SPECIAL TOPIC
Synthesis of an 1’-Azasugar Analogue of Maltose
341
tained on a Micromass LCT instrument. Concentrations were per-
formed on a rotary evaporator at a temperature below 40 °C.
1H NMR (CDCl3): d = 4.8 (d, 1 H, H-1), 3.79 (dd, 1 H, H-5”a, J5’,5”a
= 3.5 Hz, J5”a,5”b = 11.5 Hz), 3.75-3.53 (m, 6 H, H-2, H-3, H-5,
H-5”b, H-6a, H-6b), 3.46 (dt, 1-H, H-3’, J3’,2’eq = 4.0 Hz,
J3’,2’ax = J3’,4’ = 10.0 Hz), 3.37 (s, 3 H, OCH3), 3.22 (t, 1 H, H-4’, J4’,3’
= 10.0 Hz, J4’,5’ = 10.0 Hz), 3.10 (br dd, 2 H, H-2’eq, H-6’eq,
J2’eq,3’ = J6’eq,5’ = 4.0 Hz, J2’eq,2’ax = J6’eq,6’ax = 11.5 Hz), 2.70 (dd, 1 H,
H-4”a, J4”a,4 = 6.5 Hz, J4”a,4”b = 13.0 Hz), 2.54 (dd, 1 H, H-4”b, J4”a,4
= 6.5 Hz, J4”a,4”b = 13.0 Hz), 2.04 (t, 2 H, H-2’ax, H-6’ax,
J2’ax,2’eq = J6’ax,6’eq = J2’ax,3’ = J6’ax,5’ = 11.0 Hz), 1.90 (m, 1 H, H-4),
1.71 (m, 1 H, H-5’).
13C NMR (CDCl3): d = 100.2 (C-1); 74.1, 72.6, 72.4, 71.9, 71.8 (C-
2, C-3, C-5, C-3’, C-4’), 62.7, 61.2 (C-6, C-5”), 58.3, 57.4 (C-2’, C-
6’), 55.6 (OCH3), 55.2 (C-4”), 44.0, 41.3 (C-4, C-5’).
HRMS (ES): m/z calcd for C14H27NO8 (M + Na+): 360.1634, Found:
360.1632.
Methyl 4-Aminomethyl-2,3,6-tri-O-benzyl-4-deoxy-a-D-glu-
copyranoside (5)
To a stirred solution of 4 (0.200 g, 0.42 mmol) in anhyd Et2O (3.5
mL) at 0 °C and under anhydrous conditions was added dropwise a
suspension of LiAlH4 (0.065 g, 1.71 mmol) in anhyd Et2O (2 mL).
After stirring at r.t. for 3.5 h, the reaction was quenched with a mix-
ture of H2O (1 mL) and 1 N NaOH (1 mL). The aqueous phase was
extracted repeatedly with Et2O. The combined organic phases were
washed with H2O (4 mL), dried (Na2SO4), filtered and evaporated.
The crude product was subjected to chromatography with EtOAc
containing 1% Et3N as eluent; yield: 0.126 g (62%); [a]D20 +4.6 (c =
1, CHCl3).
1H NMR (CDCl3): d = 7.42-7.28 (m, 15 H, C6H5), 5.05-4.45 (m, 7
H, PhCH2, H-l), 3.94-3.81 (m, 2 H, H-3, H-5), 3.65-3.58 (m, 3 H,
Determination of Enzyme Inhibition Constants
H-2, H-6a, H-6b), 3.39 (s, 3 H, OCH3), 2.89 (dd, 1 H, H-4'a, J4,4’a
3.5 Hz, J4a,4b = 13.5 Hz), 2.65 (dd, 1 H, H-4'b, J4,4b = 3.5 Hz, J4a,4b
13.5 Hz), 1.79 (tt, 1 H, H-4, J4,4’a/4’b = 3.0 Hz, J4,3/5 = 11.0 Hz).
=
=
Each glycosidase assay was performed by preparing ca 2 mL
samples in cuvettes consisting of 1 mL of sodium phosphate buffer
(0.1 M) of pH 6.8 (or as noted in the Table), 0.2 mL to 0.8 mL of a
1.0 mM or 10 mM solution of either 4-nitrophenyl a-D-glucopyra-
noside or 4-nitrophenyl b-D-glucopyranoside, and 0.1 mL of a solu-
tion of either the potential inhibitor or H2O, and distilled H2O to a
total volume of 1.9 mL. Eight of the samples contained the potential
inhibitor at fixed concentration, but with variant nitrophenyl glyco-
side concentration. Other 8 samples contained no inhibitor, but also
variant nitrophenyl glycoside concentration. Finaly the reaction was
started by adding 0.1 mL of a diluted solution of either a-glucosi-
dase from bakers yeast (EC 3.2.1.20, Sigma G-5003), isomaltase
from yeast, glucoamylase from aspargillus niger or b-glucosidase
from almonds (EC 3.2.1.21, Sigma G-0395), and the formation of
4-nitrophenol was followed for 2 min at 25 °C (or as noted in the
Table) by measuring absorbance at 400 nm. Initial velocities were
calculated from the slopes for each of the eight reactions and used
to construct two Hanes plots; one with and without inhibitor. From
the two Michaelis-Menten constants (Km) thus obtained, the inhi-
bition constant (Ki) was calculated.
13C NMR (CDCl3): d = 138.5, 138.2, 137.9, 128.3-127.6 (several
peaks, C6H5), 98.3 (C-l), 81.7 (C-2), 75.3 (PhCH2), 75.1 (C-3), 75.3,
72.7 (PhCH2), 70.1 (C-6), 68.5 (C-5), 55.1 (OCH3), 45.2 (C-4), 38.4
(C-4').
HRMS (ES): m/z calcd for C29H35NO5 (M + H+): 478.2593, Found:
478.2560.
Methyl 2,3,6-Tri-O-benzyl-4-deoxy-4-formyl-a-D-glucopyrano-
side (6)
Compound 4 (0.400 g, 0.84 mmol) was dissolved in a mixture of
THF/CH2Cl2 (7:23, 2.4 mL) under anhydrous conditions. Then 1.5
M DIBAL in toluene (0.9 mL, 1.35 mmol) was added to the solution
over 1 min at -20 °C. The reaction mixture was stirred at -20 °C
for 2 h, and then stirred at 20 °C for 5 h. Subsequently, a mixture of
THF/MeOH (1:1, 1.6 mL) and 1 M HCl (1.6 mL) were carefully
added at -10 °C. The mixture was then stirred for 10 min at r.t. Et2O
(50 mL) was added, and the mixture was washed with 1 M HCl
(2 î 12 mL) and H2O (3 î 12 mL). The organic phase was dried
(Na2SO4), filtered and evaporated. Column chromatography was
performed with CH2Cl2 containing 1% EtOAc as eluent to afford 67;
yield: 0.290 g (72%).
1H NMR (CDCl3): d = 9.64 (d, 1 H, H-4', J4,4’ = 2.5Hz), 7.40-7.23
(m, 15 H, C6H5), 4.95-4.43 (m, 6 H, PhCH2), 4.66 (d, 1 H, H-1, J1,2
= 3.5 Hz), 4.25 (dd, 1 H, H-3, J2,3 = 9.5 Hz, J3,4 = 11.0 Hz), 4.04 (dt,
1 H, H-5, J5,6a/6b = 4.0 Hz, J4,5 = 10.5 Hz), 3.61 (dd, 1 H, H 2, J1,2=3.5
Hz, J2,3 = 9.5 Hz), 3.55 (d, 2 H, H-6a, H-6b, J5,6a = J5,6b 4.0 Hz), 3.39
(s, 3 H, OCH3), 2.98 (dt, 1 H, H-4, J4,4’ = 2.5 Hz, J4,3 = J4,5 10.5 Hz).
Glucoamylase was also assayed with a maltose substrate. Glu-
coamylase G2 from A. niger prepared as described earlier9,10 was
added to a final concentration of 0.070 mM to preincubated 1 mM
maltose containing 12 different concentrations of inhibitor in the
range 0- 2 mM in 0.1 M NaOAc (pH 4.5) at 44 °C. Linear rates of
hydrolysis were determined by measuring glucose released in ali-
quots removed at 3 min intervals using the glucose oxidase method
essentially as described.11 The Ki was calculated from the equation
1/v = i/Vmax + (Km/Vmax[S]Ki)[I], where (S) and (I) are substrate
and inhibitor concentrations, respectively, Km was 1.7 mM and
kcat (Vmax/[E]) was 11.3 s-1 as determined from the Michaelis-
Menten equation.
13C NMR (CDCl3): d = 200.5 (C-4'), 138.1, 138.0, 137.6, 128.6-
127.8 (C6H5), 98.4 (C-l), 80.7 (C-2), 75.4 (2 C, C-3, PhCH2), 73.6,
73.2 (PhCH2), 70.3 (C-6), 67.1 (C-5), 56.8, 55.5 (C-4, OCH3).
Acknowledgement
MS (ES): m/z = 531.0 (M + MeOH + Na+).
We thank the Danish National Research Councils for support throu-
gh the THOR program and Sielsel Ehlers for glycoamylase inhibi-
tion experiments.
Methyl 4-Deoxy-4-[(3R, 4R, 5R)-3,4-dihydroxy-5-hydroxyme-
thyl-piperidin-1-yl)-methylen]-a-D-glucopyranoside (3)
A solution of 9 (98 mg, 0.41 mmol) in MeOH (6 mL) and 5% Pd/C
(200 mg) was added to a solution of 6 (280 mg, 0.55 mmol) in
MeOH (6 mL). The reaction mixture was hydrogenated for 72 h at
2.5 bar. Subsequently, 1 M HCl (0.6 mL) and 10% Pd/C (100 mG)
were added, and the mixture was hydrogenated for another 72 h at
4 bar. The mixture was filtered through Celite® and ion-exchanged
with Amberlite IR 120 (plus). The product was liberated with 2.5%
NH4OH (250 mL). It was concentrated and then chromatographed
using EtOH/EtOAc (1:4) as eluent; yield: 51 mg (37%); [a]D +0.98
(c = 1, MeOH).
References
(1) Heightman, D.; Vasella, A. T. Angew. Chem., Int. Ed. 1999,
38, 750.
(2) Zechel, L.; Withers, S. G. Acc. Chem. Res. 2000, 33, 11.
(3) Stütz, E. Iminosugars as Glycosidase Inhibitors: Nojirimycin
and Beyond; Wiley-VCH: Weinheim, 1999.
(4) For examples of azasugar disaccharides see:
Johns, B. A.; Pan, Y. T.; Elbein, A. D.; Johnson, C. R. J. Am.
Synthesis 2001, No. 2, 339–342 ISSN 0039-7881 © Thieme Stuttgart · New York