Synthesis with glycosynthases: Cello-oligomers of isofagomine and a tetrahydrooxazine as cellulase inhibitors

Article abstract of DOI:10.1071/CH02165

Isofagomine and a carbohydrate-like tetrahydrooxazine, as their N-benzyloxycarbonyl derivatives, have been subjected to a glycosynthase in the presence of α-D-glucopyranosyl fluoride as a glucosyl donor. In each case, after protecting group removal, a mixture of 1,4-β-linked di-, tri-, and tetra-'saccharides' was obtained. These novel oligosaccharide derivatives were tested as inhibitors of the endo-glycanase Cex from Cellulomonas fimi. Affinities increased progressively as additional D-glucosyl residues were incorporated, which is consistent with the known substrate specificity of this enzyme.

Full text of DOI:10.1071/CH02165

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Aust. J. Chem. 2002, 55, 747–752
Synthesis with Glycosynthases: Cello-Oligomers of Isofagomine and a
Tetrahydrooxazine as Cellulase Inhibitors
James M. Macdonald,A Robert V. Stick,A,B D. Matthew G. TilbrookA and Stephen G. WithersB,C
A School of Biomedical and Chemical Sciences, Faculty of Life and Physical Sciences, The University of Western
Australia, Crawley W.A. 6009, Australia.
B Authors to whom correspondence should be addressed (e-mail: rvs@chem.uwa.edu.au; withers@chem.ubc.ca).
C Protein Engineering Network of Centres of Excellence and Department of Chemistry, University of British
Columbia, Vancouver BC V6T 1Z1, Canada.
Isofagomine and a carbohydrate-like tetrahydrooxazine, as their N-benzyloxycarbonyl derivatives, have been
subjected to a glycosynthase in the presence of α-D-glucopyranosyl fluoride as a glucosyl donor. In each case, after
protecting group removal, a mixture of 1,4-β-linked di-, tri-, and tetra-‘saccharides’ was obtained. These novel
oligosaccharide derivatives were tested as inhibitors of the endo-glycanase Cex from Cellulomonas fimi. Affinities
increased progressively as additional D-glucosyl residues were incorporated, which is consistent with the known
substrate specificity of this enzyme.
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Manuscript received: 26 August 2002.
Final version: 17 October 2002.
The detailed understanding of the mechanism of action of
retaining glycoside hydrolases,[1–3] coupled with the ability
to identify[4,5] and subsequently mutate[6] the catalytic
nucleophile, greatly contributed to the invention of
‘glycosynthases’.[7] Such mutant enzymes, in which the
nucleophilic carboxylate has been replaced by a smaller,
non-carboxylate residue, are able to glycosylate an acceptor
alcohol using a glycosyl fluoride donor of the ‘wrong’
configuration, but are unable to cause hydrolysis of the
product glycoside. Since the initial discovery, a number of
other glycosynthases have been developed, of which most are
specific for the construction of 1,4-β-D-glycosides.[8–13]
We have recently reported syntheses of isofagomine (1)
and the tetrahydrooxazine (2)[14] (Diagram 1). Isofagomine,
an unnatural molecule,[15] is a potent inhibitor of several
glycoside hydrolases; the tetrahydrooxazine (2), while not
nearly as potent as isofagomine, is also a reasonable
inhibitor.[16]
it would be possible to use conventional glycosidation
techniques to synthesize such molecules, we decided to use
enzyme-assisted procedures. Glycosynthases seemed like
ideal candidates.
As with other iminosugars, isofagomine is generally
accepted to bind to glycosidases in its protonated form.
Therefore, in order to minimize the potential problem of
inhibition of the glycosynthase through unwanted binding of
the isofagomine moiety in the –1 site, we decided to
derivatize it in a form that is not protonated under
physiological conditions: as an N-acyl derivative. Further, it
is well established that aryl glycosides are particularly good
acceptors in enzyme-assisted glycoside bond formation as
the aromatic ring seems to provide good (hydrophobic)
binding in the +2 site of the catalytic domain.[17,18]
We first converted (1) and (2) into the corresponding
carbamates (3) and (4) to satisfy both needs. The carbamate
(3) and α-D-glucopyranosyl fluoride in aqueous ammonium
bicarbonate solution (pH 7.9) were then incubated with a
second-generation glycosynthase developed from
a
OH
OH
O
β-glucosidase from Agrobacterium sp. (Glu358Ser).[11]
After a few days, thin-layer chromatography (TLC) analysis
showed the formation of three new compounds, ostensibly
di-, tri-, and tetra-‘saccharides’ of carbamate (3).
HO
HO
HO
HO
NR
NR
(1) R = H
(3) COOBn
(2) R = H
(4) COOBn
After workup and acetylation of the reaction mixture,
careful chromatography on silica gel provided the expected
series of compounds (5)–(7) (Diagram 2). The complexity of
the nuclear magnetic resonance (NMR) spectra of these
compounds, arising presumably from the partial
double-bond character within the carbamate functionality,
Diagram 1
It occurred to us that it might be worthwhile to produce
glycosylated versions of both (1) and (2) in the hope of
improving the binding to target glycan hydrolases. Although
© CSIRO 2002
10.1071/CH02165
0004-9425/02/120747

748
J. M. Macdonald et al.
frustrated the acquisition of convincing evidence concerning
the 1,4-β-D-linkage that would be expected on the basis of
previous observations with this enzyme.[7,11] Each of the
products (5)–(7) was therefore selectively deprotected
(transfer hydrogenolysis) to provide the acetylated amines
In these glycosylations, mixtures of oligosaccharides of
different lengths were obtained. Such were our intentions,
since the full series of inhibitors was required for the
inhibition studies, and it was anticipated that
chromatographic separation of these products would not be
too problematic. However, should single products of defined
lengths be desired it should be possible to adopt the strategy
employed previously on another endo-glycanase in which a
temporary protecting group is attached to the C4 hydroxyl of
the non-reducing sugar of the glycosyl fluoride donor.[12,20]
Inhibition studies were carried out with a well-analysed
endo-glycanase (Cex from Cellulomonas fimi) for which
many kinetic studies have been performed, and for which a
number of three-dimensional structures have been
determined, both of the pure enzyme and of complexes with
various inhibitors.[21,22] The majority of these inhibitor
studies to date have been with xylo-oligosaccharides, thus
comparative studies with cello-oligosaccharide derivatives
would be of interest.
As can be seen in Figure 1, which depicts inhibition by the
trisaccharide analogue cellobiosyl isofagomine (12), these
compounds act as good competitive inhibitors, in this case
with a Ki of 0.8 µM. Inhibition parameters for the two series
of inhibitors with the endo-glycanase Cex from
Cellulomonas fimi are presented in Table 1. These data
reveal some interesting changes in Ki values across the
series. The monosaccharide analogue, isofagomine (1) is a
very modest (millimolar) inhibitor, but addition of a single
β-glucosyl residue to the 4-position improves binding
1000-fold. However, further additions of β-glucosyl moieties
provides very little additional binding enhancement, with Ki
values dropping only approximately two-fold for the next
sugar residue cellobiosyl isofagomine (12), and a further
1.5-fold for the third addition (13). These increases in
affinity correlate with kinetic parameters for the cleavage of
1
(8)–(10). A combination of H and 13C NMR spectroscopy
then established the various 1,4-β-D-linkages in (8)–(10). For
1
example, two-dimensional H NMR spectroscopy of the
amine (8) clearly identified H4 as an apparent triplet with an
expected greater upfield chemical shift (δ 3.62) relative to all
other protons directly adjacent to acetoxy groups.
Furthermore, heteronuclear single quantum correlation
(HSQC) NMR spectroscopy showed H4 to be coupled to
downfield C4 (δ 79.1). Lastly, the J1′,2′ coupling constant
(7.9 Hz) was indicative of a β-D-glycosidic linkage. The
amines (8)–(10) were then separately deprotected (sodium
methoxide in methanol) to give the putative inhibitors
(11)–(13). 13C NMR spectroscopy of these fully deprotected
products showed chemical shifts [δ 79.1 for C4′ of (12) and
δ 79.0, 79.1 for C4′,4′′ of (13)] characteristic for
1,4-β-D-glycosylation.[19]
OR
O
OR
OR
4
RO
RO
RO
O
1'
O
RO
O
NR'
OR
OR
n
(5) R =
R' = COOBn
COOBn
n =
0
Ac
Ac
Ac
1
2
(6)
(7)
COOBn
(8)
0
1
2
Ac
Ac
Ac
H
H
H
(9)
(10)
(11)
(12)
(13)
0
1
2
H
H
H
H
H
H
oligosaccharide
substrates.
Thus,
p-nitrophenyl
Diagram 2
β-D-glucopyranoside is only a slow substrate (kcat/Km
=
0.0041 s–1 mM–1) compared with p-nitrophenyl cellobioside
The whole process was then repeated on carbamate (4) to
provide the oxazine suite of intermediates (14)–(16)
(Diagram 3). No line broadening was observed for these
compounds, possibly owing to the reduced basicity of the
nitrogen atom, and sound NMR spectral analysis was
possible. Therefore, one-pot deprotection by way of basic
hydrolysis (sodium hydroxide in aqueous methanol) gave the
putative inhibitors (17)–(19).
OR
O
OR
O
OR
5
RO
RO
RO
O
1'
O
RO
O
NR'
OR
OR
n
(14) R =
R' = COOBn
COOBn
n = 0
Ac
Ac
Ac
–
(15)
(16)
(17)
(18)
(19)
1
2
0
1
2
COOBn
12
H
H
H
H
H
H
Fig. 1. Dixon plot of inhibition of C. fimi endo-glycanase by the
cellobiosyl isofagomine (12). The concentrations of the substrate,
2,4-dinitrophenyl β-cellobioside, used were 0.059 (᭢), 0.113 (᭜),
0.117 (᭺), and 0.234 (᭝) mM.
Diagram 3

Synthesis with Glycosynthases
749
Table 1. Inhibition of the endo-glycanase Cex from
Cellulomonas fimi
Experimental
Syntheses
Inhibitor
Isofagomine (1)
Ki (µM)
General experimental procedures for syntheses have been given
previously.[25]
2000
Glucosyl Isofagomine (11)
Cellobiosyl Isofagomine (12)
Cellotriosyl Isofagomine (13)
Oxazine (2)
Glucosyl Oxazine (17)
Cellobiosyl Oxazine (18)
Cellotriosyl Oxazine (19)
2.0
0.8
0.6
(3R,4R,5R)-N-Benzyloxycarbonyl-3,4-dihydroxy-5-(hydroxymethyl)-
piperidine (3)
No Inhibition
1900
Isofagomine (1) (hydrogen chloride salt)[14] (156 mg, 0.85 mmol) was
treated with NaHCO3 (210 mg, 2.5 mmol) and benzyl chloroformate
(160 µL, 1.1 mmol) in H2O/MeOH/THF (2:1:1, 15 mL), and the
mixture was stirred (30 min, rt). The mixture was treated with
hydrochloric acid (1 mL of 1 M) and then concentrated, co-evaporating
with toluene. Flash chromatography (EtOAc/petrol, 1:1 then
MeOH/CHCl3, 1:9) of the residue gave the carbamate (3) (192 mg,
80%) as a colourless oil, [a]D +3.8° (MeOH) (Found: C, 59.7; H, 7.1%.
1000
80
(kcat/Km = 26 s–1 mM–1).[23] Indeed, Cex has been shown to
have three glycone sites and two aglycone sites.[24] Thus,
cleavage of oligosaccharides by Cex never occurs to release
a monosaccharide from the non-reducing end, but rather di-
or trisaccharides.[24] Large increases in inhibitor affinity
upon the addition of a single sugar residue to a
‘monosaccharide’ iminosugar have been seen for Cex
previously,[21] and three-dimensional structures have been
determined.[22] In those cases, addition of a single xylose
residue was shown to increase affinity by several orders of
magnitude, with the xylosyl xylo-isofagomine (21) binding
with a Ki value of 0.13 µM, while the ‘monosaccharide’
iminosugar, xylo-isofagomine (20) binds only very weakly
(Diagram 4). Clearly, interactions in the –2 site are very
important for effective binding and catalysis. It is interesting,
however, that the glucosyl isofagomine (11) studied here
binds only 15-fold more weakly to Cex than does the xylo
analogue (21). This stands somewhat in contrast to the
approximately thousand-fold faster hydrolysis of
xylo-oligosaccharide substrates than of the corresponding
cello-oligosaccharides.[24]
1
C14H19NO5 requires C, 59.8; H, 6.8%). H NMR (300 MHz, D2O) δ
1.40–1.54, m, H5; 2.42–2.64, m, H2,6; 3.13, t, J3,4 ≈ J4,5 9.7 Hz, H4;
3.20–3.30, m, H3; 3.41, dd, J5,H 6.9, JH,H 11.5 Hz, CH2O; 3.61, dd, J5,H
3.2 Hz, CH2O; 3.84–4.09, m, H2,6; 4.94, s, PhCH2; 7.15–7.28, m, Ph.
13C NMR (75.5 MHz, D2O) δ 44.3, C5; 45.8, 48.4, C2,6; 60.8, CH2O;
68.6, PhCH2; 71.5, 74.2, C3,4; 128.5, 129.2, 129.5, 137.1, Ph; 157.3,
NCO. High-resolution mass spectrum (HRMS) fast-atom
bombardment (FAB) m/z 282.1354. C14H20NO5 [M+H]+• requires
282.1341.
(4R,5S,6R)-N-Benzyloxycarbonyl-4,5-dihydroxy-6-hydroxymethyl-3,4,
5,6-tetrahydro-2H-1,2-oxazine (4)
The tetrahydrooxazine (2)[14] (340 mg, 2.3 mmol) was treated with
NaHCO3 (370 mg, 4.5 mmol) and benzyl chloroformate (420 µL, 2.9
mmol) in H2O/MeOH/THF (2:1:1, 20 mL), and the mixture was stirred
(30 min, rt). The mixture was treated with hydrochloric acid (3 mL of 1
M) and then concentrated, co-evaporating with toluene. Flash
chromatography (EtOAc/petrol, 1:1 then MeOH/CHCl3, 1:9) of the
residue gave the carbamate (4) (580 mg, 90%) as a colourless oil, [α]D
+71.9° (MeOH) (Found: C, 54.7; H, 5.6; N, 4.7%. C13H17NO6 requires
1
C, 55.1; H, 6.0; N, 4.9%). H NMR (300 MHz, D2O) δ 2.93, dd, J3,3
13.4, J3,4 10.3 Hz, H3; 3.35, t, J4,5 ≈ J5,6 8.9 Hz, H5; 3.39–3.51, m, H4,6;
3.57, dd, J6,H 5.4, JH,H 10.0 Hz, CH2O; 3.72, dd, J6,H 1.8 Hz, CH2O;
4.03, dd, J3,4 5.3 Hz, H3; 4.89, 4.94, ABq, J 12.2 Hz, PhCH2; 7.09–7.21,
m, Ph. 13C NMR (75.5 MHz, D2O) δ 50.4, C3; 59.9, CH2O; 69.1,
PhCH2; 69.4, 70.3, C4,5; 85.1, C6; 128.7, 129.3, 129.4, 136.1, Ph;
156.7, NCO. HRMS (FAB) m/z 284.1151. C13H18NO6 [M+H]+•
requires 284.1134.
OH
HO
HO
HO
HO
O
NH
HO
O
NH
(20)
(21)
Diagram 4
(3R,4R,5R)-3-Acetoxy-5-acetoxymethyl-N-benzyloxycarbonyl-4-
[(tetra-O-acetyl-β-D-glucopyranosyl)oxy]piperidine (5), (3R,4R,5R)-
3-acetoxy-5-acetoxymethyl-N-benzyloxycarbonyl-4-{[(tetra-O-
acetyl-β-D-glucopyranosyl)-(1→4)-O-(tri-O-acetyl-β-D-glucosyl)]
oxy}piperidine (6) and (3R,4R,5R)-3-acetoxy-5-acetoxymethyl-N-
benzyloxycarbonyl-4-{[(tetra-O-acetyl-β-D-glucopyranosyl)-(1→4)-O
-(tri-O-acetyl-β-D-glucosyl)-(1→4)-O-(tri-O-acetyl-β-D-
glucosyl)]oxy}piperidine (7)
The oxazines bind much more weakly, which is consistent
with earlier findings in regards to monosaccharidases.[14]
The parent oxazine (2) shows no significant inhibition at
6.6 mM concentrations, while weak inhibition (Ki = 1.9 mM
and 1.0 mM, respectively) is seen for the glucosyl (17) and
cellobiosyl (18) derivatives. Only when the third glucosyl
residue is added is significant additional binding observed,
this third glucosyl residue improving binding approximately
12-fold. It is interesting that useful binding is only observed
in this case when four sites are apparently filled by the
inhibitor. This would suggest that the oxazine moiety, in
contrast to the isofagomine, provides no special affinity
through binding in the –1 site, and that the increase in
affinity upon reaching a ‘tetrasaccharide’ might be a
consequence of binding across the active site. This would be
completely consistent with the earlier observation that
cleavage of tetrasaccharides is much faster than that of
trisaccharides.[24]
A
mixture of the carbamate (3) (130 mg, 0.47 mmol) and
α-D-glucopyranosyl fluoride (140 mg, 0.76 mmol) in aqueous
NH4HCO3 (10 mL of 0.15 M) was treated with AbgGlu358Ser (2 mg)
and the mixture was then incubated (7 days, rt). The mixture was
concentrated, co-evaporating with pyridine, and then taken up in
pyridine (5 mL) and treated with Ac2O (2 mL) (20 h, rt). This mixture
was quenched upon the addition of MeOH (3 mL), and then
concentrated to give a residue. Flash chromatography (EtOAc/petrol,
gradient from 3:7 to 3:2) of this residue gave, firstly, the
pseudo-disaccharide (5) (87 mg, 26%) as a colourless gum, [α]D –6.4°.
1H NMR (300 MHz) δ 1.87–1.93, m, H5; 1.97, 1.99, 2.01, 2.05, 4 × s,
18H, CH3; 3.41–3.76, m, 5H; 4.00–4.30, m, 5H; 4.57, d, J1′,2′ 7.9 Hz,
H1′; 4.81–5.23, m, H2′,3,3′,4′,PhCH2; 7.26–7.40, m, Ph. 13C NMR
(75.5 MHz) δ 20.5, 20.6, 20.7, 6C, CH3; 39.2, C5; 41.6, 41.9, 43.9,
C2,6; 61.8, 62.1, C6′,CH2O; 67.4, PhCH2; 68.2, 69.3, 71.4, 71.9, 72.6,

750
J. M. Macdonald et al.
76.3, C2′,3,3′,4,4′,5′; 100.9, C1′; 127.9, 128.1, 128.5, 136.4, Ph; 155.5,
NCO; 169.1, 169.3, 169.5, 170.2, 170.6, 6C, CO. HRMS (FAB) m/z
696.2524. C32H42NO16 [M+H]+• requires 696.2504.
(3R,4R,5R)-3-Acetoxy-5-acetoxymethyl-4-{[(tetra-O-acetyl-β-D-
glucopyranosyl)-(1→4)-O-(tri-O-acetyl-β-D-glucosyl)-(1→4)-O-(tri-
O-acetyl-β-D-glucosyl)]oxy}piperidine (10)
Next to elute was the pseudo-trisaccharide (6) (127 mg, 27%),
which was isolated as a colourless gum, [α]D –19°. 1H NMR (500 MHz)
δ 1.86–1.92, m, H5; 1.96, 1.99, 1.99, 2.01, 2.07, 2.10, 6 × s, 27H, CH3;
3.34, m, 8H; 3.98–4.22, m, 4H; 4.35, dd, J 4.3, 12.4 Hz, 1H; 4.42–4.49,
m, 2H; 4.55, d, J 7.9 Hz, 1H; 4.83–5.20, m, H2′,2′′,3,3′,3′′,4′′,PhCH2;
7.26–7.37, m, Ph. 13C NMR (125.5 MHz) δ 20.4, 20.5, 20.6, 20.6, 20.7,
20.8, 9C, CH3; 39.1, C5; 41.4, 41.7, 43.6, 43.8, C2,6; 61.5, 61.8, 62.0,
62.1, C6′,6′′,CH2O; 67.3, 67.4, PhCH2; 67.6–76.3, 10C; 100.7, 100.8,
C1′,1′′; 127.9, 128.1, 128.5, 136.4, Ph; 155.5, NCO; 169.0–170.6, 9C,
CO. HRMS (FAB) m/z 984.3396. C44H58NO24 [M+H]+• requires
984.3349.
The carbamate (7) (44 mg, 34 µmol) was treated as (5) previously to
give, after flash chromatography (EtOAc/petrol/Et3N, 9:9:2 then
9:0:1), the amine (10) (38 mg, 97%) as a colourless glass, [α]D –12.2°.
1H NMR (500 MHz) δ 1.83–1.92, m, H5; 1.94–2.12, 11 × s, 36H, CH3;
2.46, dd, J5,6 9.1, J6,6 13.2 Hz, H6; 2.50, dd, J2,2 13.0, J2,3 8.5 Hz, H2;
3.03, dd, J5,6 3.9 Hz, H6; 3.20, dd, J2,3 4.4 Hz, H2; 3.52–3.63, m, 4H;
3.70–3.76, m, H4′,4′′; 3.98–4.10, m, 4H, CH2; 4.25, dd, J 3.8, 11.1 Hz,
1H, CH2; 4.33, dd, J 4.3, 12.5 Hz, 1H, CH2; 4.35–4.40, m, CH2; 4.44,
4.45, 4.54, 3 × d, J 7.8, 7.9, 7.9 Hz, H1′,1′′,1′′′; 4.75, dt, J3,4 8.2 Hz, H3;
4.78–4.90, m, H2′,2′′,2′′′; 5.02, t, J 9.7 Hz, 1H; 5.05–5.13, m, 3H. 13
C
NMR (125.8 MHz) δ 20.4–20.9, 12C, CH3; 42.0, C5; 46.2, C6; 47.8,
C2; 61.4, 62.0, 62.2, C6′,6′′,6′′′,CH2O; 67.6, C4′′′; 71.5–72.8, 10C;
76.0, 76.3, C4′,4′′; 78.8, C4; 100.5, 100.7, C1′,1′′,1′′′; 169.0–170.6,
12C, CO. HRMS (FAB) m/z 1138.3864. C48H68NO30 [M+H]+• requires
1138.3826.
Last to elute was the pseudo-tetrasaccharide (7) (45 mg, 8%), which
1
was isolated as a colourless gum, [α]D –12.6°. H NMR (500 MHz) δ
1.86–1.92, m, H5; 1.96–2.13, 10 × s, 36H, CH3; 3.25–3.85, m, 9H;
3.27–3.98, m, 6H; 4.31–4.53, m, 5H; 4.54, d, J 7.9 Hz, 1H; 4.75–5.21,
m, 10H; 7.27–7.38, m, Ph. 13C NMR (125.5 MHz) d 20.4–20.7, 12C,
CH3; 39.1, C5; 41.4, 41.8, 43.6, 43.8, C2,6; 61.4, 61.7, 62.1,
C6′,6′′,6′′′,CH2O; 67.7, PhCH2; 67.6–76.3, 14C; 100.5, 100.6, 100.7,
C1′,1′′,1′′′; 127.9, 128.1, 128.5, 136.4, Ph; 155.5, NCO; 169.0–171.2,
12C, CO. HRMS (FAB) m/z 1272.4184. C56H74NO32 [M+H]+• requires
1272.4194.
(3R,4R,5R)-4-(β-D-Glucopyranosyl)oxy-3-hydroxy-5-(hydroxymethyl)
piperidine (11)
The per-O-acetylated amine (8) (34 mg) in MeOH (5 mL) was treated
with sodium metal (5 mg) (2 h, rt). The reaction mixture was
concentrated, taken up in H2O (5 mL) and brought to pH 5 by the
addition of hydrochloric acid (0.1 M). The mixture was applied to a
column of cation-exchange resin (Dowex 50W-X2, H+ form), washed
with water and then eluted with aqueous NH3 (3 M). The eluate was
concentrated, taken up in H2O (1 mL), applied to an anion-exchange
column (Sephadex-DEAE A-25) and eluted with H2O. Freeze-drying of
the eluate afforded the glucosyl isofagomine (11) (17 mg, 91%) as a
colourless foam, [α]D –3.3° (H2O). 1H NMR (500 MHz, D2O) α
1.79–1.87, m, H5; 2.39, dd, J2,2 12.4, J2,3 10.7 Hz, H2; 2.43, t, J5,6 ≈ J6,6
12.3 Hz, H6; 3.05–3.11, m, H6; 3.16–3.22, m, H2; 3.34, dd, J1′,2′ 8.0,
J2′,3′ 9.4 Hz, H2′; 3.43, dd, J3′4′ 9.7 Hz, H3′; 3.48–3.55, m, H4,4′,5′;
3.59, ddd, J2,3 5.0, J3,4 8.7 Hz, H3; 3.73, dd, J5,H 6.0, JH,H 11.4 Hz,
CH2O; 3.72, dd, J5′,6′ 6.0, J6′,6′ 12.4 Hz, H6′; 3.85, dd, J5,H 3.4 Hz,
CH2O; 3.91, dd, J5′,6′ 2.2 Hz, H6′; 4.52, d, H1′. 13C NMR (125.8 MHz,
D2O) δ 44.6, C5; 47.0, C6; 49.7, C2; 60.7, CH2O; 61.3, C6′; 70.2, C3′;
71.5, C3; 74.0, C2′; 76.3, 76.6, C4′,5′; 85.4, C4; 103.5, C1′. HRMS
(FAB) m/z 310.1486. C12H24NO8 [M+H]+• requires 310.1502.
(3R,4R,5R)-3-Acetoxy-5-acetoxymethyl-4-[(tetra-O-acetyl-β-D-
glucopyranosyl)oxy]piperidine (8)
A mixture of cyclohexene (200 µL), Et2NH (10 µL) and Pd(OH)2/C (18
mg) in EtOH (4 mL) was heated under reflux (10 min) and then allowed
to cool. The carbamate (5) (61 mg, 88 µmol) in EtOAc (4 mL) was
added and then this mixture was heated under reflux (15 min) and
allowed to cool. The mixture was filtered, washing with EtOAc, and the
combined filtrate and washings were concentrated to give a residue.
Flash chromatography (EtOAc/petrol/Et3N, 9:9:2 then 9:0:1) gave the
1
amine (8) (45 mg, 91%) as a colourless glass, [α]D +3.3°. H NMR
(500 MHz) δ 1.88–1.96, m, H5; 1.99, 2.01, 2.04, 2.07, 2.08, 5 × s, 18H,
CH3; 2.48, dd, J5,6 9.4, J6,6 13.1 Hz, H6; 2.52, dd, J2,2 12.8, J2,3 8.6 Hz,
H2; 3.07, dd, J5,6 3.8 Hz, H6; 3.25, dd, J2,3 4.4 Hz, H2; 3.62, t, J3,4 ≈ J4,5
8.4 Hz, H4; 3.66, ddd, J4′,5′ 9.5, J5′,6′ 2.4, 4.6 Hz, H5′; 4.04, dd, J6′,6′
12.3 Hz, H6′; 4.07, dd, J5,H 7.1, JH,H 11.1 Hz, CH2O; 4.29, dd, J5,H 3.8
Hz, CH2O; 4.33, dd, H6′; 4.60, d, J1′,2′ 7.9 Hz, H1′; 4.79, dt, H3; 4.96,
dd, J2′,3′ 9.4 Hz, H2′; 5.08, t, J3′,4′ 9.5 Hz, H4′; 5.17, t, H3′. 13C NMR
(125.8 MHz) δ 20.6, 20.6, 20.8, 21.0, 6C, CH3; 42.3, C5; 46.4, C6; 48.0,
C2; 61.8, C6′; 62.3, CH2O; 68.1, C4′; 71.71, 71.74, C2′,5′; 72.0, C3;
72.9, C3′; 79.1, C4; 100.8, C1′; 169.2, 169.3, 169.8, 170.3, 170.6,
170.8, 6C, CO. HRMS (FAB) m/z 562.2172. C24H36NO14 [M+H]+•
requires 562.2136.
(3R,4R,5R)-4-[(β-D-Glucopyranosyl)-(1→4)-O-(β-D-glucosyl)]oxy-3-
hydroxy-5-(hydroxymethyl)piperidine (12)
The per-O-acetylated amine (9) (70 mg) was treated as (8) above to
give, after freeze-drying, the cellobiosyl isofagomine (12) (33 mg, 85%)
as a colourless, amorphous solid, [α]D –10.4° (H2O). 1H NMR (500
MHz, D2O) δ 1.85–1.93, m, H5; 2.49, t, J2,2 ≈ J2,3 11.8 Hz, H2; 2.54, t,
J5,6 ≈ J6,6 12.6 Hz, H6; 3.16, dd, J5,6 3.6 Hz, H6; 3.26, dd, J2,3 4.6 Hz,
H2; 3.33, dd, J1′,2′ 8.0, J2′,3′ 9.3 Hz, H2′; 3.37–3.45, m, 2H; 3.48–3.54,
m, 2H; 3.58, dd, J 8.7, 9.2 Hz, 1H; 3.62–3.74, m, 6H; 3.80–3.88, m, 3H;
3.93, dd, J 2.2, 12.4 Hz, 1H, CH2; 3.98, dd, J 2.1, 12.4 Hz, 1H, CH2;
4.52, 5.57, 2 × d, J 7.9, 8.0 Hz, H1′,1′′. 13C NMR (125.8 MHz, D2O) δ
44.1, C5; 46.7, C6; 49.3, C2; 60.4, 60.6, 61.3, C6′,6′′,CH2O; 70.1, 70.9,
C3,3′′; 73.8–76.7, 6C; 79.1, C4′; 84.6, C4; 103.26, 103.32, C1′,1′′.
HRMS (FAB) m/z 472.2008. C18H34NO13 [M+H]+• requires 472.2030.
(3R,4R,5R)-3-Acetoxy-5-acetoxymethyl-4-{[(tetra-O-acetyl-β-D-
glucopyranosyl)-(1→4)-O-(tri-O-acetyl-β-D-glucosyl)]oxy}
piperidine (9)
The carbamate (6) (91 mg, 93 µmol) was treated as (5) above to give,
after flash chromatography (EtOAc/petrol/Et3N, 9:9:2 then 9:0:1), the
amine (9) (75 mg, 96%) as a colourless glass, [α]D –7.9°. 1H NMR
(500 MHz) δ 1.85–1.94, m, H5; 1.96–2.12, 9 × s, 27H, CH3; 2.47, dd,
J5,6 9.1, J6,6 13.3 Hz, H6; 2.51, dd, J2,2 13.0, J2,3 8.6 Hz, H2; 3.04, dd,
J5,6 4.0 Hz, H6; 3.22, dd, J2,3 4.8 Hz, H2; 3.55–3.65, m, H4,5′,5′′; 3.76,
t, J3′,4′ ≈ J4′,5′ 9.5 Hz, H4′; 4.02, dd, J 2.1, 12.4 Hz, 1H, CH2; 4.05, dd, J
7.1, 11.2 Hz, 1H, CH2; 4.10, dd, J 5.2, 12.0 Hz, 1H, CH2; 4.28, dd, J 3.9,
11.2 Hz, 1H, CH2; 4.34, dd, J 4.3, 12.4 Hz, 1H, CH2; 4.39, dd, J 1.9,
12.0 Hz, 1H, CH2; 4.48, 4.57, 2 × d, J 7.9, 7.9 Hz, H1′,1′′; 4.76, dt, J3,4
8.1 Hz, H3; 4.87, 4.90, 2 × dd, J 9.5, 9.3 Hz, H2′,2′′; 5.04, 5.11, 5.14, 3
× t, J 9.3, 9.3, 9.5 Hz, H3′,3′′,4′′. 13C NMR (125.8 MHz) δ 20.5–20.9,
9C, CH3; 42.0, C5; 46.2, C6; 47.8, C2; 61.5, 62.2, 62.3, C6′,6′′,CH2O;
67.7, C4′′; 71.5–72.9, 7C; 76.3, C4′; 78.8, C4; 100.5, 100.8, C1′,1′′;
169.0–170.6, 9C, CO. HRMS (FAB) m/z 850.3004. C36H52NO22
[M+H]+• requires 850.2981.
(3R,4R,5R)-4-[(β-D-Glucopyranosyl)-(1→4)-O-(β-D-glucosyl)-(1→4)
-O-(β-D-glucosyl)]oxy-3-hydroxy-5-(hydroxymethyl)piperidine (13)
The per-O-acetylated amine (10) (33 mg) was treated as (8) previously
to give, after freeze-drying, the cellotriosyl isofagomine (13) (15 mg,
82%) as a colourless, amorphous solid, [α]D –1.6° (H2O). 1H NMR (500
MHz, D2O) δ 1.81–1.91, m, H5; 2.43, t, J2,2 ≈ J2,3 11.6 Hz, H2; 2.47, t,
J5,6 ≈ J6,6 12.4 Hz, H6; 3.11, dd, J5,6 3.9 Hz, H6; 3.22, dd, J2,3 4.7 Hz,
H2; 3.31–3.46, m, 4H; 3.48–3.59, m, 3H; 3.60–3.77, m, 9H; 3.81–3.89,
m, 3H; 3.93, dd, J 1.8, 12.2 Hz, 1H; 3.96–4.02, m, 2H; 4.51–4.59, m,
H1′,1′′,1′′′. 13C NMR (125.8 MHz, D2O) δ 44.5, C5; 46.9, C6; 49.7, C2;
60.6, 61.3, C6′,6′′,6′′′,CH2O; 70.1, 71.3, C3,3′′′; 73.6–76.7, 9C; 79.0,
79.1, C4′,4′′; 85.1, C4; 103.0, 103.3, 103.4, C1′,1′′,1′′′. HRMS (FAB)
m/z 634.2609. C24H44NO18 [M+H]+• requires 634.2558.

Synthesis with Glycosynthases
751
(4R,5S,6R)-4-Acetoxy-6-acetoxymethyl-N-benzyloxycarbonyl-5-
(tetra-O-acetyl-β-D-glucopyranosyl)oxy-3,4,5,6-tetrahydro-2H-1,2-
oxazine (14), (4R,5S,6R)-4-acetoxy-6-acetoxymethyl-N-
benzyloxycarbonyl- 5-[(tetra-O-acetyl-β-D-glucopyranosyl)-(1→4)-O-
(tri-O-acetyl-β-D-glucosyl)]oxy-3,4,5,6-tetrahydro-2H-1,2-
oxazine (15) and (4R,5S,6R)- 4-acetoxy-6-acetoxymethyl-N-
benzyloxycarbonyl-5-[(tetra-O-acetyl-β-D-glucopyranosyl)-(1→4)-O-
(tri-O-acetyl-β-D-glucosyl)-(1→4)-O-(tri-O-acetyl-β-D-glucosyl)]oxy-
3,4,5,6-tetrahydro-2H-1,2-oxazine (16)
acid (2.5 M). The mixture was applied to a column of cation-exchange
resin (Dowex 50W-X2, H+ form), washed with water and then eluted
with aqueous NH3 (3 M). The eluate was concentrated, taken up in H2O
(1 mL), applied to an anion-exchange column (Sephadex-DEAE A-25)
and eluted with H2O. Freeze-drying of the eluate afforded the glucosyl
oxazine (17) (24 mg, 85%) as a colourless, amorphous solid, [α]D
–14.2° (H2O). 1H NMR (500 MHz, D2O) δ 2.94, dd, J3,3 13.2, J3,4 10.7
Hz, H3; 3.29–3.37, m, H2′,3; 3.41, dd, J3′,4′ 9.2, J4′,5′ 9.8 Hz, H4′;
3.47–3.54, m, H3′,5′; 3.64, t, H5; 3.68, ddd, J5,6 9.7, J6,H 1.8, 5.1 Hz,
H6; 3.72, dd, J5′,6′ 6.1, J6′,6′ 12.3 Hz, H6′; 3.79, dd, JH,H 12.6 Hz, CH2O;
3.83, ddd, J3,4 5.5, J4,5 8.0 Hz, H4; 3.92, dd, J5′,6′ 2.2 Hz, H6′; 3.96, dd,
CH2O; 4.51, d, J1′,2′ 8.0 Hz, H1′. 13C NMR (125.8 MHz, D2O) δ 52.6,
C3; 59.8, CH2O; 61.3, C6′; 69.7, C4; 70.2, C4′; 73.8, C2′; 76.1, 76.6,
C3′,5′; 80.3, C5; 82.6, C6; 103.3, C1′. HRMS (FAB) m/z 312.1294.
C11H22NO9 [M+H]+• requires 312.1295.
A
mixture of the carbamate (4) (138 mg, 0.49 mmol) and
α-D-glucopyranosyl fluoride (142 mg, 0.78 mmol) in aqueous
NH4HCO3 (6 mL of 0.15 M) was treated with AbgGlu358Ser (2 mg)
and was then incubated (8 days, rt). The mixture was concentrated,
co-evaporating with pyridine, and then taken up in pyridine (5 mL) and
treated with Ac2O (2 mL) (20 h, rt). This reaction mixture was quenched
upon the addition of MeOH (3 mL) and then concentrated to give a
residue. Flash chromatography (EtOAc/petrol, gradient from 3:7 to 3:2)
of this residue gave, firstly, the pseudo-disaccharide (14) (135 mg, 40%)
as a colourless solid. A small portion was recrystallized to give
colourless, fine needles, m.p. 126–131°C (CH2Cl2/Et2O), [α]D +18.0°
(Found: C, 53.2; H, 5.6; N, 1.8%. C31H39NO17 requires C, 53.4; H, 5.6;
N, 2.0%). 1H NMR (500 MHz) δ 1.96, 1.99, 2.00, 2.00, 2.03, 2.06, 6 ×
s, 18H, CH3; 3.36, dd, J3,3 13.5, J3,4 8.6 Hz, H3; 3.65, ddd, J4′,5′ 9.9, J5′,6′
2.4, 4.6 Hz, H5′; 3.77, t, J4,5 ≈ J5,6 7.7 Hz, H5; 3.96–4.01, m, H6; 4.03,
dd, J6′,6′ 12.4 Hz, H6′; 4.21, dd, J6,H 6.5, JH,H 12.5 Hz, CH2O; 4.25, dd,
J3,4 5.0 Hz, H3; 4.29, dd, H6′; 4.38, dd, J6,H 2.5 Hz, CH2O; 4.56, d, J1′,2′
7.9 Hz, H1′; 4.92, dd, J2′,3′ 9.5 Hz, H2′; 5.01, ddd, H4; 5.05, t, J3′,4′ 9.7
Hz, H4′; 5.11–5.21, m, 3H, H3′,PhCH2, 7.28–7.35, m, Ph. 13C NMR
(125.8 MHz) δ 20.4, 20.4, 20.5, 20.5, 20.6, 6C, CH3; 47.1, C3; 60.8,
CH2O; 61.6, C6′; 67.9, 68.0, C4,4′; 68.2, PhCH2; 71.3, C2′; 71.9, C5′;
72.6, C3′; 75.1, C5; 79.9, C6; 100.4, C1′; 128.1, 128.4, 128.5, 135.4, Ph;
154.8, NCO; 169.0, 169.19, 169.23, 170.1, 170.3, 170.4, 6C, CO. HRMS
(FAB) m/z 698.2305. C31H40NO17 [M+H]+• requires 698.2296.
(4R,5S,6R)-5-[(β-D-Glucopyranosyl)-(1→4)-O-(β-D-glucosyl)]oxy-4-
hydroxy-6-hydroxymethyl-3,4,5,6-tetrahydro-2H-1,2-oxazine (18)
The per-O-acetylated carbamate (15) (90 mg) was treated as (14) above
to give, after freeze-drying, the cellobiosyl oxazine (18) (28 mg, 65%)
1
as a colourless glass, [α]D –10.4° (H2O). H NMR (500 MHz, D2O) δ
3.00, dd, J3,3 13.2, J3,4 10.8 Hz, H3; 3.31, dd, J 5.6, 8.0 Hz, 1H;
3.34–3.45, m, 3H; 3.46–3.54, m, 2H; 3.59–3.78, m, 6H; 3.79–3.89, m,
3H; 3.91, dd, J 2.0, 12.1 Hz, 1H; 3.95–4.01, m, 2H; 4.50, 4.55, 2 × d, J
7.9, 8.0 Hz, H1′,1′′. 13C NMR (125.8 MHz, D2O) δ 52.1, C3; 59.6,
CH2O; 60.6, C6′; 61.2, C6′′; 69.0, 70.1, C4,4′′; 73.6–76.6, 6C; 79.1,
79.6, C4′,5; 82.7, C6; 103.0, 103.2, C1′,1′′. HRMS (FAB) m/z
474.1841. C17H32NO14 [M+H]+• requires 474.1823.
(4R,5S,6R)-5-[(β-D-Glucopyranosyl)-(1→4)-O-(β-D-glucosyl)-(1→4)
-O-(β-D-glucosyl)]oxy-4-hydroxy-6-hydroxymethyl-3,4,5,6-tetrahydro-
2H-1,2-oxazine (19)
The per-O-acetylated carbamate (16) (33 mg) was treated as (14)
previously to give, after freeze-drying, the cellotriosyl oxazine (19) (9.5
mg, 58%) as a colourless glass, [α]D +5.4° (H2O). 1H NMR (500 MHz,
D2O) δ 2.93, dd, J3,3 13.2, J3,4 10.7 Hz, H3; 3.23–3.44, m, 5H;
3.46–3.53, m, 2H; 3.58–3.70, m, 9H; 3.73, dd, J 5.8, 12.5 Hz, 1H;
3.76–3.85, m, 3H; 3.91, dd, J 2.2, 12.4 Hz, 1H; 3.93–4.00, m, 3H; 4.50,
4.53, 4.54, 3 × d, J 7.9, 7.9, 8.0 Hz, H1′,1′′,1′′′. 13C NMR (125.8 MHz,
D2O) δ 52.6, C3; 59.8, CH2O; 60.5, 60.6, C6′,6′′; 61.2, C6′′′; 69.7, 70.1,
C4,4′′′; 73.6–76.6, 9C; 78.98, 79.02, C4′,4′′; 80.2, C5; 82.7, C6; 103.0,
103.1, 103.2, C1′,1′′,1′′′. HRMS (FAB) m/z 636.2349. C23H42NO19
[M+H]+• requires 636.2351.
Next to elute was the pseudo-trisaccharide (15) (131 mg, 27%),
which was isolated as a colourless solid. A small portion was
recrystallized to give colourless, fine needles, m.p. 153–162°C
(CH2Cl2/Et2O), [α]D +2.1° (Found: C, 52.3; H, 5.4; N, 1.2%.
C43H55NO25 requires C, 52.4; H, 5.6; N, 1.4%). 1H NMR (300 MHz) δ
1.94–2.08, 5 × s, 27H, CH3; 3.36, dd, J3,3 13.4, J3,4 8.1 Hz, 1H, H3;
3.50–3.65, m, 2H; 3.68–3.79, m, 2H; 3.92–4.09, m, 3H; 4.12–4.24, m,
2H; 4.26–4.42, m, 3H; 4.45, 4.51, 2 × d, J 7.8, 7.9 Hz, H1′,1′′;
4.78–4.90, m, 2H; 4.91–5.20, m, 6H; 7.27–7.38, m, Ph. 13C NMR (75.5
MHz) δ 20.5, 20.6, 20.7, 9C, CH3; 47.0, C3; 60.8, CH2O; 61.4, 61.9,
C6′,6′′; 67.7, 67.9, C4,4′′; 68.1, PhCH2; 71.5, 71.9, 72.4, 72.8, 6C;
75.0, C5; 76.1, C4′; 79.9, C6; 100.2, 100.7, C1′,1′′; 128.1, 128.4, 128.5,
135.4, Ph; 154.8, NCO; 169.0–170.4, 9C, CO. HRMS (FAB) m/z
986.3165. C43H56NO25 [M+H]+• requires 986.3141.
Enzymology
The serine nucleophile mutant of the Agrobacterium sp. β-glucosidase
(Abg E358S) was created and purified as described previously.[11]
α-D-Glucopyranosyl fluoride was synthesized according to literature
protocols.[7,26] Glycosynthase reactions were carried out as described
above. Inhibition constants for Cex were determined as described
previously[21] using 2,4-dinitrophenyl β-cellobioside as substrate and a
range of concentrations of the inhibitors from approximately one fifth
of the Ki value ultimately determined to five times that value.
Last to elute was the pseudo-tetrasaccharide (16) (46 mg, 7.5%),
which was isolated as a colourless solid. A small portion was
recrystallized to give colourless, fine needles, m.p. 195–199°C
(CH2Cl2/Et2O), [α]D −4.0° (Found: C, 51.7; H, 5.3; N, 0.8%.
C55H71NO33 requires C, 51.8; H, 5.6; N, 1.1%). 1H NMR (300 MHz) δ
1.93–2.11, 10 × s, 36H, CH3; 3.37, dd, J3,3 13.4, J3,4 8.1 Hz, H3;
3.50–3.63, m, 3H; 3.67–3.77, m, 3H; 3.93–4.10, m, 4H; 4.14–4.23, m,
2H; 4.29–4.40, m, 4H; 4.42, 4.43, 4.50, 3 × d, J 7.7, 8.0, 7.8 Hz,
H1′,1′′1′′′; 4.76–4.90, m, 3H; 4.92–5.20, m, 7H; 7.27–7.36, m, Ph. 13
C
Acknowledgments
NMR (75.5 MHz) δ 20.5, 20.6, 20.7, 12C, CH3; 47.1, C3; 60.8, CH2O;
61.5, 61.9, 62.0, C6′,6′′,6′′′; 67.7, 67.9, C4,4′′′; 68.2, PhCH2;
71.5–72.8, 9C; 75.0, C5; 76.0, 76.1, C4′,4′′; 80.0, C6; 100.2, 100.5,
100.7, C1′,1′′,1′′′; 128.1, 128.4, 128.6, 135.5, Ph; 154.9, NCO;
169.0–170.4, 12C, CO. HRMS (FAB) m/z 1274.4037. C55H72NO33
[M+H]+• requires 1274.3987.
We thank the Australian Research Council and the Protein
Engineering Network Centres of Excellence of Canada
(PENCE) for financial assistance.
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