Enzymes in Carbohydrate Synthesis - American Chemical Society

Chapter 6

Use of Glycosyltransferases for Drug Modification 1

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Downloaded by UNIV MASSACHUSETTS AMHERST on August 1, 2012 | http://pubs.acs.org Publication Date: June 24, 1991 | doi: 10.1021/bk-1991-0466.ch006

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Daniel Gygax , Mario Hammel , Robert Schneider , Eric G . Berger , and Hiltrud Stierlin 1

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Central Research Laboratories and Research and Development Department of Pharmaceuticals Division, Ciba-Geigy Ltd., 4002 Basel, Switzerland Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland

Glycosyltransferases are enzymes which form glycosidic linkages regio- and stereospecifically by transferring the carbohydrate residues of activated monosaccharides to suitable acceptors. In this paper we describe the use of glycosyltransferases for the in vitro modification of drugs. Glycosyltransferases were used for the modification of glycoprotein glycans expressed in yeast. A continuous synthesis of β -D-glucuronides catalyzed by glucuronyltransferases was carried out in a membrane reactor. UDP-glucuronic acid was regenerated in situ by using a multi-enzyme system. Modification of glycan chains was performed by combined use of glycosidases and glycosyltransferases. Sialylated glycoproteins showed extended plasma residence time compared to galactosylated glycoproteins. The selective synthesis of β - g l u c u r o n i d e s with glucuronyltransferases provides a method to construct β-D-glucuronides of xenobiotics such as drugs, pesticides and endogenous steroid hormones which are widespread biotransformation products. These conjugates are needed for analytical, toxicological and pharmacological investigations. To perform these studies, amounts between 50 to 100 mg of radioactively labeled or unlabeled glucuronide are required. The enzyme-catalyzed synthesis of β-D-glucuronides is an alternative to the chemical synthesis or the isolation of these conjugates from biological fluids. The UDP-glucuronyltransferases (EC 2.4.1.17) are a family of enzymes located predominantly in endomembranes of the hepatocyte (7). They catalyze the transfer of glucuronic acid from uridine 5'-diphospho-a-D-glucuronic acid to a suitable aglycon with inversion of the configuration at the anomeric center of glucuronic acid (Figure 1) (7). The reaction is stereoselective; only the β-glucuronide is formed. None of the functional groups of glucuronic acid have to be protected. The scope of the reaction is broad: glucuronyltransferase was found to conjugate aglycons containing phenols, amines, alcohols, thiols, carbamates and carboxylic acids (2).

0097-6156/91/0466-0079$06.00/0 © 1991 American Chemical Society

In Enzymes in Carbohydrate Synthesis; Bednarski, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

ENZYMES IN CARBOHYDRATE SYNTHESIS

80


Synthesis

of

Glucuronides

Glucuronides have been synthesized batch-wise or in a hollow fiber system using microsomal or soluble enzyme preparations (5-5). Furthermore, they have been prepared with enzymes immobilized to polymeric supports (6). Here we describe the continuous synthesis of glucuronide conjugates in a 10-mL membrane reactor (7). Experimental Details. Figure 1 depicts the general reaction scheme. Fresh liver from guinea pig was homogenized and centrifuged. A 1-mL portion of the supernatant was injected directly into the reactor (Figure 2). There it was retained by a semi-permeable membrane with a molecular weight limit of 10 kD. The substrate solution, containing 5 mM of the C-labeled aglycon (1 or 2), 20 mM of UDP-glucuronic acid, 10 mM of M g C l 2 and 50 mM of HEPES buffer (pH 7.4) was pumped through the reactor at a flow rate of 6 mL/h. The glucuronide production profile of 1 reached a plateau corresponding to 95% conversion within 8 hours. This high level of conversion was maintained throughout the whole reaction period of 20 hours. Compound 2 was a poorer substrate that 2, and, consequently, resulted in only about 50% conversion over the reaction period of 20 h. Analysis by *H-NMR spectroscopy revealed that the enzyme catalyzes the conjugation of 2 stereo- and regioselectively by differentiating between the aryl- and the alkylhydroxy groups. The formation of l a and 2a were followed by T L C and quantified by radioactivity scanning. After purification using reversed-phase HPLC, 37 mg (50%) of l a and 96 mg (37%) of 2a were isolated. Structural and stereochemical assignments were based on analysis of the ^H-NMR and MS spectra. The glucuronyltransferase-catalyzed reaction is species dependent (Table 1). We used a crude liver homogenate from various animal species as the source of the enzymes. Regeneration of UDP-Glucuronic Acid. The substrate of enzymatic glucuronidation, UDP-glucuronic acid, is very costly and unstable. Therefore, we established a multi-enzyme system to regenerate UDP-glucuronic acid in situ from uridine 5'-diphosphate (UDP) and glucose 1-phosphate (Figure 3). A similar type of multi-catalyst system with immobilized enzymes was used for the synthesis of oligosaccharides (8-9). Instead of using individually immobilized enzymes, we used a crude liver homogenate from guinea pig containing all enzymes involved in the multicatalyst system. The glucuronidation is started with 0.05 equivalents of UDP-glucose compared to the aglycon. UDP-glucose is oxidized by the NAD -dependent UDP-glucose dehydrogenase to UDP-glucuronic acid (Step II). Two products are generated during UDP-glucuronyltransferase-catalyzed transfer of glucuronic acid to the aglycon (Step I), namely the glucuronide and UDP. The phosphorylation of UDP to UTP is catalyzed by pyruvate kinase (Step IV) using phosphoenolpyruvate (PEP) as phosphoryl-group donor. Finally, the cycle is closed by the UDP-glucose pyrophosphorylase-catalyzed transfer of UTP to glucose 1-phosphate (Step III). Scale-up of Procedure. The multi-catalyst synthesis of l a was established and optimized on an analytical scale. The formation of l a was followed by T L C and quantified by radioactivity scanning. Optimal conditions were: 0.5 mM of 1, 0.025 mM of UDP-glucose, 3.3 mM of glucose 1-phosphate, 2 mM of PEP, 2 mM of N A D and 20 mg/mL of liver homogenate in 0.025 M of HEPES buffer (pH 8.0). In preparative scale 14

+

+



6.

GYGAX ET AL.

Drug

81

Modification

NHCHO HO 6*

V

UT

1

Ν

0

OH 14

R= l

* = C-labels

R =2

Figure 1. Enzymatic synthesis of β - D - g l u c u r o n i d e s with crude liver homogenate from guinea pig as source of the glucuronyltransferase. 1: CGS 5649B. 2: CGP25 827A.

EMR Pump

-CKH 4°C Substrate

37° C

Product Enzyme (liver homogenate)

Figure 2. Schematic representation of the enzyme membrane (EMR) system used for enzymatic synthesis of β - D - g l u c u r o n i d e s .


reactor

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ENZYMES IN CARBOHYDRATE SYNTHESIS

Table 1.


Order

Species dependency of enzymatic

glucuronidation

Conversion (%)

Species

C G S 5649

C G P 361

Man

Enzymes in Carbohydrate Synthesis - American Chemical Society

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