Chemical synthesis and preliminary biological evaluation of C-6-O-methyl-1-deoxynojirimycin as a potent α-glucosidase inhibitor

Article abstract of DOI:10.1080/07328303.2019.1700995

A facile and efficient synthesis of 6-O-methyl-1-deoxynojirimycin 4 from commercially available methyl α-D-glucopyranoside in 10 steps and 25% overall yield was reported. The synthetic strategy was based on the regioselective protection/deprotection at 6-

Full text of DOI:10.1080/07328303.2019.1700995

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: https://www.tandfonline.com/loi/lcar20
Chemical synthesis and preliminary biological
evaluation of C-6-O-methyl-1-deoxynojirimycin as
a potent α-glucosidase inhibitor
Lin Wang, Tingting Liang & Zhijie Fang
To cite this article: Lin Wang, Tingting Liang & Zhijie Fang (2019): Chemical synthesis and
preliminary biological evaluation of C-6-O-methyl-1-deoxynojirimycin as a potent α-glucosidase
inhibitor, Journal of Carbohydrate Chemistry, DOI: 10.1080/07328303.2019.1700995
To link to this article: https://doi.org/10.1080/07328303.2019.1700995
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JOURNAL OF CARBOHYDRATE CHEMISTRY
https://doi.org/10.1080/07328303.2019.1700995
Chemical synthesis and preliminary biological
evaluation of C-6-O-methyl-1-deoxynojirimycin as a
potent a-glucosidase inhibitor
Lin Wanga, Tingting Liangb, and Zhijie Fanga
aSchool of Chemical Engineering, Nanjing University of Science and Technology, Nanjing,
b
P.R. China; College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, P.R. China
ABSTRACT
ARTICLE HISTORY
Received 21 May 2019
Accepted 1 December 2019
A facile and efficient synthesis of 6-O-methyl-1-deoxynojirimy-
cin 4 from commercially available methyl a-D-glucopyranoside
in 10 steps and 25% overall yield was reported. The synthetic
strategy was based on the regioselective protection/deprotec-
tion at 6-O-position and cyclization as the main reaction steps.
Compound 4 was evaluated for its inhibitory effect (IC50) on
a-glucosidase using an in vitro assay. It showed improved
KEYWORDS
a-Glucosidase; Chemical
synthesis; IC50; Inhibitor;
1-deoxynojirimycin
inhibitory
activity
compared
to
1-deoxynojirimycin
and acarbose.
Introduction
Iminosugars (also termed azasugars) are carbohydrate mimetics with the
endocyclic oxygen atom replaced by a nitrogen atom and they can inhibit
many carbohydrate-processing enzymes, including glycosidases and glyco-
syltransferases.[1–5] Intestinal a-glucosidase, which is a carbohydrate hydro-
lase located in the cell membranes of small intestinal epithelial cells,
catalyzes the hydrolysis of oligo- and disaccharides into monosaccharides
as glucose at the cleavage site.[6,7] The inhibition of its catalytic activity
results in retardation of glucose absorption and the decrease in postpran-
dial blood glucose levels. This indicates that effective a-glucosidase inhibi-
tors may serve as therapeutic agents in the treatment of diabetes
and obesity.[8–10]
The best-known member of the extensive family of iminosugars is 1-
deoxynojirimycin (DNJ, 1, Figure 1). Resembling D-glucose in structure, as
well as mimicking an oxocarbeniumion-like transition state, DNJ showed
good inhibitory activity against a-glucosidase in a competitive man-
ner.[7,11,12] Miglitol (2, Figure 1), a N-hydroxyethylation derivative of DNJ,
was introduced into the market in the early 1980s to treat type II diabetes
CONTACT Zhijie Fang
zjfang@njust.edu.cn
School of Chemical Engineering, Nanjing University of Science
& Technology, 200 Xiaolingwei Street, Nanjing, Jiangsu, P.R. China
ß 2019 Taylor & Francis Group, LLC

2
L. WANG ET AL.
Figure 1. Structures of biologically active iminosugars 1–3 and the designed analog 4.
mellitus.[13,14] However, the efficacy of DNJ was reported to be rather mod-
erate in vivo.[10] Miglitol has side effects such as flatulence, diarrhea, and
abdominal pain.[14] Thus, it is necessary to search for new potent DNJ
derivatives as a-glucosidase inhibitors.
In order to study the effects of lipophilicity on molecular biological
activity and pharmacokinetics of DNJ, our group has done works on the
modification of DNJ,[15] especially the synthesis of C-6-deutero DNJ (3,
Figure 1), which was obtained from natural material DNJ in six steps and
had better inhibitory effect than DNJ and acarbose. As part of this series
of studies, 6-O-methyl-1-deoxynojirimycin 4 was designed and synthesized
in this work, and its a-glucosidase inhibitory activity was evaluated.
In the reported synthesis of DNJ derivatives (Scheme 1), compound 7 has
been utilized as the key intermediate. For example, Wu et al. described a 12-
step synthesis of 7 starting from 2,3,4,6-tetra-O-benzyl-D-glucopyranose 6 in
a total yield of 29%.[16] Cheng et al. obtained the same molecule in a 7-step
synthesis starting from the same material in a yield of 43%.[17] Vincent et al.
described its preparation from DNJ in a yield of 40%over four steps.[18] The
synthesis of 6 commenced from methyl a-D-glucopyranoside 5 in a yield of
46%over two steps.[19] DNJ was prepared from L-sorbose in a yield of 18%
over eight steps,[20] from 6 in a yield of 65% over four steps[21] or plants.[22]
Although these routes could be used to produce 4 from 7 by methylation
and deprotection, we deemed them unpractical for the synthesis of 4. The
commonality of the three routes was to build up the DNJ scaffold first and
then modify at the 6-O-position, which led to long synthetic routes with
low overall yields. Moreover, the natural material DNJ is expensive and dif-
ficult to obtain.[10] In this context, we developed a convenient synthetic
method to generate 6-O-methyl-1-deoxynojirimycin. The overall design of
this new synthetic method was to first methylate the 6-O-position of gluco-
pyranoside and then build up the DNJ scaffold.
Results and discussion
Synthesis of 6-O-methyl-1-deoxynojirimycin 4
Our synthetic route for 4 relied on the oxidative amination of 12, which was
easily obtained in five steps from methyl a-D-glucopyranoside 5 by slight

JOURNAL OF CARBOHYDRATE CHEMISTRY
3
OTr
5 steps
OH
OH
6 steps
NCBz
BnO
BnO
BnO
BnO
OAc
OBn
OBn
Cheng et al.
2 steps
OH
NCBz
OH
NH
OH
OBn
NH
BnO
BnO
OBn
O
3 steps
3 steps
OH
HO
HO
O
OBn
BnO
BnO
BnO
BnO
BnO
BnO
OBn
OBn
HO
OBn
7
OMe
5
6
Vincent et al.
OMMTr
OMMTr
NCBz
2 steps
OH
NCBz
6 or 7 steps
BnO
BnO
NH
OH
HO
HO
HO
HO
5
OBn
OH
DNJ
plants
8 steps
L-sorbose
Scheme 1. Previous synthetic approaches to the key intermediate 7.
Scheme 2. Synthesis of the key intermediate 12.
modifications of previously published procedures (Scheme 2). The transform-
ation started with selective tritylation of the 6-O-position to get 8, benzylation
to form 9, and hydrolysis of the trityl group to produce 10,[23,24] in which only
the 6-OH was free. Methylation of compound 10 using NaH and MeI yielded
11, which was followed by acidic hydrolysis of the glycosidic bond to eventually
provide 12.[23,25] The 6-OH in 10 could be modified by various reactions such
as alkylation, halogenation, and oxidation, which could potentially allow the
synthesis of other 6-O-substituted iminosugar derivatives.
Compound 12 was successfully converted into d-hydroxy amide 13 by
oxidative amination with iodine in 30% aqueous ammonia, as shown in
Scheme 3, and this reaction was scaled-up (19-g scale) to get a 94% yield.
It was worth noting that nitrogen iodide monoamine (NI3ꢀNH3), a sensitive
contact explosive material when dry, could be formed by iodine and
ammonia,[26,27] thus care should be taken when the reaction was stirred.

4
L. WANG ET AL.
Scheme 3. Synthesis of 6-O-methyl-1-deoxynojirimycin 4.
Table 1. The effect of oxidation reagents on the reaction yield.
Entry
Oxidation reagent
Temperature (ꢁC)
Time (h)
Reductive aminationa
Yieldb (%)
c
d
1
2
3
Ag2CO3
rt
rt
rt
48
48
6
NaCNBH3
HCOOH
CH3CN
–
–
DMSO-DCCe
PCCf
d
50g
33h
44
reflux, 3 h
4
DMSO-Ac2Oi
rt
18
aNaCNBH3 (3 equiv), HCOOH (90 equiv).
bIsolated yield.
cAg2CO3 (5 equiv).
dNot detected.
eDMSO (excess), DCC (4 equiv), TFA (0.75 equiv), Pyridine (4 equiv).
fPCC (2 equiv).
g200 mg of 13.
h10 g of 13.
iDMSO (40 equiv), Ac2O (20 equiv).
Keto amide 14 was obtained by oxidation of 13 with DMSO-Ac2O; as 14
was unstable, it should be directly used for next step without further purifi-
cation. Intramolecular reductive amination of 14 using formic acid and
sodium cyanoborohydride afforded lactam 15 in a yield of 58% over
two steps.
Prior to the preparation of 14, the effect of the critical reaction parame-
ters such as temperature and various oxidation reagents on the reaction
was examined. In the initial study, we examined various mild oxidation
reagents, including Ag2CO3,[28] DMSO-DCC,[29] PCC,[30] and DMSO-
Ac2O.[26] The results were summarized in Table 1. The oxidation of 13 did
not work in the presence of Ag2CO3 at room temperature for 2 d (Table 1,
entry 1). The transformation of 11 into 12 was incomplete using DMSO-
DCC as an oxidation reagent (Table 1, entry 2). It was observed that a
yield of 50% was obtained when 13 (200 mg) was subjected to PCC oxida-
tion and subsequently reductive amination (Table 1, entry 3). During the
course of the latter oxidation, chemical reaction system was facile and clean
and the only byproduct was chromium tar. However, when we carried out

JOURNAL OF CARBOHYDRATE CHEMISTRY
5
Table 2. The effect of temperature on the reaction yield.
Entry
Oxidation reagenta
Temperature (ꢁC)
Time (h)
Reductive aminationb
Yieldc (%)
1
2
3
4
5
DMSO-Ac2O
0
10
20
30
40
48
22
15
8
NaCNBH3
HCOOH
CH3CN
ND
58
52
41
19
reflux, 3 h
8
aDMSO (40 equiv), Ac2O (20 equiv).
bNaCNBH3 (3 equiv), HCOOH (90 equiv).
cIsolated yield.
Figure 2. The concentration-inhibition rate relationship for various compounds.
on a 10-g batch of 13, this condition only provided 15 in a reduced yield
of 33% (Table 1, entry 3). As the scale of this process was increased, the
chromium tar contained a large amount of keto amide 14 and; as a result,
significantly lower yield of 15 was obtained. Employing DMSO-Ac2O as
the oxidation reagent, 15 was produced in a yield of 44% over two steps
(Table 1, entry 4).
We found that elevated temperature could result in degradation of inter-
mediate 14. To further optimize, the reaction conditions for the synthesis
of 15, we examined the effect of temperature on the reaction yield. The
results were summarized in Table 2. When the temperature was decreased
from 40 to 10 ꢁC (Table 2, entries 2–5), the product yield of the target
compound 15 increased from 19% to 58% over two steps. The keto amide
14 was used without further purification and was proved stable when
stored at 10 ꢁC.
Compound 16 was obtained by reduction of 15 with lithium aluminum
hydride (LAH). The last step of this synthetic route was debenzylation of
16. Hydrogenolysis with 10%Pd/C or Pd (OH)2/C in neutral MeOH-THF
failed, probably due to catalyst poisoning by the amino group. Eventually,

6
L. WANG ET AL.
fully deprotected compound 4 was obtained by hydrogenolysis under acidic
condition, as outlined in Scheme 3.
Biological assays
The inhibitory activity of 4 to a-glucosidase was assessed using the proced-
ure reported by Hakamata et al.[31] with modifications. The assays were
carried out in 96-well plates with 4-nitrophenyl-a-D-glucopyranoside as the
enzyme substrate. The results, as shown in Figure 2, revealed that
the inhibition ratio and the concentration of 4 were correlated and under
the same conditions, its maximum inhibition rate 89.10% was higher than
that of DNJ (70.53%) and acarbose (82.73%). The observed IC50 of DNJ
and acarbose were 192.74 and 105.42 mg/L, respectively, whereas the IC50
of 4 was 72.65 mg/L. Consequently, compound 4 showed the strongest
inhibitory effect toward a-glucosidase.
Conclusion
In this study, 6-O-methyl-1-deoxynojirimycin 4 was synthesized from com-
mercially available methyl a-D-glucopyranoside in 10 steps and a 25%
overall yield. Compound 4 exhibited strong inhibitory potency against
a-glucosidase. This result suggested that 6-O-mythylation, which increased
the lipophilicity of DNJ, could be beneficial for the a-glucosidase inhibitory
activity. This synthetic route developed herein was efficient to provide vari-
ous iminosugar derivate that have potential biological significance, as it
allows for flexible modifications at 6-C-position. Compound 4 could be the
lead agent for designing new a-glucosidase inhibitors.
Experimental section
General
All available commercially solvents and reagents were used without further
purification. Reactions progression was monitored by thin-layer chromatog-
raphy (TLC) using silica gel GF254 plates (0.2 mm thickness), spots were
detected under UV-light (k ¼ 254 nm). Visualization of the deprotected
iminosugar was accomplished by exposure to iodine vapor. Flash column
1
chromatography was carried out by silica gel (300–400 mesh). H NMR
and 13C NMR were recorded on a Bruker Avance III 500 MHz spectrom-
1
eter (500 MHz for H; 126 MHz for 13C) using CDCl3, CD3OD or D2O as
solvents. Chemical shifts were reported in ppm. High resolution mass spec-
tra (HRMS) were recorded by direct injection on a mass spectrometer

JOURNAL OF CARBOHYDRATE CHEMISTRY
7
(Thermo Scientific LTQ Orbitrap XL) equipped with an electrospray ion
source in positive mode.
a-glucosidase assay
The a-glucosidase inhibition assay was determined using the procedure
reported by Hakamata et al.[31] with modifications for carrying out in 96-
well plates. Briefly, each well of the plates contained 110 mL phosphate buf-
fer (0.05 molꢀLꢂ1, pH ¼ 6.8), 10 mL of sample at varying concentrations
and 20 mL of the enzyme solution (0.2 UꢀmLꢂ1 from yeast a-glucosidase).
The plates were incubated at 37 ꢁC for 15 min, and then 20 mL of 2.5 mmol
Lꢂ1 p-Nitrophenyl-a-D-glucopyranoside (PNPG) was added to the mixture.
After further incubation at 37 ꢁC for 15 min, the reaction was terminated
by adding 80 mL Na2CO3 (0.2 molꢀLꢂ1). Enzymatic activity was quantified
by measuring the absorbance at 405 nm. Each experiment was performed
in triplicate. The increased absorbance (DA) was compared with that of the
control (DMSO in place of sample solution) to calculate the inhibition.
% inhibition ¼ ðDAcontrol ꢂ DAsampleÞ ⁄ DAcontrol
Syntheses
Methyl 6-O-trityl-a-D-glucopyranoside (8)
A solution of methyl a-D-glucopyranoside 5 (15 g, 77.24 mmol), trityl
chloride (26 g, 1.2 eq), DMAP (1.9 g, 0.2 eq), and triethylamine (20 mL) in
DMF (120 mL) was stirred at rt for 12 h. Then, the reaction mixture was
poured into ice-water and extracted with ethyl acetate. The organic phase
was washed with water, dried (NaSO4), filtered, and concentrated. The
crude product can be directly used for next reaction without further purifi-
cation. And 100 mg crude product was purified by silica gel column chro-
matography to obtain compound 8[24] as a white solid. Rf ¼ 0.25(CHCl3:
1
acetone ¼ 2:1). M.p. 150–151 ꢁC (lit.[32] 143–145 ꢁC). H NMR (500 MHz,
CD3OD_SPE) d 7.44–7.37 (m, 6 H, ArH), 7.23–7.20 (m, 6 H, ArH),
7.17–7.14 (m, 3 H, ArH), 4.69 (d, J ¼ 3.7 Hz, 1 H, H1), 3.72–3.66 (m, 1H,
H6a), 3.54 (t, J ¼ 9.2 Hz, 1 H, H3), 3.45 (s, 3 H, OCH3), 3.36 (m, 2 H, H2,
H4), 3.22–3.14 (m, 2 H, H5, H6b). 13C NMR (126 MHz, CDCl3) d 143.0,
127.8, 126.9, 126.1, 98.3, 85.8, 76.4, 76.1, 75.9, 73.4, 71.0, 70.3, 69.5, 62.9,
54.1 ppm. HRMS(ESI) m/z calcd for C26H28O6Naþ (M þ Na)þ 459.1778,
found 459.1781.

8
L. WANG ET AL.
Methyl 2,3,4-tri-O-benzyl-6-O-trityl-a-D-glucopyranoside (9)
To a stirred solution of 8 (31 g, 0.07 mol) in DMF (150 mL) was added
NaH (60% in mineral oil, 12.8 g, 0.32 mol) portion-wise at 0 ꢁC. After
30 min BnBr (38 mL, 0.32 mol) was added. Then the reaction mixture was
allowed to be stirred at rt. After 18 h, the reaction mixture was quenched
with water and washed with ethyl acetate, the combined organic was dried
(Na2SO4), filtered, and concentrated in vacuo. The crude product can be
directly used for next reaction without further purification. And 100 mg
crude product was purified by silica gel column chromatography to obtain
1
the syrupy compound 9.[24] Rf ¼ 0.7 (EtOAc:PE ¼ 1:6). H NMR (500 MHz,
CDCl3) d 7.78–7.06 (m, 28 H, ArH), 6.87 (d, J ¼ 6.7 Hz, 2 H, ArH), 4.96 (d,
J ¼ 10.7 Hz, 1 H, PhCH2), 4.82 (dd, J ¼ 11.4, 6.3 Hz, 2 H, PhCH2), 4.77 (d,
J ¼ 3.5 Hz, 1 H, H1), 4.72 (dd, J ¼ 11.1, 9.3 Hz, 2 H, PhCH2), 4.30 (d,
J ¼ 10.5 Hz, 1 H, PhCH2), 3.99 (t, J ¼ 9.3 Hz, 1 H, H3), 3.82 (dd, J ¼ 10.0,
3.4 Hz, 1 H, H5), 3.67–3.60 (m, 2 H, H2, H4), 3.54–3.48 (m, 1 H, H6a), 3.44
(s, 3 H, OCH3), 3.21 (dd, J ¼ 10.0, 4.7 Hz, 1 H, H6b) ppm. 13C NMR
(126 MHz, CDCl3) d 143.2, 138.0, 137.6, 137.2, 128.0, 127.7, 127.4, 127.3,
127.1, 126.8, 126.2, 97.2, 85.6, 81.6, 79.5, 77.4, 76.7, 76.4, 76.2, 75.2, 74.2,
72.6, 69.5, 61.9, 54.2 ppm. HRMS (ESI) m/z calcd for C47H46O6Naþ
(M þ Na)þ 729.3187, found 729.3185.
Methyl 2,3,4-tri-O-benzyl-a-D-glucopyranoside (10)
To a stirred solution of 9 in CH3OH (500 mL) was added sulfuric acid
(17 mL) slowly, then the reaction mixture was stirred at rt for 9 h. TLC
showed complete conversion of starting material to a major product. Then
the mixture were diluted in water and crude product was extracted using
ethyl acetate, the combined organic was washed with saturated aqueous sol-
utions of NaHCO3, followed by saturated sodium chloride aqueous solu-
tion, dried over NaSO4, filtered, and concentrated to dryness. The crude
product was purified by silica gel column chromatography (eluent gradient,
1/10 to 1/2; EtOAc:PE) to give colorless syrup 10[33] (31.2 g). Rf ¼0.2
1
(EtOAc:PE ¼ 1:3). The three-step total yield was 87%. H NMR (500 MHz,
CDCl3) d 7.36–7.26 (m, 15 H, ArH), 4.98 (d, J ¼ 10.9 Hz, 1 H, PhCH2),
4.92–4.75 (m, 3 H, PhCH2), 4.64 (dd, J ¼ 11.5, 7.9 Hz, 2 H, PhCH2), 4.56 (d,
J ¼ 3.5 Hz, 1 H, H1), 4.00 (t, J ¼ 9.3 Hz, 1 H, H3), 3.75–3.61 (m, 3 H, H6,
H5), 3.56–3.47 (m, 2 H, H2, H4), 3.35 (s, 3 H, OCH3), 1.84 (s, 1 H, 6-OH)
ppm; 13C NMR (126 MHz, CDCl3) d 137.9, 137.3, 137.3, 127.6, 127.6,
127.2, 127.1, 127.0, 126.8, 97.3, 81.1, 79.1, 76.7, 76.5, 76.4, 76.2, 74.9, 74.1,
72.5, 69.9, 60.8, 54.3 ppm. HRMS (ESI) m/z calcd for C28H32O6Naþ
(M þ Na)þ 487.2091, found 487.2094.

JOURNAL OF CARBOHYDRATE CHEMISTRY
9
Methyl 2,3,4-tri-O-benzyl-6-O-methyl-a-D-glucopyranoside (11)
To a stirring solution of 10 (30 g, 65 mmol) in DMF (300 mL) was added
NaH (60% in mineral oil, 7.8 g, 195 mmol) portion-wise at 0 ꢁC after half
an hour, MeI (12.1 mL, 195 mmol) was added, and the reaction mixture
was stirred at rt for 18 h. Then the mixture was quenched by water and
washed with ethyl acetate. The organic phase was washed with brine, dried
(NaSO4), filtered, and concentrated. The crude product can be directly
used for next reaction without further purification. And 100 mg crude
product was purified by silica gel column chromatography to provide
1
11.[25] Rf ¼0.4 (EtOAc:PE ¼ 1:6). H NMR (500 MHz, CDCl3) d 7.44–7.27
(m, 15 H, ArH), 5.01 (d, J ¼ 10.9 Hz, 1 H, PhCH2), 4.95–4.78 (m, 3 H,
PhCH2), 4.65 (d, J ¼ 12.1 Hz, 1 H, PhCH2), 4.65–4.59 (m, 2 H, PhCH2, H1),
3.97 (t, J ¼ 9.3 Hz, 1 H, H3) , 3.80–3.70 (m, 1 H, H5), 3.64–3.48 (m, 4 H, H6,
H4, H2), 3.37 (s, 3 H, 1-OCH3), 3.33 (s, 3 H, 6-OCH3) ppm; 13C NMR
(126 MHz, CDCl3) d 138.0, 137.5, 137.3, 127.5, 127.3, 127.0, 126.8, 126.7,
97.4, 81.2, 78.9, 76.7, 74.8, 74.2, 72.5, 70.1, 69.0, 58.3, 54.3 ppm. HRMS
(ESI) m/z calcd for C29H34O6Naþ (M þ Na)þ 501.2248, found 501.2250.
2,3,4-tri-O-benzyl-6-O-methyl-D-glucopyranose (12)
A solution of 11 (30 g, 62.68 mmol) in glacial acetic acid (300 mL) was
added 1 M H2SO4(120 mL), and the reaction mixture was stirred at 85 ꢁC
for 6 h. The reaction mixture was cooled to rt and left-over night for crys-
tallization of product. The crystals were filtered, washed with water and air
dried to afford a white power 12[34] (19.5 g, 65%). Rf ¼0.3 (EtOAc:PE ¼
1
1:3). M.p. 123–125 ꢁC. H NMR (500 MHz, CDCl3) d 7.30 (m, 15 H, ArH),
5.18 (d, J ¼ 3.5 Hz, 1 H, H1), 4.95 (d, J ¼ 11.0 Hz, 1 H, PhCH2), 4.86 (t,
J ¼ 11.9 Hz, 2 H, PhCH2), 4.75 (d, J ¼ 11.7 Hz, 1 H, PhCH2), 4.68 (d,
J ¼ 11.8 Hz, 1 H, PhCH2), 4.60 (d, J ¼ 10.9 Hz, 1 H, PhCH2), 4.00 (m, 2 H,
H3, H5), 3.61–3.49 (m, 5 H, H2, H4, H6), 3.31 (s, 3 H, 6-OCH3) ppm; 13C
NMR (126 MHz, CDCl3) d 137.8, 137.4, 137.0, 127.5, 127.2, 127.0, 126.9,
126.7, 90.4, 80.8, 79.0, 76.8, 76.4, 76.2, 75.9, 74.8, 74.1, 72.3, 70.3, 69.0,
58.2 ppm. HRMS (ESI) m/z calcd for C28H32O6Naþ (M þ Na)þ 487.2091,
found 487.2093.
2,3,4-tri-O-benzyl-6-O-methyl-D-gluconamide (13)
A solution of 12 (19 g, 40.89 mmol), iodine (14.5 g, 57.25 mmol), 30% aque-
ous NH3 solution (400 mL) in THF (100 mL) was stirred at rt for 16 h. A
5% aqueous Na2S2O3 solution was added until brown color disappeared.
The resulting mixture was extracted with ethyl acetate. The combined
organic layers were dried (Na2SO4), filtered, and concentrated. The residue

10
L. WANG ET AL.
was purified by silica gel column chromatography (1/10 to 1/1; EtOAc:PE)
to provide 13 (18.5 g, 38.58 mmol) in 94% yield as a colorless oil. Rf ¼0.2
1
(EtOAc:PE ¼ 1:1). H NMR (500 MHz, CDCl3) d 7.40–7.17 (m, 15 H,
ArH), 6.67 (d, J ¼ 2.9 Hz, 1 H, NH2), 6.13 (d, J ¼ 2.8 Hz, 1 H, NH2),
4.75–4.65 (m, 2 H, PhCH2), 4.65–4.51 (m, 4 H, PhCH2), 4.24 (d, J ¼ 3.2 Hz,
1 H, H2), 4.11–4.02 (m, 1 H, H3), 3.92–3.78 (m, 2 H, H5, H4), 3.53 (dd,
J ¼ 9.8 Hz, 2.8 Hz, 1 H, H6a), 3.47 (dd, J ¼ 9.8, 5.3 Hz, 1 H, H6b), 3.33 (s,
3 H, 6-OCH3), 2.99 (s, 1 H, OH) ppm. 13C NMR (126 MHz, CDCl3) d
173.5, 137.3, 136.8, 135.8, 127.7, 127.4, 127.1, 126.9, 126. 8, 79.6, 78.8, 76.7,
76.5, 76.2, 76.0, 74.3, 73.2, 72.8, 72.6, 70.2, 58.1 ppm. HRMS (ESI) m/z
calcd for C28H33NO6Naþ(M þ Na)þ 502.2200, found 502.2202.
2,3,4-tri-O-benzyl-6-O-methyl-D-glucono-d-lactam(15)
Under argon atmosphere a solution of the compound 13 (18 g, 37.5 mmol)
in DMSO (148.8 mL, 1.56 mol) was added acetic anhydride (74.4 mL,
0.78 mol). The mixture was stirred for 22 h at 10 ꢁC. The reaction mixture
was diluted with diethyl ether, washed thoroughly with water, 10% aqueous
solution of NaHCO3, and saturated NaCl solution. After drying on
Na2SO4, the organic layer was concentrated in vacuo to give a yellow syrup,
which can be directly used for the next reaction without further
purification.
A solution of the resulting mixture, formic acid (131.7 mL, 3.48 mol),
NaCNBH3 (7.3 g, 116 mmol) in CH3CN (240 mL) was heated under reflux
for 3 h. TLC analysis showed the complete disappearance of the starting
material to a major product. The mixture was then cooled at 0 ꢁC and 2 M
HCl (50 mL) was added. After stirring for 15 min, the mixture was poured
into 1:1 mixture of ethyl acetate/saturated aqueous NaHCO3 (500 mL). The
aqueous layer was separated and extracted with ethyl acetate. The com-
bined organic layers were dried (Na2SO4), filtered, and concentrated. The
residue was purified by silica gel column chromatography (1/10 to 1/1;
EtOAc:PE) to provide 15 (10 g, 21.7 mmol) in 58% yield as a white solid.
1
Rf ¼0.5 (EtOAc:PE ¼ 1:1). M.p. 81–83 ꢁC. H NMR (500 MHz, CDCl3) d
7.38–7.23 (m, 15 H, ArH), 6.06 (s, 1 H, NH), 5.17 (d, J ¼ 11.2 Hz, 1 H,
PhCH2), 4.86 (dd, J ¼ 11.2, 5.9 Hz, 2 H, PhCH2), 4.75 (dd, J ¼ 21.2, 11.1 Hz,
2 H, PhCH2), 4.54 (d, J ¼ 11.2 Hz, 1 H, PhCH2), 4.00 (d, J ¼ 8.0 Hz, 1 H,
H2), 3.91 (t, J ¼ 7.9 Hz, 1 H, H3), 3.58–3.44 (m, 3 H, H5, H6), 3.29 (s, 3 H,
6-OCH3), 3.20 (dd, J¼ 9.2, 7.3 Hz, 1 H, H4) ppm. 13C NMR (126 MHz,
CDCl3) d 169.6, 137.1, 136.9, 136.7, 127.5, 127.4, 127.1, 127.1, 126.84, 81.4,
77.8, 76.4, 76.1, 75.9, 73.8, 73.7, 71.6, 58.0, 52.7 ppm. HRMS (ESI) m/z
þ
calcd for C28H32NO5 (M þ H)þ 462.2275, found 462.2279.

JOURNAL OF CARBOHYDRATE CHEMISTRY
11
2,3,4-tri-O-benzyl-6-O-methyl-1,5-dideoxy-1,5-imino-D-glucitol(16)
A solution of 15 (7 g, 15.17mmol) in dry THF (112 mL) was added dropwise
to a suspension of LAH (1.7 g, 45.51mmol) in dry THF (84 mL) at 0 ꢁC. The
reaction mixture was heated under reflux for 3 h, then cooled at 0ꢁC. H2O
(1.7 mL) and a 15% aqueous NaOH solution (1.7 mL) were successively added
to the mixture. After, 15 min, an additional portion of H2O (12 mL) was added.
The resulting mixture was filtered through a pad of celite and concentrated.
The residue was purified by silica gel column chromatography (1/10 to 1/1;
EtOAc:PE) to provide 16 (5.8g, 12.9 mmol) in 85% yield as a yellow oil.
Rf ¼0.4 (EtOAc:PE ¼ 1:1). 1H NMR (500MHz, CDCl3) d 7.47–7.25 (m, 15 H,
ArH), 5.03 (d, J ¼ 10.9 Hz, 1 H, PhCH2), 4.89 (d, J ¼ 11.0 Hz, 1 H, PhCH2), 4.84
(d, J ¼ 11.0 Hz, 1 H, PhCH2), 4.74 (q, J ¼ 11.6 Hz, 2 H, PhCH2), 4.62 (d,
J ¼ 11.0 Hz, 1 H, PhCH2), 3.59–3.44 (m, 3 H, H6, H2), 3.42–3.37 (m, 1 H, H3),
3.32–3.30 (m, 1 H, H4), 3.29 (s, 3 H, 6-OCH3), 3.24 (dd, J ¼ 12.2, 4.9 Hz, 1 H,
H1b), 2.69 (m, 1 H, H5), 2.62–2.51 (m, 1 H, H1a) ppm. 13C NMR (126 MHz,
CDCl3) d137.6, 127.5, 127.1, 127.0, 126.9, 126.7, 126.6, 86.4, 79.6, 79.1, 76.4,
76.1, 75.9, 74.7, 74.2, 71.9, 71.7, 58.8, 58.0, 47.2 ppm. HRMS (ESI) m/z calcd for
C28H34NO4þ (M þ H)þ 448.2482, found 448.2485.
C-6-O-methyl-1-Deoxynojirimycin (4)
A suspension of 16 (1 g, 2.2 mmol) and Pd/C (10 wt%, 200 mg) in MeOH-
THF (30 mL, 5:1) was acidified with 2 M aqueous HCl. The reaction mix-
ture was stirred vigorously under a H2(g) atmosphere for 24 h at rt. The
reaction mixture was filtered over celite and washed with MeOH and con-
centrated in vacuo. The residue was purified by silica gel column chroma-
tography (isocratic 3:1:0.1, n-propanol: H2O: NH4OH) to provide 4
(376 mg, 2.1 mmol) in 95% yield as a colorless oil. Rf ¼0.6 (1:1:0.3; EtOAc:
1
MeOH: NH4OH). H NMR (500 MHz, D2O) d 3.70 (d, J ¼ 10.4 Hz, 1 H,
H6b), 3.54 (m, 2 H, H6a, H2), 3.38 (s, 3 H, OCH3), 3.34 (t, J ¼ 9.2 Hz, 1 H,
H4), 3.27 (t, J ¼ 9.5 Hz, 1 H, H3), 3.16 (dd, J ¼ 12.3, 5.1 Hz, 1 H, H1b), 2.77
(m, 1 H, H5), 2.53 (t, J ¼ 11.6 Hz, 1 H, H1a) ppm. 13C NMR (126 MHz,
D2O) d 77.0 (C3), 70.6 (C6), 70.1 (C4), 69.2 (C2), 57þ.7 (C5, C-6-OMe), 47.2
(C1) ppm. HRMS(ESI) m/z calcd for C7H16NO4 (M þ H)þ 178.1074,
found 178.1074.
Disclosure statement
No potential conflict of interest was reported by the authors.

12
L. WANG ET AL.
Funding
This work was supported by grants from the prospective joint project of Production,
Education and Research in Jiangsu Province, PR China [Grant No. KS03008].
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