Glycosynthase-Mediated Assembly of Xylanase Substrates and Inhibitors

Article abstract of DOI:10.1002/cbic.201100229

An exo-β-xylosidase mutant with glycosynthase activity was created to aid in the synthesis of xylanase substrates and inhibitors. Simple monosaccharides were easily elaborated into di-, tri- and tetrasaccharides by using this enzyme. Some products proved

Full text of DOI:10.1002/cbic.201100229

DOI: 10.1002/cbic.201100229
Glycosynthase-Mediated Assembly of Xylanase Substrates
and Inhibitors
Ethan D. Goddard-Borger,[a] Brigitte Fiege,[b] Emily M. Kwan,[a] and Stephen G. Withers*[a]
An exo-b-xylosidase mutant with glycosynthase activity was
created to aid in the synthesis of xylanase substrates and in-
hibitors. Simple monosaccharides were easily elaborated into
di-, tri- and tetrasaccharides by using this enzyme. Some prod-
ucts proved to be surprisingly potent inhibitors of xylanases
from glycoside hydrolase families 10 and 11.
Introduction
The present interest in biofuels and renewable chemical feed-
stocks has inspired a resurgence of interest in plant polysac-
charide-degrading enzymes. While enzymes that degrade cellu-
lose remain the primary focus of attention, those that act on
other cell-wall polysaccharides, such as hemicellulose and
pectin, are also of great interest. Indeed, enzymes acting upon
these heterogeneous polysaccharides have long been exploit-
ed in various industrial processes.[1,2]
Synthesising xylanase substrates and inhibitors can be a la-
borious task, since these di- or trisaccharides are usually chemi-
cally assembled from monosaccharide synthons.[13,14] A similar
issue concerning the preparation of oligosaccharide cellulase
inhibitors was resolved some time ago with the introduction
of the glycosynthase paradigm,[17–19] which suggested that an
analogous approach might simplify the synthesis of xylanase
substrates and inhibitors.
Xylan is a common component of hemicellulose, and an
abundant plant cell wall polysaccharide. It is a polymer of d-
xylose linked in a b-(1!4) fashion and is often modified with
acetate esters and branching glycosides of l-arabinose and d-
glucuronic acid.[2] Some algae also synthesise xylans, although
these linear biopolymers typically contain a mixture of b-(1!3)
and b-(1!4) glycosidic bonds, or might even be assembled
entirely with b-(1!3) linkages.[3,4]
Glycosynthases are hydrolytically incompetent GH mutants
that can catalyse efficient glycoside bond formation using acti-
vated glycosyl donors. For a conventional retaining glycosi-
dase, this is usually accomplished by mutating the catalytic
nucleophile residue to a small, non-nucleophilic residue, such
as glycine or alanine.[19] This mutation ablates the enzyme’s hy-
drolytic activity, but also creates sufficient room for a glycosyl
fluoride with an anomeric configuration opposite to that of
the natural substrate to bind. This complex mimics the glyco-
syl–enzyme intermediate of the wild-type enzyme and, with
the aid of base catalysis from the remaining catalytic residue,
might glycosylate an acceptor alcohol occupying the aglycone
binding site (Scheme 1).
Xylanases, glycoside hydrolases (GHs) responsible for the hy-
drolysis of xylans into smaller oligosaccharides, are ubiquitous
among bacteria and fungi that subsist on plant matter.[5] The
most common, and best studied of these enzymes are the
endo-b-(1!4)-xylanases (E.C. 3.2.1.8). In accordance with the
sequence-based classification system defined by the Carbohy-
drate Active enZyme (CAZy) database (http://www.cazy.org)[6]
these enzymes hail from GH families 5, 8, 10, 11 and 43, and
facilitate hydrolysis with either inversion (GH families 8 and
43)[7,8] or retention (GH families 5, 10 and 11)[9,10] of the sub-
strate’s anomeric configuration. The less common endo-b-(1!
3)-xylanases (E.C. 3.2.1.32) are all of GH family 26, and so pre-
sumably hydrolyse their algal xylan substrates with retention
of anomeric configuration.[11]
Thus, a b-xylosidase-derived glycosynthase should catalyse
the addition of d-xylosyl units onto a host of xylo-configured
acceptors, making it ideal for the preparation of xylanase sub-
strates and inhibitors. Early attempts to generate glycosynthas-
es from GH family 39 xylosidases were unsuccessful. The weak
xylosidase activity of a GH family 1 glucosidase was exploited
to produce a glycosynthase capable of forming xylosidic
bonds, although it had rather poor activity and provided the
undesired b-(1!3) regioisomers.[20] Efficient glycosynthase mu-
tants of an exo-b-xylosidase were recently reported by Shoham
The mechanisms by which xylanases, particularly those of
GH families 10 and 11, accomplish catalysis are well studied.[5,12]
Synthetic substrates, competitive and mechanism-based inhibi-
tors, as well as activity-based probes have played crucial roles
in such studies.[13–16] These compounds will no doubt feature
prominently in future efforts to obtain a better understanding
of how the various components of cellulolytic enzyme cocktails
secreted by many organisms work in concert to deconstruct
the heterogeneous polysaccharide matrix that is the plant cell
wall.
[a] Dr. E. D. Goddard-Borger, E. M. Kwan, Prof. S. G. Withers
Department of Chemistry, University of British Columbia
2036 Main Mall, Vancouver, British Columbia, V6T 1Z1 (Canada)
E-mail: withers@chem.ubc.ca
[b] B. Fiege
Institute of Chemistry, University of Luebeck
Ratzeburger Allee 160, 23562 Luebeck (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100229.
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S. G. Withers et al.
literature procedures.[23,24] Glycosynthase-mediated glycosyla-
tion of xyloside 1 would provide xylanase substrates, while a
similar elaboration of thioglycoside 2 would presumably give a
substrate-like competitive inhibitor. The 2-deoxy-2-fluoro-xylo-
side 3 was prepared by using a modified literature procedure
(Scheme 2).[13] Enzymatic conversion of compound 3 into
xylan-like oligosaccharides would provide a simple route to
mechanism-based inactivators of retaining xylanases.
Scheme 2. Conditions: a) HBr/AcOH, CH2Cl2, b) 3,4-DNP, 2,6-collidine, Ag2CO3,
MeCN, CaSO4, c) HCl, MeOH.
Scheme 1. Retaining glycoside hydrolase and glycosynthase mechanisms.
and co-workers.[21] This particular xylosidase belonged to GH
family 52—a relatively new GH family.[22]
To generate glycosylated iminosugars using a glycosynthase,
the corresponding N-benzyloxycarbonyl (Cbz) derivative of the
iminosugar is commonly used as a glycosyl acceptor.[25] Con-
verting the iminosugar to the carbamate ablates the iminosu-
gar’s basicity, thereby, reducing its affinity for the donor-bind-
ing subsite of the enzyme and minimising substrate inhibition.
Additionally, the aromatic substituent increases the iminosu-
gar’s affinity for the acceptor binding subsite. The benzyloxy-
carbonyl group also aids in product purification by ensuring
that the desired products carry no charge, have a hydrophobic
“handle” and a chromophore for product detection. Of course,
after glycosylation, this protecting group is easily removed by
hydrogenolysis.
Results and Discussion
Glycosynthase mutants of Bacillus halodurans C-125
xylosidase
Bacillus halodurans C-125 possesses open reading frames
(ORFs) encoding putative retaining b-xylosidases from both GH
family 39 and 52—the latter being a prime candidate for con-
version into a glycosynthase. This gene was cloned, recombi-
nantly expressed in E. coli and, as anticipated, produced pro-
tein with b-xylosidase activity. Site directed mutagenesis was
performed on this B. halodurans xylosidase (Bhx) to substitute
the putative catalytic nucleophile (E334, predicted based on
sequence homology) with a glycine, alanine or serine residue
to provide three mutant enzymes. None of the Bhx mutants
catalysed the hydrolysis of 4-nitrophenyl b-d-xylopyranoside;
this demonstrates that E334 was indeed crucial for catalysis
and supports the assertion that it was the catalytic nucleo-
phile. The Bhx mutants were then evaluated as glycosynthases
by incubating each one with a-d-xylopyranosyl fluoride in
buffer (this compound functions as both donor and acceptor),
and the reactions were analysed by thin layer chromatography
and mass spectrometry to see if xylobiosyl fluoride was pro-
duced.[21] All mutants demonstrated the requisite activity, but it
was the E334G mutant that proved to be the most competent.
To assess the synthetic utility of the E334G Bhx glycosynthase,
a number of d-xylo-configured glycosyl acceptors were pre-
pared—acceptors that, once glycosylated, could serve as xyla-
nase substrates or inhibitors.
Thus, iminosugar derivatives 4 and 5 were prepared from di-
acetone glucose by way of aldehyde 6 (Scheme 3).[26] Hydroly-
sis of the acetonide from this intermediate, followed by reduc-
tive amination and N-acylation provided the desired carbamate
4. A Henry reaction of the aldehyde 6 with nitromethane, fol-
lowed by dehydration and reduction gave the nitro compound
7.[27] Removal of the acetonide group and treatment of the
product with periodate yielded the unstable aldehyde 8. This
product was immediately subjected to hydrogenation, fol-
lowed by N-acylation, presumably with concomitant transes-
terification, to provide the carbamate 5 in good yield.
Glycosynthase reactions
Each of the putative acceptors 1–5 was dissolved, along with
donor substrate (a-d-xylopyranosyl fluoride), in phosphate
buffer at pH 7 and incubated with the E334G Bhx mutant at
room temperature for one day. Although the products from
these glycosynthase reactions could have been isolated with-
out further derivatisation, each was per-O-acetylated to simpli-
fy product isolation and characterisation. To this end, each gly-
cosynthase reaction was first passed through a short column
packed with reverse-phase C-18 silica to separate the desired
Synthesis of acceptors
Simple acceptors, like 4-nitrophenyl b-d-xylopyranoside (1) and
phenyl 1-thio-b-d-xylopyranoside (2) were obtained by using
acceptor-derived products (all possessing
a hydrophobic
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Assembly of Xylanase Substrates and Inhibitors
fied products was dissolved in pyridine and acetylat-
ed by the action of acetic anhydride and a catalytic
amount of 4-dimethylaminopyridine. Subsequent pu-
rification by flash chromatography on silica gel af-
forded each individual oligosaccharide. The results
for each glycosynthase reaction are provided in
Table 1.
The proficiency of this particular glycosynthase is
demonstrated by the high total yield of glycosylated
products for each acceptor tested, ranging from 64
to 86%. For the xyloside 1 (Table 1, entry 1), the b-
(1!4) linked disaccharide 9 and trisaccharide 10
were obtained in good yields, as expected. However,
a small amount of the b-(1!3) linked disaccharide
11 was also isolated. In the case of the thioglycoside
2 (entry 2), only the b-(1!4) linked disaccharide 12
and trisaccharide 13 were obtained. The iminosugar-
derived carbamate 4 (entry 3) gave the (1!4) xylan-
like di-, tri- and tetrasaccharides 14, 15 and 16, re-
spectively. Likewise, the protected iminosugar
5
Scheme 3. Conditions: a) i: NaH, BnBr, DMF, ii: AcOH/H2O (9:1); b) NaIO4, MeOH, H2O;
c) 6n HCl, THF; d) BnNH2, H2 (3 atm), Pd/C, MeOH, 1n HCl; e) CbzCl, K2CO3, H2O/MeOH
(1:1); f) MeNO2, EtOH, 1n NaOH; g) MsCl, Et3N, CH2Cl2; h) NaBH4, EtOH; i) H2 (3 atm), Pd/C,
MeOH, 1n HCl.
(entry 4) was elaborated to the desired disaccharide
17 and trisaccharide 18.
In the case of the 2-deoxy-2-fluoro-xyloside 3
(entry 5) both disaccharide 19 and trisaccharide 20
group) from the glycosyl fluorides, their hydrolysis products,
and buffer salts. This quick, preliminary purification step made
the subsequent acetylation reaction and separation of oligo-
saccharide products much simpler. Each mixture of semipuri-
were obtained, here the first glycosylation reaction proceeded
in a b-(1!3) manner and the second in a b-(1!4) sense (see
NMR COSY experiments in the Supporting Information). The
reasons for this lapse in fidelity are unclear.
Table 1. BhxE334G-catalysed glycosylation of various acceptors.
Acceptor
Product
n
Compound
number
Yield [%]
glycosylated
isolated
5
products
1
–
11
64
0
1
2
–
9
10
19
36
23
0
1
2
–
12
13
14
32
35
2
3
4
67
80
71
0
1
2
3
–
14
15
16
7
13
53
14
0
1
2
–
17
18
11
24
47
0
1
2
–
19
20
2
63
23
5
86
Conditions: A solution of 0.2 mmol acceptor and 0.5 mmol a-d-xylopyranosyl fluoride in sodium phosphate buffer (pH 7) was treated with 480 mg
BhxE334G glycosynthase and incubated at room temperature for 24 h. Products were isolated after per-O-acetylation by treatment with excess Ac2O and
catalytic DMAP in pyridine.
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S. G. Withers et al.
The products of these glycosynthase reactions were subse-
quently deprotected by using conventional procedures
(Scheme 4). The 4-nitrophenyl glycosides 9–11 and thioglyco-
sides 12–13 were deprotected by a Zemplꢁn transesterification
to give the corresponding polyols 21–25. Similar treatment of
the carbamates 14–18, with a subsequent hydrogenation pro-
vided the iminosugars 26–29 (Scheme 4). Acid catalysed trans-
esterification, effected by methanolic hydrogen chloride, was
sufficient for the deprotection of the 2-deoxy-2-fluoro-xylosides
19–20 to provide the corresponding polyols 30–31.
Table 2. Competitive inhibition constants.
Compound
26
27
88ꢁ6
28
29
Ki for Bcx [mm]
Ki for Cex [mm]
1500[14]
1100[14]
63ꢁ4
5.8[14]
0.16ꢁ0.03
0.13[14]
0.018ꢁ0.002
for the corresponding disaccharides 26 and 28, respectively
(Table 2). Both trisaccharides are relatively good inhibitors of
the GH family 11 enzyme Bcx—in fact they are the most
potent competitive inhibitors of this xylanase reported to date.
Similarly, both 27 and 29 are excellent inhibitors of Cex, al-
though 29, with a Ki of just 18 nm, is clearly the better of the
two. The large difference in the inhibitory potency of imino-
sugars like 26–29 against the two different xylanases has pre-
viously been rationalised by the fact that these enzymes facili-
tate hydrolysis using different conformational itineraries, and
so have evolved to stabilise quite different transition
states.[14,30] It would appear then, that 26–29 more closely re-
semble the transition state of Cex than of Bcx.
The di- and trisaccharide 2-fluoro-xylosides 30 and 31 were
incapable of inactivating Bcx or Cex. However, these molecules
might prove to be mechanism-based inhibitors of the GH
family 26 xylanases.[31,32] Likewise, iminosugars 28 and 29
might also serve as inhibitor candidates for this less-common
class of xylanases.
Conclusions
In support of previous studies,[21] this body of work has illus-
trated the synthetic utility and limitations of an exo-b-xylosi-
dase-derived glycosynthase. This enzyme can efficiently cata-
lyse the glycosylation of xylose-like acceptors; for the most
part forming b-(1!4)-linked products in good to excellent
yield. Kinetic studies demonstrated that trisaccharide iminosu-
gars are better inhibitors than the corresponding disaccharides
for two xylanases of different GH families. The reasons for en-
hanced binding of the trisaccharides 27 and 29 relative to the
disaccharides 26 and 28 are unclear; this matter is the subject
of ongoing structural studies. The b-(1!3)-linked oligosacchar-
ides synthesised by an apparent lapse in glycosynthase fidelity
might yet prove to be useful as substrates and inhibitors of
the (1!3)-xylanases from GH family 26.
Scheme 4. Deprotection of oligosaccharide products. Conditions: a) i:
NaOMe, MeOH, ii: Amberlite IR-120 (H+); b) i: H2 (1 atm), Pd/C, MeOH, ii: 1n
HCl; c) HCl, MeOH.
Inhibition of xylanases
The disaccharide iminosugars 26 and 28 have been synthes-
ised previously—both are relatively good inhibitors of Cellulo-
monas fimi xylanase (Cex), a GH family 10 enzyme, but rather
poor inhibitors of the GH family 11 xylanase from Bacillus circu-
lans (Bcx; Table 2).[14] The structures of both enzymes have
been solved by using X-ray diffraction methods, and in neither
is there any obvious ꢀ3 subsite in the substrate-binding
cleft.[28,29] Thus, one might expect that the novel trisaccharides
27 and 29 would be little better as inhibitors than their disac-
charide counterparts 26 and 28, respectively. Intriguingly, the
inhibition constants for trisaccharides 27 and 29 against both
enzymes (determined by using the 4-nitrophenyl glycoside
substrate 22 prepared with the Bhx glycosynthase) were found
to be over an order of magnitude smaller than those reported
Experimental Section
General materials and methods: All chemicals were obtained
from Sigma–Aldrich and were of reagent grade. Thin layer chroma-
tography (TLC) was performed on Merck silica gel 60 F254 plates.
Flash chromatography was performed with Merck silica gel 60
1
(230–400 mesh). H and 13C NMR (referenced by using the solvent
peak, or an internal MeOH standard in the case of 13C for D2O)
spectra were recorded on a 400 MHz Bruker instrument. High-res-
olution mass spectra of all compounds were obtained in the mass
spectrometry laboratory of the Chemistry Department at UBC. Bcx
and Cex were obtained as described previously.[33,34] Inhibition con-
stants were determined at 378C by using a NaH2PO4/Na2HPO4
buffer (50 mm, pH 7.5) containing NaCl (150 mm), and 4-nitrophen-
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Assembly of Xylanase Substrates and Inhibitors
yl b-xylobioside (22) as a substrate. Reactions were initiated by the
addition of xylanase, and the increase in absorption at 400 nm
over time, in a continuous assay, gave the initial reaction rate. This
increase was linear for all measurements (3 min). Michaelis parame-
ters (Vmax and KM) were extracted from these data by best fit to the
Michaelis–Menten equation by using the program GraFit 5.0.13.
Full Ki determinations were performed by measurement of rates at
a series of five substrate concentrations for a number of inhibitor
concentrations (typically seven concentrations bracketing the Ki
value ultimately determined such that 0.2 Ki <[I]<5 Ki). Ki values
were then calculated from these data by nonlinear regression anal-
ysis by using the GraFit program.
Waters tC18 SepPak cartridge (2 g) and eluted under gravity with a
H2O/MeOH gradient (1:0–1:4). The fractions containing glycosylated
acceptor were combined, concentrated and co-evaporated with
toluene (2ꢂ10 mL). Acetic anhydride (0.38 mL, 4.0 mmol) was
added to a solution of the residue and DMAP (5 mg) in C5H5N
(10 mL) and left at room temperature (16 h). Methanol (1 mL) was
added to the solution at 08C and the mixture warmed to room
temperature. The mixture was concentrated to dryness, the residue
was dissolved in EtOAc (50 mL) and washed with HCl (1n, 50 mL),
water (50 mL) and sat. NaHCO3 (50 mL). The organic solution was
dried over MgSO4 and filtered. Concentration of the filtrate and
flash chromatography (EtOAc/hexanes, 1:2 to 2:1) enabled individ-
ual isolation of the per-O-acetylated acceptor and its glycosylation
products.
Cloning, mutagenesis and expression of Bhx
Cloning: The DNA sequence encoding Bhx, BAB05833.1 was ampli-
fied by PCR (Bio-Rad, MyCycler) from genomic DNA of B. halo-
durans C-125, ATCC BAA-125D, by using PWO DNA polymerase
(Roche) with primers 5’-GCATCG GCTAGC ATGTTT ACACCT AAAAAT
ATATTT TTTAAC GCACAT CATTCA CC-3’ and 5’-GCAGTC TGCGGC
CGCTTA TTATGT TATTTC CTCTAG CCAGAG AAT-3’ including restric-
tion sites for NheI and NotI, respectively. The PCR product was
double digested with NheI and NotI (Fermentas) and cloned into
the pET-28a(+) vector (Novagen) resulting in an N-terminal His-tag.
The pET-28a(+)-Bhx construct was transformed into BL21(DE3) cells
(Novagen) by electroporation.
3,4-Dinitrophenyl 2-deoxy-2-fluoro-b-d-xylopyranoside (3): Tri-O-
acetyl-2-deoxy-2-fluoro-d-xylopyranose[15] (1.1 g, 3.8 mmol) was
converted into 3,4-di-O-acetyl-2-deoxy-2-fluoro-a-d-xylopyranosyl
bromide according to the procedure of Ziser et al.[13] Silver carbon-
ate (2.1 g, 7.6 mmol) and anhydrous CaSO4 (2.0 g) were added to a
solution of anhydrous 3,4-dinitrophenol (1.0 g, 5.7 mmol) and 2,6-
lutidine (0.89 mL, 7.6 mmol) in anhydrous MeCN (40 mL) and the
mixture was stirred under Ar in the dark (room temperature,
10 min). A solution of the crude 3,4-di-O-acetyl-2-deoxy-2-fluoro-a-
d-xylopyranosyl bromide in anhydrous MeCN (10 mL) was added
drop-wise and the mixture was stirred (room temperature, 30 min).
The mixture was filtered, the residue was washed with EtOAc, and
the combined filtrate was concentrated. The residue was subjected
to flash chromatography (EtOAc/hexanes; 1:7). A suspension of
this product (1.1 g, 2.6 mmol) in dry MeOH (30 mL) was treated
with AcCl (0.30 mL) at 08C, and the solution was stirred (room tem-
perature, 24 h). The solution was concentrated to dryness and the
residue was recrystallised (MeOH/Et2O) to give the diol 3 as pale
yellow needles (630 mg, 52% yield). 1H NMR (400 MHz, CD3OD):
d=3.51 (dd, J=9.6, 11.6 Hz, 1H; H-5), 3.63 (ddd, J=5.2, 9.6, 9.6 Hz,
1H; H-4), 3.72 (ddd, J=8.8, 9.6, 15.2 Hz, 1H; H-3), 3.97 (dd, J=5.2,
11.6 Hz, 1H; H-5), 4.32 (ddd, J=8.4, 8.8, 51.2 Hz, 1H; H-2), 5.44 (dd,
J=4.0, 8.4 Hz, 1H; H-1), 7.44 (dd, J=2.4, 8.8 Hz, 1H; ArH), 7.62 (d,
J=2.4 Hz, 1H; ArH), 8.13 (d, J=8.8 Hz, 1H; ArH); 13C NMR
(100.6 MHz, CD3OD): d=67.1 (C-5), 70.5 (d, J=7.0 Hz, C-4), 75.8 (d,
J=18.1 Hz, C-3), 93.0 (d, J=186.2 Hz, C-2), 99.7 (d, J=24.2 Hz, C-1),
114.1, 121.0, 128.8, 137.6, 146.6, 162.0 (Ar). HRMS (ES): m/z
341.0390; [M+Na]+ requires 341.0397.
Mutagenesis: Site-directed mutagenesis of the wild-type Bhx gene
was carried out following the QuickChange protocol (Stratagene)
by using Pfu DNA polymerase (Stratagene), with pET-28a(+)-Bhx
wild type as template. Mutagenic primers were: 5’-CCATTA
TGGGTC GTGAAT GCGGGC GAATAC AGAATG-3’ and 5’-CATCAT
TCTGTA TTCGCC CGCATT CACGAC CCATAA TG-3’ for the E334A
mutant; 5’-CCATTA TGGGTC GTGAAT GGGGGC GAATAC AGAATG-3’
and 5’-CATCAT TCTGTA TTCGCC CCCATT CACGAC CCATAA TG-3’ for
the E334G mutant (mutated nucleotides in bold type). The E334S
mutant was created by using a four primer protocol with: 5’-
CGACCA TTATGG GTCGTG AATTCG GGCGAA TACAGA ATG-3’ and
5’-GTATTC ATCATT CTGTAT TCGCCC GAATTC ACGACC CATAAT G-3’
as mutagenic primers as well as the two primers used for cloning
of the wild-type gene. The mutants were transformed into
BL21(DE3) cells (Novagen) as for the wild type. Correct mutagene-
sis was validated by DNA sequencing.
Protein expression: Wild-type and mutant Bhx clones in BL21(DE3)
cells were cultured in TYP medium at 378C. When cultures reached
an OD600 of approximately 1.0, isopropyl b-d-thiogalactopyranoside
(IPTG) was added (final concentration 0.1 mm) and the cultures
were incubated for a further 16 h. Cells were harvested by centrifu-
gation (4500 g, 30 min) and suspended in buffer A (60 mL per liter
of culture; buffer A: 20 mm potassium phosphate, pH 7.4, 0.5m
NaCl, 5 mm imidazole). Cell extracts were prepared by French
press. The soluble extracts were loaded onto HisTrap FF columns
(GE Healthcare) on a FPLC system (GE Healthcare), washed with
15 column volumes of buffer A, and then eluted with a gradient of
buffers A and B (buffer B: 20 mm potassium phosphate, pH 7.4,
0.5m NaCl, 0.5m imidazole). The yield of wild-type and mutant Bhx
N-Benzyloxycarbonyl-1,5-dideoxy-1,5-imino-d-xylitol (4): Hydro-
chloric acid (6n, 5 mL) was added to a solution of the aldehyde
6[26] (1.5 g, 5.5 mmol) in THF (10 mL) and the mixture was stirred
(room temperature, 16 h). The solution was concentrated to dry-
ness. A mixture of the residue, Pd/C (10%, 100 mg), benzylamine
(0.72 mL, 6.6 mmol) and hydrochloric acid (6n, 1 mL) in MeOH
(20 mL) was stirred under H2 (3 atm, 48 h). The mixture was filtered
and concentrated. Potassium carbonate (3.0 g, 22 mmol) and CbzCl
(1.2 mL, 8.2 mmol) were added to a solution of the residue in
MeOH/H2O (1:1, 20 mL) and the mixture was stirred (16 h). The
mixture was concentrated, and the residue was repeatedly washed
with Me2CO (5ꢂ10 mL). The Me2CO washings were combined and
concentrated to dryness. The residue was subjected to flash chro-
matography (EtOAc/hexanes; 19:1–1:0) to return the triol 5 as col-
protein was around 70 mgLꢀ1
.
Glycosynthase reaction protocol: Glycosynthase (50 mL,
9.6 mgmLꢀ1 BhxE334G) was added to a solution of the acceptor
(0.20 mmol) and a-d-xylopyranosyl fluoride[35] (76 mg, 0.50 mmol)
in NaH2PO4/Na2HPO4 buffer (15 mL, 350 mm, pH 7) and the solution
was left at room temperature (24 h). Methanol (2 mL) was added,
then the solution was concentrated to a third of its volume and fil-
tered (Millex GV 0.22 mm filter unit). The filtrate was applied to a
1
ourless needles (832 mg, 57% yield). H NMR (400 MHz, D2O): d=
2.65–2.88 (m, 2H; H-1,5), 3.29–3.54 (m, 3H; H-2,3,4), 4.02–4.31 (m,
2H; H-1,5), 5.12–5.19 (m, 2H; CH2Ph), 7.35–7.55 (m, 5H; Ph);
13C NMR (100.6 MHz, D2O): d=47.6 (C-1,5), 68.1 (CH2Ph), 69.2 (C-
2,4), 77.9 (C-3), 127.9, 128.6, 128.9, 136.3 (Ph), 156.7 (NCO2). HRMS
(ES): m/z 290.1006; [M+Na]+ requires 290.1004.
ChemBioChem 2011, 12, 1703 – 1711
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1707

S. G. Withers et al.
Benzyl (3R,4R)-3,4-dihydroxy-piperidine-1-carboxylate (5): A mix-
ture of Pd/C (10%, 100 mg), the aldehyde 8[27] (0.77 g, 2.7 mmol)
and hydrochloric acid (1n, 0.5 mL) in MeOH (20 mL) was stirred
under H2 (3 atm, 48 h). The mixture was filtered and concentrated.
Potassium carbonate (0.75 g, 5.4 mmol) and CbzCl (0.59 mL,
4.1 mmol) were added to a solution of the residue in MeOH/H2O
(1:1, 10 mL) and the mixture was stirred (16 h). The mixture was
concentrated, and the residue was repeatedly washed with Me2CO
(5ꢂ10 mL). The Me2CO washings were combined and concentrated
to dryness. The residue was subjected to flash chromatography
(EtOAc/hexanes; 17:3–9:1) to return the diol 5 as a colourless foam
5’’), 62.7 (C-5’), 66.4 (C-5), 68.5 (C-4’’), 70.2 (C-2), 70.5, 70.6 (C-2’’,3’’),
71.2 (C-2’), 72.1 (C-3’), 73.4 (C-3), 74.3 (C-4’), 75.2 (C-4), 86.6 (C-1),
99.6 (C-1’’), 100.6 (C-1’), 128.4, 129.2, 132.4, 132.9 (Ph), 169.3, 169.5,
169.7, 169.9, 169.98, 170.01, 170.1 (COCH3). HRMS (ES): m/z
823.2108; [M+Na]+ requires 823.2095.
Glycosylation of N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-d-
xylitol (4): The carbamate 4 was subjected to the glycosynthase re-
action protocol described above. The following products were ob-
tained after flash chromatography and are listed in order of elution
(yields are presented in Table 1).
1
(482 mg, 70% yield). H and 13C NMR spectra obtained for this ma-
2,3,4-Tri-O-acetyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-d-xylitol:
1H NMR (400 MHz, CDCl3 at 318 K): d=2.00, 2.04 (2s, 9H; COCH3),
3.22 (dd, J=8.4, 13.6 Hz, 2H; H-1,5), 4.08 (dd, J=4.4, 13.6 Hz, 2H;
H-1,5), 4.85 (ddd, J=4.4, 7.6, 8.4 Hz, 2H; H-2,4), 5.09 (t, J=7.6 Hz,
1H; H-3), 5.16 (brs, 2H; CH2Ph), 7.28–7.37 (m, 5H; Ph); 13C NMR
(100.6 MHz, CDCl3 at 318 K): d=20.76, 20.79 (COCH3), 45.0 (C-1,5),
67.9 (CH2Ph), 68.4 (C-2,4), 72.2 (C-3), 128.2, 128.4, 128.7, 136.5 (Ph),
155.3 (NCO2), 169.7, 169.8 (COCH3). HRMS (ES): m/z 416.1325;
[M+Na]+ requires 416.1321.
terial were in agreement with the data previously reported.[14]
Glycosylation of 4-nitrophenyl b-d-xylopyranoside (1): The xylo-
side 1[23] was subjected to the glycosynthase reaction protocol
described above. The following products were obtained after flash
chromatography and are listed in order of elution (yields are pre-
sented in Table 1).
1
4-Nitrophenyl 2,3,4-tri-O-acetyl-b-d-xyloside: H and 13C NMR spectra
obtained for this compound were in agreement with the data pre-
viously reported.[36]
Benzyl (3S,4S,5R)-3,4-diacetoxy-5-(tri-O-acetyl-b-d-xylopyranosyloxy)-
piperidine-1-carboxylate (14): 1H NMR (400 MHz, CDCl3 at 318 K):
d=2.01, 2.02, 2.04 (3s, 15H; COCH3), 2.87–3.06 (m, 2H; H-2,6), 3.37
(dd, J=8.0, 11.6 Hz, 1H; H-5’), 3.71 (ddd, J=4.8, 8.8, 8.8 Hz, 1H; H-
5), 4.02–4.25 (m, 3H; H-2,6,5’), 4.59 (d, J=6.0 Hz, 1H; H-1’), 4.75–
4.95 (m, 3H; H-3,2’,4’), 5.01–5.22 (m, 4H; CH2Ph, H-4,3’), 7.29–7.41
(m, 5H; Ph); 13C NMR (100.6 MHz, CDCl3 at 318 K): d=20.7, 20.8,
21.0 (COCH3), 45.2 (C-2), 45.3 (C-6), 62.0 (C-5’), 68.0 (CH2Ph), 68.8 (C-
3,4’), 70.8 (C-2’), 71.1 (C-3’), 73.6 (C-4), 74.4 (C-5), 99.6 (C-1’), 128.2,
128.5, 128.8, 136.4 (Ph), 155.1 (NCO2), 169.3, 169.8, 169.9, 170.0
(COCH3). HRMS (ES): m/z 632.1964; [M+Na]+ requires 632.1955.
4-Nitrophenyl tri-O-acetyl-b-d-xylopyranosyl-(1!4)-2,3-di-O-acetyl-b-
1
d-xyloside (9): H and 13C NMR spectra obtained for this compound
were in agreement with the data previously reported.[37]
4-Nitrophenyl tri-O-acetyl-b-d-xylopyranosyl-(1!3)-2,4-di-O-acetyl-b-
d-xyloside (11): 1H and 13C NMR spectra obtained for this com-
pound were in agreement with those data previously reported.[20]
4-Nitrophenyl tri-O-acetyl-b-d-xylopyranosyl-(1!4)-2,3-di-O-acetyl-b-
d-xylosyl-(1!4)-2,3-di-O-acetyl-b-d-xyloside (10): 13C NMR spectra
obtained for this compound were in agreement with the data pre-
viously reported.[38]
Benzyl (3S,4S,5R)-3,4-diacetoxy-5-[tri-O-acetyl-b-d-xylopyranosyl-(1!
4)-di-O-acetyl-b-d-xylosyloxy]-piperidine-1-carboxylate (15): 1H NMR
(400 MHz, CDCl3 at 318 K): d=2.00, 2.018, 2.021, 2.03, 2.04, 2.05
(6s, 21H; COCH3), 2.89–3.09 (m, 2H; H-2,6), 3.30 (dd, J=8.4,
11.6 Hz, 1H; H-5’), 3.39 (dd, J=7.6, 12.0 Hz, 1H; H-5’’), 3.68 (ddd,
J=4.4, 8.8, 8.8 Hz, 1H; H-5), 3.80 (ddd, J=4.8, 8.4, 8.4 Hz, 1H; H-4’),
3.94 (dd, J=4.8, 11.6 Hz, 1H; H-5’), 4.02–4.20 (m, 3H; H-2,6,5’’),
4.51 (d, J=6.4 Hz, 1H; H-1’), 4.56 (d, J=5.6 Hz, 1H; H-1’’), 4.72–
4.84 (m, 3H; H-3,2’,2’’), 4.88 (ddd, J=4.4, 7.6, 7.6 Hz, 1H; H-4’’),
4.99–5.20 (m, 5H; CH2Ph, H-4,3’,3’’), 7.28–7.40 (m, 5H; Ph); 13C NMR
(100.6 MHz, CDCl3 at 318 K): d=20.7, 20.78, 20.84, 20.9, 21.0
(COCH3), 45.1 (C-2), 45.3 (C-6), 61.8 (C-5’’), 62.8 (C-5’), 68.0 (CH2Ph),
68.6 (C-3,4’’), 70.66, 70.74 (C-2’’,3’’), 71.3 (C-2’), 72.3 (C-3’), 73.5 (C-
4), 74.5 (C-4’), 74.8 (C-5), 99.6 (C-1’’), 100.3 (C-1’), 128.2, 128.5,
128.8, 136.3 (Ph), 155.1 (NCO2), 169.2, 169.5, 169.7, 169.91, 169.93,
170.0, 170.1 (COCH3). HRMS (ES): m/z 848.2582; [M+Na]+ requires
848.2589.
Glycosylation of phenyl 1-thio-b-d-xylopyranoside (2): The xylo-
side 2[24] was subjected to the glycosynthase reaction protocol
described above. The following products were obtained after flash
chromatography and are listed in order of elution (yields are pre-
sented in Table 1).
1
Phenyl 2,3,4-tri-O-acetyl-1-thio-b-d-xyloside: H and 13C NMR spectra
obtained for this compound were in agreement with the data pre-
viously reported.[39]
Phenyl tri-O-acetyl-b-d-xylopyranosyl-(1!4)-2,3-di-O-acetyl-1-thio-b-
1
d-xyloside (12): H NMR (400 MHz, CDCl3): d=2.01, 2.03, 2.04, 2.07
(4s, 15H; COCH3), 3.30–3.43 (m, 2H; H-5,5’), 3.81 (ddd, J=5.2, 9.2,
9.2 Hz, 1H; H-4), 4.02–4.16 (m, 2H; H-5,5’), 4.54 (d, J=6.0 Hz, 1H;
H-1’), 4.71 (d, J=9.2 Hz, 1H; H-1), 4.77 (dd, J=6.0, 7.6 Hz, 1H; H-
2’), 4.82–4.92 (m, 2H; H-2,4’), 5.03–5.18 (m, 2H; H-3,3’), 7.27–7.51
(m, 5H; Ph); 13C NMR (100.6 MHz, CDCl3): d=20.78, 20.81, 20.89,
20.93, 21.0 (COCH3), 61.7 (C-5’), 66.5 (C-5), 68.5 (C-4’), 70.3 (C-2),
70.5, 70.6 (C-2’,3’), 73.5 (C-3), 74.7 (C-4), 86.6 (C-1), 99.7 (C-1’),
128.4, 129.2, 132.3, 132.9 (Ph), 169.3, 169.7, 169.9, 170.0, 170.1
(COCH3). HRMS (ES): m/z 607.1451; [M+Na]+ requires 607.1461.
Benzyl (3S,4S,5R)-3,4-diacetoxy-5-[tri-O-acetyl-b-d-xylopyranosyl-(1!
4)-di-O-acetyl-b-d-xylosyl-(1!4)-di-O-acetyl-b-d-xylosyloxy]-piperi-
dine-1-carboxylate (16): 1H NMR (400 MHz, CDCl3 at 318 K): d=
1.99, 2.00, 2.01, 2.02, 2.026, 2.029, 2.04 (7s, 27H; COCH3), 2.88–3.07
(m, 2H; H-2,6), 3.22–3.42 (m, 3H; H-5’,5’’,5’’’), 3.67 (ddd, J=4.4, 8.8,
8.8 Hz, 1H; H-5), 3.71–3.84 (m, 2H; H-4’,4’’), 3.86–3.98 (m, 2H; H-
5’,5’’), 3.99–4.19 (m, 3H; H-2,6,5’’’), 4.47 (d, J=6.8 Hz, 1H; H-1’’),
4.50 (d, J=6.8 Hz, 1H; H-1’), 4.55 (d, J=6.0 Hz, 1H; H-1’’’), 4.68–
4.82 (m, 4H; H-3,2’,2’’,2’’’), 4.87 (ddd, J=4.8, 7.6, 7.6 Hz, 1H; H-4’’’),
4.97–5.19 (m, 6H; CH2Ph,H-4,3’,3’’,3’’’), 7.29–7.39 (m, 5H; Ph);
13C NMR (100.6 MHz, CDCl3 at 318 K): d=20.71, 20.73, 20.80, 20.84,
20.9, 21.0 (COCH3), 45.1 (C-2), 45.2 (C-6), 61.8 (C-5’’’), 62.6, 62.7 (C-
5’,5’’), 68.0 (CH2Ph), 68.6 (C-3,4’’’), 70.7, 70.8 (C-2’’’,3’’’), 71.1, 71.2 (C-
2’,2’’), 72.1, 72.2 (C-3’,3’’), 73.4 (C-4), 74.4, 74.9 (C-4’,4’’), 74.7 (C-5),
Phenyl tri-O-acetyl-b-d-xylopyranosyl-(1!4)-2,3-di-O-acetyl-b-d-xylo-
1
syl-(1!4)-2,3-di-O-acetyl-1-thio-b-d-xyloside (13): H NMR (400 MHz,
CDCl3): d=2.01, 2.02, 2.03, 2.04, 2.05, 2.07 (6s, 21H; COCH3), 3.26–
3.43 (m, 3H; H-5,5’,5’’), 3.72–3.84 (m, 2H; H-4,4’), 3.93 (dd, J=4.8,
12.0 Hz, 1H; H-5’), 4.03–4.15 (m, 2H; H-5,5’’), 4.47 (d, J=6.8 Hz, 1H;
H-1’), 4.55 (d, J=5.6 Hz, 1H; H-1’’), 4.69–4.76 (m, 2H; H-1,2’), 4.79
(dd, J=5.6, 6.0 Hz, 1H; H-2’’), 4.83–4.92 (m, 2H; H-2,4’’), 5.01–5.16
(m, 3H; H-3,3’,3’’), 7.28–7.50 (m, 5H; Ph); 13C NMR (100.6 MHz,
CDCl3): d=20.80, 20.82, 20.91, 20.93, 20.96, 21.00 (COCH3), 61.7 (C-
1708
www.chembiochem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1703 – 1711

Assembly of Xylanase Substrates and Inhibitors
99.7 (C-1’’’), 100.1, 100.3 (C-1’,1’’), 128.2, 128.4, 128.8, 136.3 (Ph),
155.1 (NCO2), 169.2, 169.4, 169.5, 169.7, 169.8, 169.87, 169.90
(COCH3). HRMS (ES): m/z 1064.3199; [M+Na]+ requires 1064.3223.
(Ar), 169.5, 169.9, 170.0, 170.1 (COCH3). HRMS (ES): m/z 641.1238;
[M+Na]+ requires 641.1242.
3,4-Dinitrophenyl 4-O-acetyl-2-deoxy-2-fluoro-3-[tri-O-acetyl-b-d-xylo-
pyranosyl-(1!4)-di-O-acetyl-b-d-xylosyl]-b-d-xyloside (20): 1H NMR
(400 MHz, CDCl3): d=2.04, 2.05, 2.07, 2.12 (4s, 18H; COCH3), 3.32–
3.45 (m, 2H; H-5’,5’’), 3.61 (dd, J=5.6, 12.4 Hz, 1H; H-5), 3.86 (ddd,
J=5.2, 8.8, 8.8 Hz, 1H; H-4’), 3.97–4.16 (m, 4H; H-3,5,5’,5’’), 4.44–
4.62 (m, 2H; H-2,1’’), 4.65 (d, J=7.2 Hz, 1H; H-1’), 4.80 (dd, J=6.0,
7.6 Hz, 1H; H-2’’), 4.86–5.04 (m, 3H; H-4,2’,4’’), 5.05–5.19 (m, 2H; H-
3’,3’’), 5.47 (dd, J=4.4, 8.8 Hz, 1H; H-1), 7.34 (dd, J=2.4, 9.2 Hz,
1H; ArH), 7.49 (d, J=2.4 Hz, 1H; ArH), 8.23 (d, J=9.2 Hz, 1H; ArH);
13C NMR (100.6 MHz, CDCl3): d=20.84, 20.85, 20.87, 20.95, 20.99,
21.03 (COCH3), 61.4 (C-5), 61.7 (C-5’’), 63.2 (C-5’), 68.1 (d, J=4.0 Hz,
C-4), 68.4 (C-4’’), 70.49, 70.51 (C-2’’,3’’), 71.3 (C-2’), 72.2 (C-3’), 74.6
(C-4’), 76.3 (d, J=23.1 Hz, C-3), 87.8 (d, J=184.2 Hz, C-2), 97.0 (d,
J=31.2 Hz, C-1), 99.6 (C-1’’), 102.5 (C-1’), 113.1, 119.8, 127.5, 136.7,
145.3, 159.8 (Ar), 169.4, 169.8, 169.98, 170.01, 170.04, 170.1
(COCH3). HRMS (ES): m/z 857.1869; [M+Na]+ requires 857.1876.
Glycosylation of benzyl (3R,4R)-3,4-dihydroxy-piperidine-1-car-
boxylate (5): The carbamate 5 was subjected to the glycosynthase
reaction protocol described above. The following products were
obtained after flash chromatography and are listed in order of elu-
tion (yields are presented in Table 1).
Benzyl (3R,4R)-3,4-diacetoxy-piperidine-1-carboxylate: 1H and 13C
NMR spectra obtained for this compound were in agreement with
the data previously reported.[40]
Benzyl (3R,4R)-4-acetoxy-3-(tri-O-acetyl-b-d-xylopyranosyloxy)-piperi-
dine-1-carboxylate (17): 1H NMR (400 MHz, CDCl3 at 318 K): d=
1.51–1.63 (brm, 1H; H-5), 1.88–2.12 (brm, 13H; COCH3, H-5), 3.20–
3.79 (brm, 5H; H-2,2,3,6,5’), 3.81–3.90 (brm, 1H; H-6), 4.04–4.18
(brm, 1H; H-5’), 4.58–4.69 (brm, 1H; H-1’), 4.78–5.01 (brm, 3H; H-
4,2’,4’), 5.05–5.23 (brm, 3H; CH2Ph, H-3’), 7.24–7.43 (brm, 5H; Ph).
The 13C NMR (100.6 MHz, CDCl3 at 318 K) was so broad as to be
uninterpretable. HRMS (ES): m/z 574.1913; [M+Na]+ requires
574.1900.
Deprotection of 9–13: The 4-nitrophenyl glycosides 9–11 and
phenyl thioglycosides 12–13, were subjected to a base-catalysed
transesterification. Sodium methoxide in MeOH (0.1m) was added
drop-wise to a stirred solution (or suspension) of the per-O-acetate
in anhydrous MeOH (0.5 mm) until the solution remained basic.
After completion of the reaction (as determined by TLC), the solu-
tion was neutralised with cation-exchange resin (Amberlite IR-120,
H+ form), filtered and concentrated. The residue was recrystallised
or subjected to chromatography, as described below or in accord-
Benzyl (3R,4R)-4-acetoxy-3-[tri-O-acetyl-b-d-xylopyranosyl-(1!4)-di-
O-acetyl-b-d-xylosyloxy]-piperidine-1-carboxylate
(18):
1H NMR
(400 MHz, CDCl3 at 318 K): d=1.48–1.64 (brm, 1H; H-5), 1.90–2.10
(brm, 19H; COCH3, H-5), 3.10–3.73 (brm, 7H; H-2,2,3,6,6,5’,5’’),
3.73–3.87 (brm, 1H; H-4’), 3.88–4.02 (brm, 1H; H-5’), 4.02–4.16
(brm, 1H; H-5’’), 4.49–4.62 (brm, 2H; H-1’,1’’), 4.72–4.96 (brm, 4H;
H-4,2’,2’’,4’’), 5.00–5.20 (brm, 4H; CH2Ph, H-3’,3’’), 7.28–7.40 (brm,
5H; Ph). The 13C NMR (100.6 MHz, CDCl3 at 318 K) was so broad as
to be uninterpretable. HRMS (ES): m/z 790.2547; [M+Na]+ requires
790.2534.
1
ance with literature procedures. H and 13C NMR spectra obtained
for products 21–23 were in agreement with the data previously re-
ported.[37,41,42]
Phenyl b-d-xylopyranosyl-(1!4)-1-thio-b-d-xyloside (24): This com-
pound was eluted under gravity on a Waters tC18 SepPak cartridge
(2 g) with a H2O/MeOH gradient (1:0–1:4) and was lyophilised to
give a colourless foam (21 mg, 88% yield). 1H NMR (400 MHz,
CDCl3): d=3.21–3.45 (m, 5H; H-2,5,2’,3’,5’), 3.53–3.75 (m, 3H; H-
3,4,4’), 3.94 (dd, J=5.6, 11.6 Hz, 1H; H-5’), 4.07 (dd, J=5.2, 11.6 Hz,
1H; H-5), 4.37 (d, J=8.0 Hz, 1H; H-1’), 4.70 (d, J=10.0 Hz, 1H; H-1),
7.28–7.56 (m, 5H; Ph); 13C NMR (100.6 MHz, CDCl3): d=65.3 (C-5’),
66.6 (C-5), 69.3 (C-4’), 71.7 (C-2), 72.8 (C-2’), 75.3 (C-3), 75.7 (C-3’),
76.1 (C-4), 88.0 (C-1), 101.8 (C-1’), 128.4, 129.5, 131.6, 132.3 (Ph).
HRMS (ES): m/z 397.0937; [M+Na]+ requires 397.0933.
Glycosylation of 3,4-dinitrophenyl 2-deoxy-2-fluoro-b-d-xylopyr-
anoside (3): The xyloside 3 was subjected to the glycosynthase re-
action protocol described above. The following products were ob-
tained after flash chromatography and are listed in order of elution
(yields are presented in Table 1).
3,4-Dinitrophenyl
3,4-di-O-acetyl-2-deoxy-2-fluoro-b-d-xyloside:
1H NMR (400 MHz, CDCl3): d=2.11, 2.17 (2s, 6H; COCH3), 3.68 (dd,
J=6.8, 12.4 Hz, 1H; H-5), 4.18 (dd, J=4.0, 12.4 Hz, 1H; H-5), 4.64
(ddd, J=5.2, 6.8, 47.6 Hz, 1H; H-2), 4.98 (ddd, J=4.0, 6.8, 6.8 Hz,
1H; H-4), 5.37 (ddd, J=6.8, 6.8, 12.8 Hz, 1H; H-3), 5.46 (dd, J=5.2,
7.2 Hz, 1H; H-1), 7.33 (dd, J=2.4, 8.8 Hz, 1H; ArH), 7.44 (d, J=
2.4 Hz, 1H; ArH), 8.04 (d, J=8.8 Hz, 1H; ArH); 13C NMR (100.6 MHz,
CDCl3): d=20.89, 20.91 (COCH3), 62.1 (C-5), 67.6 (d, J=4.0 Hz, C-4),
69.4 (d, J=23.1 Hz, C-3), 87.0 (d, J=188.2 Hz, C-2), 97.8 (d, J=
29.2 Hz, C-1), 113.2, 119.9, 127.6, 136.9, 145.3, 159.8 (Ar), 169.6,
170.1 (COCH3). HRMS (ES): m/z 425.0615; [M+Na]+ requires
425.0608.
Phenyl b-d-xylopyranosyl-(1!4)-b-d-xylosyl-(1!4)-1-thio-b-d-xylo-
side (25): This compound was eluted under gravity on a Waters
tC18 SepPakꢃ cartridge (2 g) with a H2O/MeOH gradient (1:0–1:4)
and lyophilised to give a colourless foam (32 mg, 91% yield).
1H NMR (400 MHz, CDCl3): d=3.22–3.47 (m, 7H; H-2,5,2’,5’,2’’3’’,5’’),
3.51–3.83 (m, 5H; H-3,4,3’,4’,4’’), 3.97 (dd, J=5.6, 11.6 Hz, 1H; H-
5’’), 4.06–4.17 (m, 2H; H-5,5’), 4.42–4.49 (m, 2H; H-1’,1’’), 4.77 (d,
J=9.9 Hz, 1H; H-1), 7.38–7.60 (m, 5H; Ph); 13C NMR (100.6 MHz,
CDCl3): d=63.1, 65.3 (C-5’,5’’), 66.6 (C-5), 69.3 (C-4’’), 71.8 (C-2),
72.8, 72.9 (C-2’,2’’), 73.8, 75.3, 75.7 (C-3,3’,3’’), 76.1 (C-4), 76.5 (C-4’),
88.0 (C-1), 101.7, 101.9 (C-1’,1’’), 128.5, 129.5, 131.5, 132.2 (Ph).
HRMS (ES): m/z 529.1352; [M+Na]+ requires 529.1356.
3,4-Dinitrophenyl 4-O-acetyl-2-deoxy-2-fluoro-3-(tri-O-acetyl-b-d-xylo-
pyranosyl)-b-d-xyloside (19): 1H NMR (400 MHz, CDCl3): d=2.02,
2.03, 2.05, 2.09 (4s, 12H; COCH3), 3.41 (dd, J=8.4, 12.0 Hz, 1H; H-
5’), 3.58 (dd, J=6.4, 12.4 Hz, 1H; H-5), 4.02–4.17 (m, 3H; H-3,5,5’),
4.54 (ddd, J=4.8, 4.9, 47.6 Hz, 1H; H-2), 4.72 (d, J=6.8 Hz, 1H; H-
1’), 4.87–5.02 (m, 3H; H-4,2’,4’), 5.16 (dd, J=8.4, 8.4 Hz, 1H; H-3’),
5.42 (dd, J=4.9, 8.0 Hz, 1H; H-1), 7.33 (dd, J=2.6, 9.2 Hz, 1H; ArH),
7.46 (d, J=2.6 Hz, 1H; ArH), 8.00 (d, J=9.2 Hz, 1H; ArH); 13C NMR
(100.6 MHz, CDCl3): d=20.77, 20.80, 20.82, 20.9 (COCH3), 61.7 (C-5),
62.2 (C-5’), 68.1 (d, J=5.0 Hz, C-4), 68.8 (C-4’), 70.7 (C-2’), 71.0 (C-
3’), 76.5 (d, J=21.1 Hz, C-3), 88.4 (d, J=185.2 Hz, C-2), 97.3 (d, J=
30.2 Hz, C-1), 101.8 (C-1’), 113.0, 119.9, 127.5, 136.6, 145.2, 159.8
Deprotection of 14–15 and 17–18: The carbamates 14–15 and
17–18 were subjected to a base-catalysed transesterification, fol-
lowed by hydrogenolysis. Sodium methoxide in MeOH (0.1m) was
added drop-wise to a stirred solution of the per-O-acetate in anhy-
drous MeOH (0.5 mm) until the solution remained basic. After com-
pletion of the reaction (as determined by TLC) the mixture was
neutralised with cation-exchange resin (Amberlite IR-120, H+ form),
filtered and concentrated. A mixture of Pd/C (10%, 15 mg) and the
ChemBioChem 2011, 12, 1703 – 1711
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1709

S. G. Withers et al.
residue in anhydrous MeOH (5 mL) was stirred under H2 (1 atm,
4 h). The mixture was filtered, concentrated, and the residue was
purified by ion-exchange chromatography (BioRad AG 50Wꢂ2, H+
form, eluted with H2O then 1m NH3). Fractions containing product
Acknowledgements
Monique Oberlander is thanked for technical assistance. E.D.G.-B.
acknowledges the CIHR for a postdoctoral fellowship.
1
were combined and lyophilised. H and 13C NMR spectra obtained
for products 26 and 28 were in agreement with the data previous-
ly reported.[14]
Keywords: carbohydrates · enzymatic synthesis · enzyme
catalysis · enzyme inhibitors · glycosides
(3R,4R,5S)-4,5-Dihydroxy-3-[b-d-xylopyranosyl-(1!4)-b-d-xylosyloxy]-
piperidine (27): The product was obtained as a colourless foam
(32 mg, 77% yield). H NMR (400 MHz, D2O): d=2.40–2.55 (m, 2H;
H-2,6), 3.06–3.69 (m, 12H; H-2,3,5,6,2’,3’,4’,5’,2’’,3’’,4’’,5’’), 3.73–3.84
(m, 1H; H-4), 3.93–4.15 (m, 2H; H-5’,5’’), 4.42–4.53 (m, 2H; H-1’,1’’);
13C NMR (100.6 MHz, D2O): d=47.6, 49.9 (C-2,6), 64.0, 66.3 (C-5’,5’’),
70.3, 71.7, 73.8, 73.9, 74.8, 76.7, 76.9, 77.5, 79.0 (C-
3,4,5,2’,3’,4’,2’’,3’’,4’’), 102.4, 102.9 (C-1’,1’’). HRMS (ES): m/z
398.1672; [M+H]+ requires 398.1662.
1
[1] D. Shallom, Y. Shoham, Curr. Opin. Microbiol. 2003, 6, 219–228.
[2] Q. K. Beg, M. Kapoor, L. Mahajan, G. S. Hoondal, Appl. Microbiol. Biotech-
nol. 2001, 56, 326–338.
[3] I. M. Mackie, E. Percival, J. Chem. Soc. 1959, 1151–1158; Y. Iriki, T.
Suzuki, K. Nisizawa, T. Miwa, Nature 1960, 187, 82–83.
[4] E. Percival, R. H. McDowell, Chemistry and Enzymology of Marine Algal
Polysaccharides, Academic Press, London, 1967, pp. 88–96, 134–137.
[5] T. Collins, C. Gerday, G. Feller, FEMS Microbiol. Rev. 2005, 29, 3–23.
[6] B. L. Cantarel, P. M. Coutinho, C. Rancurel, T. Bernard, V. Lombard, B.
Henrissat, Nucleic Acids Res. 2009, 37, D233–238.
[7] T. Collins, M. A. Meuwis, I. Stals, M. Claeyssens, G. Feller, C. Gerday, J.
Biol. Chem. 2002, 277, 35133–35139.
[8] H. Kersters-Hilderson, M. Claeyssens, E. van Doorslaer, C. K. de Bruyne,
Carbohydr. Res. 1976, 47, 269–273.
[9] S. G. Withers, D. Dombroski, L. A. Berven, D. G. Kilburn, R. C. Miller, Jr.,
R. A. Warren, N. R. Gilkes, Biochem. Biophys. Res. Commun. 1986, 139,
487–494.
[10] J. Gebler, N. R. Gilkes, M. Claeyssens, D. B. Wilson, P. Bꢁguin, W. W. Wa-
karchuk, D. G. Kilburn, R. C. Miller, Jr., R. A. Warren, S. G. Withers, J. Biol.
Chem. 1992, 267, 12559–12561.
[11] D. N. Bolam, N. Hughes, R. Virden, J. H. Lakey, G. P. Hazlewood, B. Henris-
sat, K. L. Braithwaite, H. J. Gilbert, Biochemistry 1996, 35, 16195–16204.
[12] Some examples: D. Tull, S. G. Withers, N. R. Gilkes, D. G. Kilburn, R. A.
Warren, R. Aebersold, J. Biol. Chem. 1991, 266, 15621–15625; A. M. Ma-
cLeod, T. Lindhorst, S. G. Withers, R. A. Warren, Biochemistry 1994, 33,
6371–6376; S. Miao, L. Ziser, R. Aebersold, S. G. Withers, Biochemistry
1994, 33, 7027–7032; L. P. McIntosh, G. Hand, P. E. Johnson, M. D. Joshi,
M. Korner, L. A. Plesniak, L. Ziser, W. W. Wakarchuk, S. G. Withers, Bio-
chemistry 1996, 35, 9958–9966; M. D. Joshi, G. Sidhu, J. E. Nielsen, G. D.
Brayer, S. G. Withers, L. P. McIntosh, Biochemistry 2001, 40, 10115–
10139; M. L. Ludwiczek, M. Heller, T. Kantner, L. P. McIntosh, J. Mol. Biol.
2007, 373, 337–354; J. Wicki, J. Schloegl, C. A. Tarling, S. G. Withers, Bio-
chemistry 2007, 46, 6996–7005.
[13] L. Ziser, I. Setyawati, S. G. Withers, Carbohydr. Res. 1995, 274, 137–153.
[14] S. J. Williams, R. Hoos, S. G. Withers, J. Am. Chem. Soc. 2000, 122, 2223–
2235.
[15] S. J. Williams, O. Hekmat, S. G. Withers, ChemBioChem 2006, 7, 116–124.
[16] Some examples: V. Notenboom, C. Birsan, R. A. J. Warren, S. G. Withers,
D. R. Rose, Biochemistry 2000, 39, 11553–11563; G. P. Connelly, S. G.
Withers, L. P. McIntosh, Protein Sci. 2000, 9, 512–524; O. Hekmat, Y.-W.
Kim, S. J. Williams, S. He, S. G. Withers, J. Biol. Chem. 2005, 280, 35126–
35135.
(3R,4R)-4-Hydroxy-3-[b-d-xylopyranosyl-(1!4)-b-d-xylosyloxy]-piperi-
dine (29): Product was obtained as a colourless foam (26 mg, 72%
yield). 1H NMR (400 MHz, D2O): d=1.39–1.59 (m, 1H; H-5), 1.94–
2.07 (m, 1H; H-5), 2.50–2.70 (m, 2H; H-2,6), 2.92–3.81 (m, 12H; H-
2,3,4,6,2’,3’,4’,5’,2’’,3’’,4’’,5’’), 3.89–4.11 (m, 2H; H-5’,5’’), 4.39–4.56
(m, 2H; H-1’,1’’); 13C NMR (100.6 MHz, D2O): d=31.7 (C-5), 43.0,
47.0 (C-2,6), 64.1, 66.3 (C-5’,5’’), 70.3, 70.4, 73.87, 73.90, 74.8, 76.7,
76.9, 77.5, 79.4 (C-3,4,5,2’,3’,4’,2’’,3’’,4’’), 102.6, 103.0 (C-1’,1’’). HRMS
(ES): m/z 382.1720; [M+H]+ requires 382.1713.
Deprotection of 19–20: The dinitrophenyl glycosides 19–20 were
all subjected to an acid-catalysed transesterification. Acetyl chloride
(0.10 mL) was added drop-wise to a stirred suspension of the per-
O-acetate in anhydrous MeOH (3 mL) at 08C. The mixture was
warmed to room temperature and left for 24 h. The solution was
concentrated and the residue recrystallized or subjected to flash
chromatography, as described below.
3,4-Dinitrophenyl b-d-xylopyranosyl-(1!3)-2-deoxy-2-fluoro-b-d-xylo-
pyranoside (30): This was recrystallised from MeOH/Et2O to give
pale yellow needles (47 mg, 83% yield). 1H NMR (400 MHz,
CD3OD): d=3.20–3.39 (m, 3H; H-2’,4’,5’), 3.48–3.61 (m, 2H; H-5,3’),
3.69–3.78 (m, 1H; H-4), 3.89–4.07 (m, 3H; H-3,5,5’), 4.52 (d, J=
7.6 Hz, 1H; H-1’), 4.61 (ddd, J=6.8, 8.0, 50.0 Hz, 1H; H-2), 5.51 (dd,
J=4.8, 6.8 Hz, 1H; H-1), 7.46 (dd, J=2.4, 8.8 Hz, 1H; ArH), 7.46 (d,
J=2.4 Hz, 1H; ArH), 8.15 (d, J=8.8 Hz, 1H; ArH); 13C NMR
(100.6 MHz, CD3OD): d=66.6 (C-5), 67.1 (C-5’), 68.9 (d, J=7.0 Hz,
C-4), 71.2 (C-3’), 75.0 (C-4’), 77.7 (C-2’), 82.3 (d, J=17.1 Hz, C-3),
92.3 (d, J=186.2 Hz, C-2), 99.5 (d, J=19.1 Hz, C-1), 105.0 (C-1’),
114.2, 121.0, 128.8, 137.6, 146.6, 161.9 (Ar). HRMS (ES): m/z
473.0824; [M+Na]+ requires 473.0820.
[17] L. F. Mackenzie, Q. Wang, R. A. J. Warren, S. G. Withers, J. Am. Chem. Soc.
1998, 120, 5583–5584.
3,4-Dinitrophenyl b-d-xylopyranosyl-(1!4)-b-d-xylosyl-(1!3)-2-de-
oxy-2-fluoro-b-d-xylopyranoside (31): Flash chromatography (EtOAc/
MeOH; 9:1–8:2) gave a pale yellow foam (20 mg, 76% yield).
1H NMR (400 MHz, D2O): d=3.23–4.22 (m, 14H; H-
3,4,5,5,2’,3’,4’,5’,5’,2’’,3’’,4’’,5’’,5’’), 4.41–4.74 (m, 3H; H-2,1’,2’), 5.20–
5.38 (m, 1H; H-1’), 7.10–7.42 (m, 2H; ArH), 7.79–7.92 (m, 1H; ArH);
13C NMR (100.6 MHz, D2O): d=64.1, 66.1, 66.4, 68.0, 70.3, 73.8,
74.1, 74.8, 76.7, 77.5 (C-4,5,2’,3’,4’,5’,2’’,3’’,4’’,5’’), 81.7 (d, J=1.5 Hz,
C-3), 91.9 (d, J=186.2 Hz, C-2), 98.8 (d, J=2.3 Hz, C-1), 103.9, 102.9
(C-1’,1’’), 114.0, 120.6, 128.8, 136.5, 145.2, 160.9 (Ar). HRMS (ES): m/z
605.1230; [M+Na]+ requires 605.1242.
[18] C. Malet, A. Planas, FEBS Lett. 1998, 440, 208–212.
[19] G. Perugino, A. Trincone, M. Rossi, M. Moracci, Trends Biotechnol. 2004,
22, 31–37; S. M. Hancock, M. D. Vaughan, S. G. Withers, Curr. Opin.
Chem. Biol. 2006, 10, 509–519.
[20] Y.-W. Kim, H. Chen, S. G. Withers, Carbohydr. Res. 2005, 340, 2735–2741.
[21] A. Ben-David, T. Bravman, Y. S. Balazs, M. Czjzek, D. Schomburg, G.
Shoham, Y. Shoham, ChemBioChem 2007, 8, 2145–2151.
[22] T. Bravman, G. Zolotnitsky, V. Belakhov, G. Shoham, B. Henrissat, T.
Baasov, Y. Shoham, Biochemistry 2003, 42, 10528–10536.
[23] F. G. Loontiens, C. K. de Bruyne, Naturwissenschaften 1964, 51, 359.
[24] R. Lꢄpez, A. Fernꢅdez-Mayoralas, J. Org. Chem. 1994, 59, 737–745.
[25] J. M. MacDonald, R. V. Stick, D. M. G. Tilbrook, S. G. Withers, Aust. J.
Chem. 2002, 55, 747–752.
[26] R. C. Anderson, B. Fraser-Reid, J. Org. Chem. 1985, 50, 4781–4786.
[27] S. Torii, T. Inokuchi, R. Oi, K. Kondo, T. Kobayashi, J. Org. Chem. 1986, 51,
254–256.
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1703 – 1711

Assembly of Xylanase Substrates and Inhibitors
[28] A. White, D. Tull, K. Johns, S. G. Withers, D. R. Rose, Nat. Struct. Biol.
1996, 3, 149–154.
[29] G. Sidhu, S. G. Withers, N. T. Nguyen, L. P. McIntosh, L. Ziser, G. D. Brayer,
Biochemistry 1999, 38, 5346–5354.
[36] J. M. Kim, R. Roy, J. Carbohydr. Chem. 1997, 16, 1281–1292.
[37] L. Ziser, S. G. Withers, Carbohydr. Res. 1994, 265, 9–17.
[38] K. Takeo, Y. Ohguchi, R. Hasegawa, S. Kitamura, Carbohydr. Res. 1995,
277, 231–244.
[30] J. Wicki, S. J. Williams, S. G. Withers, J. Am. Chem. Soc. 2007, 129, 4530–
4531.
[31] M. Kiyohara, K. Sakaguchi, K. Yamaguchi, T. Araki, T. Nakamura, M. Ito,
Biochem. J. 2005, 388, 949–957.
[32] M. Kiyohara, Y. Hama, K. Yamaguchi, M. Ito, J. Biochem. 2006, 140, 369–
373.
[33] W. L. Sung, C. K. Luk, D. M. Zahab, W. Wakarchuk, Protein Expression
Purif. 1993, 4, 200–206.
[39] S.-S. Weng, Y.-D. Lin, C.-T. Chen, Org. Lett. 2006, 8, 5633–5636.
[40] L. F. Solares, I. Lavandera, V. Gotor-Fernꢅndez, R. Brieva, V. Gotor, Tetra-
hedron 2006, 62, 3284–3291.
[41] C. Rꢁmond, R. Plantier-Royon, N. Aubry, E. Maes, C. Bliard, M. J. O’Do-
nohue, Carbohydr. Res. 2004, 339, 2019–2025.
[42] E. V. Eneyskaya, H. Brumer III, L. V. Backinowsky, D. R. Ivanen, A. A. Kul-
minskaya, K. A. Shabalin, K. N. Neustroev, Carbohydr. Res. 2003, 338,
313–325.
[34] N. R. Gilkes, M. L. Langford, D. G. Kilburn, R. C. Miller, R. A. J. Warren, J.
Biol. Chem. 1984, 259, 10455–10459.
Received: April 6, 2011
[35] F. Micheel, A. Klemer, M. Nolte, H. Noordiek, L. Tork, H. Westermann,
Chem. Ber. 1957, 90, 1612.
Published online on June 27, 2011
ChemBioChem 2011, 12, 1703 – 1711
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
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