, March 15, 1998
The software used for molecular modeling studies was Alchemy III, obtained from Tripos Associates (St. Louis, MO). The program allows computation of the structural conformation of the tubocurarine macrocyclic compound corresponding to the minimal energy while taking into consideration the atom size, electrostatic and dipole interactions, bond lengths, and bond angles in three dimensions. RESULTS AND DISCUSSION d-(+)-Tubocurarine chloride (DTC, MW ) 681.66, structure I) is one of the alkaloids of tube curare from South America. It is
a neuromuscular blocking drug20,21 and has been used in surgery and intensive care units in very small doses as a complement to anesthetics to produce skeletal muscle relaxation as well as in the treatment of various neuromuscular disorders. DTC has also been suggested as a potentially good model or mimetic agent for cyclophanes since it resembles that class of compounds in some respects.19 The d-(+)-tubocurarine molecule is chiral, containing two stereogenic centers (structure I). The asymmetric carbon (C37, structure I) adjacent to the quaternary ammonium nitrogen atom is levorotatory (-) and has R as its absolute configuration, while the carbon (C-19, structure I) adjacent to the tertiary ammonium nitrogen atom is dextrorotatory (+) and has the S configuration.25,26 Despite the fact that DTC is a bulky molecule, it has a fairly compact structural conformation in the solid state and in solution. In agreement with NMR and X-ray crystallographic analysis of the molecular structure of DTC,27-31 the modeling study indicates that the tubocurarine molecule assumes a twisted or folded conformation (Figure 1a), with the two tetrahydroisoquinoline rings turned toward the center of the molecule (Figure 1b), creating a hydrophobic cleft. The distance between the two nitrogen atoms of the tetrahydroisoquinoline rings is about 10.0 Å (compared to a value of ∼9.0 Å based on the previous X-ray and NMR studies27-31). The phenol ring protrudes from the compact bulk of the molecule at an angle of 123° with respect to the tertiary tetrahydroisoquinoline ring (Figure 1b). Some binding studies suggest that, if a solute molecule binds to the positively charged hydrophobic domain of tubocurarine, a “cuplike” conformation is assumed, which serves to bring the two ammonium (25) Everett, A. J.; Lowe, L. A.; Wilkinson, S. Chem. Commun. 1970, 10201021. (26) Pedersen, S. E. J. Biol. Chem. 1995, 270, 31141-31150. (27) Codding, P. W.; James, M. N. G. J. Chem. Soc., Chem. Commun. 1972, 1174-1175. (28) Codding, P. W.; James, M. N. G. Acta Crystallogr. 1973, B29, 935-942. (29) Reynolds, C. D.; Palmer, R. A. Acta Crystallogr. 1976, B32, 1431-1439. (30) Fraenkel, Y.; Gershoni, J. M.; Navon, G. Biochemistry 1994, 33, 644-650. (31) Egan, R. S.; Stanaszek, R. S.; Williamson, D. W. J. Chem. Soc., Perkin Trans. 2 1973, 716-717.
Figure 2. UV-visible absorption spectrum of d-tubocurarine (1.0 × 10-4 M) in water at 25 °C.
Figure 1. Views of the structure of the d-tubocurarine chloride. Space-filling molecular models produced through energy minimization show (a) the side view of the molecule and (b) the view looking down into the molecule’s hydrophobic cleft. The colored atoms denote the hydrophilic atoms (red represents the oxygen atoms and orange the nitrogen atoms), while black represents the hydrophobic regions (carbon atoms of the aromatic rings and connecting carbons), with blue denoting the hydrogen atoms.
ions closer together (∼6-Å separation) and causes a separation of the hydrophobic cluster from the outer hydrophilic shell comprised of the six oxygen atoms.32 Based upon results of this work, the solubility of DTC in aqueous solution was estimated to be ∼25 mM. The solubility of DTC is variously reported as ∼35-70 mM in water (25 °C)23,33,34 and ∼20-43 mM in ethanol.23,34,35 It is also soluble to a similar extent in methanol but insoluble in chloroform, benzene, acetone, and ether.23,34 Although some literature reports indicated that very dilute aqueous solutions of DTC (∼0.02 mM) were stable for at least 4 months,36 it was observed in this work that DTC is easily oxidized in the presence of air, with its solutions turning a brownish-yellow color. As expected for a species containing several aromatic moieties, DTC absorbs in the UV spectral region. The UV-visible spectrum of an aqueous solution of tubocurarine chloride (Figure 2) shows a strong end absorption, with a wavelength maximum at 280 nm (32) Fraenkel, Y.; Gershoni, J. M.; Navon, G. Biochemistry 1994, 33, 644-650. (33) Dutcher, J. D. J. Am. Chem. Soc. 1952, 74, 2221-2225. (34) Tsunakawa, N.; Tamura, B. Iyakuhin Kenkya 1986, 17, 124-130 [Chem. Abstr. 1986, 104, 365 (213099s). (35) Pedersen, S E.; Papineni, R. V. L. J. Biol. Chem. 1995, 270, 31141-31150. (36) Annan, R. S.; Kim, C.; Martyn, J. J. Chromatogr. 1990, 526, 228-234.
and a minimum at 255 nm in the UV. The molar absorptivity for DTC in water was determined to be 10 200 M-1 cm-1 (λmax 280 nm), which is in agreement with values of 8600-11 800 M-1 cm-1 reported in the literature.33,37-39 The 280-nm peak of DTC shifts to higher wavelengths (290-300 nm) with increased pH, in agreement with previous reports.35,39 Although the DTC chiral selector absorbs in the UV region, direct detection of all analytes examined was possible at the low concentrations (15 mM) normally employed in the running buffer. More importantly the detection wavelength (254 nm) employed for the analytes in this study is very close to the absorption minimum of DTC (255 nm). Although not examined, indirect detection using the 280-nm peak of DTC should also be possible for many analytes. DTC exhibits only meager luminescence (φf ) 0.01) in solution.40 Thus, it should be possible to employ fluorescence detection schemes with this chiral selector. In fact, the presence of DTC is reported to elicit fluorescence enhancements for some aromatic compounds.19 As is evident from DTC’s structure, it contains three ionizable moieties. The three reported pKs for this compound are 7.6, 8.18.65, and 9.1-9.65, which are thought to be due to the loss of a proton from the p-phenolic group (12′-position), the ammonium group (N at position 2), and the phenolic group (7′-position), respectively.39,41,42 The electrophoretic mobility of DTC as a function of pH (using phosphate buffer) is shown in Figure 3. In this system, the point of zero electrophoretic mobility occurs at a pH of ∼9.3. As previously reported for other cationic quaternary ammonium-containing species, such as hexadecyltrimethylammonium chloride (or bromide) surfactants, poly(diallyldimethylammonium) chloride, poly(2-aminoethyl) methacrylate hydrochloride, [3,22]- and [3,6]-ionenes (Polybrene), etc.,43-49 when d-tub(37) Klein, D.; Gordon, S. M. J. Am. Pharm. Assoc. 1949, 38, 438-443. (38) Naghaway, J.; Stone, T. O. J. Pharm. Sci. 1979, 655-656. (39) Kalow, W. J. Pharmacol. 1954, 110, 433-442. (40) Gibson, E. P.; Turnbull, J. H. Analyst 1979, 104, 582-583. (41) Barlow, R. B. Br. J. Pharmacol. 1982, 75, 503-512. (42) Pedersen, S. E.; Papineni, R. V. L. J. Biol. Chem. 1995, 270, 31141-31150. (43) Terabe, S.; Isemura, T. Anal. Chem. 1990, 62, 650-652. (44) Ramsey, R. S.; Kerchner, G. A.; Cadet, J. J. J. High Resolut. Chromatogr. 1994, 17, 4-8. (45) Crosby, D.; El Rassi, Z. J. Liq. Chromatogr. 1993, 16, 2161-2187. (46) Hinze, W. L.; Moreno, B.; Quina, F. H.; Suzuki, Y.; Wang, H. Anal. Chem. 1994, 66, 3449-3457. (47) Cordova, E.; Gao, J.; Whitesides, G. M. Anal. Chem. 1997, 69, 1370-1379. (48) Yashimi, T.; Tsuchiya, A.; Morita, O.; Terabe, S. Anal. Chem. 1992, 64, 2981-2984. (49) Liu, Q.; Lin, F.; Hartwick, R. A. J. Liq. Chromatogr. Relat. Technol. 1997, 20, 707-718.
Analytical Chemistry, Vol. 70, No. 6, March 15, 1998
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Figure 3. Plot illustrating the effect of pH on the effective electrophoretic mobility of d-tubocurarine. Zero electrophoretic mobility occurs at approximately pH 9.3 in this system (0.10 M phosphate buffer).
Figure 4. Plot showing the effect of variation of the chiral selector, d-tubocurarine, concentration upon the resolution for the separation of the optical isomers of amethopterin (9) and di-O,O′-toluoyltartaric acid (2). The run buffer contained 0.05 M phosphate buffered to pH 7.0.
ocurarine is used in the running buffer, the positively charged DTC molecules sorb on the fused silica walls, resulting in formation of a positively charged surface. As a consequence, the bulk d-tubocurarine running buffer solution is electroosmotically driven toward the positive electrode (anodal electroosmotic flow). Thus, the CE runs were all conducted in the reversed-polarity mode. The electroosmotic flow (EOF) ranged from ∼1.2 to 0.90 × 10-4 cm2 V-1 s-1 for DTC solutions in the pH 5-7 region. The effect of variation of the chiral selector concentration (1.020.0 mM range) in the run buffer on the resolution of several racemates was investigated. This is illustrated in Figure 4 for the separation of racemic mixtures of di-O,O′-toluoyltartaric acid and amethopterin. Increasing the DTC concentration from 1 to 20 mM served to increase the resolution of di-O,O′-toluoyltartartic 1062 Analytical Chemistry, Vol. 70, No. 6, March 15, 1998
Figure 5. Electropherograms showing the resolution of racemic (A) di-O,O′-toluoyltartaric acid, (B) 2,4-dinitrophenylethionine, and (C) amethopterin. The running buffer was composed of 20:80 (v/v) methanol/aqueous 50 mM phosphate (buffered to pH 5.0, 5.0, and 6.0, respectively) containing 15.0 mM d-tubocurarine at 25 °C. A 40cm capillary was used with a run voltage of -15 kV.
Table 1. Chiral Compounds Resolved Using Tubocurarinea pH
t1b
t2b
µ1c
µ2c
Rs
amethopterin
6
20.92
21.60
0.54
0.35
1.3
proglumide
7
15.07
15.22
2.49
2.41
0.8
di-O,O′-p-toluyltartaric acid
5
14.20
15.07
1.45
0.94
1.6
ketoprofen
7
17.54
17.91
1.32
1.17
0.9
N-(R-methylbenzyl)phthalic acid monoamide
6
15.21
16.34
1.64
1.07
2.05
dansylmethionine
7
14.65
14.98
2.73
2.54
1.4
dansylglutamic acdi
7
12.69
12.80
4.05
3.96
0.7
dansylvaline
7
18.76
19.04
0.85
0.75
1.0
dansylnorvaline
7
14.43
14.74
2.86
2.67
1.0
dansylnorleucine
7
14.72
15.15
2.69
2.44
1.4
dansylphenylalanine
7
16.22
16.51
1.90
1.76
1.1
compound
Analytical Chemistry, Vol. 70, No. 6, March 15, 1998
1063
Table 1 (Continued) pH
t1b
t2b
µ1c
µ2c
Rs
dansyl-R-amino-n-butyric acid
7
13.92
14.14
3.17
3.03
1.0
(2,4-dinitrophenyl)norvaline
7
13.49
13.75
3.46
3.29
1.3
(2,4-dinitrophenyl)ethionine
5
17.46
18.20
1.73
1.44
1.9
(2,4-dinitrophenyl)-R-amino-n-butyric acid
7
12.55
12.82
4.16
3.95
1.0
(3,5-dinitropyridyl)norleucine
7
17.78
17.97
1.22
1.15
0.6
(3,5-dinitropyridyl)leucine
6
15.07
15.35
1.72
1.57
0.6
(3,5-dinitropyridyl)phenylalanine
7
15.39
15.62
2.31
2.19
1.0
compound
a Separations were done using 15 mM solution of tubocurarine dissolved in 20% methanol and 80% 0.05 M phosphate buffer (V ) -15 kV, pressure injection time ) 1 s). b The migration times of the enantiomers, t1 and t2, are given in minutes. c The effective mobilities, µ1 and µ2, are given in cm2 kV-1 min-1.
acid by a factor of ∼2, whereas, in the case of amethopterin, no separation was observed at a DTC concentration of 1 mM and baseline resolution at 20 mM. In general, the resolution increased with increases in the d-tubocurarine concentration up until ∼15.0 mM, after which it essentially leveled off. A combination of factors probably accounts for this observation. First, the difference in mobilities between the two enantiomers decreases with an increase in DTC concentration beyond 15 mM. Second, the electroosmotic flow tends to decrease more at the higher concentrations of chiral selector. Solubility restrictions of the selector did not permit studies at concentrations much higher than 25 mM. Consequently, for most of the CE separations reported, a DTC concentration of 15.0 mM was considered optimal and employed. A study of the effect of variation of the percent methanol modifier in the running buffer was conducted. Alteration of the amount of methanol in the running buffer in the range 10.0-40.0% (v/v) did not have any marked effect on optical isomeric resolution. However, 50% or greater amounts of methanol in the running buffer substantially degraded or destroyed the resolution. Based 1064 Analytical Chemistry, Vol. 70, No. 6, March 15, 1998
on all of the data, 20.0% methanol in the running buffer appeared to be optimum in terms of yielding satisfactory resolutions of most enantiomers within a reasonable time frame. Since the buffer pH governs the net charge of the chiral selector and analytes, the effect of pH on the optical resolutions was also examined. Electropherograms were obtained for the separation of the enantiomers of 2,4-dinitrophenylethionine at pH 5.0 and 8.0 (50 mM phosphate) using 15 mM DTC in 20:80 (v/v) methanol/water. Whereas baseline separation was achieved at pH 5.0 (Rs ) 1.9), the enantiomers were only partially resolved at pH 8.0 (Rs ) ∼1.2). For the aforementioned separation, the migration times for the enantiomers were 17.5 and 18.3 min at pH 5.0 vs 15.9 and 16.1 min at pH 8.0. This was typical of the pH effect noted over the pH 5-8 region examined, namely that the use of running buffers containing low pH seemed to give better separations at the expense of slightly increased run times. No CE separations were attempted under very basic conditions, where the DTC chiral selector would exist in its monoanionic form. Some representative electropherograms illustrating the CE separations obtained under optimum conditions are presented in
Figure 5. Although there is wall adsorption of the DTC chiral selector, the migration time reproducibility was good. The series of prerinses between sample runs (as outlined in the Experimental Section) seems to ensure reproducible electroosmotic flow. In addition, the use of HCl (a good solvent for the amine) and the presence of methanol in the running buffer appears to help to prevent wall buildup of the chiral DTC selector. Some tailing (typical of amine selectors) was observed. However, reconditioning of the capillary between sample runs also helped to minimize tailing. Table 1 summarizes the migration times, effective mobilities, and resolution obtained for the separation of the enantiomers of 18 different compounds via use of d-tubocurarine as the chiral running buffer additive. Examination of the structure of these compounds reveals that all contained a carboxylate group within 2-3 atoms of the stereogenic center. The only exception was N-(R-methylbenzyl)phthalic acid monoamide, in which case the carboxylate group was four atoms removed from the asymmetric carbon. However, closer examination indicates that the carboxylate group is probably closer in space to the chiral center since it is ortho to the substituent bearing the asymmetric carbon (Table 1, compound 5). Although all compounds examined also had an aromatic group (for detection purposes) within two or three atoms of the chiral center, this is probably not a strict requirement for optical resolution, as a difference in the binding constants between the enantiomers of a nonaromatic derivatized amino acid with
d-tubocurarine has been reported. For example, the equilibrium constant for the interaction of D-N-acetylalanine was 80 M-1, whereas it was
