Escherichia coli glucuronylsynthase: An engineered enzyme for the synthesis of β-glucuronides

Article abstract of DOI:10.1021/ol8002767

The glycosynthase derived from E. coli β-glucuronidase catalyzes the glucuronylation of a range of primary, secondary, and aryl alcohols with moderate to excellent yields. The procedure provides an efficient, stereoselective, and scalable single-step synthesis of β-glucuronides under mild conditions.

Full text of DOI:10.1021/ol8002767

ORGANIC
LETTERS
2008
Vol. 10, No. 8
1585-1588
Escherichia coli Glucuronylsynthase:
An Engineered Enzyme for the
Synthesis of
â-Glucuronides
|
Shane M. Wilkinson,† Chu W. Liew,‡ Joel P. Mackay,‡ Hamzah M. Salleh,§,
Stephen G. Withers,§ and Malcolm D. McLeod*,†
School of Chemistry, UniVersity of Sydney, NSW 2006, Australia, School of Molecular
and Microbial Biosciences, UniVersity of Sydney, NSW 2006, Australia, and
Department of Chemistry, UniVersity of British Columbia, VancouVer,
BC V6T 1Z1, Canada
m.mcleod@rsc.anu.edu.au
Received February 6, 2008
ABSTRACT
The glycosynthase derived from E. coli â-glucuronidase catalyzes the glucuronylation of a range of primary, secondary, and aryl alcohols with
moderate to excellent yields. The procedure provides an efficient, stereoselective, and scalable single-step synthesis of
â-glucuronides under
mild conditions.
The formation of glucuronide conjugates during phase II
metabolism is a major pathway for the elimination of xeno-
biotic and some endobiotic compounds from the body.1 The
identification, quantification, and pharmacological evaluation
of these metabolites is essential in many fields including
sports drug testing,2 the detection of agricultural residues,3
and drug development,1 leading to a significant demand for
glucuronide standards.
The preparation of glucuronides presents significant chal-
lenges for existing methods of glucuronylation.4 Chemical
methods5 of glucuronylation are based on the Koenigs-
Knorr reaction or related procedures but often suffer from
poor yields and side reactions due to the low reactivity of
glucuronic acid derived glycosyl donors4,5 and require one
or more deprotection steps to liberate free glucuronide.
Enzymatic methods6 of synthesis employ uridine 5′-diphos-
phoglucuronosyl transferases (UGTs): a superfamily of
† School of Chemistry, University of Sydney. Current address: Research
School of Chemistry, Australian National University, Canberra, ACT 0200,
Australia.
(4) (a) Kaspersen, F. M.; Van Boeckel, C. A. A. Xenobiotica 1987, 17,
1451. (b) Stachulski, A. V.; Jenkins, G. N. Nat. Prod. Rep. 1998, 15, 173.
(c) Stachulski, A. V.; Harding, J. R.; Lindon, J. C.; Maggs, J. L.; Park, B.
K.; Wilson, I. D. J. Med. Chem. 2006, 49, 6931.
(5) For recent chemical synthesis, see: (a) Engstrom, K. M.; Daanen, J.
F.; Wagaw, S.; Stewart, A. O. J. Org. Chem. 2006, 71, 8378. (b) Harding,
J. R.; King, C. D.; Perrie, J. A.; Sinnott, D.; Stachulski, A. V. Org. Biomol.
Chem. 2005, 3, 1501. (c) Pola´kova´, M.; Pitt, N.; Tosin, M.; Murphy, P. V.
Angew. Chem., Int. Ed. 2004, 43, 2518.
‡ School of Molecular and Microbial Biosciences, University of Sydney.
§ University of British Columbia.
| Current address: Department of Biotechnology Engineering, Interna-
tional Islamic University Malaysia, Kuala Lumpur, 53100, Malaysia.
(1) (a) Shipkova, M.; Wieland, E. Clin. Chim. Acta 2005, 358, 2. (b)
Wells, P. G.; Mackenzie, P. I.; Chowdhury, J. R.; Guillemette, C.; Gregory,
P. A.; Ishii, Y.; Hansen, A. J.; Kessler, F. K.; Kim, P. M.; Chowdhury, N.
R.; Ritter, J. K. Drug Metab. Dispos. 2004, 32, 281.
(2) Scha¨nzer, W.; Donike, M. Anal. Chim. Acta 1993, 275, 23.
(3) (a) Kim, H.-J.; Ahn, K. C.; Ma, S. J.; Gee, S. J.; Hammock, B. D. J.
Agric. Food Chem. 2007, 55, 3750. (b) Hebestreit, M.; Flenker, U.; Buisson,
C.; Andre, F.; Le Bizec, B.; Fry, H.; Lang, M.; Weigert, A. P.; Heinrich,
K.; Hird, S.; Schanzer, W. J. Agric. Food Chem. 2006, 54, 2850.
(6) For recent enzymic synthesis, see: (a) Ja¨ntti, S. E.; Kiriazis, A.;
Reinila¨, R. R.; Kostiainen, R. K.; Ketola, R. A. Steroids 2007, 72, 287. (b)
Khymenets, O.; Joglar, J.; Clape´s, P.; Parella, T.; Covas, M.-I.; de la Torre,
R. AdV. Synth. Catal. 2006, 348, 2155. (c) Kuuranne, T.; Aitio, O.; Vahermo,
M.; Elovaara, E.; Kostiainen, R. Bioconjugate Chem. 2002, 13, 194.
10.1021/ol8002767 CCC: $40.75
© 2008 American Chemical Society
Published on Web 03/18/2008

enzymes responsible for glucuronylation in the body.7,8 This
procedure provides a mild and stereospecific synthesis in a
single step. However, UGTs are substrate-specific to the
acceptor alcohol and practical considerations often limit this
procedure to small scale syntheses. Given the limitations
associated with existing methods, the development of
improved glucuronylation protocols is an important goal.
Escherichia coli â-glucuronidase (EC 3.2.1.31) is a
member of the retaining â-glycosidase family 2 and catalyzes
the hydrolytic cleavage of terminal â-glucuronide residues.
The enzyme has found application in the field of analytical
chemistry for the deconjugation of a broad range of glucu-
ronide metabolites. The enzyme active site contains two key
catalytic residues. The side chain of glutamic acid 504
(E504)9 acts as a nucleophile, and glutamic acid 413 (E413)
is responsible for general acid/base catalysis in a double-
displacement mechanism (Scheme 1a). As demonstrated for
(E504A), or serine (E504S) residue disables the hydrolytic
pathway (Scheme 1b). However, the resulting glycosynthase
enzyme can catalyze the formation of glucuronide product
from R-D-glucuronyl fluoride (1) and an acceptor alcohol
substrate. In this paper, we report the development of a con-
ceptually distinct approach, based on the glycosynthase10,11
derived from E. coli â-glucuronidase, for the mild, single-
step synthesis of glucuronides that is liberated from the many
drawbacks associated with contemporary procedures.
The E504G, E504A, and E504S glucuronylsynthase
mutants were created using standard overlap mutagenesis,
subcloned into the pET28a(+) expression vector and trans-
formed into a â-glucuronidase-deficient strain of E. coli
(GMS407(DE3)). The wild-type (WT) E. coli â-glucu-
ronidase was also expressed in the same manner.
The aglycon specificity of E. coli glucuronidase and of
the putative glycosynthase enzymes was determined using
an established spectrophotometric screening protocol.12,13
This screen employs WT â-glucuronidase inactivated with
2-deoxy-2-fluoro-â-D-glucuronyl fluoride.14 A panel of 123
alcohol acceptors was evaluated in parallel for their ability
to reactivate this inactivated enzyme by glucuronyl trans-
fer (Scheme 2).12 Surprisingly, of the 54 carbohydrate
Scheme 1. Proposed Mechanism of Action of (a) E. coli WT
â-Glucuronidase and (b) the E. coli E504G
â-Glucuronylsynthase
Scheme 2. Acceptor Screening by Reactivation of Inactivated
E. coli WT â-Glucuronidase Enzyme
acceptors screened, none gave significant reactivation of
the inactivated enzyme relative to control. In contrast, thir-
teen of the 69 alcohol acceptors screened significantly
enhanced reactivation rates (2-8 × control). The thirteen
alcohols identified by this screen included a range of pri-
mary and cyclic secondary aliphatic alcohols, substituted
benzyl alcohols, and isomeric naphthalene methanols (Table
1, 2a-m). The screening suggested an unusual preference
for non-carbohydrate based acceptors to occupy the aglycon
binding site of WT â-glucuronidase and glycosynthase
enzymes.
Microgram scale reactions to identify glycosynthase activ-
ity were performed with the 13 alcohols (2a-m), R-D-
glucuronyl fluoride 1,15 and the three potential glucuronyl-
a range of other retaining â-glycosidase enzymes,10,11 muta-
tion of E504 to a non-nucleophilic glycine (E504G), alanine
(11) For a review on glycosynthases, see: Williams, S. J.; Withers, S.
G. Aust. J. Chem. 2002, 55, 3.
(12) Blanchard, J. E.; Withers, S. G. Chem. Biol. 2001, 8, 627.
(13) The details of the screening protocol and the full list of alcohols
used in the screen is reported in the Supporting Information.
(14) Wong, A. W.; He, S.; Grubb, J. H.; Sly, W. S.; Withers, S. G. J.
Biol. Chem. 1998, 273, 34057.
(7) Mulder, G. J. Annu. ReV. Pharmacol. Toxicol. 1992, 32, 25.
(8) Werschkun, B.; Wendt, A.; Thiem, J. J. Chem. Soc., Perkin Trans.
1 1998, 3021.
(9) Wong, A. W.; He, S.; Withers, S. G. Can. J. Chem. 2001, 79, 510.
(10) Mackenzie, L. F.; Wang, Q.; Warren, R. A. J.; Withers, S. G. J.
Am. Chem. Soc. 1998, 120, 5583.
(15) Prepared by TEMPO-mediated oxidation of R-D-glucopyranosyl
fluoride. Synthetic procedures and spectroscopic data are reported in the
Supporting Information.
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Org. Lett., Vol. 10, No. 8, 2008

absence of enzyme or for the serine mutant (E504S), which
was not examined further.
Table 1. E. coli Glucuronylsynthase-Catalyzed
To explore the synthetic potential of the glycosynthase
enzymes, reactions involving the 13 alcohol acceptors
(2a-m) were performed on a preparative scale (5-10 mg)
in the presence of the E504G or E504A glucuronylsynthase
mutants. The E504G mutant cleanly afforded the â-glucu-
ronide products 3a-m in 37-93% isolated yield (Table 1,
entries a-m), with the E504A mutant affording lower yields
in the cases examined (entries i and m). The low aqueous
solubility of a number of acceptor alcohols was overcome
in two ways. Dimethyl sulfoxide (DMSO) was used as
cosolvent in concentrations of up to 25% v/v (entries b-e,
h, j, l, m), with the enzyme retaining useful activity at these
levels. The nonionic detergent, n-dodecyl â-maltoside (DDM),
was also found to be effective at low concentrations resulting
in improved yields (entries j, k, m). No oligomer formation
was detected in these reactions, consistent with the observa-
tion that carbohydrates do not serve as efficient acceptors
in the screen of aglycon specificity. The glucuronylation of
phenylethanol 2f on a large scale afforded the desired
glucuronide 3f in 96% yield (718 mg).
Glucuronylation
The synthesis reaction was also attempted with ethanol
and phenol; two potential acceptors that formed part of the
screen but that were not identified as hits. Indeed, ethanol
failed to afford the glucuronide product (entry n), while a
small quantity of phenyl â-D-glucuronide (3m) was isolated
in 13% yield (entry o) using the E504G glucuronylsynthase.
The low yield of this reaction can be rationalized by the poor
nucleophilicity of phenol but also hints at a broader substrate
scope for the glycosynthase mutants.
To further demonstrate the scope of the engineered
glycosynthase enzyme, the reaction was performed on the
steroid hormone dehydroepiandrosterone (DHEA, 2p). This
substrate was only partially soluble in 25% DMSO or 2.5%
DDM solutions, so the reaction was performed with the
steroid as a suspension (Table 1, entry p). The glucuronide
(3p) was obtained in 5% yield in the absence of additives,
17% yield using DMSO and 26% using DDM with 73%
recovery of residual starting material. Although of modest
yield, these results illustrate the promise of the glucuronyl-
synthase enzyme for the synthesis of a range of important
glucuronide products on a scale sufficient for modern
analytical methods.
This glucuronylsynthase, the latest addition to the glyco-
synthase range, has been shown to catalyze the glucurony-
lation of a range of alcohols with moderate to excellent
yields. This new procedure provides an efficient, stereose-
lective, and scalable single-step synthesis of â-glucuronides
under mild conditions. It is envisaged that this alternative
method of synthesis will assist to meet the demand for
glucuronide standards required for analytical purposes.
Further engineering will be directed to increasing the
substrate scope and improving the catalytic efficiency of the
glucuronylsynthase enzyme.
a Reactions were performed using R-D-glucuronyl fluoride 1 (1.1-1.2
equiv), E504G glucuronylsynthase (0.1 mg mL1) in 50 mM sodium
phosphate buffer, pH 7.5 unless otherwise stated. b Yield in parentheses
denotes the yield obtained from the alanine glucuronylsynthase (E504A).
c 12.5% v/v. d 25% v/v. e Reaction performed on a 2.5 mmol scale. f 2%
w/v. g 0.5% w/v. h 1% w/v. i Saturated acceptor (1.4 mg mL-1), E504G (0.2
mg mL-1), and 100 mM sodium phosphate buffer, pH 7.5 was used. j 2.5%
w/v.
synthase enzymes (E504A, E504G, and E504S). Glucuronide
products 3a-m were identified by ESI-MS for all 13
alcohols in the presence of the glycine (E504G) and alanine
(E504A) mutants. No product formation was observed in the
Acknowledgment. Dr. Kelvin Picker, School of Chem-
istry, University of Sydney, is acknowledged for assistance
Org. Lett., Vol. 10, No. 8, 2008
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with affinity chromatography and MS. We thank the Uni-
versity of Sydney and the Natural Sciences and Engineering
Council of Canada for financial support.
1H and 13C NMR spectra for compounds 1 and 3a-p. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Supporting Information Available: Protein expression
procedures, experimental procedures, spectroscopic data and
OL8002767
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Org. Lett., Vol. 10, No. 8, 2008