D-glucosylated derivatives of isofagomine and noeuromycin and their potential as inhibitors of β-glycoside hydrolases

Article abstract of DOI:10.1071/CH07188

While isofagomine and noeuromycin have previously been demonstrated to be effective inhibitors of a range of exo-acting glycosidases, they are usually only very weak inhibitors of endo-glycosidases. However, the disaccharide-like 3- and 4-O-?-d-glucopyran

Full text of DOI:10.1071/CH07188

Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2007, 60, 549–565
D-Glucosylated Derivatives of Isofagomine and Noeuromycin
and Their Potential as Inhibitors of β-Glycoside Hydrolases
Peter J. Meloncelli,A Tracey M. Gloster,B Victoria A. Money,B Chris A. Tarling,C
Gideon J. Davies,B Stephen G. Withers,C and Robert V. StickA,D
AChemistry M313, School of Biomedical, Biomolecular and Chemical Sciences,
University of Western Australia, Crawley WA 6009, Australia.
BStructural Biology Laboratory, Department of Chemistry, University of York,
Heslington, York, YO10 5YW, UK.
CDepartment of Chemistry, University of British Columbia, Vancouver,
British Columbia, V6T 1Z1, Canada.
DCorresponding author. Email: rvs@chem.uwa.edu.au
While isofagomine and noeuromycin have previously been demonstrated to be effective inhibitors of a range of exo-acting
glycosidases, they are usually only very weak inhibitors of endo-glycosidases. However, the disaccharide-like 3- and
4-O-β-d-glucopyranosylisofagomines have proven to be strong inhibitors of these endo-acting enzymes that utilize mul-
tiple sub-sites. In an attempt to emulate these successes, we have prepared 3- and 4-O-β-d-glucopyranosylnoeuromycin,
the former by a selective glycosylation (at O2) of benzyl 4-C-cyano-4-deoxy-α-d-arabinoside (also leading to another
synthesis of 3-O-β-d-glucopyranosylisofagomine), the latter by a non-selective glycosylation of benzyl 4-O-allyl-β-
l-xyloside with subsequent introduction of the required nitrile group (also leading to another synthesis of 4-O-β-d-
glucopyranosylisofagomine). 3-O-β-d-Glucopyranosylnoeuromycin was evaluated as an inhibitor of a family 26 lichenase
from Clostridium thermocellum, and 4-O-β-d-glucopyranosylnoeuromycin as an inhibitor of both a family 5 endo-
glucanase from Bacillus agaradhaerans and a family 10 endo-xylanase from Cellulomonas fimi. We also report X-ray
structural investigations of 3- and 4-O-β-d-glucopyranosylnoeuromycin in complex with the family 26 and family 5
β-glycoside hydrolases, respectively. The two d-glucosylated noeuromycins were indeed able to harness the additional
binding energy from the sub-sites of their endo-glycoside hydrolase targets, and were thus excellent inhibitors (in the
nanomolar range), binding as expected in the −1 and −2 sub-sites of the enzymes.
Manuscript received: 4 June 2007.
Final version: 27 June 2007.
Introduction
itself subsequently discovered as a natural product. The abil-
ity of these two imino sugars to inhibit the action of vari-
ous carbohydrate-processing enzymes, particularly those that
hydrolyse an α-glycosidic linkage, prompted a wider search
for related molecules: so were discovered swainsonine,[5]
castanospermine,[6] and fagomine,[7] to name but a few. It was
not surprising then for chemists to ‘tinker’ with these natural
structures, in an effort to improve their ability to inhibit the act-
ion of carbohydrate-processing enzymes or to broaden or even
change the range of enzymes inhibited.
The natural imino sugar fagomine 5 is an effective inhibitor
of some α-glycosidases. Lundt and Bols sought to rearrange
this molecule into the aza∗ sugar isofagomine 6,[8] and Bols
took a step further by inventing noeuromycin 7 (Fig. 2).[9]
These molecules were duly synthesized and found to be potent
inhibitors of glycosidases: 6 of β-glycosidases, 7 of both α- and
β-glycosidases.[10] We have been able to utilize both isofagomine
6 and noeuromycin 7 to study the mechanism of action of various
Imino sugars came of age with the use of 1-deoxy-N-(2-
hydroxyethyl)nojirimycin 1 (Miglitol/Glyset)[1] and N-butyl-
1-deoxynojirimycin 2 (Miglustat, Zavesca)[2] for the clinical
treatment of non-insulin-dependent (type II) diabetes melli-
tus, and type I Gaucher’s disease, respectively (Fig. 1). Imino
sugar 1 interferes with the action of both α-amylases and
α-glucosidases, thus minimizing the burst of d-glucose that nor-
mally follows a high-starch meal. Imino sugar 2 acts, somewhat
curiously, as an inhibitor of, among others, N-acylsphingosine
d-glucosyltransferase, the enzyme responsible for catalyzing the
firstglycosylationinthebiosynthesisofglycosphingolipids, thus
reducing the formation of these lipids in individuals who cannot
process them.
These two major advances in medicine came about by
fundamental discoveries in chemistry: nojirimycin 3 was first
discovered in Streptomyces nojiriensis,[3] then converted by
chemical curiosity into 1-deoxynojirimycin 4,[4] which was
∗ According to IUPAC-IUBMB nomenclature recommendations for carbohydrates, ‘Use of the terms ‘aza sugar’, ‘phospha sugar’, etc. should be restricted to
structures where carbon, not oxygen, is replaced by a heteroatom. The term ‘imino sugar’ may be used as a class name for cyclic sugar derivatives in which
the ring oxygen atom has been replaced by nitrogen.’
© CSIRO 2007
10.1071/CH07188
0004-9425/07/080549

550
P. J. Meloncelli et al.
HO
HO
HO
HO
HO
HO
HO
HO
N
N
NH
NH
HO
OH
HO
HO
HO
HO
OH
HO
HO
HO
1
2
3
4
Fig. 1. Structures 1–4.
HO
HO
was confirmed in part by a downfield shift of C2 relative to C3 in
the 13C NMR spectrum (δ 80.20 and δ 68.49, respectively); also,
acetylation of the product gave the penta-acetate 15 that showed
H3 at δ 5.09 in the 1H NMR spectrum. In retrospect, the result of
this glycosidation was not surprising – the 3-OH of 12 is more
sterically encumbered than the 2-OH and, as well, suffers the
electron-withdrawing effect of the neighbouring nitrile group.
Unfortunately, deacetylation of 14 under either basic or acidic
conditions, as a prelude to subsequent transformations, proved
unsuccessful.
HO
HO
HO
HO
NH
HO
NH
HO
HO
NH
HO
5
6
7
Fig. 2. Structures 5–7.
HO
O
HO
HO
HO
HO
HO
HO
HO
O
HO
O
HO
O
NH
NH
HO
HO
Inanefforttoreducethenumberofestergroupsintheproduct
of glycosylation, the diol 12 was treated with the trichloro-
acetimidate 16 to yield only the disaccharide 17 (C2, δ 79.98;
C3, δ 68.66; Scheme 2). Deacetylation of 17 was now pos-
sible under acidic conditions, to yield the diol 18. Reduction
of 18 with alane, so successful in our revamped synthesis of
isofagomine,[14] followed by the addition of di(tert-butyl) dicar-
bonate, gave the carbamate 19. Acetylation of 19 gave the
diacetate 20, the 1H NMR spectrum of which (H3, δ 5.07) again
confirmed the original structural assignment to 17.
The carbamate 19 was now used for the synthesis of
3-O-β-d-glucopyranosylisofagomine 9 and 3-O-β-d-gluco-
pyranosylnoeuromycin 11 (Scheme 3). Thus, N-deprotection
of 19, followed by treatment with hydrogen, yielded the
previously reported 9 as the hydrochloride.[17] Alternatively,
treatment of 19 first with hydrogen, which presumably
formed the hemiacetal, followed by dilute acid, gave 3-O-β-d-
glucopyranosylnoeuromycin 11, also as the hydrochloride.
Fig. 5 shows the NMR spectra of 9.[17]
8
9
Fig. 3. Structures 8 and 9.
HO
O
O
HO
HO
HO
HO
HO
HO
O
HO
O
HO
HO
NH
NH
HO
HO
HO
HO
10
Fig. 4. Structures 10 and 11.
11
β-glycosidases[11,12] and, naturally, have chosen to improve their
synthesis.[13,14] It also seemed logical to develop aza sugars that
were more substrate-like; we therefore prepared 4- and 3-O-β-d-
glucopyranosylisofagomine, 8 and 9, respectively (Fig. 3) with
the aim of inhibiting glucoside hydrolases that utilize extended
glycon binding sites. The synthesis of 8 profited from the glyco-
synthase technology developed by Withers and coworkers;[15]
at the time of the work we did not have access to a glycosyn-
thase capable of constructing a β-1,3-glycosidic linkage[16] and
so were forced to synthesize 9 along conventional lines.[17] We
were very pleased to find that both 8 and 9 were appreciably
better inhibitors against endo-acting enzymes, where multiple
sub-sites can be harnessed to increase the binding potential, than
isofagomine itself.[15,17,18]
In light of the fact, shown by Bols, that noeuromycin 7 is
sometimes a better inhibitor of glycosidases than isofagomine
6, which may be attributable to the presence of a hydroxyl group
at C2 in the former,[10] we were keen to prepare 4- and 3-O-β-
d-glucopyranosylnoeuromycin, 10 and 11, respectively (Fig. 4).
This was always going to be a challenge as the final ring closure
to form the hemiaminal has to be performed on a glycosylated
precursor, not accessible by any glycosynthase-assisted synthe-
sis. We now report the synthesis of 10 and 11, a study of their
ability to inhibit the action of some β-glycoside hydrolases, and
an X-ray structural investigation of their complexes with two
of these glycoside hydrolases. Along the way, we developed an
alternative route to the glycosylated isofagomines 8 and 9.
Fig. 6a shows the 1H NMR spectrum of 11, with characteris-
tic resonances for H5, H6glc, H1ꢀglc, and H2man.[9] H1ꢀman
and H2glc are obscured by the solvent signal at δ 4.7 but
were detectable by standard two-dimensional techniques. Also
apparent in the spectrum is the contribution from the pyranose
tautomer of 11 (Scheme 4).[9] Fig. 6b shows the 13C NMR spec-
trum of 11. The resonances for C3 are both well downfield,
as expected, from those for C4. Again obvious are small but
significant contributions from the pyranose form of 11.
We next started on a synthesis of 4-O-β-d-glucopyranosylno-
euromycin 10, naturally from our readily available diol 12. We
reasoned, in light of the exclusive glycosylation of 12 at the
2-OH group, that a standard protection reaction (ester or ether)
on 12 would give a derivative with just the 3-OH now available
for a subsequent glycosylation. Much to our surprise, treat-
ment of 12 with benzoyl chloride gave only the 3-O-benzoyl
derivative 21 (Scheme 5). It is possible that the basic conditions
employed here promote formation of an oxyanion at a position
(C3) closest to the electron-withdrawing nitrile group. Never-
theless, we saw some hope of retrieving the situation by now
protecting the 2-OH of 21 so that a subsequent debenzoylation
would give the required alcohol. However, treatment of 21 with
benzyl bromide under standard conditions proved fruitless. The
use of less basic conditions (silver carbonate) gave some of the
required benzyl ether 22 but most had apparently suffered an
elimination of benzoic acid, which furnished the unwanted
Synthesis of 8, 9, 10, and 11
In our first approach towards the aza sugars 10 and 11, we treated
the diol 12[19] with the trichloroacetimidate 13, which produced
just the disaccharide 14 (Scheme 1). The structure of the product

d-Glucosylated Derivatives of Isofagomine and Noeuromycin
551
O
OBn
AcO
AcO
O
O
OBn
OH
O
(a)
O
AcO
AcO
OAc
OAc
ϩ
OR
OC(NH)CCl3
NC
OH
AcO
NC
AcO
13
12
14 R ϭ H
15 R ϭ Ac
(b)
Scheme 1. (a) Et2OBF3, CH2Cl2, 76%; (b) Ac2O, pyridine, DMAP, CH2Cl2, 55%.
O
OBn
BnO
BnO
BnO
O
O
(a)
O
OR
OBn
20 R ϭ Ac
19 R ϭ H
ϩ
12
OH
OC(NH)CCl3
NC
BnO
(d)
AcO
BnO
16
17 R ϭ Ac
18 R ϭ H
O
OBn
O
O
(b)
(c)
OR
OBn
OR
BnO
BocHN
BnO
Scheme 2. (a) Et2OBF3, CH2Cl2, 74%; (b) HCl, MeOH, 77%; (c) (i) AlH3, THF; (ii) 1 M NaOH, (Boc)2O, 76%; (d) Ac2O, pyridine,
DMAP, CH2Cl2, 85%.
O
OBn
BnO
end of the synthesis, when the structure of the final product was
obvious, were the assignments made.The result of the glycosyla-
tion seems explicable in terms of the lack of steric encumbrance
around either hydroxyl group, and the absence of the nitrile.
We obviously had amounts of the 2-O-β-d-glucopyranosyl
derivative 33 and sought to make some use of it. Removal of the
allyl group from 33 gave the alcohol 35, and a standard sequence
as developed for our synthesis of isofagomine provided a some-
what unstable intermediate imidazylate, and then the nitrile 36
(Scheme 10).[13,14] Reduction of 36 with alane, followed by the
addition of di(tert-butyl) dicarbonate, gave the carbamate 37.
Treatment of 37 with hydrogen, and then dilute acid, again gave
3-O-β-d-glucopyranosylnoeuromycin 11, as the hydrochloride.
This result confirmed the earlier structural assignment of 33.
A similar sequence on the 3-O-β-d-glucopyranosyl deriva-
tive 34 gave the alcohol 38, the nitrile 39, and the carbamate
40 (Scheme 11). N-Deprotection of 40, followed by treat-
ment with hydrogen, yielded the previously reported 4-O-β-
d-glucopyranosylisofagomine 8 (Scheme 12).[15] Alternatively,
treatment of 40 first with hydrogen, then with dilute acid, gave
4-O-β-d-glucopyranosylnoeuromycin 10, as the hydrochloride.
Fig. 7 shows the NMR spectra of 8.[14]
O
O
(a)
(b)
OH
OBn
9
11
OH
BnO
BocHN
19
Scheme 3. (a) (i) Et2OBF3, CH2Cl2; (ii) Amberlite IRA 400 (OH−);
(iii) H2, Pd/C, AcOH, MeOH, 66%; (b) (i) H2, Pd/C, THF, H2O; (ii) 1 M
HCl, 79%.
alkene 23 as the major product. The use of benzyl trichloro-
acetimidate under acidic conditions improved the yield of 22
somewhat but the subsequent debenzoylation under basic con-
ditions (sodium methoxide) was too harsh. Some success was
achieved with the less basic potassium cyanide in methanol,
which formed the alcohol 24 (Scheme 6).
With the variable results and less than satisfactory yields
encountered in the above transformations, we turned to other
possible protecting groups for the 2-OH of 21. Although 21
could be converted into the silyl ether 25 or the acetal 26, the sub-
sequent debenzoylations proved fruitless (Scheme 7). A direct
treatment of the diol 12 with tert-butyldimethylsilyl chloride
gave mainly the desired 2-O-silyl ether 27, with the ether 28 a
minor component of the mixture (Scheme 8). The more acidic
nature of the 3-OH seems to have been outweighed by the sig-
nificant steric encumbrances associated with the hydroxyl group
and the reagent. The structures of 27 and 28 were confirmed by
conversion into the acetates 29 (δH3 4.81) and 30 (δH2 4.81),
respectively. Unfortunately, glycosylation of the alcohol 27 with
either the trichloroacetimidate 16 or the corresponding phenyl
thioglycoside was not successful.
It seemed at this stage of our work that the early intro-
duction of a nitrile was causing problems in the subsequent
synthetic steps. Therefore, we decided to protect the 4-OH and
proceed with the glycosylation first. Treatment of the readily
available alcohol 31[13] with allyl bromide, followed by mild
acidhydrolysis, gavethediol 32(Scheme9). Glycosylationof 32
with the trichloroacetimidate 16, followed by deacetylation and
benzylation to facilitate what was a difficult chromatographic
separation, gave the 2-O-β-d-glucopyranosyl 33 (39%) and 3-
O-β-d-glucopyranosyl 34 (40%) derivatives. An unequivocal
assignment of structure at this stage was not possible. Only at the
1
Fig. 8a shows the H NMR spectrum of 10, with charac-
teristic resonances for H5, H6glc, H4man, H1ꢀ, and H2.[9] The
contribution from the pyranose tautomer 41 (Scheme 12) seems
increased over that for 11 (Fig. 6a), with resonances clearly vis-
ible for H4, H3, H1ꢀ, and H1α.[9] Fig. 8b shows the 13C NMR
spectrum of 10. The resonances for C4 are both well downfield,
as expected, from those for C3. Again there are obvious and sig-
nificant contributions from the pyranose tautomer 41, in which
the α-anomer predominates.
We have depicted the various molecules in the above schemes
and figures in the conformations expected or observed. It is well
known that α-d-arabinose prefers to exist in aqueous solution in
the 1C4 conformation, and simple derived glycosides should be
little different (Scheme 13).[20] One way of distinguishing the
two chair conformations is to compare the values of the various
vicinal coupling constants. For α-d-arabinose in the 4C1 confor-
mation, one value for J4,5 must be large (8–10 Hz), with all other
1
vicinal values small; in the C4 conformation, there can be no
large value for J4,5, but J1,2 and J2,3 are now required to be around

552
P. J. Meloncelli et al.
(a)
HO
HO
HO
HO
O
HO
O
H1Ј
NH
HO
9
H6
H2, H6
H5
4.00
3.50
3.00
2.50
2.00
ppm
(b)
C3Ј, C5Ј
C1Ј
C2Ј
C4Ј, C4
C6Ј, CH2O
C2, C6
C5
C3
100
90
80
70
60
50
40
ppm
Fig. 5. (a) 1H (600 MHz) and (b) 13C (150.9 MHz) NMR spectra of 3-O-β-d-glucopyranosylisofagomine 9 hydrochloride in D2O.
8 Hz. For molecules that possess the β-l-xylo configuration, it
is the 1C4 conformation that is expected, with all but one of the
vicinal coupling constants large. Table 1 summarizes the values
of the vicinal coupling constants for some of the molecules in
the above schemes.
conformation, while 24, 25, 26 (one diastereoisomer), 28, 30,
and 36 exist in a 4C1 conformation, with 12, 15, 22, 26 (other
diastereoisomer), 29, 32, and 39 showing contributions from
both conformations.Any rationalization of these conformational
preferences seems fraught with danger; for example, the 2-O-
silyl ether 27 seems to prefer a 1C4 conformation, whereas the
isomeric 3-O-silyl ether 28 exists in a 4C1 conformation.
It is apparent from a perusal of the data in Table 1 that 14,
17, 19, 21, 27, 33, and 35 exist in chloroform solution in a 1C4

d-Glucosylated Derivatives of Isofagomine and Noeuromycin
553
(a)
HO
HO
O
HO
O
HO
HO
NH
HO
HO
11
H1Јman, H2glc
H6glc
H1Јglc
H2man
H5
H1pyr
H3pyr
CH2Npyr
5.50
ppm
5.00
4.50
4.00
3.50
3.00
2.50
2.00
(b)
C6Ј, CH2O
C2Ј
C3man
C5, C6man
C1Јman
C2glc, C3glc
C1Јglc
C6glc
100
90
80
70
60
50
40
ppm
Fig. 6. (a) 1H (600 MHz) and (b) 13C (150.9 MHz) NMR spectra of 3-O-β-d-glucopyranosylnoeuromycin 11 hydrochloride in D2O.
Resonances emanating from the diastereoisomer with an equatorial 2-OH are labelled ‘glc’, those with an axial 2-OH as ‘man’, and
those from the pyranose tautomer as ‘pyr’.
Enzyme Inhibition Studies with 10 and 11
as substrates, respectively, and for 11 with a family 26 lichenase
(CtLic26A) from Clostridium thermocellum using β-d-Glcp-
(1→4)-β-d-Glcp-(1→3)-β-d-GlcpMU. The noeuromycin ana-
logues were shown to be highly potent inhibitors of the enzymes,
with Ki values of 24 and 28 nM for 10 with Cel5A and Cex,
Inhibition constants for 10 with a family 5 endo-glucanase
(Cel5A) from Bacillus agaradhaerens and a family 10 endo-
xylanase (Cex) from Cellulomonas fimi were determined using
2,4-dinitrophenyl β-lactoside and 4-nitrophenyl β-xylobioside

554
P. J. Meloncelli et al.
HO
HO
OH
HO
HO
O
O
O
HO
HO
11
12
OH
NH2
O
HO
O
HO
HO
CHO
HO
H2N
Scheme 4.
NC
OBn
OBn
O
OBn
O
O
(a)
(b)
OH
ϩ
OBz
NC
OBz
NC
OBn
OBn
23
21
22
15%
54%
Scheme 5. (a) BzCl, Et3N, CH2Cl2, 78%; (b) BnBr, Ag2CO3, CH2Cl2.
BnO
been observed, suggestions have been made that this could be
due to any, or all, of the following: a slow ‘induced fit’ of
the enzyme due to difficulties in directly binding a transition
state analogue; slow proton transfer events in the active site;[12]
slow encounter of enzyme and inhibitor at the very low con-
centrations that are necessarily employed. There is, as yet, no
indication regarding which of these explanations is the more
likely in this case. It is interesting to note that while isofagomine
6 has been shown to be around a 4-fold better inhibitor than
noeuromycin 7 with the family 1 beta-glucoside from Thermo-
toga maritima TmGH1,[12] the noeuromycin-derived inhibitor
10 is actually 30-fold more potent than its isofagomine counter-
part 8 with Cel5A,[18] and likewise 11 is 7-fold more potent
than 9 with CtLic26A.[21] These considerable enhancements
in inhibition with noeuromycin-derived compounds over the
comparative isofagomine compounds presumably arise from
interactions with the C2 hydroxyl group. The inhibition data
clearly show the potency that can be derived from making
inhibitors more ‘substrate-like’ in order for multiple sub-sites
in the enzyme to be harnessed. Indeed, one should consider
the potential of compounds with additional sugar moieties. The
inhibition constant for the ‘disaccharide 8’ with Cel5A was
determined to be 700 nM, whereas the trisaccharide version was
considerably more potent with a Ki value of 5 nM, which reflects
important interactions that are formed at the −3 sub-site.[18] If a
similar factor was observed with a trisaccharide version of 10,
then potentially a picomolar inhibitor could result.
O
(a)
(b)
NC
21
22
OH OBn
24
Scheme 6. (a) CCl3C(NH)OBn, CF3SO3H, CH2Cl2, 50%; (b) KCN,
MeOH, 67%.
O
Et3 SiO
O
(b)
(a)
NC
O
21
O
NC
OBz OBn
OBz OBn
26
25
Scheme 7. (a) Et3SiCl, DMAP, pyridine, 60%; (b) 3,4-dihydro-2H-pyran,
CSA, CH2Cl2, 94%.
HO
OBn
O
O
(a)
NC
OSiMe2But
ϩ
12
OR
NC
ButMe2SiO
OBn
27 R ϭ H, 70%
29 R ϭ Ac
28 R ϭ H, 29%
30 R ϭ Ac
(b)
(c)
Scheme 8. (a) ButMe2SiCl, imidazole, DMF; (b)Ac2O, DMAP, pyridine,
65%; (c) Ac2O, DMAP, pyridine, 95%.
Structural Investigations with 10 and 11
The structures of 10 in complex with Cel5A and 11 in complex
with CtLic26A, respectively, were solved using X-ray crystal-
lography, with data to 1.50 and 1.20 Å resolution, respectively.
The Cel5A complex was refined to an Rcryst of 0.11 and Rfree of
0.14, and the CtLic26A structure was refined to an Rcryst of 0.12
and Rfree of 0.14 (Table 3).
respectively, and 168 nM for 11 with CtLic26A (Table 2). In
cases where full Ki determinations were employed (Cel5A, Cex,
and CtLic26A), competitive inhibition was observed. In the
other case (TmGH1), by analogy, competitive inhibition was
assumed for data analysis.
Slow-onset inhibition, which has previously been observed
with 7 and several isofagomine-derived compounds, was
observed with 10 and 11. Indeed, the slow-onset inhibition of
10 with Cel5A and Cex was among the most dramatic observed
with the different enzyme systems where it has been reported,
with equilibration times to a constant rate of inhibition taking
in the order of 1 h; this phenomenon may in part be a conse-
quence of the four tautomeric forms (two hemiaminal and two
pyranose) that are available for a molecule such as 10 (Scheme
12). In other cases, where such slow binding behaviour has
In both structures the noeuromycin moiety is found in an
4
undistorted C1 conformation, in a ‘gluco’ form with a clear
equatorial hydroxyl group at C2 (Fig. 9).[22]
The majority of the interactions in the Cel5A complex with
10 are similar to those described for the complex with 8.[18] The
nitrogen atom (N1) at the ‘anomeric’ position hydrogen bonds
with the Oε2 atom of the acid/base residue, Glu139, and also
weakly (at a distance of 3.2 Å) with the Oε1 atom. The same
nitrogen atom also interacts with the Oε2 atom of the nucleophile
residue, Glu228. The N1 atom does not, however, interact with a

d-Glucosylated Derivatives of Isofagomine and Noeuromycin
555
BnO
BnO
OAll
O
O
BnO
O
BnO
33
34
BnO
O
OBn
BnO
HO
OBn
OH
O
O
(a)
(b)
AllO
ϩ
O
HO
BnO
BnO
BnO
BnO
O
O
BnO
O
31
32
BnO
AllO
Scheme 9. (a) (i) CH2CHCH2Br, NaH, DMF; (ii) CSA, MeOH, 89%; (b) (i) 16, Et2OBF3, CH2Cl2; (ii) KCN, MeOH;
(iii) BnBr, NaH, DMF.
BnO
BnO
OBn
OBn
BnO
BnO
BnO
OH
O
O
(a)
(b)
O
BnO
O
BnO
O
33
O
NC
BnO
OBn OBn
35
36
BnO
O
BnO
OBn
OBn
(c)
(d)
O
11
O
BocHN
OBn OBn
37
Scheme 10. (a) (i) (Ph3P)3RhCl, EtOH; (ii) 1 M HCl, 91%; (b) (i) (Me3Si)2NLi, Im2SO2, THF; (ii) Me3SiCN,
Bu4NF, MeCN, 66%; (c) (i) AlH3, THF; (ii) 1 M NaOH; (iii) (Boc)2O, 65%; (d) (i) H2, Pd/C, THF, H2O; (ii) 1 M
HCl, 97%.
BnO
BnO
BnO
BnO
BnO
BnO
BnO
OBn
O
NC
(a)
(b)
O
O
O
BnO
O
BnO
O
34
BnO
HO
BnO
38
39
BocHN
BnO
BnO
BnO
OBn
O
(c)
O
BnO
O
BnO
40
Scheme 11. (a) (i) (Ph3P)3RhCl, EtOH; (ii) 1 M HCl, 98%; (b) (i) (Me3Si)2NLi, Im2SO2, THF; (ii) Me3SiCN, Bu4NF,
MeCN, 43%; (c) (i) AlH3, THF; (ii) 1 M NaOH; (iii) (Boc)2O, 81%.
HO
HO
O
HO
1Ј
4
(a)
(b)
HO
O
HO
8
40
HO
HO
O
HO
O
OH
HO
O
NH
HO
HO
H2N
HO
10
41
Scheme 12. (a) (i) CF3CO2H; (ii) Amberlite IRA 400 (OH−), MeOH; (iii) H2, Pd/C, AcOH, MeOH, 75%; (b) (i) H2, Pd/C,
THF, H2O; (ii) 1 M HCl, 72%.
watermoleculeasseeninthecomplexwith 8;instead, inasimilar
position there is an oxygen atom of a glycerol molecule (present
in the crystal cryo-protectant) but this is at a distance of 3.8 Å.
The presence of the C2 hydroxyl group in 10 means hydrogen
bonds are formed with the Oε1 atom of Glu228 and the amine
group of Asn138, which may well contribute to the enhanced
affinity compared to the isofagomine-related ‘disaccharide’ 8.
The importance of interactions made between the enzyme and
the C2 hydroxyl group has been illustrated by numerous kinetic
studies using substrates where the group has been removed. For
example, with the Clan GHA endo-xylanase, Cex, it was shown
that the C2 hydroxyl group contributes up to 10 kcal mol−1
to stabilizing interactions at the transition state. Furthermore
X-ray crystallographic analyses of the covalent glycosyl enzyme
intermediate of Cex, and of its complexes with transition state
analogues, confirm key interactions between the C2 hydroxyl
group and the enzyme, notably to the carbonyl oxygen of the
nucleophile and the amide side chain of a conserved asparagine
preceding the acid/base residue. [23,24]
The interactions between 11 and CtLic26A are again simi-
lar to those observed for the complex with 9.[21] The nitrogen
atom forms hydrogen bonds with the Oε2 atom of the acid/base
residue, Glu109, and with the Oε1 atom of the nucleophile
residue, Glu222.ThepresenceofthehydroxylgroupatC2allows
additional interactions with the Oε2 atom of Glu222, and His108
and Tyr115, which are not possible with 9.

556
P. J. Meloncelli et al.
(a)
HO
O
O
HO
HO
HO
HO
NH
HO
8
H1Ј
H3Ј
H2Ј
H6
H2, H6
H5
4.50
4.00
3.50
3.00
2.50
(b)
C4Ј, C5Ј
C2Ј
C3Ј
C1Ј
C5
C6Ј, CH2O
C3
C4
C2
C6
100
90
80
70
60
50
40
Fig. 7. (a) 1H (600 MHz) and (b) 13C (150.9 MHz) NMR spectra of 4-O-β-d-glucopyranosylisofagomine 8 hydrochloride in D2O.
Conclusions
the productive interaction at O2, contributes to their enhanced
affinities.
Glycosylation of the diol 12 is regioselective (at O2) and leads
to successful syntheses of 3-O-β-d-glucopyranosylisofagomine
9 and 3-O-β-d-glucopyranosylnoeuromycin 11. However, other
functionalizations of 12 were not so selective and a change
in strategy with late introduction of the requisite nitrile group
was required to provide 4-O-β-d-glucopyranosylisofagomine
8 and 4-O-β-d-glucopyranosylnoeuromycin 10. Both of the
d-glucosylated noeuromycins 10 and 11 were excellent (nano-
molar) inhibitors of two endo-glycosidases. Subsequent struc-
tural investigations of 10 and 11 in complex with the appropriate
glycosidase clearly show that the compounds do harness the
adjacent sub-sites, as expected, and that this, together with
Experimental
Synthesis of 8, 9, 10, and 11
General experimental procedures have been given previously.[25]
Benzyl 4-C-Cyano-4-deoxy-2-O-(tetra-O-acetyl-
β-D-glucopyranosyl)-α-D-arabinoside 14
A mixture of tetra-O-acetyl-β-d-glucopyranosyl trichloro-
acetimidate 13 (254 mg, 0.531 mmol),[26] the diol 12 (110 mg,
0.443 mmol), and 4 Å molecular sieves (100 mg) in dry CH2Cl2

d-Glucosylated Derivatives of Isofagomine and Noeuromycin
557
(a)
HO
1Ј
4
HO
O
HO
HO
HO
O
HO
HO
HO
O
O
OH
HO
O
NH
HO
H N
2
HO
HO
10
41
H2glc
H1Јα, H1Јβ
CH2Nα, CH2Nβ
H1Јglc, H1Јman
H6glc
H4man
H2man
H5glc,
H5man
H1β
H3α
H3β
H4α, H4β
5.50
5.00
4.50
4.00
3.50
3.00
2.50
2.00
(b)
C6man
CH2Nα
C1Јglc
C1Јman
C4glc
C2glc
C5glc, C5man
C6glc
C2man
C4man
C6Јglc, C6Јman
CH2Oglc, CH2Oman
C5α
C6Јα
C1Јα C1α
C3α
C4α
C1β
100
90
80
70
60
50
40
Fig. 8. (a) 1H (600 MHz) and (b) 13C (150.9 MHz) NMR spectra of 3-O-β-d-glucopyranosylnoeuromycin 10 hydrochloride in
D2O. Resonances emanating from the diastereoisomer with an equatorial 2-OH are labelled ‘glc’, those with an axial 2-OH as ‘man’,
and those from the two anomers of the pyranose tautomer as either ‘α’ or ‘β’.
was stirred (room temp., 3 h). The mixture was cooled (−60◦C),
treated with Et2OBF3 (20 µL), and allowed to warm (room
temp.). Treatment with Et3N (100 µL), followed by filtra-
tion, concentration of the filtrate, and flash chromatography
(EtOAc/petrol, 1/1) gave the alcohol 14 (195 mg, 76%) as a
colourless oil, [α]D +25.1◦. δH (600 MHz) 1.83, 1.98, 2.02, 2.10
(4 × s, 12H, CH3), 3.20 (m, H4), 3.58 (dd, J5,5 12.3, J4,5 2.9,
H5), 3.66 (dd, J2,3 7.7, J1,2 6.2, H2), 3.76–3.79 (m, H5ꢀ), 3.85
J5 ,6 4.2, H6ꢀ), 4.20 (dd, J5 ,6 2.4, H6ꢀ), 4.45 (d, H1), 4.5ꢀ4, 4.87
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(AB, J 11.6, PhCH2), 4.68 (d, J1 ,2 8.1, H1 ), 4.99 (m, H2 , H4 ),
5.20 (dd, J 9.5, 9.5, H3ꢀ), 7.31–7.37 (m, Ph). δC (150.9 MHz)
20.50, 20.63, 20.75 (CH3), 34.21 (C4), 60.44 (C5), 61.85 (C6ꢀ),
68.38 (C4ꢀ), 68.49 (C3), 70.91 (PhCH2), 71.08 (C2ꢀ), 72.18
(C5ꢀ), 72.54 (C3ꢀ), 80.20 (C2), 99.94 (C1), 101.05 (C1ꢀ), 117.69
(CN), 127.77–136.50 (Ph), 169.44, 169.52, 170.15, 170.89 (4C,
C=O). m/z (FAB) 580.2045 (C27H34NO13 [M + H]+ requires
580.2030).
ꢀ
ꢀ
(dd, J3,4 5.0, H3), 4.13 (dd, J4,5 4.2, H5), 4.14 (dd, J6 ,6 12.3,

558
P. J. Meloncelli et al.
HO
Table 2. Summary of kinetic data with isofagomine- and noeuro-
mycin-derived compounds with the Thermotoga maritima family GH1
β-glycosidase TmGH1,[12] Cel5A, and Cex (ref. [18] and this work), and
CtLic26A (ref. [21] and this work)
OH
OH
O
O
HO
OH
HO
HO
OH OH
4C1
1C4
α-D-arabinose
Test
Isofagomine-derived
compounds
Noeuromycin-derived
compounds
HO
HO
OH
OH
O
O
TmGH1 with 6 and 7
Cel5A with 8 and 10
Cex with 8 and 10
23 5 nM
700 40 nM
3.5 0.1 µM
1.2 0.1 µM
88 20 nM
26 2 nM
28 2 nM
OH
OH OH
4C1
1C4
CtLic26A with 9 and 11
168 20 nM
β-L-xylose
Scheme 13.
Table 1. Vicinal coupling constants (Hz) for various compounds described in the schemes
Compound
J1,2
J2,3
J3,4
J4,5
Conformation
4
12
14
15
17
18
19
20
21
22
24
25
26
4.9
6.2
4.5
6.0
6.9
6.5
6.8
7.7
6.4
7.7
3.9
5.0
4.3
5.0
3.9, 6.2
2.9, 4.2
3.5, 6.7
2.9, 4.1
4.0
5.1
9.3
2.9, 4.3
3.5, 6.6
4.4, 9.7
3.7, 8.2
1C4 ꢀ C1
1C4
4
1C4 ꢀ C1
1C4
(1C4)A
1C4
7.7
5.0
(4C1)
1C4
5.9
4.5
3.0
3.3
2.1
4.1
6.4
4.7
2.0
2.1
4.9
8.0
7.8
6.1
2.1
8.0
6.4
4.6
5.0
3.8
6.1
7.8
6.6
3.8
3.4
5.8
8.1
8.3
7.3
3.5
3.8
7.7
5.9
5.8
4.9
4.4
4
1C4 ꢀ C1
4C1
4C1
4C1
4.8
3.7
4.2
5.3
4.4
2.7
3.4
6.1
11.1
4
(1C4 ꢀ C1)
27
29
28
30
32
33
34
35
36
37
38
39
40
2.7, 3.2
3.4, 6.2
4.7, 10.9
3.7, 9.5
5.3
1C4
4
1C4 ꢀ C1
4C1
4C1
4
1C4 ꢀ C1
4.3, 9.8
1C4
(1C4)
1C4
7.4
3.5
3.8
4.6
4.4
10.0
4C1
(4C1)
(1C4)
6.3
4.2
4
3.4
(1C4 ꢀ C1)
5.8
AThe assignment is tentative, with not enough data to support the claim.
(PhCH2), 71.41 (C2ꢀ), 72.12 (C5ꢀ), 72.85 (C3ꢀ), 74.00 (C2),
99.53 (C1), 100.33 (C1ꢀ), 116.78 (CN), 127.96–136.73 (Ph),
169.30, 169.55, 170.13, 170.33, 170.81 (5C, C=O). m/z (FAB)
622.2147 (C29H36NO14 [M + H]+ requires 622.2135).
Benzyl 3-O-Acetyl-4-C-cyano-4-deoxy-2-O-(tetra-O-acetyl-
β-D-glucopyranosyl)-α-D-arabinoside 15
The disaccharide 14 (40 mg) in CH2Cl2 (1.5 mL) was
treated with pyridine (200 µL), Ac2O (100 µL) and 4-
dimethylaminopyridine (DMAP, 2 mg) and kept (room temp.,
2 h).The solution was treated with MeOH, concentrated and sub-
jected to flash chromatography (EtOAc/petrol, 1/1) to give the
pentaacetate 15 (32 mg, 55%) as a colourless oil, [α]D +9.8◦.
δH (600 MHz) 1.95, 1.99, 2.02, 2.08, 2.09 (5 × s, 15H, CH3),
3.36–3.39 (m, H4), 3.68 (dd, J5,5 11.8, J4,5 3.5, H5), 3.68–
Benzyl 2-O-(2-O-Acetyl-3,4,6-tri-O-benzyl-β-D-glucosyl)-
4-C-cyano-4-deoxy-α-D-arabinoside 17
A mixture of 2-O-acetyl-3,4,6-tri-O-benzyl-β-d-glucosyl tri-
chloroacetimidate 16 (1.165 g, 1.835 mmol),[27] the diol 12
(457 mg, 1.84 mmol), and 4 Å molecular sieves (1.0 g) in dry
CH2Cl2 (15 mL) was stirred (room temp., 3 h). The mixture was
cooled (−60◦C), treated with Et2OBF3 (100 µL), and allowed to
warm (room temp.). Treatment with Et3N (100 µL), followed by
filtration, concentration of the filtrate, and flash chromatography
(EtOAc/petrol, 1/1) gave the disaccharide 17 (910 mg, 74%)
as a colourless oil, [α]D +8.5◦. δH (600 MHz) 1.77 (s, CH3),
3.20–3.22 (m, H4), 3.53–3.57 (m, H5ꢀ), 3.58 (dd, J5,5 12.2,
3.73 (m, H5ꢀ), 3.90 (dd, J2,3 6.4, J1,2 4.5, H2), 4.08 (dd, J6 ,6
ꢀ
ꢀ
12.3, J5 ,6 2.3, H6ꢀ), 4.19 (dd, J4,5 6.7, H5), 4.30 (dd, J5 ,6 5.7,
ꢀ
ꢀ
ꢀ
ꢀ
H6ꢀ), 4.51, 4.83 (AB, J 11.4, PhCH2), 4.54 (d, H1), 4.73 (d,
J1 ,2 8.0, H1ꢀ), 4.96 (dd, J2 ,3 9.6, H2ꢀ), 5.03 (dd, J3 ,4 ≈ J4 ,5
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
9.8, H4ꢀ), 5.09 (dd, J3,4 4.3, H3), 5.17 (dd, H3ꢀ), 7.25–7.35 (m,
Ph). δC (150.9 MHz) 20.70, 20.72, 20.74, 20.80 (CH3), 30.88
(C4), 58.85 (C5), 62.01 (C6ꢀ), 67.75 (C4ꢀ), 68.45 (C3), 70.77

d-Glucosylated Derivatives of Isofagomine and Noeuromycin
559
Table 3. Data processing and refinement statistics for Cel5A in complex with 10, and
CtLic26A in complex with 11
Parameters
Cel5A
CtLic26A
Space group
P212121
P212121
a, b, c [Å]
α, β, γ [◦]
a 54.9, b 69.6, c 76.7
90, 90, 90
a 49.8, b 63.5, c 78.2
90, 90, 90
Resolution (outer shell)
Rmerge (outer shell)
<I/σI > (outer shell)
20–1.50 (1.55–1.50)
0.055 (0.260)
28.5 (6.3)
50–1.20 (1.26–1.20)
0.046 (0.114)
21.6 (9.7)
Completeness [%] (outer shell)
Redundancy (outer shell)
Number of unique reflections
Rcryst/Rfree
99.9 (100.0)
5.5 (5.4)
45273
98.0 (98.0)
4.3 (4.1)
72588
0.11/0.14
0.12/0.14
Number of atoms
Protein
Ligand
2532
22
2305
22
Water
409
505
B-factors
Protein
9.9
7.2
Ligand
7.1
5.0
Water
26.3
21.0
R.M.S. deviations
Bond lengths [Å]
Bond angles [◦]
PDB code
0.012
1.44
2V38
0.014
1.53
2V3G
(a)
Ala234
Ala234
Glu139
Glu139
Lys267
(b)
Tyr66
Lys267
Gln138
Glu228
Tyr66
Gln138
Glu228
Trp262
Trp262
His35
His35
His101
His101
Glu261
Glu261
Tyr115
Lys260
Tyr115
Lys260
Glu109
Glu109
Glu70
Glu70
Glu258
Glu258
His108
Glu222
His108
Glu222
Fig. 9. Divergent stereo ball-and-stick representation of: (a) Cel5A in complex with 10; and (b) CtLic26A in
complex with 11. Observed electron density for the maximum likelihood weighted 2Fobs-Fcalc map is contoured at
1σ (Cel5A) and 1.5σ (CtLic26A); figures were drawn using BOBSCRIPT.[22]
J4,5 2.9, H5), 3.64–3.75 (m, 5H, H2, H3ꢀ, H4ꢀ, H6ꢀ), 3.88 (dd,
J3,4 5.0, J2,3 7.7, H3), 4.13 (dd, J4,5 4.1, H5), 4.46 (d, J1,2 6.0,
H1), 4.52, 4.88 (AB, J 11.8,ꢀPhCH2), 4.54, 4.78 (AB, J 10.9,
127.82–138.10 (Ph), 169.64 (C=O). m/z (FAB) 722.2936
(C42H44NO10 [M − H]+ requires 722.2965).
ꢀ
ꢀ
PhCH2), 4.56 (d, J1 ,2 7.8, H1 ), 4.57, 4.60 (AB, J 12.2, PhCH2),
4.67, 4.79 (AB, J 11.4, PhCH2), 5.01 (dd, J2 ,3 9.1, H2ꢀ), 7.25–
7.37, 7.16–7.19 (2 × m, Ph). δC (150.9 MHz) 20.78 (CH3), 34.22
(C4), 60.45 (C5), 68.37 (C6ꢀ), 68.66 (C3), 70.83, 73.65, 75.17,
75.19 (4C, PhCH2), 72.96 (C2ꢀ), 74.83 (C5ꢀ), 77.64, 82.69
(C3ꢀ, C4ꢀ), 79.98 (C2), 100.03 (C1), 101.27 (C1ꢀ), 117.73 (CN),
Benzyl 4-C-Cyano-4-deoxy-2-O-(3,4,6-tri-O-benzyl-
β-D-glucosyl)-α-D-arabinoside 18
ꢀ
ꢀ
Thedisaccharide17 (270 mg)inMeOH(40 mL)wastreatedwith
HCl in MeOH (1 M, 2 mL) and stirred (40◦C, 14 h). The solu-
tion was neutralized with Et3N, concentrated, and subjected to

560
P. J. Meloncelli et al.
flash chromatography (EtOAc/petrol, 1/1) to give the diol 18 as
a colourless oil (195 mg, 77%), [α]D −4.0◦. δH (600 MHz) 3.22–
3.26 (m, H4), 3.50–3.69 (m, 8H, H2, H2ꢀ, H3, H3ꢀ, H4ꢀ, H5, H5ꢀ,
(C3ꢀ), 98.24 (C1), 100.72 (C1ꢀ), 127.32–138.03 (Ph), 155.72
(NC=O), 169.26, 170.38 (2C, CH3C=O). m/z (FAB) 870.4078
(C49H60NO13 [M + H]+ requires 870.4065).
H6ꢀ), 3.87 (dd, J6 ,6 7.8, J5 ,6 5.0, H6ꢀ), 4.18 (dd, J5,5 12.2, J4,5
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
4.0, H5), 4.40 (d, J1 ,2 7.6, H1 ), 4.50, 4.57 (AB, J 12.2, PhCH2),
4.51 (d, J1,2 6.9, H1), 4.53, 4.92 (AB, J 11.0, PhCH2), 4.57, 4.81
(AB, J 12.0, PhCH2), 4.82, 4.88 (AB, J 11.8, PhCH2), 7.15–7.18,
7.25–7.38 (2 × m, Ph). δC (150.9 MHz) 34.27 (C4), 60.71 (C5),
68.54 (C6ꢀ), 68.80 (C3), 70.92, 73.65, 75.18, 75.29 (4C, PhCH2),
74.75, 75.02 (C2ꢀ, C5ꢀ), 77.13, 80.99, 84.22 (C2, C3ꢀ, C4ꢀ), 99.83
(C1), 103.90(C1ꢀ), 117.91(CN), 127.91–138.64(Ph). m/z (FAB)
681.2979 (C40H43NO9 [M]+• requires 681.2938).
(3R,4R,5R)-3-(β-D-Glucopyranosyloxy)-
5-(hydroxymethyl)piperidin-4-ol (3-O-β-D-
Glucopyranosylisofagomine) 9 Hydrochloride
The carbamate 19 (25 mg) in CH2Cl2 (1 mL) was treated with
Et2OBF3 (100 µL) and stirred (room temp., 2 h). The solution
was then treated with resin (Amberlite IRA 400, OH−) until
neutral, filtered, and concentrated. The residue was taken up in
MeOH/AcOH (100/1, 15 mL), and then treated with Pd/C (10%,
5 mg) and H2 (1 atm, 12 h). Filtration followed by concentra-
tion of the filtrate gave a colourless foam that was dissolved
in hydrochloric acid (1 M, 1 mL) and applied to a cation-
exchange column (Dowex 50W-X2, H+ form). The column
was washed with water and eluted with aqueous NH3 (1.5 M),
the eluate was concentrated and the residual foam taken up in
a little hydrochloric acid (1 M, 1 mL) and again concentrated
to give 3-O-β-d-glucopyranosylisofagomine 9 hydrochloride
(6.5 mg, 66%) as a colourless glass. The 1H (600 MHz) and
13C (150.9 MHz) NMR spectroscopic data (Fig. 5) were in good
agreement with those reported.[17]
Benzyl 4-C-[(tert-Butoxycarbonyl)amino]methyl-4-deoxy-
2-O-(3,4,6-tri-O-benzyl-β-D-glucosyl)-α-D-
arabinoside 19
[28]
Freshly prepared AlH3
in tetrahydrofuran (THF) (1.23 M,
2.0 mL, 2.5 mmol) was added to the diol 18 (160 mg,
0.236 mmol) in dry THF (4 mL, 0◦C) and the solution stirred
(2 h). The mixture was quenched by the addition of NaOH
(1 mL, 1.0 M), followed by treatment with (Boc)2O (180 mg,
0.826 mmol) (room temp., 2 h). The mixture was then filtered,
diluted with EtOAc (100 mL), dried, concentrated, and subjected
to flash chromatography (EtOAc/petrol, 1/1) to give the carba-
mate 19 (140 mg, 76%) as a colourless solid.A small amount was
further purified by recrystallization, mp 128–129◦C (MeOH)
(Found: C 68.7, H 7.1, N 1.9. C45H55NO11 requires C 68.8, H
7.0, N 1.9%.) [α]D +20.8◦. δH (600 MHz) 1.44 (s, 9H, CH3),
2.19–2.23 (m, H4), 3.30–3.37 (br m, CH2N), 3.48–3.60 (m, 6H,
(2R/2S,3S,4R,5R)-3-(β-D-Glucopyranosyloxy)-
5-(hydroxymethyl)piperidine-2,4-diol (3-O-β-D-
Glucopyranosylnoeuromycin) 11 Hydrochloride
(a) The carbamate 19 (25 mg) in THF/H2O (1/1, 15 mL) was
treated with Pd/C (10%, 10 mg) and H2 (1 atm, 48 h). The mix-
ture was filtered, concentrated, and subjected to flash chroma-
tography (THF/H2O, 20/1) to give presumably the hemiacetal as
a colourless oil. This oil was then dissolved in hydrochloric acid
(1 M, 1.5 mL) and left for 15 min. Concentration of the mixture
then gave 3-O-β-d-glucopyranosylnoeuromycin 11 hydrochlo-
ride (9.1 mg, 79%) as a colourless glass. δH (600 MHz, D2O)
1.85–1.95 (m, H5), 2.90 (dd, J 13.1, 13.1, H6glc), 3.22–3.85
(H2ꢀ, H3, H3ꢀ, H4, H4ꢀ, H5ꢀ, H6, H6man, H6ꢀ, CH2O), 4.51 (d,
H2, H2ꢀ, H3ꢀ, H4ꢀ, H5, H5ꢀ), 3.62 (dd, J6 ,6 10.7, J5 ,6 5.0, H6ꢀ),
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3.68 (dd, J5 ,6 2.0, H6 ), 3.89 (dd, J2,3 7.7, J3,4 5.0, H3), 3.91 (dd,
J5,5 12.6, J4,5 5.1, H5), 4.35 (d, J1 ,2 7.6, H1ꢀ), 4.51, 4.54 (AB,
J 12.2, PhCH2), 4.52 (d, J1,2 6.5, H1), 4.54, 4.83 (AB, J 12.2,
PhCH2), 4.58, 4.83 (AB, J 11.5, PhCH2), 4.80, 4.94 (AB, J 11.2,
PhCH2), 7.15–7.17, 7.25–7.37 (2 × m, Ph). δC (150.9 MHz)
28.33(CH3), 39.05(C4), 39.18(CH2N), 62.33(C5), 68.53(C6ꢀ),
70.35, 73.37, 74.95, 74.96 (4C, PhCH2), 70.72 (C3), 74.71,
74.86 (C2ꢀ, C5ꢀ), 76.99, 79.06, 84.07 (C2, C3ꢀ, C4ꢀ), 80.22
(CH3C), 99.67 (C1), 103.56 (C1ꢀ), 127.59–138.49 (Ph), 156.00
(C=O). m/z (FAB) 786.3897 (C45H56NO11 [M + H]+ requires
786.3853).
ꢀ
ꢀ
J1 ,2 7.9, H1ꢀglc), 4.67–4.73 (m, H1ꢀman, H2glc), 5.34 (d, J2,3
2.4, H2man). δC (150.9 MHz, D2O) 38.23 (C6glc), 40.42, 40.71
(C5), 41.08 (C6man), 58.75, 58.85, 60.52 (C6ꢀ, CH2O), 65.18,
68.25, 69.32, 69.42, 75.36, 75.45, 75.80, 75.96 (C3ꢀ, C4, C4ꢀ,
C5ꢀ), 72.69, 73.11 (C2ꢀ), 76.00 (C2man), 77.97 (C3man), 80.64
(C3glc), 80.75 (C2glc), 101.00 (C1ꢀglc), 102.19 (C1ꢀman). m/z
(FAB) 326.1454 (C12H24NO9 [M + H]+ requires 326.1451).
(b) The carbamate 37 (40 mg) was treated as for the carba-
mate 19 to again yield 3-O-β-d-glucopyranosylnoeuromycin 11
hydrochloride (14 mg, 97%) as a colourless glass.
ꢀ
ꢀ
Benzyl 3-O-Acetyl-2-O-(2-O-acetyl-3,4,6-tri-O-benzyl-
β-D-glucosyl)-4-C-[(tert-butoxycarbonyl)amino]methyl-
4-deoxy-α-D-arabinoside 20
The carbamate 19 (6.0 mg) in CH2Cl2 (1.5 mL) was treated
with pyridine (100 µL), Ac2O (50 µL), and DMAP (0.5 mg),
and stirred (room temp., 2 h). The solution was treated with
MeOH, concentrated, and subjected to flash chromatography
(EtOAc/petrol, 1/1) to give the diacetate 20 (5.9 mg, 85%) as a
colourless oil, [α]D +60.0◦. δH (600 MHz) 1.41 (s, 9H, CH3C),
1.88, 1.98 (2 × s, 6H, CH3C=O), 2.27–2.33 (m, H4), 3.05–3.10
Benzyl 3-O-Benzoyl-4-C-cyano-4-deoxy-
α-D-arabinoside 21
The diol 12 (39 mg, 0.16 mmol) in dry CH2Cl2 (2 mL) was
treated with BzCl (26 mg, 0.19 mmol) and Et3N (19 mg,
0.19 mmol) and stirred (room temp., 12 h). The solution was
then concentrated and subjected to flash chromatography
(EtOAc/petrol, 1/1) to give the benzoate 21 (42 mg, 78%) as
a colourless oil, [α]D −24.8◦. δH (600 MHz) 3.48–3.51 (m, H4),
3.75 (dd, J5,5 12.1, J4,5 2.9, H5), 4.02–4.04 (m, H2), 4.29 (dd,
J4,5 4.3, H5), 4.50 (d, J1,2 5.9, H1), 4.60, 4.92 (AB, J 11.5,
PhCH2), 5.21 (dd, J2,3 8.0, J3,4 4.9, H3), 7.20–7.30 (m, Ph).
δC (150.9 MHz) 32.11 (C4), 60.46 (C5), 68.81, 70.02 (C2, C3),
70.76 (PhCH2), 101.28 (C1), 116.81 (CN), 128.00–136.48 (Ph),
(m, CH2N), 3.45–3.48 (m, 2H, H5, H5ꢀ), 3.62 (dd, J3 ,4 9.3,
ꢀ
ꢀ
J2 ,3 8.7, H3ꢀ), 3.69–3.81 (m, 3H, H2, H4ꢀ, H6ꢀ), 3.87 (dd, J5,5
11.4, J4,5 9.3, H5), 4.42, 4.80 (AB, J 11.7, PhCH2), 4.50 (d,
ꢀ
ꢀ
J1 ,2 8.1, H1ꢀ), 4.55, 4.64 (AB, J 12.2, PhCH2), 4.56, 4.64 (AB,
J 10.7, PhCH2), 4.59–4.62 (m, H1, NH), 4.65, 4.77 (AB, J 11.4,
PhCH2), 4.96 (dd, H2ꢀ), 5.06–5.08 (m, H3), 7.16–7.18, 7.25–
7.34 (2 × m, Ph). δC (150.9 MHz) 20.69, 20.78 (2C, CH3C=O),
28.66 (CH3C), 35.29 (C4), 37.90 (CH2N), 58.90 (C5), 68.44
(C6ꢀ), 68.64 (C3), 69.33, 73.44, 74.85, 74.90 (4C, PhCH2), 72.91
(C2ꢀ), 73.31 (C2), 75.00 (C5ꢀ), 77.64 (C4ꢀ), 79.20 (CH3C), 82.64
ꢀ
ꢀ

d-Glucosylated Derivatives of Isofagomine and Noeuromycin
561
165.73 (C=O). m/z (FAB) 354.1347 (C20H20NO5 [M + H]+
mixture, followed by flash chromatography (EtOAc/petrol, 1/3),
gave the silyl ether 25 (15 mg, 60%) as a colourless oil, [α]D
+3.1◦. δH (600 MHz) 0.60 (t, 9H, J 7.9, CH3), 0.91 (q, 6H,
CH2Si), 3.54 (m, H4), 3.80 (dd, J5,5 11.5, J4,5 3.7, H5), 3.99 (dd,
J2,3 5.0, J1,2 3.3, H2), 4.33 (dd, J4,5 8.2, H5), 4.51, 4.83 (AB, J
11.5, PhCH2), 4.59 (d, H1), 5.20 (dd, J3,4 4.8, H3), 7.26–7.28,
7.35–7.40, 7.55–7.59, 7.99–8.03 (4 × m, Ph). δC (150.9 MHz)
4.82 (CH3), 6.78 (CH2Si), 30.05 (C4), 57.94 (C5), 67.36 (C3),
69.83 (C2), 70.44 (PhCH2), 100.37 (C1), 117.15 (CN), 127.99–
137.00(Ph), 165.72(C=O). m/z (FAB)468.2176(C26H34NO5Si
[M + H]+ requires 468.2206).
requires 354.1341).
Benzyl 3-O-Benzoyl-2-O-benzyl-4-C-cyano-4-deoxy-
α-D-arabinoside 22 and
(5S,6S)-5,6-Di(benzyloxy)-5,6-dihydro-2H-
pyran-3-carbonitrile 23
(a) The benzoate 21 (43 mg, 0.12 mmol) and benzyl bromide
(BnBr, 31 mg, 0.18 mmol) in CH2Cl2 (2 mL) were treated with
Ag2CO3 (50 mg, 0.18 mmol) and stirred (reflux, 12 h). The sus-
pension was filtered, the filtrate was concentrated, and subjected
to flash chromatography (EtOAc/petrol, 1/3) to give the alkene
23 (21 mg, 54%) as a colourless oil, [α]D +80.5◦. δH (600 MHz)
3.86 (m, H5), 4.23 (ddd, J2,2 16.3, J 1.5, 1.5, H2), 4.27 (ddd, J 2.1,
2.1, H2), 4.61, 4.68 (AB, J 11.8, PhCH2), 4.61, 4.81 (AB, J 11.9,
PhCH2), 4.93 (d, J5,6 2.4, H6), 6.56–6.58 (m, H4), 7.28–7.39
(m, Ph). δC (150.9 MHz) 60.10 (C2), 70.09 (C5), 70.44, 72.44
(2C, PhCH2), 97.74 (C6), 115.24, 115.61 (C3, CN), 128.04–
127.37 (Ph), 139.05 (C4). m/z (FAB) 321.1348 (C20H19NO3
[M]+• requires 321.1365).
Benzyl 3-O-Benzoyl-4-C-cyano-4-deoxy-2-O-(tetrahydro-
2H-pyran-2-yl)-α-D-arabinoside 26
The benzoate 21 (50.6 mg, 0.143 mmol) in dry CH2Cl2 (2 mL)
was treated with 3,4-dihydro-2H-pyran (19.3 mg, 0.229 mmol)
and camphor-10-sulfonic acid (2 mg) and the solution stirred
(1 h). The solution was neutralized with Et3N (200 µL), diluted
with CH2Cl2 (50 mL), and washed with saturated NaHCO3.
The organic layer was dried, concentrated, and subjected to
flash chromatography (EtOAc/petrol, 1/3) to give the acetal 26
(58 mg, 94%) as a colourless oil and as a mixture of diastereoiso-
mers. Further purification enabled partial separation of the
diastereoisomers.
Further elution (EtOAc/petrol, 1/2) gave the benzyl ether 22
(8 mg, 15%) as a colourless oil, [α]D −19.8◦. δH (600 MHz)
3.50–3.57 (m, H4), 3.78 (dd, J5,5 11.8, J4,5 3.5, H5), 3.83 (dd,
J2,3 6.4, J1,2 4.5, H2), 4.31 (dd, J4,5 6.6, H5), 4.57, 4.88 (AB,
J 11.6, PhCH2), 4.71 (d, H1), 4.74, 4.78 (AB, J 11.6, PhCH2),
5.33 (dd, J3,4 4.4, H3), 7.25–7.30, 7.37–7.41, 7.55–7.60, 7.96–
8.00 (4 × m, Ph). δC (150.9 MHz) 31.40 (C4), 58.99 (C5), 68.48
(C3), 70.71, 74.19(2C, PhCH2), 74.32(C2), 100.26(C1), 116.96
(CN), 128.02–137.40 (Ph), 165.71 (C=O). m/z (FAB) 444.1806
(C27H26NO5 [M + H]+ requires 444.1811).
Diastereoisomer A: δH (600 MHz) 1.45–1.60, 1.70–1.75,
1.78–1.85 (3 × m, 6H, CH2), 3.46–3.50 (m, 1H, CH2O), 3.54
(m, H4), 3.82–3.87 (m, 2H, H5, CH2O), 4.04 (dd, J2,3 3.8, J1,2
2.1, H2), 4.37 (dd, J4,5 ≈ J5,5 11.1, H5), 4.51, 4.81 (AB, J 11.5,
PhCH2), 4.84 (dd, J 4.6, 3.0, OCHO), 4.94 (d, H1), 5.34 (dd,
J3,4 3.7, H3), 7.24–7.35, 7.53–7.57, 7.95–7.97 (3 × m, Ph). δC
(150.9 MHz) 19.60, 25.27, 30.87 (3C, CH2), 29.32 (C4), 56.53
(C5), 63.20 (CH2O), 67.38 (C2), 70.20 (C3), 70.26 (PhCH2),
99.70 (C1), 99.87 (OCHO), 116.83 (CN), 127.90–137.19 (Ph),
165.66 (C=O).
Further elution (EtOAc/petrol, 1/2) gave unreacted starting
material 21 (8 mg).
(b) The benzoate 21 (83.6 mg, 0.237 mmol) and benzyl
trichloroacetimidate[29] (238 mg, 0.947 mmol) in dry CH2Cl2
(3 mL) were stirred with 4 Å molecular sieves (200 mg) (room
temp., 1 h). The mixture was treated with CF3SO3H (20 µL)
(room temp., 4 h), diluted with CH2Cl2, filtered, and the filtrate
washed with saturated NaHCO3 and brine. Concentration of the
organic layer followed by flash chromatography (EtOAc/petrol,
1/3) gave only the benzyl ether 22 (49.5 mg, 50%).
Diastereoisomer B: δH (600 MHz) 1.38–1.45, 1.46–1.59,
1.65–1.75 (3 × m, 6H, CH2), 3.44–3.49 (m, H5), 3.51–3.54 (m,
H4), 3.79 (dd, 1H, J 11.7, 3.5, CH2O), 3.80–3.82 (m, H5), 4.08
(dd, J2,3 6.1, J1,2 4.1, H2), 4.33 (dd, 1H, J 7.3, 11.7, CH2O),
4.55, 4.85 (AB, J 11.6, PhCH2), 4.69 (d, H1), 4.89 (dd, J 3.7,
3.7, OCHO), 5.42 (dd, J3,4 4.2, H3), 7.27–7.30, 7.35–7.38, 7.54–
7.57, 8.03–8.04 (4 × m, Ph). δC (150.9 MHz) 19.30, 25.25, 30.59
(3C, CH2), 31.04 (C4), 58.58 (CH2O), 62.79 (C5), 68.52 (C3),
70.62 (PhCH2), 71.20 (C2), 99.03 (OCHO), 99.64 (C1), 117.02
(CN), 127.96–133.58 (Ph), 165.69 (C=O). m/z (FAB) 438.1887
(C25H28NO6 [M + H]+ requires 438.1917).
Benzyl 2-O-Benzyl-4-C-cyano-4-deoxy-
α-D-arabinoside 24
The benzyl ether 22 (20 mg, 0.045 mmol) in MeOH (2 mL)
was treated with KCN (3.0 mg, 0.046 mmol) and the mixture
stirred (room temp., 4 h). The solution was then concentrated,
adsorbed onto silica and subjected to flash chromatography
(EtOAc/petrol, 1/2) to afford the alcohol 24 (10 mg, 67%) as a
colourless oil, [α]D +134.3◦. δH (600 MHz) 3.30–3.40 (m, H4),
3.55 (dd, J2,3 4.6, J1,2 3.0, H2), 3.78 (dd, J5,5 11.8, J4,5 4.4, H5),
4.05 (m, H3), 4.11 (dd, J4,5 9.7, H5), 4.55, 4.81 (AB, J 11.7,
PhCH2), 4.58, 4.68 (AB, J 11.8, PhCH2), 4.78 (d, H1), 7.20–
7.30 (m, Ph). δC (150.9 MHz) 31.16 (C4), 56.23 (C5), 66.56
(C3), 70.24, 73.00 (2C, PhCH2), 73.82 (C2), 97.70 (C1), 117.51
(CN), 127.76–130.77 (Ph). m/z (FAB) 340.1545 (C20H32NO4
[M + H]+ requires 340.1549).
Benzyl 2-O-(tert-Butyldimethylsilyl)-4-C-cyano-4-deoxy-
α-D-arabinoside 27 and
Benzyl 3-O-(tert-Butyldimethylsilyl)-4-C-cyano-
4-deoxy-α-D-arabinoside 28
The diol 12 (20 mg, 0.080 mmol) in N,N-dimethylformamide
(DMF) was treated with imidazole (27 mg, 0.40 mmol) and tert-
butyldimethylsilyl chloride (37 mg, 0.24 mmol) and the solution
stirred (room temp., 14 h). The solution was then concentrated
and subjected to flash chromatography (EtOAc/petrol, 1/3) to
give the 3-O-silyl ether 28 (8.1 mg, 29%) as a colourless oil,
[α]D +4.2◦. δH (600 MHz) 0.01, 0.05 (2 × s, 6H, CH3Si), 0.09
(s, 9H, CH3C), 3.34 (ddd, J4,5 10.9, 4.7, J3,4 2.7, H4), 3.75 (dd,
J2,3 3.8, J1,2 2.0, H2), 3.79 (dd, J5,5 11.6, J4,5 4.7, H5), 3.90–
3.92 (m, H3), 4.09 (dd, J4,5 10.9, H5), 4.53, 4.79 (AB, J 11.7,
PhCH2), 4.63 (br m, H1), 7.31–7.38 (m, Ph). δC (150.9 MHz)
−5.15 (CH3Si), 17.83 (CH3C), 25.50 (CH3C), 30.00 (C4), 55.22
Benzyl 3-O-Benzoyl-4-C-cyano-4-deoxy-
2-O-triethylsilyl-α-D-arabinoside 25
The benzoate 21 (18.6 mg, 0.053 mmol) in pyridine (1 mL) was
treated with DMAP (0.6 mg) and Et3SiCl (15.9 mg, 0.105 mmol)
and the mixture stirred (room temp., 4 h). Concentration of the

562
P. J. Meloncelli et al.
(C5), 67.30(C2), 68.56(C3), 70.08(PhCH2), 98.63(C1), 117.79
(CN), 128.05–135.81 (Ph). m/z (FAB) 364.1937 (C19H24NO4Si
[M + H]+ requires 364.1944).
J4,5 5.3, H5), 4.13–4.15 (m, CH2O), 4.62 (d, H1), 4.58, 4.84 (AB,
J 11.7, PhCH2), 5.21–5.23, 5.28–5.32 (2 × m, CH2CH), 5.87–
5.94 (m, CH2CH), 7.30–7.37 (m, Ph). δC (150.9 MHz) 60.44
(C5), 70.62 (PhCH2), 70.88 (C2), 71.01 (C3), 71.39 (CH2O),
76.44 (C4), 101.09 (C1), 117.99 (CH2CH), 128.26–136.79 (Ph),
134.25 (CH2CH). m/z (FAB) 281.1367 (C15H21O5 [M + H]+
requires 281.1389).
Further elution gave the 2-O-silyl ether 27 (19.6 mg, 70%),
[α]D +4.2◦. δH (600 MHz) 0.01 (s, 6H, CH3Si), 0.91 (s, 9H,
CH3C), 3.03 (ddd, J3,4 5.3, J4,5 3.2, 2.7, H4), 3.57 (dd, J5,5 12.1,
J4,5 2.7, H5), 3.66 (dd, J2,3 7.8, J1,2 6.4, H2), 3.78 (dd, H3),
4.20 (dd, J4,5 3.2, H5), 4.33 (d, H1), 4.58, 4.89 (AB, J 11.7,
PhCH2), 7.32–7.36 (m, Ph). δC (150.9 MHz) −5.17, −4.64 (2C,
CH3Si), 17.92 (CH3C), 25.50 (CH3C), 35.91 (C4), 60.90 (C5),
70.40 (C3), 70.60 (PhCH2), 72.26 (C2), 101.62 (C1), 117.69
(CN), 127.91–136.79 (Ph). m/z (FAB) 364.1939 (C19H24NO4Si
[M + H]+ requires 364.1944).
Benzyl 4-O-Allyl-3-O-benzyl-2-O-(tetra-O-benzyl-
β-D-glucopyranosyl)-β-L-xyloside 33 and
Benzyl 4-O-Allyl-2-O-benzyl-3-O-(tetra-O-benzyl-
β-D-glucopyranosyl)-β-L-xyloside 34
The trichloroacetimidate 16 (3.95 g, 6.23 mmol) and the allyl
ether 32 (1.55 g, 5.56 mmol) in dry CH2Cl2 (15 mL) were
stirred with 4 Å molecular sieves (1.0 g) (room temp., 3 h). The
mixture was cooled (−60◦C), treated with Et2OBF3 (200 µL),
and allowed to warm (room temp., 1 h). Treatment with Et3N
(400 µL), followed by filtration and flash chromatography
(EtOAc/petrol, 1/1), gave a colourless oil. This oil in MeOH
(15 mL) was stirred with KCN (50 mg) (50◦C, 12 h). Con-
centration of the solution followed by flash chromatography
(EtOAc/petrol, 2/1) gave a colourless oil. This oil in dry DMF
(25 mL, 0◦C) was stirred with NaH (60% in mineral oil,
290 mg, 12 mmol) and BnBr (3.48 g, 20.3 mmol) (0◦C, 30 min).
Treatment with MeOH (2 mL), followed by concentration of
the mixture, an aqueous workup, and flash chromatography
(EtOAc/toluene, 1/20) yielded the 2-O-β-d-glucosyl derivative
33 (1.92 g, 39%) as a colourless oil, [α]D +23.0◦. δH (600 MHz)
3.24 (dd, J5,5 11.4, J4,5 9.8, H5), 3.38–3.40 (m, H5ꢀ), 3.46 (dd,
Benzyl 3-O-Acetyl-2-O-(tert-butyldimethylsilyl)-
4-C-cyano-4-deoxy-α-D-arabinoside 29
The 2-O-silyl ether 27 (4.5 mg) was treated with pyridine
(100 µL), Ac2O (50 µL), and DMAP (0.5 mg) and left at room
temp. for 2 h. The solution was then treated with MeOH, con-
centrated, and subjected to flash chromatography (EtOAc/petrol,
1/1) to give the acetate 29 (3.3 mg, 65%) as a colourless oil, [α]D
+22.0◦. δH (600 MHz) 0.04, 0.10 (2 × s, 6H, CH3Si), 0.85 (s,
9H, CH3C), 2.11 (s, CH3C=O), 3.37–3.42 (m, H4), 3.67 (dd,
J5,5 11.8, J4,5 3.4, H5), 3.85 (dd, J2,3 6.6, J1,2 4.7, H2), 4.20 (dd,
J4,5 6.2, H5), 4.44 (d, H1), 4.52, 4.84 (AB, J 11.7, PhCH2),
4.81 (dd, J3,4 4.4, H3), 7.32–7.35 (m, Ph). δC (150.9 MHz)
−5.07, −4.84 (2C, CH3Si), 17.85 (CH3C), 20.73 (CH3C=O),
25.50 (CH3C), 30.76 (C4), 58.92 (C5), 67.96, 70.22 (C2, C3),
70.35 (PhCH2), 100.82 (C1), 116.97 (CN), 127.80–136.80 (Ph),
170.11 (C=O). m/z (FAB) 406.2052 (C21H32NO5Si [M + H]+
requires 406.2050).
J2 ,3 8.5, J1 ,2 8.0, H2ꢀ), 3.52–3.56 (m, H3, H4), 3.64 (dd, J3 ,4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
9.3, H3ꢀ), 3.70 (dd, J6 ,6 11.5, J5 ,6 5.3, H6ꢀ), 3.71 (dd, J4 ,5
ꢀ
ꢀ
ꢀ
ꢀ
Benzyl 2-O-Acetyl-3-O-(tert-butyldimethylsilyl)-
4-C-cyano-4-deoxy-α-D-arabinoside 30
9.6, H4ꢀ), 3.76 (dd, J5 ,6 1.7, H6ꢀ), 3.90 (dd, J2,3 8.1, J1,2 8.0,
H2), 4.03 (dd, J4,5 4.3, H5), 4.14 (dd, 1H, J 12.7, 5.9, CH2O),
4.25 (dd, 1H, J 12.7, 5.4, CH2O), 4.40 (d, H1), 4.44, 4.77 (AB,
J 11.0, PhCH2), 4.52, 4.55 (AB, J 12.2, PhCH2), 4.62, 4.85
(AB, J 10.8, PhCH2), 4.76, 5.03 (AB, J 11.3, PhCH2), 4.81,
4.92 (AB, J 11.5, PhCH2), 4.85, 4.99 (AB, J 10.8, PhCH2), 4.98
(d, H1ꢀ), 5.19 (m, 1H, CH2CH), 5.29 (m, 1H, CH2CH), 5.87–
5.95 (m, CH2CH), 7.20–7.45 (m, Ph). δC (150.9 MHz) 64.07
(C5), 69.04 (C6ꢀ), 71.67, 74.90, 74.96, 75.12, 75.85 (PhCH2),
72.50 (CH2O), 75.09 (C5ꢀ), 77.01 (C4ꢀ), 78.16, 78.27 (C2, C4),
82.40 (C3), 83.09 (C2ꢀ), 85.01 (C3ꢀ), 101.96 (C1ꢀ), 103.45 (C1),
117.23 (CH2CH), 127.46–139.11 (Ph), 134.99 (CH2CH). m/z
(FAB) 893.4271 (C56H61O10 [M + H]+ requires 893.4205).
Further elution (EtOAc/toluene, 1/20) yielded the 3-O-β-d-
glucosyl derivative 34 as a colourless solid (1.93 g, 40%). A
small amount was further purified by suspension in petrol and
filtration, mp 81–82◦C, [α]D +35.4◦. δH (600 MHz) 3.17 (dd, J
ꢀ
ꢀ
The3-O-silylether 28(5 mg)wastreatedwithpyridine(100 µL),
Ac2O (50 µL), and DMAP (0.5 mg) and maintained at room
temp. for 2 h. The solution was then treated with MeOH, con-
centrated, and subjected to flash chromatography (EtOAc/petrol,
1/1) to give the acetate 30 (5.3 mg, 95%) as a colourless oil, [α]D
+55.2◦. δH (600 MHz) 0.12, 0.15 (2 × s, 6H, CH3Si), 0.87 (s,
9H, CH3C), 2.09 (s, CH3C=O), 3.13 (ddd, J4,5 9.5, 3.7, J3,4 3.5,
H4), 3.66 (dd, J5,5 10.8, J4,5 3.7, H5), 4.04 (dd, J2,3 3.4, H3),
4.31 (dd, J4,5 9.5, H5), 4.50, 4.74 (AB, J 11.8, PhCH2), 4.64 (d,
J1,2 2.1, H1), 4.81 (dd, H2), 7.30–7.36 (m, Ph). δC (150.9 MHz)
−5.29, −4.83 (2C, CH3Si), 17.82 (CH3C), 20.80 (CH3C=O),
25.50 (CH3C), 32.39 (C4), 56.11 (C5), 66.20 (PhCH2), 69.14,
70.22 (C2, C3), 96.56 (C1), 117.41 (CN), 127.80–136.84 (Ph),
169.27 (C=O). m/z (FAB) 406.2057 (C21H32NO5Si [M + H]+
requires 406.2050).
ꢀ
ꢀ
11.1, 11.1, H5), 3.41 (dd, J2,3 8.3, J1,2 7.8, H2), 3.43 (dd, J2 ,3
8.0, J1 ,2 7.8, H2ꢀ), 3.45–3.54 (m, H4, H5ꢀ), 3.65–3.69 (m, 3H,
Benzyl 4-O-Allyl-β-L-xyloside 32
ꢀ
ꢀ
The alcohol 31 (1.97 g, 7.09 mmol) in dry DMF (25 mL, 0◦C)
was treated with NaH (60% in mineral oil, 480 mg, 12 mmol)
and allyl bromide (1.70 g, 14.2 mmol) and the suspension was
stirred (0◦C, 30 min). Methanol (5 mL) was added and the solu-
tion stirred (0◦C, 30 min), concentrated, and taken up in MeOH
(20 mL).The solution was treated with CSA (200 mg) and stirred
(room temp., 30 min), followed by treatment with Et3N (1 mL).
Concentration of the solution followed by an aqueous workup
and flash chromatography (EtOAc/petrol, 3/2) gave the allyl
ether 32 as a colourless solid (1.76 g, 89%), mp 55–57◦C, [α]D
+116.5◦. δH (600 MHz) 3.48–3.50 (m, 2H, H4, H5), 3.55 (dd,
J2,3 5.8, J1,2 4.9, H2), 3.76 (dd, J3,4 6.1, H3), 4.08 (dd, J5,5 14.4,
H3ꢀ, H4ꢀ, H6ꢀ), 3.78 (dd, J6 ,6 10.3, J5 ,6 1.2, H6 ), 3.95–4.03 (m,
4H, H3, H5, CH2O), 4.47 (d, H1), 4.51 (s, PhCH2), 4.62, 5.08
(AB, J 10.7, PhCH2), 4.65, 4.93 (AB, J 11.9, PhCH2), 4.77,
4.81 (AB, J 11.4, PhCH2), 4.84, 4.85 (AB, J 10.0, PhCH2),
4.89, 4.97 (AB, J 11.1, PhCH2), 4.98 (d, H1ꢀ), 5.06–5.07, 5.12–
5.15 (2 × m, CH2CH), 5.80–5.86 (m, CH2CH), 7.20–7.40 (m,
Ph). δC (150.9 MHz) 63.32 (C5), 69.13 (C6ꢀ), 71.22 (CH2O),
71.99, 73.55, 74.69, 74.87, 75.12, 75.83 (6C, PhCH2), 75.19
(C5ꢀ), 78.37 (C4ꢀ), 78.64 (C4), 79.37 (C3), 80.12 (C2), 83.07
(C2ꢀ), 85.07 (C3ꢀ), 102.56 (C1ꢀ), 102.88 (C1), 117.61 (CH2CH),
127.42–138.94 (Ph), 134.78 (CH2CH). m/z (FAB) 893.4244
(C56H61O10 [M + H]+ requires 893.4205).
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ

d-Glucosylated Derivatives of Isofagomine and Noeuromycin
563
Benzyl 3-O-Benzyl-2-O-(tetra-O-benzyl-
β-D-glucopyranosyl)-β-L-xylopyranoside 35
PhCH2), 4.57, 4.80 (AB, J 10.8, PhCH2), 4.67, 4.73 (AB, J
11.7, PhCH2), 4.70, 4.86 (AB, J 10.8, PhCH2), 4.78–4.82 (m,
H1, PhCH2), 4.85 (A of AB, 1H, J 11.5, PhCH2), 4.93 (A of
AB, 1H, J 11.0, PhCH2), 7.18–7.20, 7.26–7.33 (2 × m, Ph).
δC (150.9 MHz) 30.41 (C4), 56.49 (C5), 68.75 (C6ꢀ), 69.87,
72.62, 73.68, 75.19, 75.23, 75.88 (6C, PhCH2), 72.72 (C2),
73.17 (C3), 75.00 (C5ꢀ), 77.71 (C4ꢀ), 82.13 (C2ꢀ), 84.68 (C3ꢀ),
98.23 (C1), 103.92 (C1ꢀ), 117.96 (CN), 127.68–138.60 (Ph). m/z
(FAB) 862.3952 (C54H56NO9 [M + H]+ requires 862.3955).
The glycoside 33 (180 mg) in EtOH (5 mL) was treated with
Wilkinson’s catalyst (20 mg) and the solution refluxed (3 h). The
solution was then treated with hydrochloric acid (1 M, 1 mL)
and again refluxed (1 h). Treatment with Et3N (0.5 mL), fol-
lowed by concentration of the mixture and flash chromatography
(EtOAc/toluene, 1/4), yielded the alcohol 35 as a colourless oil
(155 mg, 91%), [α]D +40.8◦. δH (600 MHz) 3.36 (dd, J5,5 11.5,
J4,5 8.2, H5), 3.38–3.41 (m, H5ꢀ), 3.48 (dd, J2 ,3 8.5, J1 ,2 8.0,
ꢀ
ꢀ
ꢀ
ꢀ
H2ꢀ), 3.55 (dd, J3,4 7.4, J2,3 7.3, H3), 3.65 (dd, J3 ,4 8.9, H3ꢀ),
Benzyl 3-O-Benzyl-4-C-[(tert-butoxycarbonyl)
amino]methyl-4-deoxy-2-O-(tetra-O-benzyl-
β-D-glucopyranosyl)-α-D-arabinoside 37
ꢀ
ꢀ
3.69 (dd, J4 ,5 9.3, H4ꢀ), 3.70–3.76 (m, 3H, H4, H6ꢀ), 3.94 (dd,
J1,2 6.1, H2), 4.10 (dd, J4,5 4.4, H5), 4.47 (A of AB, 1H, J 11.2,
PhCH2), 4.53, 4.58 (AB, J 12.2, PhCH2), 4.56 (d, H1), 4.60, 4.98
(AB, J 10.8, PhCH2), 4.63, 4.98 (AB, J 10.9, PhCH2), 4.80–4.86
(m, 5H, H1ꢀ, PhCH2), 4.93 (A ofAB, J 11.3, PhCH2), 7.25–7.37
(m, Ph). δC (150.9 MHz) 64.08 (C5), 68.61 (C4), 68.99 (C6ꢀ),
71.19, 73.55, 73.82, 75.08, 75.16, 75.84 (6C, PhCH2), 75.04
(C5ꢀ), 77.20 (C2), 78.08 (C4ꢀ), 80.91 (C2ꢀ), 82.85 (C3), 84.94
(C3ꢀ), 102.18, 102.52 (C1, C1ꢀ), 127.60–138.73 (Ph). m/z (FAB)
853.3954 (C53H57O10 [M + H]+ requires 853.3952).
ꢀ
ꢀ
The nitrile 36 (210 mg) was treated as for the diol 18 to yield the
carbamate 37 (151 mg, 65%) as a colourless oil, [α]D +44.6◦. δH
(600 MHz) 1.43 (s, 9H, CH3), 2.30–2.35 (m, H4), 3.19–3.26 (m,
CH2N), 3.41–3.49 (m, 3H, H2ꢀ, H5, H5ꢀ), 3.58–3.64 (m, H3ꢀ,
H4ꢀ), 3.66 (dd, J6 ,6 10.9, J5 ,6 4.7, H6ꢀ), 3.70 (dd, J5 ,6 2.1,
H6ꢀ), 3.83 (dd, J2,3 ≈ J3,4 3.8, H3), 3.98–4.04 (m, H2, H5), 4.43,
4.50 (AB, J 11.6, PhCH2), 4.54–4.57 (m, 4H, H1ꢀ, PhCH2), 4.70
(A of AB, 1H, J 10.8, PhCH2), 4.74–4.84 (m, 5H, H1, PhCH2),
4.95, 4.97 (AB, J 10.5, PhCH2), 7.17–7.19, 7.25–7.37 (2 × m,
Ph). δC (150.9 MHz) 28.56 (CH3), 36.61 (CH2N), 39.60 (C4),
59.50 (C5), 69.02 (C6ꢀ), 69.74, 71.72, 73.60, 75.11, 75.18, 75.87
(6C, PhCH2), 73.13 (C2), 74.98 (C5ꢀ), 75.80 (C3), 77.91 (C4ꢀ),
79.15 (CH3C), 82.28 (C2ꢀ), 84.79 (C3ꢀ), 99.15 (C1), 103.42
(C1ꢀ), 127.62–138.73 (Ph), 156.13 (C=O). m/z (FAB) 966.4788
(C59H68NO11 [M + H]+ requires 966.4792).
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Benzyl 3-O-Benzyl-4-C-cyano-4-deoxy-2-O-(tetra-
O-benzyl-β-D-glucopyranosyl)-α-D-arabinoside 36
(a) Lithium bis(trimethylsilyl)amide in THF (1 M, 0.15 mL,
0.15 mmol) was added to the alcohol 35 (105 mg, 0.123 mmol)
in dry THF (5 mL, 0◦C) and the solution stirred (0◦C, 30 min).
The solution was cooled (−30◦C) and freshly prepared N,Nꢀ-
sulfuryldiimidazole[30] (30 mg, 0.15 mmol) was added, and
the solution was then stirred (35◦C, 3 h). Methanol (0.3 mL)
was added and, after 30 min, the solution was concentrated,
diluted with EtOAc, washed with saturated NaHCO3, and dried.
Flash chromatography (EtOAc/toluene, 1/9 containing 0.5%
Et3N) gave benzyl 3-O-benzyl-4-O-(imidazolyl-1-sulfonyl)-2-
O-(tetra-O-benzyl-β-d-glucopyranosyl)-β-l-xyloside (107 mg,
88%) as a colourless oil, [α]D +29.2◦ (CH2Cl2). δH (600 MHz)
3.35 (m, 3H, H2ꢀ, H5, H5ꢀ), 3.57–3.71 (m, 5H, H3, H3ꢀ, H4ꢀ,
H6ꢀ), 3.90 (dd, J2,3 6.5, J1,2 6.1, H2), 4.07 (dd, J5,5 12.2, J4,5 4.5,
H5), 4.43, 4.72 (AB, J 11.2, PhCH2), 4.47 (d, H1), 4.49–4.58
(m, 5H, H4, PhCH2), 4.76 (A of AB, 1H, J 11.1, PhCH2), 4.76
Benzyl 2-O-Benzyl-3-O-(tetra-O-benzyl-
β-D-glucopyranosyl)-β-L-xylopyranoside 38
The glycoside 34 (505 mg) was treated as for the glycoside 33
to yield the alcohol 38 as a colourless solid (472 mg, 98%). A
small sample of 38 was further purified by recrystallization,
mp 145–147◦C (CH2Cl2/petrol), [α]D +15.6◦. δH (600 MHz)
3.24 (dd, J5,5 11.6, J4,5 8.8, H5), 3.39–3.44 (m, H5ꢀ), 3.45 (dd,
J2,3 7.7, J1,2 6.3, H2), 3.54 (dd, J2 ,3 8.1, J1 ,2 8.0, H2ꢀ), 3.57–
3.61 (m, H4), 3.64–3.75 (m, 5H, H3, H3ꢀ, H4ꢀ, H6ꢀ), 3.93 (dd,
J4,5 4.6, H5), 4.40, 4.56 (AB, J 12.0, PhCH2), 4.53 (d, H1),
4.56, 4.80 (AB, J 10.8, PhCH2), 4.61 (A of AB, 1H, J 12.0,
PhCH2), 4.65 (d, H1ꢀ), 4.76 (A ofAB, 1H, J 11.2, PhCH2), 4.84–
4.89 (m, 6H, PhCH2), 7.13–7.16, 7.24–7.36 (2 × m, Ph). δC
(150.9 MHz) 64.07 (C5), 68.84 (C6ꢀ), 69.67 (C4), 70.88, 73.67,
74.30, 75.10, 75.69, 75.70 (6C, PhCH2), 75.20 (C5ꢀ), 78.08
(C4ꢀ), 78.75 (C2), 82.41 (C2ꢀ), 84.76, 85.35 (C3, C3ꢀ), 102.02
(C1), 103.98 (C1ꢀ), 127.56–138.68 (Ph). m/z (FAB) 851.3812
(C53H55O10 [M − H]+ requires 851.3795).
ꢀ
ꢀ
ꢀ
ꢀ
(d, J1 ,2 8.0, H1ꢀ), 4.81–4.85 (m, 4H, PhCH2), 4.95 (A of AB,
1H, J 10.9, PhCH2), 6.84–6.85 (m, 1H, Im), 7.16–7.18, 7.24–
7.32 (2 × m, Ph, Im), 7.89–7.91 (m, 1H, Im). δC (150.9 MHz)
61.01 (C5), 68.97 (C6ꢀ), 71.24, 73.51, 74.14, 75.14, 75.17, 75.85
(6C, PhCH2), 75.10 (C5ꢀ), 76.59 (C2), 77.08, 78.02 (C3ꢀ, C4ꢀ),
80.81 (C4), 82.82 (C3), 84.84 (C2ꢀ), 101.49 (C1), 102.24 (C1ꢀ),
118.11, 137.64 (Im), 127.65–138.78 (Ph). m/z (FAB) 983.3725
(C56H59N2O12S [M + H]+ requires 983.3789).
ꢀ
ꢀ
Benzyl 2-O-Benzyl-4-C-cyano-4-deoxy-3-O-(tetra-
O-benzyl-β-D-glucopyranosyl)-α-D-arabinoside 39
(b) The above imidazylate (459 mg, 0.467 mmol) in CH3CN
(10 mL) was treated with Me3SiCN (92.0 mg, 0.934 mmol) and
Bu4NF (1 M, 70 µL, 0.07 mmol) in CH3CN (1 mL) and the mix-
ture heated to reflux. A further solution of Bu4NF (1 M, 700 µL,
0.70 mmol) in CH3CN (1 mL) was added dropwise (30 min) and
the solution refluxed (30 min). The solution was then concen-
trated somewhat, diluted with EtOAc (100 mL), washed with
H2O, and dried. Concentration of the solution followed by flash
chromatography (EtOAc/petrol, 1/3) gave the nitrile 36 as a
colourless oil (292 mg, 75%), [α]D +35.6◦. δH (600 MHz) 3.32
(ddd, J4,5 10.0, 4.0, J3,4 3.5, H4), 3.36–3.38 (m, H5ꢀ), 3.42 (dd,
(a) The alcohol 38 (132 mg) was treated as for the alcohol 35
to yield benzyl 2-O-benzyl-4-O-(imidazolyl-1-sulfonyl)-3-O-
(tetra-O-benzyl-β-d-glucopyranosyl)-β-l-xyloside as a colour-
less oil (110 mg, 72%), [α]D +18.8◦ (CH2Cl2). δH (600 MHz)
3.32–3.39 (m, 3H, H2ꢀ, H5, H5ꢀ), 3.54–3.62 (m, H2, H3ꢀꢀ, H4ꢀ),
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3.63 (dd, J6 ,6 11.3, J5 ,6 4.8, H6 ), 3.69 (dd, J5 ,6 2.0, H6 ), 3.99
(dd, J5,5 12.8, J4,5 3.8, H5), 4.02 (dd, J2,3 ≈ J3,4 6.0, H3), 4.41 (d,
J1 ,2 7.7, H1ꢀ), 4.49, 4.53 (AB, J 12.1, PhCH2), 4.55–4.58 (m,
PhCH2), 4.61–4.70 (m, 5H, H1, H4, PhCH2), 4.76, 4.78 (AB,
J 12.3, PhCH2), 4.80, 4.83 (AB, J 10.9, PhCH2), 4.90 (A of
AB, 1H, J 11.0, PhCH2), 7.05–7.07, 7.93–7.95 (2 × m, 2H, Im),
7.18–7.32 (m, Ph, Im). δC (150.9 MHz) 59.90 (C5), 68.95 (C6ꢀ),
ꢀ
ꢀ
J2 ,3 9.5, J1 ,2 7.5, H2ꢀ), 3.58–3.65 (m, 4H, H3ꢀ, H4ꢀ, H6ꢀ), 3.70
(dd, J5,5 11.1, J4,5 4.0, H5), 3.91 (dd, J2,3 3.5, J1,2 2.1, H2),
4.11 (dd, H3), 4.33 (dd, J4,5 10.0, H5), 4.42 (d, H1ꢀ), 4.54 (s,
ꢀ
ꢀ
ꢀ
ꢀ

564
P. J. Meloncelli et al.
70.52, 73.56, 73.57, 74.88, 75.10, 75.78 (6C, PhCH2), 74.18
(C3), 75.15 (C5ꢀ), 76.75, 77.77 (C2, C4ꢀ), 80.92 (C4), 82.16
(C2ꢀ), 84.62 (C3ꢀ), 100.31 (C1), 101.98 (C1ꢀ), 118.23, 137.27
(Im), 127.82–138.60 (Ph). m/z (FAB) 983.3765 (C56H59N2O10
[M + H]+ requires 983.3789).
H3ꢀman, H3ꢀα, H3ꢀβ, H4ꢀglc, H4ꢀman, H4ꢀα, H4ꢀβ, H5ꢀglc,
H5ꢀman, H5ꢀα, H5ꢀβ, H6glc, H6man, CH2Nα, CH2Nβ), 3.57–
3.66, 3.73–3.89 (2 × m, H2α, H2β, H3glc, H3man, H4glc, H5α,
H5β, H6ꢀglc, H6ꢀman, H6ꢀα, H6ꢀβ, CH2Oglc, CH2Oman), 3.95
(dd, J 8.9, 8.8, H4man), 4.04 (dd, J3,4 5.3, J2,3 9.7, H3α), 4.14
(dd, J3,4 4.4, J2,3 7.9, H3β), 4.45–4.47 (m, H1ꢀglc, H1ꢀman,
(b)The imidazylate from the alcohol 38 (255 mg) was treated
as for the imidazylate from the alcohol 35 to yield the nitrile 39
as a colourless oil (141 mg, 60%), [α]D +14.9◦. δH (600 MHz)
3.27–3.30 (m, H4), 3.49–3.70 (m, 4H, H3ꢀ, H5ꢀ, H6ꢀ), 3.52 (dd,
H1α), 4.52 (d, J1 ,2 8.0, H1ꢀβ), 4.52 (d, J1 ,2 7.9, H1ꢀα), 4.57
(d, J2,3 9.0, H2glc), 5.11 (d, J1,2 2.9, H1β), 5.18 (d, J2,3 2.8,
H2man). δC (150.9 MHz) 36.19 (C4α), 37.11 (C6man), 38.42
(CH2Nα), 39.76, 40.08 (C5glc, C5man), 40.96 (C6glc), 58.04,
58.37, 60.43, 60.49 (C6ꢀglc, C6ꢀman, CH2Oglc, CH2Oman),
60.55, 62.64 (C5α, C6ꢀα), 69.29–75.95 (C2α, C2ꢀglc, C2ꢀman,
C2ꢀα, C3glc, C3man, C3ꢀglc, C3ꢀman, C3ꢀα, C4ꢀglc, C4ꢀman,
C4ꢀα, C5ꢀglc, C5ꢀman, C5ꢀα), 76.63 (C4man), 77.73 (C2man),
78.59 (C3α), 79.22 (C4glc), 80.88 (C2glc), 92.15 (C1β), 96.91
(C1α), 99.82 (C1ꢀα), 102.53, 102.65 (C1ꢀglc, C1ꢀman). m/z
(FAB) 326.1431 (C12H24NO9 [M + H]+ requires 326.1451).
ꢀ
ꢀ
ꢀ
ꢀ
J2 ,3 9.0, J1 ,2 7.6, H2ꢀ), 3.57 (dd, J5,5 11.6, J4,5 3.4, H5), 3.60
ꢀ
ꢀ
ꢀ
ꢀ
(dd, J3 ,4 ≈ J4 ,5 9.6, H4ꢀ), 3.81 (dd, J2,3 5.9, J1,2 4.2, H2),
4.17–4.20 (m, 2H, H3, H5), 4.50 (s, PhCH2), 4.57–4.71 (m,
8H, H1, H1ꢀ, PhCH2), 4.77, 4.91 (AB, J 11.0, PhCH2), 4.84 (A
of AB, 1H, J 11.4, PhCH2), 5.06 (A of AB, 1H, J 11.1, PhCH2),
7.19–7.21, 7.26–7.39 (2 × m, Ph). δC (150.9 MHz) 31.17 (C4),
58.52 (C5), 69.34 (C6ꢀ), 70.28, 73.57, 74.04, 75.10, 75.23, 75.73
(6C, PhCH2), 73.25 (C3), 75.31 (C5ꢀ), 76.49 (C2), 77.99 (C4ꢀ),
82.45 (C2ꢀ), 84.71 (C3ꢀ), 100.10, 102.68 (C1, C1ꢀ), 117.74
(CN), 127.71–138.74 (Ph). m/z (FAB) 862.3951 (C54H56NO9
[M + H]+ requires 862.3955).
ꢀ
ꢀ
ꢀ
ꢀ
Enzyme Inhibition Studies
Kinetic analysis of Cel5A and Cex with 10 was performed using
a spectrophotometric assay that monitored the release of the
(di)nitrophenolate ion at 400 nm. Ki values for Ce15A were
determined by measuring rates at a range of substrate concen-
trations flanking the KM value at each of a series of inhibitor
concentrations. Dixon plots of the data revealed the inhibition
mode (competitive) while actual values measured (and errors
thereon) were calculated by direct fit to the equation for com-
petitive inhibition within the program GraFit. Ki values for Cex
weredeterminedfromDixonplotsusinginhibitorconcentrations
from one-third to three times the Ki, and substrate concentra-
tions near the KM value. Assays with Cel5A were carried out
in 50 mM sodium phosphate buffer, pH 6.0, at 37◦C. The con-
centration of the substrate, 2,4-dinitrophenyl β-d-lactoside, was
varied from 0.1 to 1.0 mM and the final enzyme concentration
was 4 nM. Assays with Cex were performed in 50 mM sodium
phosphate buffer, pH 7.0, at 37◦C. The concentration of sub-
strate, 4-nitrophenyl β-d-xylobioside, was 90 µM and the final
enzyme concentration was 0.1 nM. In all cases the enzymes were
pre-incubated with 10 for 1 h before rate determination because
of the slow-onset behaviour of the inhibitor.
Kinetic studies on CtLic26A with 11 were performed
using a fluorometric assay at 37◦C and pH 7.0. CtLic26A
(100 nM) was pre-incubated with 11 (at concentrations rang-
ing from 75 to 500 nM) for 1 h before the addition of
substrate [β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-β-d-GlcpMU at
15 µM]. Aliquots were taken over a 10 min period and added
to 50 mM glycine/NaOH buffer, pH 10.4, to stop the reaction.
The amount of methylumbelliferone (MU) product was deter-
mined by fluorescence using excitation at 365 nm and emission
at 460 nm. Ki values were determined through analysis of the
apparent KM as a function of inhibitor concentration.
Benzyl 2-O-Benzyl-4-C-[(tert-butoxycarbonyl)
amino]methyl-4-deoxy-3-O-(tetra-O-benzyl-
β-D-glucopyranosyl)-α-D-arabinoside 40
The nitrile 39 (110 mg) was treated as for the diol 18 to yield the
carbamate 40 as a colourless oil (100 mg, 81%), [α]D +19.1◦.
δH (600 MHz) 1.43 (s, 9H, CH3), 2.28–2.33 (m, H4), 3.19–3.25
(m, CH2N), 3.37–3.44 (m, 2H, H5, H5ꢀ), 3.46 (dd, J 8.0, 7.9,
H2ꢀ), 3.62–3.70 (m, 5H, H2, H3ꢀ, H4ꢀ, H6ꢀ), 3.88–3.95 (m, 1H,
H5), 4.06 (dd, J2,3 ≈ J3,4 5.8, H3), 4.48–4.61 (m, 6H, H1, H1ꢀ,
PhCH2), 4.62, 4.69 (AB, J 10.3, PhCH2), 4.77 (A of AB, 1H, J
11.3, PhCH2), 4.81–4.91 (m, 5H, PhCH2), 4.93–4.97 (br s, NH),
7.18–7.40 (m, Ph). δC (150.9 MHz) 28.34 (s, CH3), 37.18 (C4),
38.71 (CH2N), 61.35 (C5), 68.68 (C6ꢀ), 69.85, 73.30, 73.59,
74.93, 74.97, 75.55(6C, PhCH2), 75.08(C5ꢀ), 76.07(C3), 77.10,
77.95 (C2, C4ꢀ), 78.85 (CH3C), 82.12 (C2ꢀ), 84.93 (C3ꢀ), 100.87
(C1ꢀ), 101.31 (C1), 127.40–138.58 (Ph), 155.88 (C=O). m/z
(FAB) 966.4780 (C59H68NO11 [M + H]+ requires 966.4792).
(3R,4R,5R)-4-(β-D-Glucopyranosyloxy)-
5-(hydroxymethyl)piperidin-3-ol (4-O-β-D-
Glucopyranosylisofagomine) 8 Hydrochloride
The carbamate 40 (26 mg) was treated with CF3COOH (2 mL,
10 min). The solution was concentrated, the residue taken up
in MeOH (5 mL), and then processed as for the preparation
of 9 (from 19) to give 4-O-β-d-glucopyranosylisofagomine
8 hydrochloride (7 mg, 72%) as a colourless glass. The 1H
(600 MHz) and 13C (150.9 MHz) NMR spectroscopic data
(Fig. 7) were in good agreement with those reported.[15]
(2R/2S,3S,4R,5R)-4-(β-D-Glucopyranosyloxy)-
5-(hydroxymethyl)piperidine-2,3-diol (4-O-β-D-
Glucopyranosylnoeuromycin) 10 Hydrochloride
Structural Investigations
The carbamate 40 (26 mg) was treated as for the car-
bamate 19 (in the preparation of 11) to yield 4-O-β-d-
glucopyranosylnoeuromycin 10 hydrochloride as a colourless
glass (7 mg, 75%), that contained a significant amount of
the pyranose tautomer 41 [4-C-aminomethyl-4-deoxy-3-O-
(β-d-glucopyranosyl)-d-arabinose] as the hydrochloride. δH
(600 MHz) 2.00–2.11 (m, H5glc, H5man), 2.41–2.48 (m, H4α,
H4β), 2.93 (dd, J 13.2, 13.2, H6glc), 3.04–3.13 (m, CH2Nα,
CH2Nβ), 3.22–3.45 (m, H2ꢀglc, H2ꢀman, H2ꢀα, H2ꢀβ, H3ꢀglc,
Cel5A and CtLic26A were co-crystallized in the presence of
10 mM 10 and 11, respectively, using crystallization conditions
described previously,[18,21] and flash frozen. Data were collected
at the European Synchrotron Radiation Facility on beamlines
ID14–1 (Cel5A) and ID14–2 (CtLic26A). Data were collected
to a resolution of 1.50 Å for the Cel5A complex and 1.20 Å
for the CtLic26A complex, and were integrated and scaled
with DENZO/SCALEPACK[31] or MOSFLM/SCALA.[32] All
subsequent manipulations used the CCP4 suite of programs.[33]

d-Glucosylated Derivatives of Isofagomine and Noeuromycin
565
Starting phases were obtained using PDB entries 1OCQ (for
Cel5A) and 2BV9 (for CtLic26A). Refinement was done
using iterative cycles of model building in COOT[34] and
REFMAC.[35]
[14] P. J. Meloncelli, R. V. Stick, Aust. J. Chem. 2006, 59, 827.
doi:10.1071/CH06241
[15] J. M. Macdonald, R. V. Stick, D. M. G. Tilbrook, S. G. Withers, Aust.
J. Chem. 2002, 55, 747. doi:10.1071/CH02165
[16] M. Hrmova, T. Imai, S. J. Rutten, J. K. Fairweather, L. Pelosi,
V. Bulone, H. Driguez, G. B. Fincher, J. Biol. Chem. 2002, 277, 30102.
doi:10.1074/JBC.M203971200
Acknowledgments
Carlos Fontes (Lisbon) and Harry Gilbert (Newcastle) and their research
groups are thanked for inspiration and provision of resources for the
CtLic26 work. The Biotechnology and Biological Sciences Research Coun-
cil (BBSRC) and the Canadian Institutes for Health Research (CIHR) are
thanked for funding. G.J.D. is the recipient of a Royal Society–Wolfson
Research Merit Award.
[17] J. M. Macdonald, M. Hrmova, G. B. Fincher, R.V. Stick,Aust. J. Chem.
2004, 57, 187. doi:10.1071/CH03227
[18] A. Varrot, C. A. Tarling, J. M. Macdonald, R. V. Stick, D. L. Zechel,
S. G. Withers, G. J. Davies, J. Am. Chem. Soc. 2003, 125, 7496.
doi:10.1021/JA034917K
[19] J. M. Macdonald, R. V. Stick, Aust. J. Chem. 2004, 57, 449.
doi:10.1071/CH03228
[20] P. L. Durette, D. Horton, Adv. Carbohyd. Chem. Biochem. 1971,
26, 49.
[21] E. J.Taylor,A. Goyal, C. I. P. D. Guerreiro, J.A. M. Prates,V.A. Money,
N. Ferry, C. Morland, A. Planas, J. A. Macdonald, R. V. Stick,
H. J. Gilbert, C. M. G. A. Fontes, G. J. Davies, J. Biol. Chem. 2005,
280, 32761. doi:10.1074/JBC.M506580200
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