2815
Anal. Chem. 1992, 64, 2815-2820
Chiral Recognition and Enantiomeric Resolution Based on Host-Guest Complexation with Crown Ethers in Capillary Zone Electrophoresis Reinhard Kuhn' and Fritz Erni Analytical Research and Development, Sandoz Pharma Ltd., CH-4002 Basel, Switzerland
Thomas Bereuter and Johannes HPusler Institute for Organic Chemistry, University of Vienna, A-1090 Wien, Austria
A chlral crown ether b successfully used as a pseudostatlonary phase In caplllary zone electrophoreds to separate optlcally actlve amlnes. On the bad8 of the results obtalned from the separatlon of more than 20 amines, two recognttlon mechanlrmr are proposed (I) the four crown ether subdttuents act as chlral barrlers for the guest molecules and (11) lateral okctrortatlclnteractlonroccur betweenhost and guest. These results are conflnned by thermodynamic dudks on the hostguest complexes. Best resolutions are achleved ll the chlral center k adjacent to the amlne functlonallty. However, even racemates wlth a chkal center In the 6-posltlon to the amlne could be resolved excellently. For the separatlon of allphatlc amlno aclds a method has been developedallowlng detectlon by an lndlrect detectlon mode.
INTRODUCTION Crown ethers discovered in 1967 by Pedersen192 are macrocyclic polyethers which are able to form stable and selective complexes with alkali, alkaline-earth, and primary ammonium cations. These ligands are usually classified in the family of coronands. A second, more sophisticated generation of macrocycles are the cryptands of Lehn394 and spherands of Cram.596 In modern chemistry all these compounds are of great importance in biological studies, e.g. as enzyme models,' in the synthesis, e.g. for phase-transfer catalysis,S and in analytical chemistry. For instance, complex formation of crown ethers can be utilized in ion chromatography! for the extraction of heavy metals! and in electrochemical sensors.1oJ1
* Author to whom correspondence and reprint requests should be addressed. (1)Pedersen, C. J. J. Am. Chem. SOC.1967,89,2495,7017. (2)Pedersen, C. J. Nobel Lecture, December 8,1987,reprinted in J. Zncl. Phenom. 1988,6,337. (3)Dietrich, J.-M.;Lehn, J.-M.; Sauvage, J.-P.TetrahedronLett. 1969, 2885 and 2889. (4)Lehn, J.-M. Nobel Lecture, December 8,1987,reprinted in J. Zncl. Phenom. 1988,6,351. (5)Kyba, E. P.; Siegel, M. G.; Soma, L. R.; Sogah, G. D. Y.; Cram, D. J. J. Am. Chem. SOC.1973,95,2691. (6) Cram, D. J. Nobel Lecture, December 8,1987,reprinted in J. Zncl. Phenom. 1988,6,397. ( 7 ) Sasaki, S.;Koga, K. J.Zncl. Phenom. 1989,7 , 267. (8)Weber, W. P., Gokel, G. W., Eds. Phase Transfer Catalysis in Organic Synthesis, Reactivity and Structure Concepts in Organic Chemistry 4 , Springer-Verlag: New York, 1977. (9)Blasius, E.; Janzen, K. P. Analytical Applications of Crown Compounds and Cryptande. In Dewar, M. J. S., Dunitz, J. D., Haffner, K., Heilbronner, E., Ito, S., Lehn, J.-M., Niedenzu, K., Raymond, K. N., Rees, C. W., Schiifer, K., Wittig, G., Eds. Topics in Current Chemistry, Vol. 98,Host Guest Complex Chemistry I; Springer-Verlag: New York, 1981. (10) Thoma, A.P.; Viviani-Nauer, A,; Schellenberg, K. H.; Bedekovic, D.; Pretach, E.; Prelog, V.; Simon, W. Helu. Chim. Acta 1979,62,2303. 0003-2700/92/0364-2815$03.00/0
Cram and co-workers12J3 were the first to recognize the potential of optically active crown ethers for the separation of enantiomers in liquid chromatography. The staggered arrangement of the binaphthyl rings in the structure of Cram's crown ethers behaves like a chiral barrier for the enantiomers. Separations of a variety of optically active amines using chiral crown ether stationary phases in HPLC have been reported subsequently.14-18 Chiral separations by capillary electrophoresis (CE) have been accomplished by utilizing solubilization with optically active micelles,lQligand-exchange complexation,20 or host-guest complexation with cyclodextrins.21 In capillary zone electrophoresis (CZE) we described the w e of a chiral crown ether for the separation of racemic amino acids for the first time.22 A comprehensive review of this topic is presented in ref 23. The focus of this study is to investigatethe chiral recognition mechanism between 18-crown-6tetracarboxylic acid (lSC6h) with several optically active amines by means of open-tube capillary zone electrophoresis. Appropriate separation methods are developed which allow "baselinen resolution for racemic mixtures.
EXPERIMENTAL SECTION Methods. Capillary electrophoresis was carried out using a P/ACE 2000 capillary electrophoresis instrument (Beckman Ins., Palo Alto, CA). Separations were performed at 25 "C in a fused silicacapillarytube(50cm X 75-pm i.d., PolymicroCorp.,Phoenix, AZ) applying a potential of 15 kV. All experiments described in Table I were carried out in Tris/citrate (10mM, pH 2.2) as carrier electrolyte with 10mM 18C6H4. For the temperature variations a system consisting of 30 mM 18C6Hd in water was used. Separations with indirect detection were performed in benzyltrimethylammonium chloride (6mM)/Tris (5mM) adjusted with citric acid to pH 2.2. To this buffer was added 15 mM 18C6H4. (11)Bussmann, W.; Lehn, J.-M.; Oesch, U.; PlumerB, P.; Simon, W. Helv. Chim. Acta 1981,134,657. (12)Kyba, E. P.; Timko, J. M.; Kaplan, L, J.; de Jong, F.; Gokel, G. W.; Cram, D. J. J. Am. Chem. SOC. 1978,100,4555. (13)S o w . L. R.:Sopah. G. D. Y.:Hoffman. D. H.: Cram, D. J. J.Am. Chem: SOC.1978,lbo, 2569. (14)Hilton, M.; Armstrong, D. W. J. Liq. Chromatogr. 1991,14 (I), 9. -.
(15)Armstrong, D. W.; Ward, T.; Czech, B.; Bartach, R. J. Org. Chem. 1986,50,5557. (16)Zukowski, J.; Pawlowska, M.; Pietraszkiewicz,M. Chromatographia 1991,32,82. (17)Udvarhelyi, P. M.; Watkins, J. C. Chirality 1990,2,200. (18)Daicel Crownpak CR(+)instruction Manual, Daicel (Europe): Diisseldorf, Germany, 1990. (19)OtaukaK.; Kowahara, J.;Tatekawa,K.;Terab,S. J.Chromatogr. 1991,559,209. (20)Gmmann, E.;Kuo, J. E.; &e, R. N. Science 1986,230,813. (21)Fanali, S. J. Chromatogr. 1991,545,437. (22)Kuhn, R.;Stoecklin, F.; Erni, F. Chromatographia 1992,33,32. (23)Kuhn, R.;Hoffstetter-Kuhn, S. Chromatographia, in press. 0 1992 American Chemical Soclety
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992
Table I. SeDaration Data for ODticallv Active Amines on Chiral 18C6Hd ~~
~
no. 1
~
name GlyPhe
structure
tR'
~
(min) a 1.141 45.13
structure
R. no. name 3.09 14 histidine
tgn (min)
a
R.
14.94
1.016 0.54
28.75
1.090 2.50
15.48
1.072 1.83
24.75
1.043 2.17
23.41
1.032 1.69
H P
2
tryptophan
25.56
1.055
3
phenylglycine
59.11
no separation
3.13
15
3-amino-3-phenylpropionic acid
16
norephedrine (lR,2S)l(lS,2R)
H
O
H,N
4
phenylalanine
29.56
1.059
3.75
17
5
tyrosine
25.92
1.051
2.65
18 threo-2-amino-3-phenylbutyric acid
P
CH,
erythro-2-amino-3-phenylbutyric acid
HIN H)c$
6 Dopa
29.18
1.053
2.78
19
threonine
24.73
1.078 3.42
3.17
20
GlyGlyLeu
46.18
1.034 0.70
21
valine
26.69
1.086 1.42
7
p-aminophenylalanine
36.86
1.076
8
ephedrine
10.30
no separation
0
phenylalaninol
13.47
1.070
10
phenylethylamine
17.36
no separation
11
naphthylethylamine
22.56
1.241
12
noradrenaline
32.99
13
normetanephrine
28.36
9
a
1.85 22
leucine
33.33
1.130 1.75
23
alanine
26.23
1.071 0.79
1.79 24
norvaline
31.73
1.054 0.79
1.023
0.59 25
isoleucine
31.52
1.106 1.68
1.037
0.60
Retention time of the first eluted enantiomer.
An appropriate amount of each sample was dissolved in diluted phosphoric acid or methanol/water and injected hydrodynamically for 1s. The solutes were detected by UV absorption at 254 nm for direct detection and at 214 nm in the indirect detection mode. A Perkin-Elmer CLAS/2000 system was used for data acquisition. Materials. All reagents were of analytical grade if not otherwisestated. Citric acid,benzyltrimethylammoniumchloride (98%), 18-crown-6 tetracarboxylic acid (18C6H4, 98%), phosphorous acid (85%), and tris(hydroxymethy1)aminomethane (Tris) were obtained from Merck (Darmstadt, Germany). All a-amino acids,p-amino-D,L-phenyldanine, ~,~-2-amino-3-phenylbutyric acid (67% threo, 33% erythro), D,L-Dopa, glyCyl-D,Lphenylalanine (GlyPhe), (i)-normetanephrine hydrochloride
(98% ), (&)-norephedrine,and (&)-2-amin0-4-phenylbutyric acid were purchased from Sigma Chemicals(St. Louis, MO). (-)-and acid (+)-ephedrine (purum), (A)-2-amino-l-phenyl-l-propionic (purum), D,L-noradrenaline (purum), (F?)-(+)- and (S)-(-)-l-(lnaphthy1)ethylamine (NEA; >99.5% 1, (It)-(+)- and (E+(-)phenylethylamine (>99 % ), 4-amino-D,L-phenylalaninehydrochloride (purum), and D- and L-phenylalaninol (purum) were from Fluka (Buchs, Switzerland). D,L-Phenylglycine(99%) was obtained from Janssen Chimica (Berse, Belgium) and D,L-3amino-3-phenylpropionicacid (98% ) and (f)-normetanephrine hydrochloride (98%)were from Aldrich Chemicals (Steinheim, Germany). Glycylglycyl-D,L-leucine(GlyGlyLeu) was purchased from Bachem Feinchemicalien (Bubendorf, Switzerland).
ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992
HOOC
A.
oYcooH COOH
/""!
CJ
Flgure 1. Chemical structure of 18trown-&tetracarboxylic acld.
RESULTS AND DISCUSSION Scope. Chirall8-crown-6tetracarboxylicacid derived from tartaric acid was first synthesized by Lehn and co-workersazs (see Figure 1). The polyether ring forms the shape of a cavity which is able to form stable complexes, particularly with potassium, ammonium, and protonated alkylamine cations. The six oxygens of the ring system are in the interior of the cavity and roughly define a plane.12 Ammonium or primary amines held inside the cavity are bound by three +NH-O hydrogen bonds in a tripod arrangement. However, since these host-guest complexes do not show chiral recognition, secondary lateral interactions between the substituents of the crown ether ring and the ligands are necessary for chiral discrimination. With respect to the structure of 18C6H4,we propose two different recognition mechanisms. First, the crown ether substituents, arranged perpendicularly to the plane formed by the ring s y ~ t e m , ~act ~ , 2like ~ steric barriers dividing the space available for the substrate into two cavities. Also perpendicular to the plane is the C*-+NHs bond of a complexed primary amine. The three remaining substituents of C* are distributed among the two cavities. If C* is chiral, transient diastereomers are formed with different complex formation constants depending on the size and steric arrangement of the substituents. On the other hand, a second mechanism can be discussed that is caused by electrostatic interactions (ionic or hydrogen bonds) of the crown ether substituents with functional groups of the guest molecule. Even repulsion forces of carboxylate groups between host and guest should have an influence on the complex formation constant and on the enantioselectivity. Resolutionof Chiral Amines. We investigated the chiral resolution of a series of different optically active amines in capillary zone electrophoresis to elucidate the recognition mechanism. In the chosen pH range, 18C6H4 added to the buffer electrolyte is slightly negatively charged and migrates as an anion in the opposite directionto the solutes. Separation is based on a different steric arrangement of the host-guest complexeswhich forms diastereomerswith disparate complex formation constants and different electrophoreticmobilities. More than 20 optically active amines were investigated and compared with respect to retention time, separation factor, and resolution. The results are summarized in Table I. It should be mentioned that better results could be obtained for a specific separation problem by individually optimizing the separation conditions. The separation factor (a)is supposed to be independent of migration time and reflects the chiral recognition. In general, only small but significant differencesof a for the various compounds could be calculated. Most enantiomers were "baseline separated" mainly because of the high efficiency achieved in CZE. (24) Behr,J.-P.;Girodeau,J.-M.;Heyward,R.C.;Lehn, J.-M.;Sauvage, J.-P. HeZu. Chim. Acta 1980, 63, 2096. (25) Behr, J.-P.;Lehn, J.-M.; Vierling, P. Helu. Chim.Acta 1982, 65, 1863. (26) Gehin, D.; Kollmann, P. A.; Wipff, G. J.Am. Chem. SOC.1989, 111, 3011.
2817
Armstrong and co-workern studied the influenceof distance between the amine function and chiral center of dipeptides in HPLC using a chiral crown ether with hydrophobic substituents as stationary phase. While good resolution was found for those solutes where the amine group was adjacent to the stereogeniccenter, dipeptides of the glycyl amino acid type were not resolved or showed poor resolution. We observed that glycyl-D,L-phenylalanine (1, GlyPhe) was excellently resolved although the chiral center is in the &position to the primary amine. Molecular models of the 18C6H4-GlyPhe complex indicate that chiral recognition occurspredominantlyby hydrogen bonds rather than by steric barrier mechanisms. A possible arrangement of the hostguest complex is shown in Figure 2a. Recognition is probably based on hydrogen bonds between the acidic functional group of the dipeptide with the crown ether side chains, indicated by the arrow in Figure 2a. Even the tripeptide GlyGlyLeu (20), which has its asymmetric center seven bonds from the amine, was recognized by the selector, although resolution was poor under the given conditions. On the other side, nonpolar substituents like methyl, phenyl, or naphthyl groups are not able to bind with the polar crown ether substituents. Recognition is based on mere steric barrier mechanisms and, therefore, dependent on the size of the substituents. (f)-Phenylethylamine (lo), for example, could not be separated because of too small substituents whereas the naphthyl analogue 11 was excellently resolved with a separation factor of 1.24. A stereoscopicimage (Figure 2b) shows spatial hindrances by the crown ether substituents indicated by an arrow. A representative electropherogram of a separation of aromatic amines with 18C6Hl is shown in Figure 3a. Chiral recognition of D,L-tryptophanis probably based on both polar interactions and steric barrier mechanisms. A second ammonium function as inp-aminophenylalanine (7)represented an additional anchoring group for the crown ether and caused, therefore, longer migration times and a slightly improved separationfactor comparedto phenylalaniie (4). The method described showed sufficient selectivity to resolve all four stereoisomersof 2-amino-3-phenylbutyricacid (17and 18)in one single run. Most aromatic amines except phenylglycine (3), phenylethylamine (lo),and histidine (14)were baseline separated. While 3 and 10 were not recognized owing to the size of substituents, 14 was poorly resolved under the given test conditions. Complete separation of hietidine was achieved by changing the pH from 2.2 to 5.0. Poor resolution was also found for the amines 12 and 13 which both have their chiral centers in the B-position to the amine. Although a values of 1.023 and 1.037 were found to be promising for satisfactory separation, incomplete resolution of enantiomers resulted in an unexpected loss in efficiency. Aliphatic amino acids and the tripeptide (compounds 1925) could be detected by an indirect detection mode using benzyltrimethylammonium chloride (BTA) as tracer in the running buffer. An example is given in Figure 3b with the separation of D,L-threonine (19). Negative peaks originate from the threonine enantiomers which displace BTA in the running buffer and thus decrease the absorbance. Amino acids with bulky Substituents showed improved separation factors in comparison with those with small substituents, e.g. leucine (22)compared to valine (21). Branched alkyl groups gave better a values than their straight-chain isomers (see leucine-isoleucine and valine-norvaline). Obviously, the complexation of the ammonium cation is mandatory for chiral recognition as demonstrated with (*)ephedrine (8). This separation failed because of an extremely (27) Hilton, M.; Armstrong, D. W. J. Liq. Chromatogr. 1991,14 (20), 3673.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992
Flguro2. Stereoscopic image of a host-guestcomplex of 18C6H4with (a) glycylphenylalanineand (b) naphthylethylamine. Arrows Indicate positlon of possible hydrogen bonds (a) and spatial hindrance by the carboxylic acids of the crown ether (b). Key for the symbols: hydrogens are white, oxygens light, carbons dark and nitrogens black.
weak complex formation of 18C6H4with the secondaryamine, which does not fit into the crown ether cavity. As a consequence ephedrine eluted faster than its demethyl analogue, norephedrine (16), which was strongly retained by the crown ether complex. Calculationof ThermodynamicParameters for Chiral Recognition. For enantiorecognition based on two mechanisms: (i) steric barrier of the host carboxylic acid groups for naphthylethylamine and (ii) Coulombic interactions in the case of GlyPhe, thermodynamic parameters of the complexationreaction should differ significantly. These parameters are accessible by the separation factor ~ ~ ~ 8which 9 % is related to A,(AG), the difference in the molar Gibbs energy of the two enantiomers, by eq 1 -A*(AG) = R T h a (1) where Tis the absolute temperature and R is the gas constant (8.3143J K-l M-l). Now, with A,(AG) = A , ( m - TA,(AS) (2) where A*(AH) is the enthalpy difference of the complex formations and A* (AS) is the difference in the entropy, it follows combining eqs 1and 2 A,(m lna=--+-
A*(m
(3) R Equation 3should enable A(AH) and A(AS) to be determined from the slope and the intercept of a plot of In a versus 1/T
RT
(28) Akanya, J. N.; Taylor, D. R. Chromatographia 1988,25,639. (29) Schurig, V.; Weber, R. J. Chromatogr. 1981,217,51.
(Van't Hoff plot). Figure 4 displays Van't Hoff plots of naphthylethylamine (NEA), GlyPhe, tryptophan, and phenylalanine in the temperature range 20-40 "C. Regression analysis gave correlation coefficients better than 0.993. An increase of temperature caused a decreased separation factor of NEA, Try, and Phe but improveda of the dipeptide. Hence, the positive and negativeslopes found with NEA and GlyPhe, respectively, confirmthat two chiral recognitionmechanisms exist. The curve obtained for GlyPhe was unexpected. So far such shapes were only measured in gas chromatography.30 Within a 95% confidence range, straight lines and thermodynamic data of Try and Phe are identical. Therefore, complex formation constants and chiral recognition for both amino acids should not differ significantly. Although the separation factor a represents the chiral recognition based on lateral interactions of the host-guest complex, mere complexationof the amine with the polyether cavity also effects a. Therefore, the total Gibb's energy of the complexation A*(AGbt) calculated by eq 2 consists of two components: (i) a polyether cavity factor A,(AGmv) and (ii) the lateral interaction A,(AGht) of the ligand without polyether participation.31 A*(AG,t)
= A*(AGmJ
+ A*(AG,,)
(4) Table I1 summarizes the thermodynamic data calculated from Figure 4 according to eqs 2 and 3. It has to be noted here that A(AH) is assumed to be independent of temperature in the range studied and that extrapolation to the intercept (30) Watabe, K.; Charles, R.; Gil-Av, E. Angew. Chem. 1989,101,195. (31) Dutton, P. J.; Fyles, T. M.; McDermid, S. J. Can. J . Chem. 1988, 66, 1097.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992 0.015
281Q
0.020
O.OO0
0.010
W
u
-0.020
f P
W
u z U
8 m
m
a
a
0 v)
m
0.005
U
-0.040
-0.060
O.OO0 1
0.00
10.00
20.00
30.00
40.00
0.00
10.00
20.00
1
30.00
40.00
TIME [minl
TIME [min]
Fburr 9. Chiral separatlon of D,L-tryptophan (a) and oc-threonine (b) with L-threonine in excess.
0.05
O.Ob32
O.Ob34
1 I T [1/K]
Flguo 4. Van't Hoff plotsof naphthylethylamine(1 ), glycylphenylalanlne (2) tryptophan (3),and phenylalanine (4). Temperatures were 20,25, 30, 35, and 40 OC.
Table 11. Thermodynamic Data Calculated from the Straight Lines in Figure 4 Ai ( A W g 8 Ai (iw) Ai (ASS) compound (J mol-') (J mol-') (J K-lmol-') naphthylethylamine -369 -1232 -2.9 GlyPhe -161 +703 +2.9
nr Phe
-140 -133
-641
-550
-1.7 -1.4
at infinite temperature is linear. While large errors were supposed to exist in such a procedure for HPLC,z*our data enabled us to estimate the thermodynamic values with sufficient precision. Though accuracy of these data does not permit a sophisticated interpretation, obvious effects can be discussed. According to eq 1 the difference in the Gibb's energy is always negative since a is defined arbitrarily by the term tdtl,where tzrepresenta themigration time of the second eluted enantiomer and tl the migration time of the antipode. A(AG) of naphthylethylamine enantiomers was the highest measured, although a large negative term T A ( W represented an unfavorable influence of the entropy on A(AG) and thus on the separation. Usually Gibbs energy of the inclusion
complex formation is associated with a favorable proportion of enthalpy and an unfavorable entropy proportion. This holds, for example, for the complex formation of \he crown ether with NEA. The entropyvalue is influenced by a number of factors. A major contribution to the entropy is that hydrate water surrounding polar groups of host and guest is highly ordered and becomes disordered in particular if lateral hydrogen bonds are formed during complex formation. Simultaneously, there are losses in the translational and rotational degrees of freedom because of the association of host and guest. These two opposite effects are usually balanced, with the consequence that the contribution of entropy to the complex formation may be very small. In the case of the 18C6HrGlyPhe complexation the contribution by the release of hydrate water dominates over that caused by the loss of a degree of freedom. This effect should be more pronounced for the enantiomer which forms the stronger lateral hydrogen bonds with the host. Since the positive term A(AH) does not contribute to the separation, a is controlled mainly by the entropic contribution, TA(AS). The positive A(AS) value in the case of GlyPhe is supposed to be characteristic for the recognition mechanism based on electrostatic interactions. If the chiral separation is based on both mechanisms, one should be able estimate from A ( W and TA(AS) which mechanism dominates. In the case of Try and Phe a barrier mechanism dominates because A(AG) is negative owing to the contribution of A(AH).
CONCLUSIONS The paper demonstrates the versatility of 18C6H4as chiral selector for the separation of enantiomers in capillary zone electrophoresis. Host-guest complexation with the crown ether depends on primary ammonium groups of the ligands and thus increases the appliciation range of CZE for chiral separations. Twenty-five optically active amines were screened with a uniform separation system. Approximately 90% of all compounds were recognized by the chiral selector and 60% were at least baseline resolved. Although separation factors of the enantiomers were small in general, most amines
2820
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992
were well separated because of the high efficiency of capillary zone electrophoresis. Best results were obtained if the chiral center of the molecule was adjacent to the primary amine, but excellent resolution was found even with a asymmetric center in the 6-position. Despite the suitability of the crown ether for the resolution of many amines, predictions based on the chemical structure of the guests are difficult. CZE has proven to be extremely useful for mechanistic studies because all measurements can be performed under defined conditions and in pure aqueous solutions. On the basis of molecular models of complexes with naphthylethylamine and glycylphenylalanine,two different recognition mechanisms were proposed. It is very likely that in most separations barrier mechanisms dominate over electrostatic
interactions as demonstrated with tryptophan and phenylalanine. By varying the pH value of the carrier electrolyte the mechanism could presumably be influenced in the opposite way. Thermodynamic studies have proven particularly useful to permit an additional insight into the chiral recognition and support the existence of two different separationmechanisms. While a temperature increase usually decreases the separation of enantiomers, it improved the separation in the case of GlyPhe.
RECEIVED for review March 5, 1992. Accepted July 31, 1992.
