Casuarine-6-O-α-d-glucoside and its analogues are tight binding inhibitors of insect and bacterial trehalases

Article abstract of DOI:10.1039/b926600c

Two novel casuarine-6-α-d-glucoside analogues, as well as the parent compound, were synthesized and tested as inhibitors towards Chironomus riparius, mammalian pig kidney and Escherichia coli trehalases. Their potent and selective activity is promising fo

Full text of DOI:10.1039/b926600c

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COMMUNICATION
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Casuarine-6-O-a-D-glucoside and its analogues are tight binding
inhibitors of insect and bacterial trehalasesw
Francesca Cardona,*a Andrea Goti,a Camilla Parmeggiani,a Paolo Parenti,b
Matilde Forcella,b Paola Fusi,c Laura Cipolla,c Shirley M. Roberts,d
d
Gideon J. Daviesd and Tracey M. Glosterz
Received (in Cambridge, UK) 16th December 2009, Accepted 10th February 2010
First published as an Advance Article on the web 23rd February 2010
DOI: 10.1039/b926600c
Two novel casuarine-6-a-D-glucoside analogues, as well as the
parent compound, were synthesized and tested as inhibitors
towards Chironomus riparius, mammalian pig kidney and
Escherichia coli trehalases. Their potent and selective activity
is promising for the development of new insecticides.
The coupling of inhibitors with structure determination is
able to provide molecular insights into enzyme mechanism and
features of the active site. The first three-dimensional structure
of a trehalase was the family 37 periplasmic enzyme from
Escherichia coli (Tre37A), which was solved in complex with 2
and a nonnatural analogue of 3.10 Furthermore, the first total
synthesis of 4 and its three-dimensional structure in complex
with Tre37A have also been described.11
Trehalose (a-D-glucopyranosyl a-D-glucopyranoside, 1) has
a multifunctional physiological role in various organisms
ranging from bacteria and fungi to invertebrates and higher
plants.1 Trehalose is particularly important for insects
(it constitutes their major blood sugar)2 as it is hydrolysed
into glucose, which is vital for insect flight. Inhibition of the
hydrolysis of trehalose is thus an interesting target for novel
insecticides. In order to utilise trehalose, insect tissues possess
an a-trehalase (EC 3.2.1.28), an inverting glycosidase which
promotes the hydrolysis of one of the two glycosidic bonds in
trehalose.2,3 Trehalose is not found in mammalian cells,
although humans do possess a trehalase in intestinal villi
and kidney brush border cells, probably to handle ingested
trehalose.1 Although insects possess two types of trehalase, a
soluble trehalase (localized in haemolymph) and a membrane-
bound trehalase (localized in tissues), very little is known
about the latter, nor the difference in function between the
two enzymes.4,5
As with the design of any inhibitor, specificity is a significant
concern; the design of a viable insecticide which targets a
trehalase would therefore require it to be safe for plants and
mammals, and also if possible for insects which are of benefit
in nature, and thus only target specific insect trehalases.
A membrane-bound isoform of trehalase from midge larvae
(Chironomus riparius) has been recently purified.12 C. riparius
represents a good model for biochemical studies, and it has
been demonstrated that its resistance to stress factors is highly
dependent on trehalose catabolism.13 Studies revealed that
the trehalase from C. riparius is specific for trehalose
and competitively inhibited by known trehalase inhibitors,12
and can therefore be considered a relevant target for new and
specific compounds with insecticidal activity.
In order to generate highly potent trehalase inhibitors with
potential insecticidal activity, we undertook the total synthesis
of two new casuarine-6-O-a-D-glucoside analogues 5 and 6
(Fig. 1). Compounds 5 and 6 bear a hydrogen atom and a
hydroxymethyl group in place of the hydroxy group at C(7) of
casuarine-6-O-a-D-glucoside, respectively. In this communication,
we report the study of the inhibitory properties of compounds
Trehalase inhibitors are valuable tools for studying the
molecular physiology of trehalase function and sugar
metabolism in insects6 and have the potential to produce novel
insecticides.7 Among the most powerful inhibitors of trehalases
are some natural pseudodisaccharides, such as validoxylamine
A (2),8 trehazolin (3),8 and casuarine-6-O-a-D-glucoside (4)9
(Fig. 1).
a Department of Chemistry ‘‘U. Schiff’’, Laboratory of Design,
Synthesis and Study of Biologically Active Heterocycles
(HeteroBioLab), University of Florence, Via della Lastruccia 3-13,
50019, Sesto Fiorentino, Florence, Italy.
E-mail: francesca.cardona@unifi.it; Fax: +39 055 4573531;
Tel: +39 055 4573504
b Department of Environmental Sciences, University of
Milano-Bicocca, Piazza della Scienza 2, 20126, Milano, Italy
c Department of Biotechnology and Biosciences, University of
Milano-Bicocca, Piazza della Scienza 2, 20126, Milano, Italy
d Structural Biology Laboratory, Department of Chemistry,
The University of York, Heslington, York, UK YO10 5YW
w Electronic supplementary information (ESI) available: Spectro-
scopic data and experimental details for the synthesis of compounds
5–6 and 11–14, details for the inhibition assays, and X-ray crystallo-
graphic studies. See DOI: 10.1039/b926600c
z Current address: Department of Chemistry, Simon Fraser University,
8888 University Drive, Burnaby,V5A 1S6, Canada.
Fig. 1
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4–6 towards C. riparius trehalase, Tre37A and commercial
porcine kidney trehalases. Additionally, we solved the structure
of Tre37A in complex with 6 to gain insights into the inter-
actions important for binding.
Table
C. riparius trehalase by compounds 4, 5 and 6
1
Inhibition of porcine kidney trehalase, Tre37A and
Porcine kidney, Ki
E. coli Tre 37A, Ki
C. riparius, Ki
4
5
6
11 nMa
138 nM
410 mM
12 nMb
86 nM
2.8 mM
0.66 nM
22 nM
157 nM
The key steps for the synthesis of glycosides 5 and 6 are a
completely selective 1,3-dipolar cycloaddition of a cyclic
nitrone to a suitable dipolarophile, a well established route
in our group for the synthesis of pyrrolizidine and indolizidine
alkaloids,14 and a selective a-glucosylation on a lactam inter-
mediate. Lactam 8, prepared through cycloaddition of nitrone
7 (synthesized from commercially available 2,3,5-tri-O-benzyl
D-arabinose)15 to dimethylacrylamide and N–O bond cleavage
of the isoxazolidine adduct, bears a free hydroxyl group on
C(6) of the pyrrolizidine ring, thus allowing selective gluco-
sylation at this position (Scheme 1). Reaction with glucosyl
donor 10 in diethyl ether in the presence of TMSOTf
selectively gave a-glucoside 11 (88% yield). Only traces of
the b-anomer, which could not be isolated, were detected in
the crude reaction mixture. Reduction of the CQO bond with
LiAlH4 (58% yield), followed by catalytic hydrogenation of 13
in the presence of a few drops of conc. HCl afforded the free
amine 5 (77% yield) after elution of the crude reaction mixture
through an ionic exchange resin (Scheme 1).
a
Ki = 18 nM for 4 isolated from natural source (purity about 60%).
b
See ref. 9. Ref. 11.
The potency of 4, 5 and 6 towards all three trehalases
showed a similar trend, with the most potent inhibition
obtained using compound 4. These direct comparisons
demonstrate that the functional group at the C7 position has
a significant effect on the inhibition of the casuarine-6-O-a-D-
glucoside analogues; a hydroxyl group appears to be preferential
to a hydrogen atom, which in turn are both more potent than a
hydroxymethyl group. It is interesting to note, however, that
compounds 4 and 6 are at least one order of magnitude more
potent towards the C. riparius trehalase than the porcine
trehalase. Compound 4 is the most potent trehalase inhibitor
described to date with a Ki value against C. riparius lower than
that displayed by validoxylamine A towards the porcine
kidney trehalase (Ki = 2 nM)8,16 and an order of magnitude
lower than that determined for trehazolin with other insect
species (Ki = 10ꢂ8 M towards locust flight muscle treha-
lase).6,8 However, in terms of selectivity, it is perhaps 6 that
is the most interesting compound reported here. This displays
more than 60-fold selectivity over the porcine kidney trehalase.
Molecular insights into casuarine-6-O-a-D-glucoside analogue
inhibition can be obtained from the three-dimensional structure
of a trehalase in complex with inhibitor. Although it would be
ideal to obtain the structure of each trehalase studied to
demonstrate the basis for selectivity, to date only the trehalase
from E. coli has been amenable to crystallization. We solved
the structure of Tre37A in complex with 6, with the aim of
dissecting which features make the inhibitor less favourable to
this trehalase compared to the C. riparius enzyme. X-ray data
for Tre37A in complex with 6 were collected to 2.1 A (see
Supplementary Material for data collection and refinement
statisticsw). Electron density clearly showed the presence of a
molecule of 6 bound in the ꢂ1 (casuarine analogue moiety)
and +1 (glucose) subsites of each molecule of Tre37A in the
asymmetric unit (Fig. 2a). Compound 6 and active site
residues overlap perfectly with Tre37A in complex with 4
(Fig. 2b). As observed with the Tre37A complex with 4, both
pyrrolidine rings were found in an envelope conformation.
The majority of the interactions with active site residues are
the same as described previously10,11 and can be seen in
Fig. 2c. The only differences lie with the hydroxymethyl group
at the C7 position. Whereas previously 4 was observed
to make a weak (3.1 A) hydrogen bond interaction with
Trp447 and with a water molecule, 6 forms two hydrogen
bonds with active site residues: Trp447 (at a distance of 2.7 A)
and the peptide carbonyl group of Asp312 (which incidentally
is the catalytic base). The observation of the formation
of additional interactions with active site residues with 6
compared to 4 makes it difficult to rationalize why it binds
more weakly.
Analogously lactam 9, prepared through cycloaddition of 7
to dimethyl maleate15 followed by N–O bond cleavage of the
isoxazolidine adduct, was used for selective a-glucosylation
with 10 in the presence of TMSOTf, affording a-glucoside 12
(75% yield, Scheme 1). As previously, only traces of the
b-anomer were detected in the crude reaction mixture. Reduction
of 12 was not a trivial task, requiring an excess of LiBH4 and
BH3ꢁTHF in refluxing THF for several days; however,
glucoside 14 was obtained in quantitative yield. Catalytic
hydrogenation afforded the desired glucosyl derivative 6
(79% yield, Scheme 1).
With the two novel glucosides 5 and 6 as well as compound
4 which was previously synthesized10 in hand, we tested their
potency against trehalases from commercial porcine kidney,
E. coli (Tre37A) and C. riparius, Table 1.
Scheme 1 (a) TMSOTf, Et2O, rt, 1 h, 88% for 11; TMSOTf,
Et2O–CH2Cl2, rt, 1 h, 75% for 12. (b) LiAlH4, THF, rt, 1 h, 58%
for 13; LiBH4, BH3ꢁTHF, THF, rt, 11 d, 98% for 14. (c) H2, Pd/C,
HCl, MeOH, rt, 18 h, 77% for 5; H2, Pd/C, HCl, MeOH, rt, 12 h, 79%
for 6.
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selectivity for the insect enzyme. In particular, compound 6
shows major potential in terms of selectivity. Such promising
binding signatures auger well for the development of biologically
active compounds with insecticidal activity in the future.
We thank Prof. S. G. Withers, University of British
Columbia, Canada and Prof. D. R. Rose, Department of
Biology, University of Waterloo, Canada, for inhibition tests
towards HPA and NtMGAM. We thank MiUR (PRIN 2008)
and Ente Cassa di Risparmio di Firenze, Italy, for financial
support. T.M.G. is a Sir Henry Wellcome postdoctoral fellow
and G.J.D. holds a Royal Society Wolfson Research Merit
Award. The work was supported in part by a grant from the
University of Milano-Bicocca (FA 2008).
Notes and references
1 A. D. Elbein, Y. T. Pan, I. Pastuszak and D. Carroll, Glycobiology,
2003, 13, 17R–27R.
2 A. Becker, P. Schloder, J. E. Steele and G. Wegener, Experientia,
1996, 52, 433–439.
¨
3 J. Defaye, H. Driguez, B. Henrissat and E. Bar-Guilloux,
Carbohydr. Res., 1983, 124, 265–273.
4 J.-H. Lee, S. Saito, H. Mori, M. Nishimoto, M. Okuyama,
D. Kim, J. Wongchawalit, A. Kimura and S. Chiba, Biosci.,
Biotechnol., Biochem., 2007, 71, 2256–2265.
5 B. Tang, X. Chen, Y. Liu, H. Tian, J. Liu, J. Hu, W. Xu and
W. Zhang, BMC Mol. Biol., 2008, 9, 51.
6 G. Wegener, V. Tschiedel, P. Schloder and O. Ando, J. Exp. Biol.,
2003, 206, 1233–1240.
¨
7 N. Asano, M. Takeuchi, Y. Kameda, K. Matsui and Y. Kono,
J. Antibiot., 1990, 43, 722–726.
8 S. V. Kyosseva, Z. N. Kyossev and A. D. Elbein, Arch. Biochem.
Biophys., 1995, 316, 821–826.
9 A. Kato, E. Kano, I. Adachi, R. J. Molyneux, A. A. Watson,
R. J. Nash, G. W. J. Fleet, M. W. Wormald, H. Kizu, K. Ikeda and
N. Asano, Tetrahedron: Asymmetry, 2003, 14, 325–331.
Fig. 2 (a) Ball-and-stick representation of Tre37A in complex with 6.
The catalytic acid Glu496 (above) and the catalytic base Asp312
(below) are shown. Observed electron density, contoured at 1s
(0.35 electrons Aꢂ3) for the maximum likelihood weighted 2Fobs ꢂ Fcalc
map is shown. (b) Overlap between Tre37A in complex with 6 (green) and
4 (yellow). (c) Active site interactions between Tre37A and 6.
10 R. P. Gibson, T. M. Gloster, S. Roberts, R. A. J. Warren, I. Storch
´
de Gracia, A. Garcıa, J. L. Chiara and G. J. Davies, Angew. Chem.,
´
Int. Ed., 2007, 46, 4115–4119.
11 F. Cardona, C. Parmeggiani, E. Faggi, C. Bonaccini, P. Gratteri,
L. Sim, T. M. Gloster, S. Roberts, G. J. Davies, D. R. Rose and
A. Goti, Chem.–Eur. J., 2009, 15, 1627–1636.
12 M. Forcella, F. Cardona, A. Goti, C. Parmeggiani, L. Cipolla,
M. Gregori, R. Schirone, P. Fusi and P. Parenti, Glycobiology,
submitted.
13 M. Forcella, E. Berra, R. Giacchini and P. Parenti, Arch. Insect
Biochem. Physiol., 2007, 65, 181–194.
14 A. Brandi, F. Cardona, S. Cicchi, F. M. Cordero and A. Goti,
Chem.–Eur. J., 2009, 15, 7808–7821.
15 F. Cardona, E. Faggi, F. Liguori, M. Cacciarini and A. Goti,
Tetrahedron Lett., 2003, 44, 2315–2318.
16 N. Asano, T. Yamaguchi, Y. Kameda and K. Matsui, J. Antibiot.,
1987, 40, 526–532.
17 L. Sim, R. Quezada-Calvillo, E. E. Sterchi, B. L. Nichols and
D. R. Rose, J. Mol. Biol., 2008, 375, 782–792.
18 G. D. Brayer, G. Sidhu, R. Maurus, E. H. Rydberg, C. Braun,
Y. Wang, N. T. Nguyen, C. M. Overall and S. G. Withers,
Biochemistry, 2000, 39, 4778–4791.
As promiscuous inhibition is a potential problem for
agricultural applications of trehalase inhibitors, the specificity
of these compounds for trehalases versus other glycosidases
was also addressed. The inhibition of 4, 5 and 6 towards two
human glycosidases, N-terminal human maltase glucoamylase
(NtMGAM)17 and human pancreatic amylase (HPA),18,19 was
assessed. All compounds were found not to inhibit HPA at a
concentration of 100 mM. Of the three compounds, only 4
inhibits NtMGAM to any significant extent, with a Ki of
280 mM;11 a value that is around 20 000-fold less than with
the insect trehalase.
In conclusion, casuarine-6-O-a-D-glucoside derivatives have
been shown to be highly potent compounds against trehalases
derived from insects, bacteria and eukaryotes. The compounds
are especially attractive since they bind only weakly to
relevant human enzymes, notably those involved in starch
metabolism (promiscuous inhibition is a potential drawback of
‘‘monosaccharide’’ inhibitors)20 whilst also displaying significant
19 S. Numao, R. Maurus, G. Sidhu, Y. Wang, C. M. Overall,
G. D. Brayer and S. G. Withers, Biochemistry, 2002, 41,
215–225.
20 A. A. Watson, G. W. J. Fleet, N. Asano, R. J. Molyneux and
R. J. Nash, Phytochemistry, 2001, 56, 265–295.
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