Chiral Separations by Liquid Chromatography Downloaded from pubs.acs.org by UNIV OF MICHIGAN ANN ARBOR on 11/20/18. For personal use only.
Chapter 8
Chiral Recognition on Biopolymer-Based HighPressure Liquid Chromatographic Chiral Stationary Phases A Case for Multiple Interaction Sites Irving W. Wainer, Terence A. G. Noctor, Enrico Domenici, and Philippe Jadaud Department of Oncology, McGill University, 3655 Drummond, Suite 701, Montreal, Quebec H3G 1Y6, Canada The c h i r a l r e c o g n i t i o n mechanisms o p e r a t i n g on two HPLC c h i r a l s t a t i o n a r y phases (CSPs) based upon immobilized biopolymers, α-chymotrypsin (ACHT-CSP) and human serum albumin (HSA-CSP) have been i n v e s t i g a t e d . The r e s u l t s of these s t u d i e s i n d i c a t e t h a t the observed s t e r e o s e l e c t i v i t y i s a measure of the d i f f e r e n c e s i n b i n d i n g affini ties a t two or more s i t e s r a t h e r than the c o n sequence of differential affinities at a s i n g l e site. The proposed m u l t i p l e site/multiple mechanism model d i f f e r s from the s i n g l e s i t e / single mechanism and single site/multiple mechanisms models proposed f o r other HPLC-CSPs. Introduction Stereochemical r e s o l u t i o n s on an HPLC c h i r a l s t a t i o n a r y phase (CSP) involve the formation of transient d i a s t e r e o m e r i c complexes between a c h i r a l s e l e c t o r on the CSP and the separate enantiomers of the solute. E n a n t i o s e l e c t i v i t y i s then a f u n c t i o n of the f r e e energy d i f f e r e n c e ( Δ Δ 6 ) between the two complexes and only s l i g h t energy d i f f e r e n c e s are necessary t o produce an e f f e c t i v e chiral resolution. For example, a A A G of 410 c a l / m o l e r e s u l t s i n a s t e r e o s e l e c t i v i t y (a) of 2.00. Within the s o l u t e / C S P complex, c h i r a l r e c o g n i t i o n by the s e l e c t o r i s based on the "3-point i n t e r a c t i o n " model proposed by D a l g l i e s h (1). According t o t h i s mechanism, t h r e e i n t e r a c t i o n s occur between the s o l u t e and the c h i r a l s e l e c t o r and, a t l e a s t one of these i n t e r a c t i o n s must be dependent on the stereochemical s t r u c t u r e of the s o l u t e . A s i m p l i f i e d v e r s i o n of t h i s process i s presented i n F i g u r e 1, (2). 0097-6156/91/0471-0141$06.00/0 © 1991 American Chemical Society
F i g u r e 1. I l l u s t r a t i o n of the "3-point i n t e r a c t i o n " model. (Reproduced with permission from reference 2. Copyright 1984 Aster Publishing Corporation.)
8.
WAINER E T A L .
Biopolymer-Based HPLC Chiral Stationary Phases 143
In t h i s i l l u s t r a t i o n , there are three p o s s i b l e points o f i n t e r a c t i o n b e t w e e n t h e c h i r a l s o l u t e s ( s i t e s A, B and C) a n d t h e c h i r a l s e l e c t o r ( s i t e s A', B' and C ) . Chiral s o l u t e I i n t e r a c t s with the c h i r a l s e l e c t o r at s i t e s A—A', B—B' and C — C , w h e r e a s i t s m i r r o r image, c h i r a l s o l u t e I I does not i n t e r a c t a t s i t e s C — C . I f the C — C interaction r e s u l t s i n the s t a b i l i z a t i o n of the diastereomeric complex b e t w e e n S o l u t e I and t h e CSP, S o l u t e I w i l l be r e t a i n e d on t h e column l o n g e r t h a n S o l u t e I I . However, i f t h e C—C i n t e r a c t i o n d e s t a b i l i z e s t h e S o l u t e I-CSP c o m p l e x , S o l u t e I w i l l elute before Solute II. I f C and C interact minimally or not a t a l l , Solute I and I I w i l l not be r e s o l v e d on t h i s CSP. Armstrong, et al. (3) have used computational chemistry techniques to demonstrate that this chiral r e c o g n i t i o n mechanism i s t h e b a s i s o f t h e r e s o l u t i o n o f ( R ) - and (S)-propranolol on a j 8 - c y c l o d e x t r i n CSP. The m a j o r d i f f e r e n c e between t h e d i a s t e r e o m e r i c (R)-propranolol//3-cyclodextrin and (S) - p r o p r a n o l o l / j S - c y c l o d e x t r i n c o m p l e x e s i s t h e p o s i t i o n o f t h e s e c o n d a r y a m i n e g r o u p on the propranolol molecule. I n t h e ( R ) - p r o p r a n o l o l complex, the n i t r o g e n i s i d e a l l y placed f o r hydrogen bonding t o both a 2- and a 3 - h y d r o x y l g r o u p on t h e j 8 - c y c l o d e x t r i n w i t h r e s p e c t i v e b o n d d i s t a n c e s o f 3.3 and 2.8 A. In the (S)p r o p r a n o l o l c o m p l e x , t h e s e bond d i s t a n c e s a r e 3.8 and 4.5 A. Thus, t h e ( R ) - p r o p r a n o l o l / j 8 - c y c l o d e x t r i n complex s h o u l d be t h e more s t a b l e o f t h e two d i a s t e r e o m e r i c c o m p l e x e s and (R)-propranolol s h o u l d be t h e more r e t a i n e d enantiomer. The calculated elution order is consistent with the experimental r e s u l t s . The c h i r a l r e c o g n i t i o n p r o c e s s d e s c r i b e d by A r m s t r o n g , e t a l . (3) i s b a s e d on a s i n g l e s i t e / s i n g l e m e c h a n i s m a p p r o a c h i n w h i c h t h e o n l y i n t e r a c t i o n s i t e on t h e CSP i s t h e j 8 - c y c l o d e x t r i n and b o t h p r o p r a n o l o l e n a n t i o m e r s b i n d i n t h e same manner. However, t h i s i s o n l y one o f t h e p o s s i b l e chiral recognition mechanisms. Two of the other possibilities are single s i t e / m u l t i p l e mechanisms and m u l t i p l e s i t e s / m u l t i p l e m e c h a n i s m s , T a b l e 1. Table
1.
Possible
chiral
recognition
processes
S i n g l e S i t e / S i n g l e Mechanism S i n g l e S i t e / M u l t i p l e Mechanisms M u l t i p l e S i t e / M u l t i p l e Mechanisms
The s i n g l e s i t e / m u l t i p l e m e c h a n i s m p r o c e s s h a s b e e n p r e v i o u s l y d i s c u s s e d by P i r k l e , e t a l . (4,5). In these s t u d i e s the c h i r a l s t a t i o n a r y phase c o n t a i n e d a single c h i r a l s e l e c t o r , (R)-N-(lO-undecenoyl)-a-(6,7-dimethyl-1naphthyl)-isobutylamine, and t h e s o l u t e s w e r e a h o m o l o g o u s
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CHIRAL SEPARATIONS BY LIQUID C H R O M A T O G R A P H Y
series of dinitrobenzoyl derivatives of 1-phenylalkylamines. The experimental results indicated that the solutes interacted with the chiral selector using two different mechanisms one based on a d i p o l e stacking i n t e r a c t i o n between t h e s o l u t e and CSP and t h e o t h e r based on a solute/CSP hydrogen-bonding interaction. This s i t u a t i o n i s d i a g r a m m e d i n F i g u r e 2. Two chiral recognition mechanisms have also been proposed for the stereochemical resolution of enantiomeric amide and a n a l i d e s on the s i n g l e site (R)-N-(3,5-dintrobenzoyl)phenylglycine C S P (6-9). Both of the mechanisms a r e based on a d i p o l e / d i p o l e i n t e r a c t i o n between the s o l u t e and chiral selector. The d i f f e r e n c e i s due t o t h e positioning of the solutes relative to the chiral selector within the solute/CSP complexes. P i r k l e and McCune (6) l a b e l l e d t h e two p o s s i b i l i t i e s as "head t o head" and "head to t a i l " dipole stacking. The d e t e r m i n a t i o n o f t h e c h i r a l r e c o g n i t i o n mechanism i s f u r t h e r c o m p l i c a t e d when b i o p o l y m e r s s u c h a s cellulose and serum a l b u m i n a r e u s e d as t h e c h i r a l s e l e c t o r s . Large chiral polymers can contain multiple complexation sites exhibiting different enatioselectivities, b i n d i n g mechanisms and s t r u c t u r a l requirements. Francotte and Wolf (10) have d e m o n s t r a t e d t h i s phenomenon on a c e l l u l o s e triacetate I C S P . We h a v e a l s o d e m o n s t r a t e d t h e e x i s t e n c e o f m u l t i p l e i n t e r a c t i o n s i t e s o n C S P s b a s e d o n a - c h y m o t r y p s i n a n d human serum a l b u m i n . These r e s u l t s are discussed below. Chiral Recognition t r y p s i n CSP
of
Amino
Acid
Esters
on
an
a-Chymo-
Wainer, et a l . (11) have r e p o r t e d t h e s y n t h e s i s o f a CSP based on the enzyme a-chymotrypsin (ACHT-CSP). The i m m o b i l i z a t i o n o f ACHT p r o d u c e d a s t a t i o n a r y p h a s e t h a t was c a p a b l e o f b i n d i n g a n d h y d r o l y z i n g s u b s t r a t e s o f ACHT s u c h as L-amino a c i d amides and e s t e r s . The enzymatic activity o f t h e i m m o b i l i z e d a n d f r e e ACHT w e r e e q u i v a l e n t (11). The ACHT-CSP was capable of resolving a number of e n a n t i o m e r i c compounds i n c l u d i n g D , L - t r y p t o p h a n amide and N-benzoyl-D,L-leucine. The r e s o l u t i o n of D,L-tryptophan a m i d e was a f u n c t i o n o f t h e a c t i v i t y o f t h e enzyme w h i c h h y d r o l y z e d L - t r y p t o p h a n amide but not the D-enantiomer. The o b s e r v e d c h r o m a t o g r a p h i c r e s o l u t i o n was a c t u a l l y the separation of L - t r y p t o p h a n and D - t r y p t o p h a n amide (12). Neither the L or D forms of N - b e n z o y l - l e u c i n e are subs t r a t e s f o r ACHT a n d t h e o b s e r v e d c h i r a l s e p a r a t i o n o f this compound was based only on d i f f e r e n t i a l binding to the protein (12). Since i t is assumed t h a t substrates and pseudosubs t r a t e s b i n d a t t h e a c t i v e s i t e o f an enzyme, t h e observed enantioselectivity o f the ACHT-CSP s h o u l d be based on a s i n g l e s i t e / m u l t i p l e mechanism c h i r a l r e c o g n i t i o n process, i . e . t h e s i n g l e s i t e i s t h e a c t i v e s i t e o f t h e ACHT a n d t h e
8.
WAINER E T A L .
Biopolymer-Based HPLC Chiral Stationary Phases 145
Dipole-stacking process
Hydrogen-bonding process
F i g u r e 2 . Two p o s s i b l e c h i r a l r e c o g n i t i o n m e c h a n i s m s for the stereochemical resolution of dinitrobenzoyl d e r i v a t i v e s o f e n a n t i o m e r i c 1 - p h e n y l a l k y l a m i n e s on an HPLC c h i r a l s t a t i o n a r y p h a s e d b a s e d u p o n ( R ) - N - ( I O undecenoyl) - a - (6,7-dimethyl-l-naphthyl) -isobutylamine. (Reproduced with permission from reference 5. Copyright 1985 Elsevier.)
146
CHIRAL SEPARATIONS BY LIQUID C H R O M A T O G R A P H Y
m e c h a n i s m s a r e enzyme a c t i v i t y a n d s o l u t e s t r u c t u r e . One method to determine whether a single site/multiple mechanism c h i r a l r e c o g n i t i o n p r o c e s s i s i n d e e d o p e r a t i n g on t h e ACHT-CSP i s t o b l o c k t h e a c t i v e s i t e o f t h e enzyme. This c a n be a c c o m p l i s h e d u s i n g N-tosyl-L-phenylalanine c h l o r o m e t h y l k e t o n e (TPCK). I f c h i r a l r e c o g n i t i o n took p l a c e s o l e l y a t t h e a c t i v e s i t e o f t h e enzyme b y e i t h e r mechanism, t h e i n a c t i v a t i o n s h o u l d r e s u l t i n a l o s s o f stereoselectivity. When t h e ACHT-CSP was treated with TPCK, the i m m o b i l i z e d enzyme was d e a c t i v a t e d (12) . T h e c o l u m n was no l o n g e r a b l e t o h y d r o l y z e L-tryptophanamide and t h e c h i r a l r e s o l u t i o n s b a s e d on e n z y m a t i c a c t i v i t y were l o s t . In a d d i t i o n , t h e TPCK i n a c t i v a t e d ACHT-CSP was a l s o u n a b l e t o stereochemically resolve N-benzoyl-D,L-leucine and t h e c h i r a l r e s o l u t i o n s b a s e d u p o n s o l u t e s t r u c t u r e were a l s o l o s t (12). These r e s u l t s support a s i n g l e s i t e / m u l t i p l e mechanism c h i r a l r e c o g n i t i o n p r o c e s s . T a b l e 2 . E n a n t i o s e l e c t i v i t y and c h r o m a t o g r a p h i c r e t e n t i o n o f amino a c i d e s t e r s on a c t i v e a n d T P C K - d e a c t i v a t e d a c h y m o t r y p s i n - b a s e d HPLC c h i r a l s t a t i o n a r y p h a s e s (ACHTCSP) . D a t a o b t a i n e d f r o m R e f e r e n c e 13
ACHT-CSP (active) k/ a
Solute
a
L-Alanine benzyl
ester
b
ACHT-CSP (TPCK) k / ct a
1.22
4.19
4. 06
1.74 D-Alanine benzyl
ester
L-Tyrosine methyl
4.95
7.28
ester
0.38
0.41
1. 98
1.0 D-Tyrosine methyl L-Tryptophan e t h y l
ester
0.72
0.42
ester
0.98
1.21
1. 46
1.26 D-Tryptophan e t h y l
ester
1.53
b
1.43
8
C a p a c i t y f a c t o r (k') o f t h e f i r s t e l u t e d e n a n t i o m e r where k' i s d e f i n e d a s ( t - t ) / t ; t = r e t e n t i o n i n s e c o n d s o f t h e i n j e c t e d compound a n d t = r e t e n t i o n i n s e c o n d s o f a non-retained solute. Stereoselectivity factor (a) where a = k' 0
0
0
b
s e c o n d
e l u t e d
enantiomer/^ first eluted enantiomer* The r e s u l t s obtained from t h e chromatography o f r a c e m i c amino a c i d e s t e r s o n t h e a c t i v e a n d T P C K - i n a c t i v e
8. WAINER ETAL.
147 Biopolymer-Based HPLC Chiral Stationary Phases
f o r m s o f t h e ACHT-CSP a r e n o t c o n s i s t e n t w i t h t h i s c h i r a l recognition process. The r e s u l t s from t h e chromatography o f t h r e e e s t e r s a r e p r e s e n t e d i n T a b l e 2. A l a n i n e i s n o t a s u b s t r a t e o f ACHT a n d t h e c h i r a l r e s o l u t i o n i s d u e t o t h e d i f f e r e n t i a l b i n d i n g o f t h e two e n a n t i o m e r s . L-Tyrosine methyl e s t e r and L - t r y p t o p h a n e t h y l e s t e r a r e s u b s t r a t e s and a r e h y d r o l y z e d b y ACHT w h i l e t h e D - f o r m s a r e n o t . I f a s i n g l e s i t e / m u l t i p l e mechanism c h i r a l r e c o g n i t i o n p r o c e s s i s o p e r a t i n g , t h e n TPCK i n a c t i v a t i o n s h o u l d r e s u l t i n t h e l o s s o f s t e r e o s l e c t i v i t y a s was o b s e r v e d f o r D,Ltryptophanamide and N - b e n z o y l - D , L - l e u c i n e . However, t h e observed enantioselectivities f o r the esters actually i n c r e a s e d o n t h e i n a c t i v e f o r m o f t h e ACHT-CSP, T a b l e 2, (12)
.
These r e s u l t s s u g g e s t t h a t s o l u t e b i n d i n g s i t e s on t h e ACHT m o l e c u l e e x i s t o u t s i d e o f t h e a c t i v e s i t e o f t h e enzyme a n d t h a t t h e b i n d i n g o f amino a c i d e s t e r s a t t h e s e sites i s stereoselective. Therefore, the observed c h i r a l r e s o l u t i o n o f t h e e s t e r s o l u t e s o n t h e ACHT-CSP must b e t h e result of a multiple site/multiple mechanism chiral recognition process. T h i s c o n c l u s i o n was s u p p o r t e d b y t h e r e s u l t s o b t a i n e d d u r i n g t h e study o f t h e chromatography o f aspartame s t e r e o i s o m e r s o n t h e ACHT-CSP (13). Aspartame, N a - a s p a r t y l phenylalanine 1-methyl ester (APME, F i g u r e 3), i s a d i p e p t i d e w h i c h e x i s t s a s 4 s t e r e o i s o m e r s : LL-APME, DDAPME, DL-APME a n d LD-APME. T h e LL-/DD-and DL-/LD- a r e e n a n t i o m e r i c p a i r s a n d t h e two p a i r s o f e n a n t i o m e r s a r e r e l a t e d t o each other as diastereomers. The e n a n t i o m e r i c and d i a s t e r e o m e r i c p a i r s c a n be r e s o l v e d o n t h e ACHT-CSP (13)
.
The e f f e c t o f t h e m o l a r i t y o f t h e p h o s p h a t e b u f f e r i n t h e m o b i l e p h a s e o n r e t e n t i o n a n d s t e r e o s e l e c t i v i t y o f APME s t e r e o i s o m e r s o n t h e ACHT-CSP was i n v e s t i g a t e d a n d t h e r e s u l t s a r e p r e s e n t e d i n F i g u r e 4, (13). I t i sof interest t o note t h a t t h e m o l a r i t y o f t h e phosphate b u f f e r had a g r e a t e r a n d o p p o s i t e e f f e c t o n t h e APME s t e r e o i s o m e r s containing L-phenylalanine than those containing Dphenylalanine. When t h e m o l a r i t y o f t h e p h o s p h a t e b u f f e r was r a i s e d f r o m 0.050 t o 0.500 M, t h e k' f o r DL-APME d r o p p e d f r o m 1.10 t o 0.32 a n d t h e k' f o r LL-APME f e l l f r o m 1.29 t o 0.33. O v e r t h e same c o n c e n t r a t i o n r a n g e , t h e k ' s f o r LD- a n d DDAPME r o s e f r o m 0.17 t o 0.20 (LD-) a n d 0.18 t o 0.22 (DD). The o b s e r v e d e n a n t i o - s e l e c t i v i t i e s (a) a l s o d e c r e a s e d - f o r LD/DL f r o m 6.67 (0.050 M) t o 1.60 (0.500 M) a n d f o r DD/LL f r o m 7.33 (0.050 M) t o 1.54 (0.500 M). T h e s e r e s u l t s s u g g e s t t h e e x i s t e n c e o f two s e p a r a t e b i n d i n g s i t e s , t h e L - p h e n y l a l a n i n e (L-Phe) a n d D - p h e n y l a l a n i n e (D-Phe) s i t e s . T h e d e c r e a s e i n k' f o r t h e DL- a n d L L - i s o m e r s i n d i c a t e s t h a t t h e p h o s p h a t e i o n e i t h e r competes f o r b i n d i n g a t t h e L-Phe s i t e o r t h a t t h e i n c r e a s e i n t h e i o n i c s t r e n g t h o f t h e mobile phase decreases t h e a f f i n i t y
American Chemical Society Library 1155 16th St., N.W. Washington, D.C. 20036
148
CHIRAL SEPARATIONS BY LIQUID C H R O M A T O G R A P H Y
0
n U * HO
*
; c - CH 2
0
H
n i
II
- CH • NH
3,
6 • CH
The molecular
1
0.0 0.0
1
0.1
#
- c - N - C H - c;
2
Figure
*
1
0.2
0.3
OCH
Q 3
2
structure
1
U
1
of
1
0.4
aspartame.
1
0.5
0.6
Molarity (M) Figure 4. The effect of the molarity of the phosphate buffer in the mobile phase on retention of aspartame stereoisomers. (Reproduced with permission from reference 13. Copyright 1990 Chirality.)
8. WAINER ET AL.
149 Biopolymer-Based HPLC Chiral Stationary Phases
of t h e s i t e f o r these isomers. I f LD- a n d DD-APME w e r e b o u n d a t t h e same s i t e , t h e i r k ' s s h o u l d b e a f f e c t e d i n t h e same manner b y t h e p h o s p h a t e b u f f e r c o n c e n t r a t i o n . Since t h e o b s e r v e d k ' s o f t h e LD- a n d D D - i s o m e r s i n c r e a s e s , i t a p p e a r s t h a t t h e p h o s p h a t e i o n d o e s n o t compete a t t h e DPhe s i t e a n d t h a t a m o b i l e p h a s e w i t h a h i g h i o n i c s t r e n g t h i n c r e a s e s t h e a f f i n i t y o f LD- a n d DD-APME f o r t h i s s i t e .
C h i r a l R e c o g n i t i o n on a Human Serum Albumin HPLC-CSP Human serum a l b u m i n (HSA) i s a g l o b u l a r , hydrophobic p r o t e i n w h i c h h a s b e e n shown t o s t e r e o s e l e c t i v e l y b i n d s m a l l e n a n t i o m e r i c m o l e c u l e s (14)• T h e r e a r e two m a j o r drug-binding sites on HSA: the warfarin-azapropazoneb i n d i n g a r e a and t h e i n d o l e and b e n z o d i a z e p i n e b i n d i n g s i t e w h i c h b o t h d i s p l a y some s t e r e o s e l e c t i v i t y (15-17). An a d d i t i o n a l one ( d i g i t o x i n , (18)) t o three s i t e s (digitoxin, b i l i r u b i n a n d f a t t y a c i d , (19)) have a l s o been proposed. The s t e r e o s e l e c t i v i t y o f t h e w a r f a r i n - a z a p r o p a z o n e b i n d i n g a r e a h a s b e e n i n v e s t i g a t e d b y a number o f l a b o r atories. S e l l e r s a n d Koch-Weser (20) have demonstrated that S-warfarin i s more h i g h l y bound t o HSA t h a n Rwarfarin. The i n d o l e and b e n z o d i a z e p i n e b i n d i n g s i t e has a l s o b e e n t h e s u b j e c t o f a number o f i n v e s t i g a t i o n s a n d a p p e a r s t o be more s t e r e o s p e c i f i c t h a n t h e w a r f a r i n azapropazone-binding area. F o r example, t h e r a t i o o f t h e affinity constants of (+)-oxazepam hemisuccinate/(-)oxazepam h e m i s u c c i n a t e i s 49.5 (21). The d i f f e r e n t b i n d i n g s i t e s o n HSA h a v e b e e n s t u d i e d u s i n g competitive b i n d i n g i n t e r a c t i o n s . I n t h i s approach, a compound known t o b i n d a t a s p e c i f i c s i t e o n t h e HSA ( t h e competitor) i s added t o t h e i n c u b a t i o n m i x t u r e c o n t a i n i n g HSA a n d t h e m o l e c u l e u n d e r i n v e s t i g a t i o n ( t h e l i g a n d ) . I f b o t h t h e l i g a n d a n d t h e c o m p e t i t o r b i n d a t t h e same s i t e , t h e n t h e r e w i l l be a r e d u c t i o n i n t h e b i n d i n g o f t h e l i g a n d t o t h e HSA. The magnitude o f t h e r e d u c t i o n w i l l depend upon t h e c o n c e n t r a t i o n o f t h e c o m p e t i t o r and i t s a f f i n i t y f o r t h e HSA. I n a c h r o m a t o g r a p h i c s y s t e m u t i l i z i n g i m m o b i l i z e d HSA, the a d d i t i o n o f a competitor t o t h e mobile phase w i l l r e s u l t i n a r e d u c t i o n o f t h e c h r o m a t o g r a p h i c r e t e n t i o n (k') of the ligand. T h e e x t e n t o f t h i s r e d u c t i o n w i l l be a r e f l e c t i o n of the d i f f e r e n t i a l binding a f f i n i t i e s of the l i g a n d a n d d i s p l a c e r a n d t h e s i t e s a t w h i c h t h e s e compounds bind. The a d d i t i o n o f a c o m p e t i t o r t o t h e m o b i l e p h a s e c o u l d a l s o have an e f f e c t on any c h i r a l r e s o l u t i o n s . The e x t e n t o f t h e i m p a c t on t h e s t e r e o s e l e c t i v i t y w i l l d e p e n d i n p a r t upon t h e a f f i n i t y o f t h e c o m p e t i t o r f o r t h e b i n d i n g s i t e o f t h e l i g a n d a n d t h e c h i r a l mechanism r e s p o n s i b l e f o r t h e separation o f t h e l i g a n d enantiomers. For t h e l a t t e r aspect, i f the l i g a n d enantiomers bind
150
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a t t h e same s i t e o n t h e p r o t e i n w i t h t h e same mechanism, a s i n g l e s i t e / s i n g l e mechanism s i t u a t i o n , t h e n t h e a d d i t i o n of a competitor should reduce t h e r e t e n t i o n o f both enantiomers and t h e s t e r e o s e l e c t i v i t y . I f single site/ m u l t i p l e mechanisms o r m u l t i p l e s i t e / m u l t i p l e mechanisms processes are operating, then the d i s p l a c e r should e f f e c t t h e chromatography o f each isomer t o a d i f f e r e n t e x t e n t . We h a v e r e p o r t e d t h e s y n t h e s i s o f a n H S A - b a s e d CSP i n w h i c h t h e p r o t e i n was c o v a l e n t l y b o n d e d t o a c o m m e r c i a l l y a v a i l a b l e d i o l HPLC c o l u m n p r e v i o u s l y a c t i v a t e d w i t h 1,1c a r b o n y l d i i m i d a z o l e (22) . T h e HSA-CSP c a n b e u s e d f o r c h i r a l s e p a r a t i o n s and i n i t i a l s t u d i e s suggested t h a t t h e separations reflected binding i n t e r a c t i o n s between t h e l i g a n d a n d t h e n a t i v e p r o t e i n (22). As p a r t o f o u r i n v e s t i g a t i o n o f t h e a p p l i c a t i o n and u s e o f t h e HSA-CSP, we h a v e s t u d i e d t h e c h i r a l r e c o g n i t i o n processes f o r t h e c h i r a l s e p a r a t i o n o f t h e enantiomers o f ( R , S ) - i b u p r o f e n (IBU) [ N o c t o r , T.A.G.; F e l i x , G.; W a i n e r , I.W. Chromatographia, i n p r e s s . ] and a b e n z o d i a z e p i n e d e r i v a t i v e , (R,S)-oxazepam h e m i s u c c i n a t e (OXH) (23). The e f f e c t s o f t h e a d d i t i o n o f d i s p l a c e r s t o t h e mobile phase on t h e k ' s o f ( R ) - a n d ( S ) - I B U w e r e i n v e s t i g a t e d u s i n g t h e i n d i v i d u a l I B U e n a n t i o m e r s a n d , i n t h e c a s e o f OXH, ( R ) and (S)-OXH w e r e u s e d . T h e s e m o d i f i e r s w e r e c h o s e n t o t e s t f o r b i n d i n g c o m p e t i t i o n between t h e e n a n t i o m e r s i n d i c a t i n g a s i n g l e s i t e / s i n g l e mechanism p r o c e s s . In addition, the (R,S)-IBU were u s e d a s c o m p e t i t o r s i n t h e chromatography o f t h e OXH e n a n t i o m e r s . The r e s u l t s o f t h e a d d i t i o n o f ( R ) - o r ( S ) - I B U t o t h e m o b i l e phase on t h e r e t e n t i o n o f ( R ) - and (S)-IBU a r e p r e s e n t e d i n T a b l e 3. (R)-IBU, w h i c h i s known t o b e more t i g h t l y bound t o p l a s m a p r o t e i n s t h a n ( S ) - I B U (24) a n d t h e observed k's r e f l e c t t h i s d i f f e r e n c e . The e f f e c t s on t h e k' o f ( R ) - I B U o f t h e a d d i t i o n o f ( R ) - I B U t o t h e m o b i l e phase r e l a t i v e t o t h e e f f e c t o f t h e a d d i t i o n o f (S)-IBU a l s o r e f l e c t t h e d i f f e r e n c e i n b i n d i n g a f f i n i t i e s between t h e two I B U e n a n t i o m e r s . I n t h i s c a s e , a 10 /iM c o n c e n t r a t i o n o f t h e ( R ) - i s o m e r r e d u c e s t h e k' o f ( R ) - I B U b y 85% w h i l e a 10 /ttM c o n c e n t r a t i o n o f ( S ) - I B U p r o d u c e s o n l y a 44% reduction. The f a c t t h a t t h e a d d i t i o n o f o n e e n a n t i o m e r a f f e c t s t h e r e t e n t i o n o f t h e o t h e r , i n d i c a t e s t h a t ( R ) - and (S)-IBU b i n d a t t h e same s i t e o n HSA a n d w i t h t h e same mechanism. T h u s , i t a p p e a r s t h a t f o r (R,S)-IBU, c h i r a l r e c o g n i t i o n o n the HSA-CSP involves a single site/single mechanism process. T h i s was n o t t h e c a s e f o r t h e OXH e n a n t i o m e r s (23) . I t h a s b e e n g e n e r a l l y assumed t h a t t h e e n a n t i o m e r s o f a c h i r a l b e n z o d i a z e p i n e b i n d a t t h e same s i t e o n HSA (21,25) . However, t h e r e s u l t s o f t h e c o m p e t i t i o n s t u d i e s c a r r i e d o u t on t h e HSA-CSP, T a b l e 3, do n o t s u p p o r t t h i s h y p o t h e s i s . The a d d i t i o n o f up t o 0.050 mM o f (S)-OXH t o t h e m o b i l e p h a s e h a d r e l a t i v e l y l i t t l e e f f e c t o n t h e k ' o f (R)-OXH
8. WAINER ETAL.
Biopolymer-Based HPLC Chiral Stationary Phases 151
and, c o n v e r s e l y , t h e a d d i t i o n o f up t o 0.020 mM o f (R)-OXH h a d o n l y a s l i g h t e f f e c t o n t h e k ' o f (S)-OXH. These r e s u l t s i n d i c a t e t h a t o n t h e HSA-CSP, (R)-OXH d o e s n o t b i n d a t t h e same s i t e a s (S)-OXH. The e f f e c t o f t h e IBU e n a n t i o m e r s o n t h e r e t e n t i o n o f ( R ) - a n d (S)-OXH i s a l s o c o n s i s t e n t w i t h t h e a s s u m p t i o n t h a t t h e OXH e n a n t i o m e r s b i n d a t d i f f e r e n t s i t e s o n HSA, F i g u r e 5, (23) . IBU i s known t o b i n d a t t h e b e n z o d i a z e p i n e b i n d i n g s i t e o n HSA w h i c h i s t h e same s i t e a t w h i c h t h e IBU e n a n t i o m e r s b i n d (21,24,25). S i n c e t h e k ' o f (S)-OXH i s r e d u c e d w h i l e t h e k ' o f (R)-OXH r e m a i n s c o n s t a n t , t h e s e studies confirm that only (S)-OXH i s bound t o the benzodiazepine binding s i t e . Table 3 . Changes i n t h e chromatographic r e t e n t i o n o f i b u p r o f e n e n a n t i o m e r s , ( R ) - I B U a n d ( S ) - I B U , a n d oxazepam h e m i s u c c i n a t e e n a n t i o m e r s , (R)-OXH a n d (S)-OXH, a s a r e s u l t of t h e a d d i t i o n o f t h e o p p o s i t e enantiomer t o t h e mobile phase Solute/Competitor
8
Competitor Concentration
k' [mM]
(S)-IBU/(R)-IBU
0.000 0.005 0.010 0.050 0.100
21.37 18.27 16.33 11.69 10.47
(R)-IBU/(S)-IBU
0.000 0.020 0.040 0.100 0.200
73.41 66.84 61.86 41.17 31.83
(R)-OXH/(S)-OXH
0.000 0.005 0.010 0.020 0.050
8.36 8.31 8.26 8.17 8.00
(S)-OXH/(R)-OXH
0.000 0.005 0.010 0.020 0.050
22.86 22.80 22.69 22.20 nd
Not d e t e r m i n e d due t o l a r g e background chromatogram and poor peak e f f i c i e n c y .
a
noise
i n the
152
CHIRAL SEPARATIONS BY LIQUID C H R O M A T O G R A P H Y
F i g u r e 5. T h e e f f e c t on t h e k' o f ( R ) - a n d (S)-OXH o f t h e a d d i t i o n o f ( R ) - and ( S ) - i b u p r o f e n t o t h e m o b i l e phase. Where: • = k' o f (S)-OXH [ ( R ) - i b u p r o f e n i n t h e m o b i l e p h a s e ] ; · = k' o f (R)-OXH [ ( R ) - i b u p r o f en] ; • = k' o f (S)-OXH [ ( S ) - i b u p r o f en] ; 0 = k' o f (R)-OXH [ ( S ) ibuprofen].
8. WAINER ET AL.
Biopofymer-Based HPLC Chiral Stationary Phases 153
These r e s u l t s i n d i c a t e t h a t the c h i r a l r e s o l u t i o n of (R)- and (S)-OXH i s the r e s u l t of a m u l t i p l e s i t e / m u l t i p l e mechanism p r o c e s s . The a c t u a l b i n d i n g process a t each s i t e has not been identified and is currently under investigation. In a d d i t i o n , (R)-IBU, which i s known t o be more t i g h t l y bound t o plasma p r o t e i n s than (S)-IBU (24), also had a g r e a t e r e f f e c t on the k ' of (S)-OXH. This result a l s o confirms the hypothesis t h a t the e n a n t i o s e l e c t i v i t y i n the chromatography of IBU on the HSA-CSP i s the r e s u l t of a s i n g l e s i t e / s i n g l e mechanism process a r i s i n g from b i n d i n g a t the benzodiazepine s i t e . Conclusion In t h e i r study of d i p o l e / d i p o l e s t a c k i n g c h i r a l r e c o g n i t i o n mechanisms, P i r k l e and McCune (6) concluded t h a t e n a n t i o d i f f e r e n t i a t i o n i s a time-weighted average o f m u l t i p l e processes and cannot be s t r i n g e n t l y a s c r i b e d t o a s i n g l e mechanism i n a l l i n s t a n c e s . " The r e s u l t s presented i n t h i s review support t h i s o b s e r v a t i o n and extend i t t o i n c l u d e the f a c t t h a t the c h i r a l r e c o g n i t i o n process can i n c l u d e m u l t i p l e s i t e s as w e l l as m u l t i p l e mechanisms. T h i s o b s e r v a t i o n w i l l become i n c r e a s i n g l y important as the number of biopolymer-based c h i r a l s t a t i o n a r y phases grows and as t h e i r a p p l i c a t i o n s expand. 11
Acknowledgements We would l i k e t o thank M a r i l y n E . Levy f o r her i n v a l u a b l e h e l p i n the p r e p a r a t i o n of t h i s manuscript. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
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RECEIVED January 30, 1991
