The synthesis of 3-O-(β-D-glucopyranosyl)- and 3-O-(β-laminaribiosyl)-isofagomines, potent inhibitors of a 1,3-β-D-glucan endo-hydrolase

Article abstract of DOI:10.1071/CH03227

The glycosylation of 4,6-O-benzylidene-N-benzyloxycarbonylisofagomine with a D-glucosyl and a laminari-biosyl trichloroacetimidate has given, after removal of protecting groups, 3-O-(β-D-glucopyranosyl)- and 3-O-(β-laminaribiosyl)-isofagomines. Also inclu

Full text of DOI:10.1071/CH03227

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CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2004, 57, 187–191
The Synthesis of 3-O-(β-d-Glucopyranosyl)- and
3-O-(β-Laminaribiosyl)-isofagomines, Potent Inhibitors of a
1,3-β-d-Glucan endo-Hydrolase
James M. Macdonald,A Maria Hrmova,B Geoffrey B. Fincher,B and Robert V. StickA,C
A Chemistry, School of Biomedical and Chemical Sciences M313, University of Western Australia,
Crawley WA 6009, Australia.
B Faculty of Sciences, School of Agriculture and Wine, University of Adelaide, Glen Osmond
SA 5064, Australia.
C Author to whom correspondence should be addressed (e-mail: rvs@chem.uwa.edu.au).
The glycosylation of 4,6-O-benzylidene-N-benzyloxycarbonylisofagomine with a d-glucosyl and a laminari-
biosyl trichloroacetimidate has given, after removal of protecting groups, 3-O-(β-d-glucopyranosyl)- and
3-O-(β-laminaribiosyl)-isofagomines. Also included are similar glycosylations of a related tetrahydrooxazine.
3-O-(β-d-Glucopyranosyl)- and 3-O-(β-laminaribiosyl)-isofagomines acted as potent inhibitors of a barley
1,3-β-d-glucan endo-hydrolase, with ID50 values of 7.8 and 3.1 µM, respectively.
Manuscript received: 1 September 2003.
Final version: 7 January 2004.
We have recently described the synthesis of 4-O-(β-d-
glucopyranosyl)- and 4-O-(β-cellobiosyl)-isofagomine,
compounds 1 and 2 (Diagram 1).[1] While both compounds
were effective inhibitors of some endo-cellulases, com-
pound 2 was strikingly so (Ki 5 nm) against Cel5A from
Bacillus agaradhaerens.[2] It occurred to us that similar suc-
cess could be obtained by glycosylating isofagomine 3 at
the C3 hydroxyl group, so generating potential inhibitors
for enzymes that hydrolyze 1,3-β-d-glucans. The target
molecules were to be 3-O-(β-d-glucopyranosyl)- and
3-O-(β-laminaribiosyl)-isofagomine, 4 and 5.
Our synthesis of compounds 1 and 2 profited from the
glycosynthase-assisted glycosylation of the isofagomine car-
bamate 6.[1] Although a related glycosynthase exists for the
construction of the 1,3-β linkage required here,[3] the glyco-
syl donor is, of necessity, α-laminaribiosyl fluoride, limiting
the synthesis to just compound 5. In any case, we did try such
a glycosynthase-assisted approach, but the carbamate 6 did
not appear to act as an acceptor for α-laminaribiosyl fluo-
ride (results not shown). Direct chemical synthesis seemed
the only viable alternative; with this decision, we also
included glycosylation of the oxazine 7 (Diagram 2), to
form compounds 8 and 9, but were aware that similar
compounds in the cello-series were only moderate inhibitors
of endo-cellulases.[1]
Towards compounds 4 and 5, we converted the iso-
fagomine carbamate 6[1] into the alcohol 10 (Diagram 3).
Subsequent glycosylation of this alcohol with the trichloro-
acetimidates 11 and 12 gave the glycosides 13 and 14,
respectively. Although the stereochemistry of the newly
formed glycosidic linkages could not be confirmed at this
stage (owing to the complexity of the NMR spectra brought
OH
OH
OH
O
OH
OH
O
O
HO
HO
HO
HO
O
O
HO
HO
HO
NH
NH
O
OH
OH
OH
1
2
OH
OH
OH
OH
OH
OH
O
O
HO
O
HO
HO
1؅
HO
HO
HO
HO
HO
HO
O
O
O
NH
NH
NR
3
HO
HO
HO
R = H
3
4
5
R = CO2Bn
6
Diagram 1.
© CSIRO 2004
10.1071/CH03227
0004-9425/04/030187

188
J. M. Macdonald et al.
Table 1. ID50 values for isofagomine and oxazine derivatives
OH
OH
OH
O
with a barley 1,3-β-d-glucan endo-hydrolase
O
O
HO
1؅
HO
HO
HO
HO
O
InhibitorA
ID50 [µM]
NH
NH
4
HO
7
8
3
4
5
7
8
9
>20 mM
7.8
OH
O
OH
OH
3.1
O
O
HO
B
HO
HO
HO
—
O
O
NH
678
197
HO
HO
9
A Using reduced laminarin as a substrate at the pH optimum
(4.75) of the enzyme.[5]
Diagram 2.
B Not determined.
OAc
O
AcO
AcO
Ph
O
O
CCl3
protons directly bound to the isofagomine ring system, and
O
AcO
HO
ꢀ
ꢀ
NCO2Bn
J1 ,2 having a value of 8.0 Hz. As well, C3 for 4 and 5 res-
onated at about δ 80, a value some 10 ppm greater than the
corresponding resonance in isofagomine itself.
NH
10
11
OAc
OAc
For compounds 8 and 9, the carbamate 15[1] was converted
into the alcohol 16 (Diagram 4), and subsequent glycosyla-
tions as described above gave the new glycosides 17 and 18.
These molecules were not plagued by phenomena associated
with restricted rotation (around the N–CO bond) and so direct
evidence was available from the associated NMR spectra to
establish the nature of the new linkages (1,4-β). Subsequent
deprotection provided compounds 8 and 9.
O
O
AcO
AcO
AcO
O
AcO
AcO
O
CCl3
NH
12
AcO
AcO
AcO
Ph
O
O
O
O
NCO2Bn
Finally, the glycosylated derivatives of isofagomine 3 and
the tetrahydrooxazine 7 were tested as inhibitors of a barley
1,3-β-d-glucan endo-hydrolase (Table 1). While compounds
8 and 9 were ineffective, compounds 4 and 5 showed good
inhibition of the hydrolase. In particular, the laminaribiosyl
isofagomine 5 is a candidate molecule for generating a binary
complex with the barley 1,3-β-d-glucan endo-hydrolase in
future X-ray crystallographic investigations.[4]
AcO
13
AcO
AcO
AcO
Ph
O
O
O
O
AcO
AcO
O
O
NCO2Bn
AcO
AcO
14
Diagram 3.
Experimental
General experimental procedures have been given previously.[6]
OH
O
Ph
O
O
HO
HO
O
HO
NCO2Bn
NCO2Bn
Methyl 2-O-Benzoyl-4,6-O-benzylidene-3-O-(tetra-O-acetyl-
β-D-glucopyranosyl)-α-D-glucoside
15
16
Tetra-O-acetyl-α-d-glucopyranosyl trichloroacetimidate[7] (5.60 g,
11.4 mmol), methyl 2-O-benzoyl-4,6-O-benzylidene-α-d-glucoside[8]
(3.38 g, 8.77 mmol), and crushed molecular sieves (4 Å, 8 g) in dry
CH2Cl2 (30 mL) were stirred under Ar (2 h) and then cooled (−20◦C).
TMSOTf (60 µL) was added, and this mixture stirred (30 min), and then
allowed to warm to room temperature and stirred for a further 30 min.
Et3N (1 mL) was added and the mixture was filtered through Celite,
washing with EtOAc. The combined filtrate/washings were concen-
trated, and flash chromatography (EtOAc/petrol, 28 : 72 then 35 : 65)
of the residue afforded a colourless solid. Recrystallization gave the
title glycoside (5.05 g, 81%) as colourless micro-cubes, mp 184–186◦C
(EtOAc/petrol; lit.[9] 187–189◦C), [α]D +34.4◦ (lit.[9] +34.8◦).
AcO
AcO
AcO
Ph
O
O
O
O
O
NCO2Bn
AcO
17
AcO
AcO
AcO
AcO
Ph
O
O
O
O
AcO
O
O
O
NCO2Bn
AcO
AcO
2,4,6-Tri-O-acetyl-3-O-(tetra-O-acetyl-β-D-glucopyranosyl)-
α-D-glucosyl Fluoride
18
Diagram 4.
In a screw-capped plastic vial, octa-O-acetyl-α-laminaribiose (obtained
from the previous glycoside;[9] 500 mg, 0.74 mmol) was treated with
HF–pyridine(7 : 3, 1 mL), andthemixturekeptfor48 h.Themixturewas
poured into EtOAc and carefully neutralized by washing with aqueous
NaHCO3. The organic layer was subjected to normal workup and flash
chromatography(EtOAc/petrol, 2 : 3then1 : 1)toaffordthe titlefluoride
(280 mg, 60%) as a colourless solid. A small portion was recrystallized
about by the inherent carbamate[1]), a two-step deprotection
protocol furnished the desired compounds 4 and 5.The NMR
spectra were now amenable to analysis, with H3 of 4, a dou-
blet of doublets of doublets, clearly downfield from all other

Synthesis of 3-O-(β-d-Glucopyranosyl)- and 3-O-(β-Laminaribiosyl)-isofagomines
189
•
to give fine needles, mp 196–198◦C (EtOH), [α]D +11.6◦ (Found: C
48.9, H 5.5. C26H35FO17 requires C 48.9, H 5.5%). δH (300 MHz) 1.95,
1.96, 1.99, 2.03, 2.05, 2.07, 2.21 (21 H, 7 s, CH3), 3.68 (1 H, ddd, J
2.3, 4.4, 9.8), 4.00–4.23 (5 H, m), 4.35 (1 H, dd, J 4.4, 12.5), 4.63 (d,
(12 C, Ph), 155.02 (C=O). m/z (FAB) 370.1691 [(M + H)+ requires
370.1654].
(1R,3R,6R,10R)-8-Benzyloxycarbonyl-3-phenyl-10-(tetra-O-acetyl-
β-D-glucopyranosyl)oxy-8-aza-2,4-dioxabicyclo[4.4.0]decane 13
J1 ,2 8.1, H1ꢀ), 4.83–4.98 (2 H, m), 5.01–5.16 (3 H, m), 5.63 (dd, J1,2
2.7, J1,F 53, H1). δC (125.8 MHz) 20.26, 20.32, 20.50, 20.57, 20.67,
20.69 (7 C, CH3), 61.29, 61.55 (C6, 6ꢀ), 66.65, 67.83 (2 C), 70.01 (1 C,
d, JC,F 24), 72.83 (1 C), 75.56 (C3), 100.73 (C1ꢀ), 103.98 (d, J1,F 228,
ꢀ
ꢀ
Tetra-O-acetyl-β-d-glucopyranosyltrichloroacetimidate11[11] (150 mg,
0.30 mmol), the alcohol 10 (90 mg, 0.23 mmol), and crushed molecular
sieves (4 Å, 1 g) in dry CH2Cl2 (10 mL) were stirred under Ar (2.5 h)
and then cooled (−30◦C). TMSOTf (30 µL) was added and this mixture
stirred (1 h). Et3N (0.5 mL) was added, and the mixture was allowed to
warmtoroomtemperatureandthenfilteredthroughCelite, washingwith
EtOAc. The combined filtrate/washings were concentrated, and flash
chromatography (EtOAc/petrol, 3 : 7 then 9 : 11) of the residue gave the
glycoside 13 (150 mg, 91%) as a colourless foam, [α]D −6.2◦ (Found:
C 60.5, H 6.3, N 1.8. C35H41NO14 requires C 60.1, H 5.9, N 2.0%). δH
(500 MHz) 1.98, 1.98, 1.99 (12 H, 3 s, CH3), 2.04–2.09 (m, H6), 2.38–
2.57 (1 H, m), 2.63–2.83 (1 H, m), 3.39–3.53(1 H, m), 3.60–4.00 (5 H,
m), 4.03–4.28 (3 H, m), 4.64–4.72 (1 H, m), 4.87–5.17 (5 H, m), 5.60
(s, H3), 7.31–7.49 (10 H, m, Ph). δC (125.8 MHz) δ 20.22, 20.54, 20.58,
20.67 (4 C, CH3), 37.56, 37.98 (C6), 43.10, 46.56 (C7, 9), 61.54 (C6ꢀ),
67.81, 68.08 (C4ꢀ, 5, PhCH2), 71.57, 71.90, 72.78 (C2ꢀ, 3ꢀ, 5ꢀ, 10), 82.72
(C1), 100.88, 101.39 (C1ꢀ, 3), 125.93, 128.17, 128.33, 128.60, 128.76,
129.06, 136.08, 137.72 (12 C, Ph), 154.90 (NC=O), 169.29, 170.27,
•
C1). m/z (FAB) 639.1931 [(M + H)+ requires 639.1937].
3-O-β-D-Glucopyranosyl-α-D-glucopyranosyl Fluoride
(α-Laminaribiosyl Fluoride)
The above per-acetylated fluoride (90 mg) in MeOH was treated with
Na (5 mg). After 1 h, the mixture was neutralized with resin (Dowex-
50W, H+), then filtered, the resin washed with MeOH, and the combined
filtrate and washings were concentrated to give the crude title fluoride.
The purity of the product was checked by TLC and NMR spectroscopic
analysis. δH (500 MHz, D2O) 3.38 (1H, dd, J 8.3, 9.4), 3.43 (1H, t,
J ≈ 9.4), 3.47–3.57 (2 H, m), 3.64 (1 H, t, J ≈ 9.7), 3.75 (1 H, dd, J
6.0, 10.3), 3.79–3.92 (4 H, m), 3.94 (1 H, dd, J 2.0, 12.4), 3.98 (1H, t,
J ≈ 9.5), 4.75 (d, J1 ,2 7.9, H1ꢀ), 5.72 (dd, J1,2 2.7, J1,F 53, H1). δC
(125.8 MHz, D2O) 60.77, 61.34 (C6, 6ꢀ), 67.72, 70.29 (2 C), 71.37 (1
C, d, JC,F 25), 74.08 (1 C), 74.65 (1 C, d, JC,F 3.0), 76.24, 76.68 (2 C),
81.71 (C3), 103.42 (C1ꢀ), 108.05 (d, J1,F 223, C1). m/z (FAB) 345.1192
ꢀ
ꢀ
•
170.68 (4 C, CH3C=O). m/z (FAB) 700.2612 [(M + H)+ requires
•
700.2605].
[(M + H)+ requires 345.1197].
(1R,3R,6R,10R)-8-Benzyloxycarbonyl-3-phenyl-10-[(tetra-O-acetyl-
β-D-glucopyranosyl)-(1→3)-O-(tri-O-acetyl-β-D-glucopyranosyl)]-
oxy-8-aza-2,4-dioxabicyclo[4.4.0]decane 14
2,4,6-Tri-O-acetyl-3-O-(tetra-O-acetyl-β-D-glucopyranosyl)-
α-D-glucosyl Trichloroacetimidate 12
Octa-O-acetyl-α-laminaribiose[9] (1.27 g, 1.87 mmol)inCH2Cl2 (5 mL)
was treated with HBr in AcOH (30% w/v, 2.5 mL) overnight. This mix-
ture was then diluted with ice–water, and normal workup (EtOAc) gave
a solid (1.28 g). This solid, in acetone/H2O (6 : 1, 30 mL), was treated
with Ag2CO3 (560 mg, 2.0 mmol), and stirred (1 h) with the exclusion
of light. The mixture was filtered (silica; washing with EtOAc) and the
combined filtrate/washings were concentrated to give a solid. Recrystal-
lization gave, presumably, the hemiacetal (740 mg, 62%) as fine needles,
mp 210–213◦C (EtOH; lit.[10] 232–234◦C). A portion of these needles
(520 mg, 0.81 mmol) in dry CH2Cl2 (15 mL) was treated with Cl3CCN
(200 µL, 2.0 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 1
drop). After 1 h, the mixture was concentrated. Flash chromatography
(EtOAc/petrol, 3 : 7 then 4 : 6) of the residue gave the trichloroacetim-
idate 12 [530 mg, 84% from the hemiacetal] as an oil, [α]D +36.4◦
(Found: C 42.8, H 4.4, N 1.6. C28H36Cl3NO18 requires C 43.1, H 4.6,
N 1.8%). δH (500 MHz) 1.97, 1.98, 2.00, 2.05, 2.07, 2.08, 2.10 (21
H, 7 s, CH3), 3.73 (1 H, ddd, J 2.4, 4.6, 10.0), 4.07–4.21 (5 H, m),
The trichloroacetimidate 12 (150 mg, 0.19 mmol) and the alcohol 10
(60 mg, 0.15 mmol)weretreatedasfor 11and 10previouslytogive, after
flash chromatography (EtOAc/petrol, 9 : 11 then 6 : 4), the glycoside 14
(110 mg, 75%) as an opaque film, [α]D −17.4◦ (Found: C 57.1, H 6.0, N
1.3. C47H57NO22 requires C 57.1, H 5.8, N 1.4%). δH (500 MHz) 1.95,
1.97, 1.97, 1.98, 2.04 (21 H, 5 s, CH3), 2.08–2.18 (m, H6), 2.35–2.56
(1 H, m), 2.59–2.83 (1 H, m), 3.35–4.25 (12 H, m), 4.31–4.37 (1 H, m),
4.49–4.51 (2 H, m), 4.80–5.17 (7 H, m), 5.57 (s, H3), 7.28–7.49 (10 H,
m, Ph). δC (125.8 MHz) 20.20, 20.34, 20.41, 20.43, 20.50, 20.60 (7 C,
CH3), 37.55, 37.91 (C6), 43.03, 46.49 (C7, 9), 61.56, 61.72 (C6ꢀ, 6ꢀꢀ),
67.70, 67.92, 67.95 (4 C), 70.95, 71.58, 71.87, 72.84 (6 C), 78.75 (C3ꢀ),
82.50, 82.61 (C1), 100.76, 101.17 (C1ꢀ, 1ꢀꢀ, 3), 125.83, 128.03, 128.25,
128.52, 128.90, 136.06, 137.72 (12 C, Ph), 154.84 (NC=O), 168.70,
168.96, 169.15, 169.24, 170.24, 170.34, 170.65 (7 C, CH3C=O). m/z
•
(FAB) 988.3436 [(M + H)+ requires 988.3450].
(3R,4R,5R)-3-(β-D-Glucopyranosyl)oxy-4-hydroxy-5-(hydroxymethyl)-
piperidine [3-O-(β-D-Glucopyranosyl)isofagomine] 4
4.35 (1 H, dd, J 4.6, 12.4), 4.66 (d, J1 ,2 8.1, H1ꢀ), 4.90 (1 H, dd, J
8.1, 9.6), 5.03–5.18 (4 H, m), 6.45 (d, J1,2 3.7, H1), 8.70 (s, NH). δC
(125.8 MHz) 20.33, 20.42, 20.51, 20.53, 20.61, 20.68 (7 C, CH3), 61.54,
61.71 (C6, 6ꢀ), 67.18, 68.10, 70.21, 71.37, 71.67, 71.73, 72.86, 76.37 (8
C), 90.77 (CCl3), 93.11 (C1), 100.93 (C1ꢀ), 160.57 (C=NH), 169.10,
169.29, 169.55, 170.33, 170.48, 170.64 (7 C, C=O).
ꢀ
ꢀ
The glycoside 13 (75 mg) was treated with aqueous AcOH (14 M,
3 mL) and heated to 70◦C (3 h). The mixture was allowed to cool and
was then concentrated. Flash chromatography (EtOAc/petrol, 6 : 4, then
MeOH/EtOAc, 1 : 9) of the residue gave an oil (63 mg), presumably
a diol. A mixture of this oil and NaOH (290 mg) in MeOH/H2O (2 : 1,
4.5 mL) was heated under reflux (2 h) and then allowed to cool.The reac-
tion mixture was concentrated, taken up in H2O (2 mL), and brought
to pH 5 by the addition of HCl (2 M). The mixture was applied to a
column of cation-exchange resin (Dowex 50W-X2, H+), washed with
water, and then eluted with aqueous NH3 (3 M). The eluate was concen-
trated, 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 d-glucosyl isofagomine 4 (31 mg, 94%) as a
colourless, amorphous solid, [α]D −18.1◦ (H2O). δH (600 MHz, D2O)
1.70–1.72 (m, H5), 2.44–2.51 (2 H, m, H2, 6), 3.12 (dd, J5,6 3.4, J6,6
(1R,3R,6R,10R)-8-Benzyloxycarbonyl-3-phenyl-8-aza-
2,4-dioxabicyclo[4.4.0]decan-10-ol 10
The carbamate
6 (110 mg, 0.39 mmol), PhCH(OEt)2 (102 µL,
0.54 mmol), and camphorsulfonic acid (CSA; 5 mg) in dry CHCl3
(5 mL) were heated under reflux (1 h) and allowed to cool. Et3N (0.5 mL)
was added and then this mixture concentrated. Flash chromatography
(EtOAc/petrol, 1 : 4 then 2 : 3) of the residue gave the benzylidene acetal
10 (120 mg, 88%) as a colourless foam, [α]D +2.0◦ (Found: C 68.0, H
6.1, N 3.7. C21H23NO5 requires C 68.3, H 6.3, N 3.8%). δH (300 MHz)
1.87–2.02 (m, H6), 2.34–2.52 (1 H, m), 2.61–2.78, (1 H, m), 2.86–2.98
(1 H, m), 3.45 (1 H, t, J 9.1), 3.57 (1 H, t, J 11.0), 3.62–3.77 (1 H, m),
3.90–4.20 (2 H, m), 4.30–4.55 (1 H, m), 5.09–5.15 (m, PhCH2), 5.55 (s,
H3), 7.28–7.50 (10 H, m, Ph). δC (75.5 MHz) 36.85 (C6), 43.30, 47.91
(C7,9), 67.56, 68.07 (C5, PhCH2), 68.19 (C10), 84.84 (C1), 101.97
(C3), 126.17, 127.95, 128.15, 128.30, 128.5, 129.18, 136.24, 137.58
13.2, H6), 3.29 (dd, J1 ,2 8.0, J2 ,3 9.4, H2ꢀ), 3.29–3.30 (m, H2), 3.40
(dd, J 9.2, 9.8, H4ꢀ), 3.45–3.51 (m, H3ꢀ, 4, 5ꢀ), 3.66 (dd, J5,H 6.5, JH,H
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
11.5, 1H, CH2O), 3.71 (ddd, J 5.0, 9.0, 10.7, H3), 3.73 (dd, J5 ,6 6.0,
J6 ,6 12.4, H6ꢀ), 3.81 (dd, J5,H 3.4, 1H, CH2O), 3.92 (dd, J5 ,6 2.2,
H6ꢀ), 4.55 (d, H1ꢀ). δC (150.9 MHz, D2O) 44.98 (C5), 46.59 (C6), 47.60
(C2), 61.08 (CH2O), 61.37 (C6ꢀ), 70.26 (C4ꢀ), 72.32 (C4), 73.57 (C2ꢀ),
ꢀ
ꢀ
ꢀ
ꢀ

190
J. M. Macdonald et al.
76.29, 76.61 (C3ꢀ,5ꢀ), 80.33 (C3), 101.57 (C1ꢀ). m/z (FAB) 310.1512
after flash chromatography (EtOAc/petrol, 4 : 6 then 6 : 4), the glycoside
18 (170 mg, 73%) as a colourless glass, [α]D +3.2◦ (Found: C 55.5, H
5.6, N 1.6. C46H55NO23 requires C 55.8, H 5.6, N 1.4%). δH (500 MHz)
1.96, 1.98, 1.98, 1.98, 1.99, 2.01, 2.04 (21 H, 7 s, CH3), 3.21 (dd, J4,4
13.6, J4,5 10.1, H4), 3.44–3.50 (1 H, m), 3.64 (1 H, ddd, J 2.4, 4.2,
9.3), 3.70–3.94 (5 H, m), 3.98 (1 H, dd, J 2.4, 12.3), 4.02 (1 H, dd, J
2.4, 12.5), 4.06 (1 H, dd, J 4.1, 12.3), 4.31–4.40 (3 H, m), 4.53, 4.54 (2
d, J 8.1, 8.1, H1ꢀ,1ꢀꢀ), 4.86, 4.89 (2 dd, J 8.1, 9.5, J 8.1, 9.6, H2ꢀ, 2ꢀꢀ),
4.92, 5.04, 5.11 (3 t, J 9.7, 9.5, 9.4, H3ꢀꢀ, 4ꢀ, 4ꢀꢀ), 5.20 (s, PhCH2), 5.59
(s, H8), 7.30–7.46 (10 H, m, Ph). δC (125.8 MHz) 20.25, 20.38, 20.47,
20.55, 20.63, 20.67 (7 C, CH3), 48.96 (C4), 61.59, 61.69 (C6ꢀ, 6ꢀꢀ), 66.52
(C10), 67.74, 67.94 (2 C), 68.35 (PhCH2), 70.97, 71.63, 71.96, 72.60,
72.85, 73.53, 74.55 (9 C), 78.71 (C3ꢀ), 80.59 (C1), 100.20, 100.81,
101.57 (C1ꢀ, 1ꢀꢀ, 8), 125.95, 128.11, 128.18, 128.53, 128.63, 129.21,
135.35, 136.77 (12 C, Ph), 155.08 (NC=O), 168.66, 168.99, 169.19,
169.27, 170.28, 170.38, 170.64 (7 C, CH3C=O). m/z (FAB) 990.3271
•
[(M + H)+ requires 310.1502].
(3R,4R,5R)-3-[(β-D-Glucopyranosyl)-(1→3)-O-(β-D-
glucopyranosyl)]oxy-4-hydroxy-5-(hydroxymethyl)piperidine
[3-O-(β-Laminaribiosyl)isofagomine] 5
The pseudo-trisaccharide 14 (90 mg) was treated as for 13 previ-
ously to give, after flash chromatography (EtOAc/petrol, 3 : 1, then
MeOH/EtOAc, 1 : 19), first an oil (80 mg), presumably a diol, and then
the laminaribiosyl isofagomine 5 (40 mg, 93%) as a colourless, amor-
phous solid, [α]D −23.4◦ (H2O). δH (500 MHz, D2O) 1.67–1.71 (m,
H5), 2.38–2.48 (2 H, m, H2, 6), 3.08 (dd, J5,6 4.3, J6,6 13.0, H6), 3.28
(dd, J2,2 5.0, J2,3 12.5, H2), 3.41 (1H, dd, J 9.1, 9.8), 3.36 (1H, dd, J 8.0,
9.4), 3.44–3.55 (6 H, m), 3.63–3.78 (5 H, m), 3.81 (1 H, dd, J 3.5, 11.5),
3.90–3.96 (2 H, m), 4.59, 4.75 (2 d, J 7.9, 8.0, H1ꢀ,1ꢀꢀ). δC (125.8 MHz,
D2O) 45.35 (C5), 46.74 (C6), 47.84 (C2), 61.22, 61.34, 61.37 (C6ꢀ,
6ꢀꢀ, CH2O), 68.79 (C4ꢀ), 70.25 (C4ꢀꢀ), 72.56 (C4), 73.38, 74.10 (C2ꢀ,
2ꢀꢀ), 76.21, 76.67 (C3ꢀꢀꢀ, 5ꢀ, 5ꢀꢀ), 80.71 (C3),•84.84 (C3ꢀ), 101.30, 103.47
(C1ꢀ, 1ꢀꢀ). m/z (FAB) 472.2042 [(M + H)+ requires 472.2030].
•
[(M + H)+ requires 990.3243].
(4R,5S,6R)-4-(β-D-Glucopyranosyl)oxy-5-hydroxy-6-hydroxymethyl-
3,4,5,6-tetrahydro-1,2(2H)-oxazine 8
The glycoside 17 (110 mg) was treated as for 13 previously to give, after
flash chromatography (EtOAc/petrol, 4 : 6, then MeOH/EtOAc 1 : 9),
first an oil (75 mg), presumably a diol, and then the d-glucosyl oxazine
8 (31 mg, 64%) as a colourless, amorphous solid, [α]D −29.3◦ (H2O).
(1R,5R,6S,8R)-3-Benzyloxycarbonyl-8-phenyl-3-aza-
2,7,9-trioxabicyclo[4.4.0]decan-5-ol 16
The carbamate 15[1] (580 mg, 2.0 mmol) was treated with PhCH(OEt)2
(520 µL, 2.8 mmol) and CSA (20 mg) in dry CHCl3 (20 mL) as for
6 previously to give, after flash chromatography (EtOAc/petrol, 1 : 4
then 2 : 3), the benzylidene acetal 16 (610 mg, 81%) as a colourless
solid. A small portion was recrystallized to give colourless needles, mp
153–156◦C (EtOAc/petrol), [α]D +54.3◦ (Found: C 65.0, H 5.8, N 4.0.
C20H21NO6 requires C 64.7, H 5.7, N 3.8%). δH (500 MHz) 3.06 (br
s, OH), 3.23 (dd, J 10.4, 13.5, H4), 3.65 (1 H, t, J 9.2), 3.71 (1 H,
t, J 10.2), 3.80 (1 H, dd, J 4.8, 9.8), 3.82–3.89 (1 H, m), 4.36–4.44
(2 H, m), 5.18–5.24 (2 H, m), 5.57 (s, H8), 7.33–7.50 (10 H, m, Ph).
δC (125.8 MHz) 50.63 (C4), 66.23 (1 C), 66.53 (C10), 68.19 (PhCH2),
74.02 (1 C), 82.63 (C1), 102.10 (C8), 126.15, 128.11, 128.32, 128.42,
128.56, 129.38, 135.42, 136.61 (12 C, Ph), 155.06 (C=O). m/z (FAB)
ꢀ
ꢀ
δH (600 MHz, D2O) 3.11 (dd, J3,3 13.2, J3,4 10.7, H3), 3.31 (dd, J1 ,2
7.9, J2 ,3 9.4, H2ꢀ), 3.42 (J3 ,4 9.1, J4 ,5 9.8, H4ꢀ), 3.47–3.53 (m, H3ꢀ,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
5ꢀ), 3.61 (dd, J3,4 5.3, H3), 3.66 (t, J4,5 ≈ J5,6 8.9, H5), 3.72 (dd, J5 ,6
ꢀ
ꢀ
6.3, J6 ,6 12.4, H6ꢀ), 3.75–3.80 (2 H, m, H6, CH2O), 3.91–3.97 (2 H,
m, H6ꢀ, CH2O), 4.01 (ddd, H4), 4.59 (d, H1ꢀ). δC (150.9 MHz, D2O)
48.89 (C3), 60.11 (CH2O), 61.36 (C6ꢀ), 68.34 (C5), 70.23 (C4ꢀ), 73.48
(C2ꢀ), 76.18 (C3ꢀ), 76.64 (C5ꢀ), •77.17 (C4), 84.11 (C6), 101.26 (C1ꢀ).
m/z (FAB) 312.1277 [(M + H)+ requires 312.1295].
ꢀ
ꢀ
(4R,5S,6R)-4-[(β-D-Glucopyranosyl)-(1→3)-O-(β-D-
glucopyranosyl)]oxy-5-hydroxy-6-hydroxymethyl-
3,4,5,6-tetrahydro-1,2(2H)-oxazine 9
•
372.1447 [(M + H)+ requires 372.1447].
The pseudo-trisaccharide 18 (110 mg) was treated as for 13 previ-
ously to give, after flash chromatography (EtOAc/petrol, 8 : 2, then
MeOH/EtOAc, 1 : 19), first an oil (90 mg), presumably a diol, and then
the laminaribiosyl oxazine 9 (36 mg, 66%) as a colourless, amorphous
solid, [α]D –39.6◦ (H2O). δH (500 MHz, D2O) 2.98 (dd, J3,3 13.3, J3,4
10.6, H3), 3.36 (1 H, dd, J 7.9, 9.4), 3.42 (1 H, dd, J 8.9, 9.8), 3.44–3.64
(8 H, m), 3.70–3.78 (4 H, m), 3.90–3.97 (4 H, m), 4.62, 4.76 (2 d, J 7.9,
8.0, H1ꢀ, 1ꢀꢀ). δC (125.8 MHz, D2O) 50.85 (C3), 60.46 (CH2O), 61.36,
61.37 (C6ꢀ, 6ꢀꢀ), 68.81, 69.05 (C4ꢀ, 5), 70.24 (C4ꢀꢀ), 73.36, 74.09 (C2ꢀ,
2ꢀꢀ), 76.20, 76.26, 76.66 (C3ꢀꢀ, 5ꢀ, 5ꢀꢀ), 78.51 (C4), 84.12, 84.74 (C3ꢀ,
6), 100.99, 103.45 (C1ꢀ, 1ꢀꢀ). m/z (FAB) 474.1859 [(M + H)+• requires
474.1823].
(1R,5R,6S,8R)-3-Benzyloxycarbonyl-8-phenyl-5-(tetra-O-acetyl-β-D-
glucopyranosyl)oxy-3-aza-2,7,9-trioxabicyclo[4.4.0]decane 17
The trichloroacetimidate 11 (160 mg, 0.32 mmol) and the alcohol 16
(100 mg, 0.27 mmol) were treated as for 11 and 10 previously to give,
after flash chromatography (EtOAc/petrol, 2 : 8 then 1 : 2), the glycoside
17 (150 mg, 82%) as a colourless foam, [α]D +23.9◦ (Found: C 57.9, H
5.6, N 2.0. C34H39NO15 requires C 58.2, H 5.6, N 2.0%). δH (500 MHz)
1.94, 1.97, 1.98, 1.99 (12 H, 4 s, CH3), 3.22 (dd, J4,4 13.7, J4,5 10.4,
H4), 3.49 (ddd, J 2.4, 4.0, 9.7, H5ꢀ), 3.74 (1 H, t, J 9.8), 3.79 (dt,
J 3.9, J ≈ 8.7, H1), 3.86 (1 H, t, J 8.9), 3.92–3.99 (2 H, m), 4.10 (1 H,
dd, J 4.0, 12.4), 4.36 (1 H, dd, J 4.0, 9.6), 4.40 (dd, J4,5 5.5, H4), 4.67 (d,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J1 ,2 8.0, H1 ), 4.97 (dd, J2 ,3 9.4, H2 ), 5.07 (t, J3 ,4 ≈ J4 ,5 9.6, H4 ),
5.12 (t, H3ꢀ), 5.20 (s, PhCH2), 5.60 (s, H8), 7.31–7.46 (m, 10 H, Ph). δC
(125.8 MHz) 20.40, 20.46, 20.49, 20.58 (4 C, CH3), 48.96 (C4), 61.42
(C6ꢀ), 66.53 (C10), 67.80 (C4ꢀ), 68.35 (PhCH2), 71.19, 71.88, 72.68,
73.68, 74.48 (C2ꢀ, 3ꢀ, 5, 5ꢀ, 6), 80.62 (C1), 100.17, 101.64 (C1ꢀ, 8),
125.94, 128.14, 128.21, 128.51, 128.61, 129.25, 135.32, 136.70 (12 C,
Ph), 155.02 (NC=O), 169.12, 169.20, 170.17, 170.54 (4 C, CH3C=O).
Enzyme Inhibition
Enzyme Isolation and Purity
Barley 1,3-β-d-glucan endo-hydrolase isoenzyme GII was purified from
a homogenate of 14-day-old seedlings as described.[5] The purity of
the enzyme was assessed by SDS–PAGE, where a single protein band
was detected at high protein loadings, and by NH2-terminal amino acid
sequence analysis.[12]
•
m/z (FAB) 702.2401 [(M + H)+ requires 702.2398].
(1R,5R,6S,8R)-3-Benzyloxycarbonyl-8-phenyl-5-[(tetra-O-acetyl-β-
D-glucopyranosyl)-(1→3)-O-(tri-O-acetyl-β-D-glucopyranosyl)]oxy-
3-aza-2,7,9-trioxabicyclo[4.4.0]decane 18
Determination of ID50 Values of the Isofagomine and
Oxazine Derivatives
The ID50 (inhibitory dose at 50%) values were determined by incu-
bating 0.5 pkat∗ of the purified enzyme at 37◦C in 50 mm sodium
acetate buffer (pH 4.75) containing 160 µg/mL BSA, and 0.1% (w/v)
The trichloroacetimidate 12 (240 mg, 0.31 mmol) and the alcohol 16
(90 mg, 0.24 mmol) were treated as for 11 and 10 previously to give,
*The unit picokatal (pkat) refers to one-trillionth of a katal (kat), where a katal is a unit of catalytic activity equal to one mole of product formed (or
substrate consumed) per second, as of the amount of enzyme that catalyses the transformation of one mole of substrate per second.

Synthesis of 3-O-(β-d-Glucopyranosyl)- and 3-O-(β-Laminaribiosyl)-isofagomines
191
reduced Laminaria digitata laminarin (Sigma Chemical Co., St Louis,
MO, USA). The ID50 values were estimated using concentrations
of inhibitors that were 0.2–3 times the ID50 values, at five concen-
trations, in duplicate. The residual enzyme activity was monitored
reductometrically.[13,14] Substrate hydrolysis never exceeded 10% of
the initial substrate concentrations. Standard deviation values for assays
were within the range 6–9%. The ID50 values were calculated using
non-linear regression analyses[15] and the GraFit program.[16] One unit
of enzyme activity is defined as 1 µmol glucose equivalents released
per min and one unit corresponds to 16.67 nkat.
[3] 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
[4] M. Hrmova, G. B. Fincher, Plant Mol. Biol. 2001, 47, 73.
doi:10.1023/A:1010619128894
[5] M. Hrmova, G. B. Fincher, Biochem. J. 1993, 289, 453.
[6] J. C. McAuliffe, R. V. Stick, Aust. J. Chem. 1997, 50, 193.
doi:10.1071/C96151
[7] R. R. Schmidt, M. Stumpp, Liebigs Ann. Chem. 1983, 1249.
[8] S. David, A. Thieffry, J. Chem. Soc., Perkin Trans. 1 1979,
1568.
[9] E. B. Rodriguez, R. V. Stick, Aust. J. Chem. 1990, 43, 665.
[10] K. Takeo, Carbohydr. Res. 1980, 86, 151. doi:10.1016/S0008-
6215(00)84592-9
[11] R. R. Schmidt, J. Michel, M. Roos, Liebigs Ann. Chem. 1984,
1343.
Acknowledgment
We thank the Australian Research Council for financial
assistance.
[12] M. Hrmova, A. J. Harvey, J. Wang, N. J. Shirley, G. P. Jones,
B. A. Stone, P. B. Høj, J. Biol. Chem. 1996, 271, 5277.
doi:10.1074/JBC.271.9.5277
References
[1] J. M. Macdonald, R. V. Stick, D. M. G. Tilbrook, S. G. Withers,
Aust. J. Chem. 2002, 55, 747. doi:10.1071/CH02165
[2] 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
[13] N. Nelson, J. Biol. Chem. 1944, 153, 375.
[14] M. Somogyi, J. Biol. Chem. 1952, 195, 19.
[15] D. W. Marquart, J. Soc. Ind. Appl. Math. 1963, 11, 431.
[16] R. J. Leatherbarrow, in GraFit User’s Guide Version 4.0 1998
(Erithacus Software: Horley).