N-Bridged 1-deoxynojirimycin dimers as selective insect trehalase inhibitors

Article abstract of DOI:10.1016/j.carres.2013.12.025

A small set of N-bridged 1-deoxynojirimycin dimers has been synthesized and evaluated as potential inhibitors of insect trehalase from midge larvae of Chironomus riparius, porcine trehalase as the mammalian counterpart and α-amylase from human saliva. All the tested compounds (2-4) proved to be active (micromolar range activity) against insect trehalase, showing selectivity toward the insect glycosidase. No activity was observed against α-amylase.

Full text of DOI:10.1016/j.carres.2013.12.025

Carbohydrate Research 389 (2014) 46–49
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
N-Bridged 1-deoxynojirimycin dimers as selective insect trehalase
inhibitors
Laura Cipolla a, Antonella Sgambato a, Matilde Forcella a, Paola Fusi a, Paolo Parenti b, Francesca Cardona c,
Davide Bini a,
⇑
a Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy
b Department of Earth and Environmental Sciences, University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy
c Department of Chemistry ‘Ugo Schiff’, University of Florence, Via della Lastruccia 3-13, I-50019 Sesto Fiorentino, Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A small set of N-bridged 1-deoxynojirimycin dimers has been synthesized and evaluated as potential
Received 13 September 2013
Received in revised form 16 December 2013
Accepted 18 December 2013
Available online 30 January 2014
inhibitors of insect trehalase from midge larvae of Chironomus riparius, porcine trehalase as the mamma-
lian counterpart and
(micromolar range activity) against insect trehalase, showing selectivity toward the insect glycosidase.
No activity was observed against -amylase.
a-amylase from human saliva. All the tested compounds (2–4) proved to be active
a
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Trehalase
Enzyme inhibitors
Trehalose
Iminosugars
Deoxynojirimycin
1. Introduction
potent inhibitors of trehalase, together with synthetic trehalose
analogues.8,13–16
Trehalase
specific glycosidase that catalyzes the hydrolysis of trehalose1
(1, -glucopyranosyl- -glucopyranoside, Fig. 1) to the two
(
a
-glucoside-1-glucohydrolase, EC 3.2.1.28) is
a
In the search for new inhibitors that might be specific toward
insect trehalase based on 1-deoxynojirimycin and its N-acyl
derivatives13 we herein propose the synthesis of N-bridged
1-deoxynojirimycin dimers (Fig. 1, 2–4) and their biological evalu-
ation toward both insect and porcine trehalase, compared to the
a-D
a-D
constituent glucose units. This disaccharide is found in many
organisms as diverse as bacteria, yeast, fungi, nematodes, plants,
insects and some other invertebrates, but is absent in mammals.
In lower organisms, trehalose may serve as a source of energy, a
carbohydrate store, or an agent for protecting proteins and cellular
membranes from inactivation or denaturation caused by a variety
of environmental stress conditions. In insects, trehalose hydrolysis
by trehalase is fundamental in various physiological processes
including chitin synthesis during molting,2 and thermotolerance
in larvae.3 Moreover, trehalase activity is the basis for flight metab-
olism,1,4 trehalose being the principal hemolymph sugar in insects5
that acts as an indispensable substrate for energy production and
macromolecular biosynthesis.6 Given these premises, insect
trehalases are attractive targets for the search of inhibitors as
potential novel and selective insecticides.7 Some natural pseudodi-
saccharides, such as validoxylamine A,8 trehazolin,9 casuarine-6-O-
human a-amylase enzyme (EC 3.2.1.1).
The synthesis of compounds 2–4 was performed straightforward
from protected 1-deoxynojirimycin,17 as outlined in Scheme 1. The
reaction of compound 5 with oxalyl or succinyl chloride success-
fully afforded compounds 6 and 8 in 66% and 49% yields, respec-
tively; in contrast, the reaction of 5 with malonyl chloride in the
same reaction conditions gave compound 7 only in traces. The
unexpected outcome of the reaction with malonyl chloride is prob-
ably due to by-products deriving from reaction of methylenic
acidic protons of malonyl chloride with the basic pyridine used
in the procedure. Dimer 7 was obtained in 70% yield by reaction
of 5 with malonic acid in the presence of DCC. Direct hydrogenol-
ysis of 6–8 quantitatively afforded the compounds 2–4.
Compounds 2–4 were tested for their inhibitory activity against
insect trehalase of midge larvae of C. riparius,18 porcine trehalase
(purchased from Sigma–Aldrich) as the mammalian counterpart
a-D
-glucoside10,11 and its analogues12 have been shown to be
and
Aldrich), as
a
-amylase from human saliva (purchased from Sigma–
⇑
Corresponding author. Tel.: +39 02 64483427; fax: +39 02 64483565.
E-mail address: davide.bini@unimib.it (D. Bini).
a
relevant glycolytic enzyme. Midge larvae are
http://dx.doi.org/10.1016/j.carres.2013.12.025
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

L. Cipolla et al. / Carbohydrate Research 389 (2014) 46–49
47
HO
HO
HO
HO
HO
OH
N
HO
HO
OH
OH
OH
HO
OH
O
OH
HO
HO
O
HO
N
H
HO
N
H
OH
OH
OH
OH
O
O
OH
OH
O
trehalose (1)
validoxylamine A
OH
H
trehazolin
HO
HO
HO
HO
HO
NH
O
HO
HO
O
OH
OH
N
OH
OH
casuarine-6-O-α-D-glucoside
1-deoxynojirimycin
OH
HO
OH
HO
OH
OH
HO
HO
O
O
HO
OH
HO
O
O
HO
N
N
N
N
N
OH
N
O
HO
OH
HO
O
OH
HO
OH
OH
OH OH
2
3
4
OH
OH
Figure 1. Structures of trehalose (1), validoxylamine A, trehazolin, casuarine-6-O-
a-
D
-glucoside, 1-deoxynojirimycin, and the synthesized dimers 2–4.
BnO
BnO
BnO
BnO
2. Experimental
O
O
n
OBn
OBn
a
NH
N
N
6 (n = 0) (66%)
8 (n = 2) (49%)
2.1. General methods
BnO
BnO
OBn
OBn
OBn OBn
c
5
All solvents were dried over molecular sieves, for at least 24 h
prior to use, when required. When dry conditions were required,
the reaction was performed under Ar or N2 atmosphere. Thin-layer
chromatography (TLC) was performed on silica gel 60F254 coated
glass plates (Merck) with UV detection when possible, or spots
were visualized by charring with a conc. H2SO4/EtOH/H2O solution
(10:45:45 v/v/v), or with a solution of (NH4)6Mo7O24 (21 g),
Ce(SO4)2 (1 g), concd H2SO4 (31 mL) in water (500 mL) and then
heating to 110 °C for 5 min. Flash column chromatography was
performed on silica gel 230–400 mesh (Merck). Routine 1H and
13C NMR spectra were recorded on a Varian Mercury instrument
b
BnO
BnO
HO
O
O
O
O
OBn
OBn
HO
HO
OH
c
N
N
N
N
0-2
BnO
OH
OBn
OBn OBn
OH
OH OH
2-4
(quant.)
7
(70%)
at 400 MHz (1H) and 100.57 MHz
(
13C). Chemical shifts are
Scheme 1. Synthesis of nojirimycin dimers 2–4. Reagents and conditions: (a)
Oxalyl or succinyl chloride, pyridine, DCM, 0 °C?rt, 3 h; (b) Malonic acid, DCC,
DMAP, p-TsOH, DCM, rt, 30 min; (c) Pd(OH)2/C, H2, EtOAc/EtOH = 1:1.
reported in parts per million downfield from TMS as an internal
standard; J values are given in Hz. Mass spectra were recorded
on a System Applied Biosystems MDS SCIEX instrument (Q TRAP,
LC/MS/MS, turbo ion spray) or on a System Applied Biosystem
MDS SCIEX instrument (Q STAR elite nanospray). Elemental analy-
ses (C, H, N) were performed with a Perkin–Elmer series II 2400
analyzer.
widespread in freshwater ecosystems, as sentinel organisms are
widely used in ecotoxicological studies and environmental
biomonitoring program and represent a good model for biochemi-
cal studies. To examine the potential of each 1-deoxynojirimycin
dimer as trehalase inhibitor, preliminary screening assays at a
fixed concentration (1 mM) of potential inhibitors were carried
out, and dose–response curves were established for most active
compounds in order to determine the IC50 values. Experiments
were performed at a fixed substrate concentration, in the presence
of increasing inhibitor concentrations. The inhibitory activity is
shown in Figure 2 as IC50 value.
2.2. General procedure for the hydrogenolysis reaction
A 0.02 m solution of the appropriate dimer dissolved in EtOAc/
EtOH (1:1) was treated with Pd(OH)2/C (100% in weight). The reac-
tion was stirred for 5 d under a H2 atmosphere. Palladium was then
removed by filtration through a Celite pad followed by washing
with EtOH and water. Evaporation of the solvents afforded the cor-
responding deprotected compounds in quantitative yields.
All the synthesized dimers were inactive against
a-amylase,
while they were similarly active against C. riparius trehalase, with
the activity in the micromolar range. Compound 2 resulted to be
the most active derivative of the series; all compounds 2–4 showed
a slight selectivity toward the insect glycosidase, resulting more
selective between mammalian and insect trehalase if compared
to the parent compound 1-deoxynojirimycin.
2.3. 1,2-Bis((2R,3R,4R,5S)-3,4,5-tris(benzyloxy)-2-
((benzyloxy)methyl)piperidin-1-yl)ethane-1,2-dione (6)
To a solution of compound 5 (108 mg, 0.21 mmol) in dry DCM
We can conclude that despite the fact the both trehalase specif-
ically hydrolyze trehalose, they might have significant differences
in the catalytic pocket that can be exploited for the design and
development of specific insect trehalase inhibitors, with potential
follow up in the development of insecticides. However, further in-
sights are needed into enzyme recognition features.
(1.1 mL), pyridine (33 lL, 0.41 mmol) and oxalyl chloride (9 lL,
0.10 mmol) were added at 0 °C. The temperature was slowly in-
creased to rt (3 h); the mixture was then concentrated and the res-
idue was purified directly on a silica gel column (petroleum ether/
EtOAc, 65:35) affording 6 (74 mg, 66% yield). 1H NMR (CDCl3):
d = 7.37–7.01 (m, 40H, ArH), 4.77–4.19 (m, 18H, OCH2Ph, H-5),

48
L. Cipolla et al. / Carbohydrate Research 389 (2014) 46–49
Figure 2. Histogram of the inhibitory activity of compounds 2–4, compared to 1-deoxynojirimycin.
3.97–3.54 (m, 12H, H-1a, H-2, H-3, H-4, H-6), 3.14–3.02 (m, 2H, H-
1b) ppm. 13C NMR (CDCl3): d = 165.0, 164.7 (C@O), 138.5, 138.5,
138.3, 138.2, 138.2, 138.1, 138.1, 137.9 (C Ar), 128.7–127.6 (CH
Ar), 82.4 (C-3), 79.1, 76.1 (C-2, C-4), 73.7–70.1 (OCH2Ph), 68.9
(C-6), 58.6 (C-5), 43.8 (C-1) ppm. MS (TOF, m/z): [M+H]+ calcd for
32.0 (CH2C@O) ppm. MS (TOF, m/z): [M+H]+ calcd for C72H77N2O10
,
1129.6; found 1129.6. C72H76N2O10 (1129.38): calcd C 76.57,
H 6.78, N 2.48; found C 76.49, H 6.80, N 2.47.
2.6. 1,2-Bis((2R,3R,4R,5S)-3,4,5-trihydroxy-2-
C70H73N2O10, 1101.5; found 1101.5. C70H72N2O10 (1101.33): calcd
(hydroxymethyl)piperidin-1-yl)ethane-1,2-dione (2)
C, 76.34, H 6.59, N 2.54; found C 76.49, H 6.58, N 2.55.
1H NMR (D2O): d = 3.75–3.65 (m, 4H, H-6), 3.60–3.53 (m, 2H,
H-2), 3.42–3.26 (m, 6H, H-1a, H-3, H-4), 3.02–2.98 (m, 2H, H-5),
2.82–2.72 (m, 2H, H-1b) ppm. 13C NMR (D2O): d = 76.2 (C-3),
67.7, 66.9 (C-2, C-4), 60.0 (C-5), 57.6 (C-6), 45.8 (C-1) ppm. MS
(TOF, m/z): [M+H]+ calcd for C14H25N2O10, 381.1; found 381.4.
2.4. 1,3-Bis((2R,3R,4R,5S)-3,4,5-tris(benzyloxy)-2-
((benzyloxy)methyl)piperidin-1-yl)propane-1,3-dione (7)
Compound
5 (67 mg, 0,12 mmol), malonic acid (6,4 mg,
0,06 mmol), DMAP (3 mg, 0.02 mmol) and p-toluenesulfonic acid
(4.7 mg, 0.02 mmol) were dissolved in DCM (1.14 mL). DCC
(32 mg, 0.15 mmol) was added and the solution was stirred for
30 min at room temperature. Then the DCC–urea was filtered off
and washed with a small volume of DCM. The solvent was evapo-
rated, and the residue was purified by flash column chromatogra-
phy (petroleum ether/EtOAc, 62.5:37.5) giving pure 7 (48 mg, 70%
yield). 1H NMR (CDCl3) d = 7.34–7.15 (m, 40H, ArH), 4.77–4.59 (m,
6H, H-5, OCH2Ph), 4.59–4.44 (m, 8H, OCH2Ph), 4.41–4.25 (m, 4H,
OCH2Ph), 4.03–3.93 (m, 4H, H-2, H-1a), 3.80–3.56 (m, 10H, H-6,
H-3, CH2C=O, H-4), 3.56–3.46 (m, 2H, H-1b) ppm. 13C NMR (CDCl3)
d = 166.9 (C@O), 142.7–137.8 (C Ar), 128.4–127.5 (CH Ar), 80.9
(C-3), 78.1, 73.9 (C-2, C-4), 72.9–70.8 (OCH2Ph), 68.0 (C-6), 54.2
(C-5), 44.4 (C-1), 41.9 (CH2C@O) ppm. MS (TOF, m/z): [M+H]+ calcd
for C71H75N2O10, 1115.5; found 1115.5. C71H74N2O10 (1115.35):
calcd C 76.46, H 6.69, N 2.51; found C 76.51, H 6.67, N 2.52.
C14H24N2O10 (380.35): calcd C 44.21, H 6.36, N 7.37; found C
44.27, H 6.34, N 7.38.
2.7. 1,3-Bis((2R,3R,4R,5S)-3,4,5-trihydroxy-2-
(hydroxymethyl)piperidin-1-yl)propane-1,3-dione (3)
1H NMR (D2O) d = 4.36 (d, 1H, J = 14.4 Hz, CH2C@O), 3.96–3.52
(m, 16H, H-1, H-2, H-3, H-4, H-5, H-6), 3.20 (d, 1H, J = 14.8, CH2-
C@O) ppm. 13C NMR (D2O) d = 74.1 (C-3), 69.3, 68.4 (C-2, C-4),
63.0 (C-5), 59.8 (C-6), 47.7 (C-1), 40.6 (CH2C@O) ppm. MS (TOF,
m/z): [M+H]+ calcd for C15H27N2O10
C
,
395.2; found 395.3.
15H26N2O10 (394.37): calcd C 45.68, H 6.65, N 7.10; found C
45.74, H 6.63, N 7.11.
2.8. 1,4-Bis((2R,3R,4R,5S)-3,4,5-trihydroxy-2-
(hydroxymethyl)piperidin-1-yl)butane-1,4-dione (4)
2.5. 1,4-Bis((2R,3R,4R,5S)-3,4,5-tris(benzyloxy)-2-
((benzyloxy)methyl)piperidin-1-yl)butane-1,4-dione (8)
1H NMR (D2O): d = 3.82–3.71 (m, 4H, H-6), 3.68–3.61 (m, 2H, H-
2), 3.48–3.34 (m, 6H, H-1a, H-3, H-4), 3.09–3.05 (m, 2H, H-5), 2.86–
2.80 (m, 2H, H-1b), 2.61–2.50 (m, 4H, CH2C@O) ppm. 13C NMR
(D2O): d = 76.0 (C-3), 67.5, 66.7 (C-2, C-4), 59.7 (C-5), 57.4 (C-6),
45.6 (C-1), 28.6 (CH2C@O) ppm. MS (TOF, m/z): [M+H]+ calcd for
To a solution of compound 5 (177 mg, 0.34 mmol) in dry DCM
(1.9 mL), pyridine (55
lL, 0.67 mmol) and succinyl chloride
(19 L, 0.17 mmol) were added at 0 °C. The temperature was
l
C16H29N2O10, 409.2; found 409.4. C16H28N2O10 (408.40): calcd C
slowly increased to rt (3 h); the mixture was then concentrated
and the residue was purified directly on a silica gel column (petro-
leum ether/EtOAc, 50:50) affording 8 (93 mg, 49% yield). 1H NMR
(CDCl3): d = 7.38–7.16 (m, 40H, ArH), 4.82–4.23 (m, 18H, OCH2Ph,
H-5), 4.05–3.41 (m, 12H, H-1a, H-2, H-3, H-4, H-6), 2.97–2.58 (m,
6H, H-1b, CH2C@O) ppm. 13C NMR (CDCl3): d = 171.8 (C@O),
138.4–137.8 (C Ar), 128.5–127.6 (CH Ar), 82.4 (C-3), 78.9, 74.3
(C-2, C-4), 73.3–71.1 (OCH2Ph), 68.3 (C-6), 54.5 (C-5), 44.0 (C-1),
47.05, H 6.91, N 6.86; found C 47.14, H 6.89, N 6.87.
2.9. Biological assays
Trehalase activity was measured through a coupled assay with
glucose-6-phosphate dehydrogenase and hexokinase according to
Wegener at al.19. To examine the potential of each compound as
a trehalase inhibitor, screening assays of potential inhibitors were

L. Cipolla et al. / Carbohydrate Research 389 (2014) 46–49
49
carried out at a fixed concentration of 1 mM and dose–response
Acknowledgments
curves were established to determine the IC50 values. Experiments
were performed at fixed substrate concentration close to the Km
value (0.5 mM for C. riparius trehalase and 2.5 mM for porcine
trehalase), in the presence of increasing inhibitor concentrations.
Initial rates as a function of inhibitor concentration were fitted to
the following equation:
We gratefully acknowledge University of Milano-Bicocca FAR
2009 and MIUR (PRIN2008/24M2HX) for financial support.
References
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mi
m
1
¼
ꢀ
ꢁ
n
½Iꢀ
1 þ
IC50
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to 0.5 mL enzyme solution (10 lg/mL). The reaction was initiated
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Units
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¼
All enzyme assays were performed in triplicates at 30 °C.
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