Probing stereoselective inhibition of the acyl binding site of cholesterol esterase with four diastereomers of 2'-N-α-methylbenzylcarbamyl-1, 1'-bi-2-naphthol
© Chiou et al; licensee BioMed Central Ltd. 2005
Received: 28 June 2005
Accepted: 22 September 2005
Published: 22 September 2005
Recently there has been increased interest in pancreatic cholesterol esterase due to correlation between enzymatic activity in vivo and absorption of dietary cholesterol. Cholesterol esterase plays a role in digestive lipid absorption in the upper intestinal tract, though its role in cholesterol absorption in particular is controversial. Serine lipases, acetylcholinesterase, butyrylcholinesterase, and cholesterol esterase belong to a large family of proteins called the α/β-hydrolase fold, and they share the same catalytic machinery as serine proteases in that they have an active site serine residue which, with a histidine and an aspartic or glutamic acid, forms a catalytic triad. The aim of this work is to study the stereoselectivity of the acyl chain binding site of the enzyme for four diastereomers of an inhibitor.
Four diastereomers of 2'-N-α-methylbenzylcarbamyl-1, 1'-bi-2-naphthol (1) are synthesized from the condensation of R-(+)- or S-(-)-1, 1'-bi-2-naphthanol with R-(+)- or S-(-)-α-methylbenzyl isocyanate in the presence of a catalytic amount of pyridine in CH2Cl2. The [α]25D values for (1R, αR)-1, (1R, αS)-1, (1S, αR)-1, and (1S, αS)-1 are +40, +21, -21, and -41°, respectively. All four diastereomers of inhibitors are characterized as pseudo substrate inhibitors of pancreatic cholesterol esterase. Values of the inhibition constant (K i ), the carbamylation constant (k2), and the bimolecular rate constant (k i ) for these four diastereomeric inhibitors are investigated. The inhibitory potencies for these four diastereomers are in the descending order of (1R, αR)-1, (1R, αS)-1, (1S, αR)-1, and (1S, αS)-1. The k2 values for these four diastereomers are about the same. The enzyme stereoselectivity for the 1, 1'-bi-2-naphthyl moiety of the inhibitors (R > S, ca. 10 times) is the same as that for 2'-N-butylcarbamyl-1, 1'-bi-2-naphthol (2). The enzyme stereoselectivity for the α-methylbenzylcarbamyl moiety of the inhibitors is also R > S (2–3 times) due to the constraints in the acyl binding site.
We are the first to report that the acyl chain binding site of cholesterol esterase shows stereoselectivity for the four diastereomers of 1.
Recently there has been increased interest in pancreatic cholesterol esterase (CEase, EC 18.104.22.168) due to correlation between enzymatic activity in vivo and absorption of dietary cholesterol [1, 2]. Physiological substrates include cholesteryl esters, retinyl esters, triacylglycerols, vitamin esters, and phospholipids [3–5]. CEase plays a role in digestive lipid absorption in the upper intestinal tract, though its role in cholesterol absorption in particular is controversial [1, 6]. A recent report indicates that CEase is directly involved in lipoprotein metabolism, in that the enzyme catalyzes the conversion of large LDL to smaller, denser, more cholesteryl ester-rich lipoproteins, and that the enzyme may regulate serum cholesterol levels [7, 8]. Serine lipases, acetylcholinesterase, butyrylcholinesterase, and CEase belong to a large family of proteins called the α/β-hydrolase fold [9, 10], and they share the same catalytic machinery as serine proteases in that they have an active site serine residue which, with a histidine and an aspartic or glutamic acid, forms a catalytic triad [11, 12]. The conservation of this catalytic triad suggests that as well as sharing a common mechanism for substrate hydrolysis, that is, formation of a discrete acyl enzyme species via the active site serine hydroxy group, serine proteases, CEase, and lipases may well be expected to be inhibited by the same classes of mechanism-based inhibitors such as phosphorothiolates , pyrones , fluoroketones , boronic acids , and carbamates [16–29].
k app = k2 [I]/(K i (1+ [S]/K m )+ [I]) (1)
In Equation 1, kapp values are first-order rate constants which can be obtained as described in Hosie et al. . Bimolecular rate constant, k i = k2/K i , is related to overall inhibitory potency.
For the first time, we synthesize four optical pure diastereomers of 1. (1R, αR)-1, (1R, αS)-1, (1S,αR)-1, and (1S, αS)-1 (Figure 2) are synthesized from the condensation of R-(+)- or S-(-)-1, 1'-bi-2-naphthanol with R-(+)- or S-(-)-α-methylbenzyl isocyanate in the presence of a catalytic amount of pyridine in CH2Cl2. The [α]25 D values for (1R, αR)-1, (1R, αS)-1, (1S,αR)-1, and (1S, αS)-1 are +40, +21, -21, and -41°, respectively.
Inhibition constants for CEase-catalyzed hydrolysis of PNPB in the presence of the four diastereomers of 1 and the two enantiomers of 2
0.20 ± 0.01
2.0 ± 0.2
10 ± 1
0.50 ± 0.03
2.0 ± 0.2
4.0 ± 0.4
2.0 ± 0.1
2.0 ± 0.2
1.0 ± 0.1
6.0 ± 0.4
1.8 ± 0.2
0.30 ± 0.03
0.8 ± 0.1
10 ± 1
12 ± 2
1.3 ± 0.1
6.0 ± 0.5
5.0 ± 0.6
Among the four diastereomers of 1, (1R, αR)-1 is the most potent inhibitor and its overall inhibitory potency (k i ) is about the same as that of R-2 (Table 1). On the other hand, (1S, αS)-1 is the least potent inhibitor of CEase and its overall inhibitory potency is about 17-fold lower than that of S-2. All k2 values for the CEase inhibition by1 are about the same (Table 1).
The stereoselectivity of CEase at ABS of the enzyme for the α-methylbenzyl group of 1 (R > S) (Table 1) is the same as that of CRL at its ABS for 2-methyl-6-(2-thienyl) hexanate . For the K i step (Figure 3), (1R, αR)-1 and (1S, αR)-1 bind to CEase 2.5 and 3 times more tightly than (1R, αS)-1 and (1S, αS)-1, respectively. The K i value with regard to the chiral center at the α-position of 1 is quite low compared to that with regard to the binaphthol chiral axis of 1 (Table 1) [20, 22] and to that with regard to the phosphorus chiral center of isomalathion . Therefore, we propose that ABS of CEase does not show high selectivity for the chiral acyl group due to a narrow and hydrophobic binding pocket for ABS [30, 31], which selectively and tightly binds to the benzyl phenyl moiety of the inhibitor and results in the discrimination of stereoselectivity by either the hydrogen atom or the methyl group at the α-position of the four diastereomers of 1 (Figure 5).
(1R, αR)-1 and (1R, αS)-1 are bound to CEase 10 and 12 times more tightly than (1S, αR)-1 and (1S, αS)-1, respectively (Table 1); however, R-2 is bound to CEase only 1.6 times more tightly than S-2 [20, 22]. The possible reason is that the binding of the phenyl moiety of the α-methylbenzylcarbamyl group of 1 to ABS (Figure 5) constrains the binaphthol moiety of 1 to a more favorable conformation to bind with LBS, on the other hand, the n-butyl carbamyl of 2 has lots of room to "breathe" in ABS and therefore the binaphthol moiety of2 has many conformations and results in loosely binding to LBS.
The k2 values for the four diastereomers of 1 are about the same. This means that the k2 step is insensitive to the stereochemistry of 1. In other words, the stereoselectivity of CEase for (1R, αR)-1 primarily results from the K i step. The k2 values for all diastereomers of1 are lower than those for the two atropisomers of 2 (Table 1). The possible reason is that the n-butylcarbamyl enzyme from both atropisomers of 2 is relatively more stable than the α-methylbenzylcarbamyl enzymes from the four diastereomers of 1.
Overall, we report that CEase has two stereoselective binding sites at LBS and ABS for the four diastereomers of 1. CEase , Chromobacterium viscosum lipase, and Rhizopus oryzal lipase  also show two stereoselective binding sites at LBS and ES for organic phosphorus compounds. Therefore, it is possible that CEase and lipase may contain totally three stereoselective binding sites at ABS, ES, and LBS for the six diastereomers of substrates or inhibitors.
Four diastereomers of 1 are synthesized and characterized as the pseudo substrate inhibitors of pancreatic cholesterol esterase. The inhibitory potencies for these four diastereomeric inhibitors are in the descending order of (1R, αR)-1, (1R, αS)-1, (1S, αR)-1, and (1S, αS)-1. The enzyme stereospecificity toward the 1, 1'-bi-2-naphthyl moiety of the inhibitors is the R-form and is the same as that for 2. The enzyme stereospecificity toward the α-methylbenzylcarbamyl moiety of the inhibitors is also R-form. For the first time, we observe that the acyl binding site of cholesterol esterase shows stereospecificity for diastereomeric inhibitors.
Porcine pancreatic CEase (ca. 70% pure since the observed K m value for this enzyme catalyzed hydrolysis of PNPB is 1.4 times higher than that for the pure enzyme ) and PNPB were obtained from Sigma; TFA and other chemicals were obtained from Aldrich. Silica gel used in liquid chromatography (Licorpre Silica 60, 200–400 mesh), medium pressure liquid chromatography column (LiChroprep Si 60) and thin layer chromatography plates (Kieselgel 60 F254) were obtained from Merck. An UV lamp as well as an UV detector (Linear UV-106 or ISCO UA-6) was used in detection. Hexane-ethyl acetate solvent gradient was used in liquid chromatography and medium pressure liquid chromatography. Other chemicals were of the highest quality available commercially. Carbamates 2 were synthesized as described before [20, 22].
1H and 13C NMR spectra were recorded at 300 and 75.4 MHz (Varian-VXR 300 spectrometer), respectively. The 1H and 13C NMR chemical shifts were referred to internal Me4Si. UV spectra were recorded on an UV-visible spectrophotometer (Hewlett Packard 8452A or Beckman DU-650) with a cell holder circulated with a water bath. High resolution mass spectra were recorded at 70 eV on a Joel JMS-SX/SX-102A mass spectrophotometer. Elemental analyses were preformed on a Heraeus instrument.
Synthesis of four diastereomers of 1
(1R, αR)-1, (1R, αS)-1, (1S, αR)-1, and (1S, αS)-1 (Figure 2) were prepared from the condensation of R-(+)- or S-(-)-α-methylbenzyl isocyanate ([α]20 D = +10° or -10°) with 1 equivalent of R-(+)- or S-(-)-1, 1'-bi-2-naphthol ([α]20 D = +34° or -34°) in the presence of a catalytic amount of pyridine in CH2Cl2 at 25°C for 24 h (80–95 % yield). All products were purified by liquid chromatography or medium pressure liquid chromatography (silica gel, hexane-ethyl acetate) and characterized by 1H and 13C NMR spectra and high resolution mass spectra.
(1R, αR)-1, (1R, αS)-1, (1S, αR)-1, and (1S, αS)-1: 1H NMR (CDCl3, 300 MHz) δ/ppm 1.02 (d, J = 6.6 Hz, 3H, CH(Ph)CH3), 4.48 (quintet, J = 7 Hz, 1H, CH(Ph)CH3), 5.27 (d, J = 8.1 Hz, 1H, NH), 7.07–8.06 (m, 17H, aromatic H); 13C NMR (CDCl3, 75.4 MHz) δ/ppm 21.88 (CH3), 50.36 (CH(Ph)CH3), 122.45, 123.51, 125.43, 125.69, 126.08, 126.48, 126.60, 127.10, 127.24, 127.91, 128.18, 128.37, 128.53, 129.40, 131.40, 133.30, 133.41, 142.98, and 147.20 (aromatic Cs), 153.91 (C = O); High resolution mass spectra: Found: 433.1674; C29H23NO3 requires 433.1678. [α]25 D = +40, +21, -21, and -41° for (1R, αR)-1, (1R, αS)-1, (1S, αR)-1, and (1S, αS)-1, respectively. The stability of these compounds is very high at -20°C (no significant change for the optical rotation in 1 month).
Enzyme kinetics and data reduction
All kinetic data were obtained by using an UV-visible spectrophotometer that was interfaced to a computer. Microcal Origin (version 6.0) was used for all least squares curve fittings. The CEase inhibition was assayed as described in Hosie et al. . The temperature was maintained at 25.0°C by a refrigerated circulating water bath. All reactions were performed in sodium phosphate buffer (pH 7.0) containing NaCl (0.1 M), acetonitrile (2% by volume), substrate PNPB (50 μM), triton X-100 (0.5 % by weight) and varying concentration of inhibitors (from 0.1 to 10 μM). The K m value for CEase-catalyzed hydrolysis of PNPB was calculated to be 140 ± 10 μM from the Michaelis-Menten equation. Requisite volumes of stock solution of substrate and inhibitors in acetonitrile were injected into reaction buffers via a pipet. CEase was dissolved in sodium phosphate buffer (0.1 M, pH 7.0). Reactions were initiated by injecting enzyme and monitored at 410 nm on the UV-visible spectrometer. First-order rate constants (the kapp values) for inhibition of CEase were determined as described by Hosie et al.  Values of K i and k2 can be obtained by the parameters of non-linear least squares curve fittings of kapp vs. [I] plot to Equation (1) (Figure 4A). Duplicate sets of data were collected for each inhibitor concentration.
List of abbreviations used
acyl chain binding site
acetylcholinesterase, BChE, butyrylcholinesterase
Candida rugosa lipase
catalytic or esteratic site
Geotrichum candidum lipase
- k app :
first-order rate constants
- k 2 :
- k i :
bimolecular rate constant
leaving group hydrophilic binding site
leaving group binding site
1'-bi-2-naphthol ((1R, αR)-1)
1'-bi-2-naphthol ((1R, αS)-1)
1'-bi-2-naphthol ((1S, αR)-1)
1'-bi-2-naphthol ((1S, αS)-1)
the oxyanion hole
Pseudomonas species lipase
Pseudomonas species lipase
the second acyl chain binding site
The authors thank the National Science Council of Taiwan for financial support.
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