Synthesis of novel iminosugar-based trehalase inhibitors by cross-metathesis reactions

Article abstract of DOI:10.1002/ejoc.201100484

The synthesis of a novel class of trehalase inhibitors composed of iminopyranose or iminofuranose residues linked at the pseudoanomeric carbon through an alkyl chain is described. A set of six novel compounds was prepared by the same reaction sequence inv

Full text of DOI:10.1002/ejoc.201100484

FULL PAPER
DOI: 10.1002/ejoc.201100484
Synthesis of Novel Iminosugar-Based Trehalase Inhibitors by Cross-Metathesis
Reactions
Davide Bini,[a] Matilde Forcella,[a] Laura Cipolla,*[a] Paola Fusi,[a] Camilla Matassini,[b] and
Francesca Cardona*[b]
Keywords: Iminosugars / Inhibitors / Glycosides / Metathesis / Disaccharide mimetics
The synthesis of a novel class of trehalase inhibitors com-
posed of iminopyranose or iminofuranose residues linked at
the pseudoanomeric carbon through an alkyl chain is de-
scribed. A set of six novel compounds was prepared by the
same reaction sequence involving the Grubbs Ru–carbene-
tected allyl C-iminoglycosides as the key step in homo- or
heterodimerization reactions. The target products, obtained
with the CM reaction, were fully hydrogenated by catalytic
hydrogenolysis, and preliminary biological screening of the
products as inhibitors of commercially available porcine tre-
catalyzed cross-metathesis (CM) of different N-Cbz-pro- halase was performed.
Introduction
Trehalase (EC 3.2.1.28) is an extremely specific inverting
glycosidase that hydrolyzes trehalose [α-d-glucopyranosyl-
α-d-glucopyranoside (1) Figure 1][1] to two glucose units.
Trehalose assumes considerable relevance in nature,[2] and
its hydrolysis is a process essential to vital functions of sev-
eral organisms, in particular fungi and insects, while being
almost absent in mammalian metabolism. Thus, trehalose
processing enzymes such as trehalases are attractive targets
for the search of inhibitors, as valuable tools for studying
the molecular physiology of trehalase function and sugar
metabolism in insects, and as potential novel insecticides.[3]
Some natural pseudodisaccharides, such as validoxyl-
amine A (2),[4] trehazolin (3),[5] casuarine-6-O-α-d-gluco-
side (4),[6,7] and its analogues (i.e., 5 and 6)[8] have been
shown to be potent inhibitors of trehalase. In addition, it
is well known that iminosugars[9,10] are potent glycosidases
inhibitors due to the presence of the endocyclic nitrogen,
Figure 1. Chemical structure of trehalose (1), validoxylamine A (2),
which is able to mimic the transition state of the enzymatic
reaction. Hence, a few iminosugar-based compounds (such
as 4–8, Figure 1) have been also proposed as trehalase in-
hibitors.[5,11,12]
trehazolin (3), casuarine-6-O-α-d-glucoside (4), its analogues (i.e.,
5, 6), and other iminosugar-based trehalase inhibitors (i.e., 7 and
8).
In the search for new inhibitors that might be specific
towards insect trehalase,[8,12] we herein propose the synthe-
sis of iminosugar-based trehalase inhibitors, possessing a
pseudodisaccharidic structure, obtained by cross-metathesis
(CM) reactions between suitably functionalized piperidine
or pyrrolidine iminosugars.
[a] Department of Biotechnology and Biosciences,
University of Milano-Bicocca,
P.za della Scienza 2, 20126 Milano, Italy
Fax: +39-02-64483565
E-mail: laura.cipolla@unimib.it
[b] Department of Chemistry “Ugo Schiff”,
University of Florence, Polo Scientifico e Tecnologico,
Via della Lastruccia 13, 50019 Sesto Fiorentino, Florence, Italy
Fax: +39-055-4573531
Results and Discussion
We report therein the synthesis and preliminary bio-
logical assays of novel trehalase inhibitors (compounds 10,
E-mail: francesca.cardona@unifi.it
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201100484.
Eur. J. Org. Chem. 2011, 3995–4000
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D. Bini, M. Forcella, L. Cipolla, P. Fusi, C. Matassini, F. Cardona
FULL PAPER
11, 18–20; Scheme 1) containing one or two iminosugar were tried (DCM at r.t., DCM at 40 °C, DCE at 80 °C,
moieties, namely, the nojirimycin core or the 1,4-dideoxy- DCE plus ultrasonication at 50 °C), and desired product
1,4-imino-d-arabinitol or the 1,4-dideoxy-1,4-imino-d-rib- 12 was obtained only in trace amounts (Scheme 2A). The
itol scaffolds. To mimic the natural disaccharidic substrates, extremely scarce reactivity observed for vinyl nojirimycin
the two units were linked by a short and flexible alkyl derivative 15 towards CM is supported also by similar ex-
bridge; the Grubbs Ru–carbene-catalyzed CM reaction[13] amples reported on vinyl-C-glycosides.[17]
was used to connect the two moieties suitably function-
alized with a double bond, as outlined in the retrosynthetic
scheme (Scheme 1).
Scheme 2. Homodimerization of pyranose structures by CM. Rea-
gents and conditions: (a) Hoveyda–Grubbs catalyst 2nd generation,
DCE, 80 °C, overnight; (b) Hoveyda–Grubbs catalyst 2nd genera-
tion, DCM, r.t., overnight; (c) Pd(OH)2/C, H2, EtOAc/EtOH = 1:1.
Scheme 1. Retrosynthetic scheme for the synthesis of trehalase in-
hibitors.
We proceeded further with the homodimerization of α-
As the starting point, we decided to synthesize nojirimy- C-allyl nojirimycin 16 (Scheme 2B), and in this case the CM
cin dimers 9 and 10, linked through a two and four carbon reaction afforded compound 13 (see general procedure) in
atom spacer, respectively, having in our hands fully pro- 51% yield as the (E) alkene (Ͼ95%). The yield, despite be-
tected α-C-vinyl nojirimycin 15[14] and protected α-C-allyl ing quite low, is comparable to those obtained in different
nojirimycin 16 (Scheme 1).[15] Each monomer was homodi- CM reactions reported on the same substrate.[18] Dimer
merized by CM using the 2nd generation Grubbs catalyst product 13 was fully deprotected by Pd-catalyzed hydrogen-
in dry dichloromethane as solvent.
ation.
It should be pointed out that the endocyclic nitrogen
To assess the relevance of the nitrogen atom in the inhibi-
atom has to be protected as a carbamate, as recent re- tion assays with iminosugar-based trehalase inhibitors, an
ports[16] showed that the CM is not compatible with the additional homodimerization by CM was performed on α-
endocyclic N-Bn group (found as protecting group in our C-allyl glucoside 17 (Scheme 2C), which afforded dimer 27
iminosugar synthesis), very likely because of the coordinat- in 65% yield; that compound in turn gave compound 26
ing ability of the latter with the Ru–carbene catalyst. Thus, after catalytic hydrogenolysis.
the N-carbobenzyloxynojirimycin was used in the present
study.
To gain better insight on the role of nitrogen, a hetero-
dimer containing one nojirimycin unit and one glucose unit
Initially, protected α-vinyl nojirimycin 15 was treated was also synthesized (Scheme 3) by CM. Due to the inert-
with 20% in weight Grubbs 1,3-dimesityl-4,5-dihydroimid- ness of vinyl derivatives, we decided to concentrate our at-
azol-2-ylideneruthenium carbene (Scheme 2, inset) in tention only on C-allyl monomers (α-C-allyl nojirimycin 16
dichloromethane at room temperature, but the reaction did and α-C-allyl glucoside 17). The CM reaction was per-
not afford the expected product. Hence, different conditions formed by using a slight excess amount of α-C-allyl gluco-
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Iminosugar-Based Trehalase Inhibitors by Cross-Metathesis Reactions
side 17, which gave desired compound 14 in 32% yield. The the anti isomer was formed, according to the expected
remaining material was composed of glucose and nojirimy- stereochemical outcome; reduction of hydroxylamine 31 to
cin homodimers 27 and 13 together with unreacted starting the corresponding amine 32 followed by carbamoylation af-
alkenes 16 and 17. Final catalytic hydrogenation afforded forded β-C-allyl iminoribofuranosyl derivative 25 suitable
compound 11.
to be employed in the CM reaction.
The two scaffolds were then used for homodimerization
and heterodimerization reactions by CM (Scheme 5). The
CM reactions were performed by following the general pro-
cedure, and afforded dimers 21, 22, and 23 in 54, 42, and
12% yield, respectively. As expected, the heterodimerization
proceeded with a similar reaction outcome to that observed
for six-membered ring derivatives (homodimerization and
starting material byproducts). Finally, hydrogenolysis af-
forded deprotected compounds 18, 19, and 20 (see the gene-
ral procedure).
Scheme 3. Heterodimerization of pyranose structures by CM. Rea-
gents and conditions: (a) Hoveyda–Grubbs catalyst 2nd generation,
DCM, r.t., overnight; (b) Pd(OH)2/C, H2, EtOAc/EtOH = 1:1.
Due to the reported activity against trehalases of some
iminofuranoses, such as compound 8[11] (Figure 1), we en-
visaged the possibility to expand our set of potential inhibi-
tors also to pseudodisaccharides obtained by CM of C-all-
yl-iminofuranoses. Two different allyl-iminofuranoses were
synthesized and used for the CM reaction (Scheme 4), pos-
sessing a 1,4-dideoxy-1,4-imino-d-arabinitol (24) or 1,4-di-
deoxy-1,4-imino-d-ribitol (25) core, respectively.
Scheme 5. Dimerization of furanose structures by CM. Reagents
and conditions: (a) Hoveyda–Grubbs catalyst 2nd generation,
DCM, r.t., overnight; (b) Pd(OH)2/C, H2, EtOAc/EtOH = 1:1.
All the deprotected compounds (i.e., 10, 11, 18–20, 26)
were tested for their inhibitory activity against porcine tre-
halase. To examine the potential of each member of the
library as trehalase inhibitor, preliminary screening assays
at a fixed concentration (1 mm) of potential inhibitors was
carried out, and dose–response curves were established for
Scheme 4. Synthesis of the iminofuranose scaffolds. Reagents and
conditions: (a) CbzCl, H2O/dioxane, NaHCO3, r.t., 16 h; (b) allyl-
magnesium bromide, THF, 0 °C, 3 h; (c) Zn, AcOH/H2O, r.t.,
40 min; (d) CbzCl, H2O/dioxane, NaHCO3, r.t., 20 h.
The synthesis of α-C-allyl iminoarabinofuranosyl deriva- the most active compounds to determine the Ki values. Ex-
tive 24 (Scheme 4A) proceeded by Grignard addition to periments were performed at a fixed substrate concentra-
nitrone 28,[19] as recently described.[20] Final carbamo- tion, close to the Km value (2.5 mm) in the presence of in-
ylation of intermediate 29 by Cbz-chloride in water/diox- creasing concentrations of the inhibitor. The inhibitory ac-
ane[21] afforded 24 (80%).[20] On the contrary, the synthesis tivity is shown as a percentage at the fixed concentration in
of β-C-allyl iminoribofuranosyl derivative 25 is not re- Figure 2.
ported in the literature and is presented here from nitrone
At a concentration of 1 mm, compounds 11, 18, 19, and
30,[22] as illustrated in Scheme 4B. The Grignard reaction 20 showed 25, 14, 28, and 43% inhibition, respectively, with
of nitrone 30 occurred with total stereoselectively and only respect to the control in the absence of an inhibitor, whereas
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FULL PAPER
Experimental Section
General Methods: All solvents were dried with molecular sieves for
at least 24 h prior to use. When dry conditions were required, the
reaction was performed under an Ar atmosphere. Thin-layer
chromatography (TLC) was performed on silica gel 60F254 coated
glass plates (Merck) with UV detection when possible, charring
with a conc. H2SO4/EtOH/H2O (10:45:45) solution, or with a solu-
tion of (NH4)6Mo7O24 (21 g), Ce(SO4)2 (1 g), conc. H2SO4 (31 mL)
in water (500 mL) and then heating to 110 °C for 5 min. Flash col-
umn chromatography was performed on silica gel 230–400 mesh
(Merck). Routine 1H and 13C NMR spectra were recorded at
400 MHz (1H) and at 100.57 MHz (13C) with a Varian Mercury
instrument or with
a Varian Gemini 200 MHz instrument,
50.29 MHz (13C) when specified. Chemical shifts are reported in
parts per million downfield from TMS as an internal standard.
Mass spectra were recorded with a System Applied Biosystems
MDS SCIEX instrument (Q TRAP, LC–MS–MS, turbon ion
spray) or with a System Applied Biosystem MDS SCIEX instru-
ment (Q STAR elite nanospray). ESI full MS were recorded with
a Thermo LTQ instrument by direct inlet; relative percentages are
shown in brackets. Elemental analyses (C, H, N) were performed
with a Perkin–Elmer series II 2400 analyzer or with a Perkin–Elmer
CHNS/O 2400 analyze, and all synthesized compounds showed a
purity of more than 95%.
General Procedure for the Cross-Metathesis Reaction: To a 0.1 m
solution of the appropriate allyl monomers in anhydrous CH2Cl2
was added the Grubbs catalyst (5% in weight). The mixture was
stirred overnight at room temperature and concentrated. The resi-
due was purified directly on a silica gel column using a suitable
eluent. All the products obtained by CM were the (E) isomers
(Ͼ95% as determined by NMR).
Figure 2. (A) Inhibition data of the synthesized compounds against
porcine trehalase observed at 1 mm inhibitor concentration.
(B) Dose–response curve of compound 10 (concentrations ranging
from 50 μm to 1 mm) on porcine trehalase.
General Procedure for the Hydrogenolysis Reaction: A 0.02 m solu-
tion of the appropriate dimer dissolved in EtOAc/EtOH (1:1) was
treated with Pd(OH)2/C (100% in weight). The reaction 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 corresponding
deprotected compounds in quantitative yields.
compound 10, which is the nojirimycin dimer, was the most
active derivative of the series, showing 100% inhibition at a
concentration of 1 mm (Figure 2A). For this compound, a
dose–response curve was performed (Figure 2B), affording
IC50 = 88 μm and Ki = 44 μm. On the other hand, reference
glucose dimer 26 did not show any inhibition at a concen-
tration of 1 mm.
1
Homodimer 10: H NMR (D2O): δ = 4.09–2.90 (m, 14 H, 1-H, 2-
H, 3-H, 4-H, 5-H, 6-H), 1.90–0.91 (m, 8 H, CH2) ppm. 13C NMR
(D2O): δ = 74.5, 71.6, 70.5, 58.0, 56.2 (C-1, C-2, C-3, C-4, C-5),
59.8 (C-6), 28.3, 26.8 (CH2) ppm. MS (TOF): m/z = 381.22 [M +
H]+; found 381.50. C16H32N2O8 (380.44): calcd. C 50.51, H 8.48,
N 7.36; found C 50.54, H 8.49, N 7.34.
Conclusions
1
A series of iminosugars was synthesized by cross-metath-
Heterodimer 11: H NMR (D2O): δ = 3.99–2.78 (m, 14 H, 1,1Ј-H,
esis reactions, and the series was preliminarily assayed for 2,2Ј-H, 3,3Ј-H, 4,4Ј-H, 5,5Ј-H, 6,6Ј-H), 1.12 (m, 4 H, CH2), 0.99
(br. s, 4 H, CH2) ppm. 13C NMR (D2O): δ = 78.2, 75.9, 75.0, 74.5,
73.9, 73.0,71.6, 70.5, 58.1, 57.6 (C-1,1Ј, C-2,2Ј, C-3,3Ј, C-4,4Ј, C-
their activity against porcine trehalase. The collected data
showed that very small inhibition was observed with imino-
arabinofuranosyl dimers, whereas a significant inhibition
could be detected with nojirimycin dimer 10. It is worth
noting that nojirimycin–glucose heterodimer 11 showed in-
hibitory activity in between that of nojirimycin homodimer
5,5Ј), 63.7, 59.8 (C-6,6Ј), 28.1, 28.0, 26.9, 25.9 (CH2) ppm. MS
(TOF): m/z = 382.21 [M + H]+; found 382.50. C16H31NO9 (381.42):
calcd. C 50.38, H 8.19, N 3.67; found C 50.34, H 8.20, N 3.65.
Protected Homodimer 13: Flash column chromatography (petro-
leum ether/EtOAc, 85:15). 1H NMR (CDCl3): δ = 7.46–7.02 (m,
10 and glucose homodimer 26, which was fully inactive.
50 H, ArH), 5.48 (br. s, 2 H, CH=CH), 5.06 (s, 4 H, CH2 Cbz),
These results highlight that the nitrogen atom is relevant
4.66–4.32 (m, 16 H, OCH2Ph), 4.30 (m, 2 H, 1-H), 4.15 (m, 2 H,
for inhibition, as usually reported for iminosugars, and that
3-H), 3.91 (m, 2 H, 4-H), 3.81 (d, J = 7.7 Hz, 2 H, 6a-H), 3.67 (m,
probably in the present case an additive effect occurs. Work
4 H, 2-H, 6b-H), 3.56 (m, 2 H, 5-H), 2.48 (m, 2 H, CH2-CH=CH),
is underway in our laboratories to expand the scope of this
reaction and to investigate the role of the length of the alkyl
chain connecting the two iminosugar moieties in more de-
tail.
2.39 (m, 2 H, CH2-CH=CH) ppm. 13C NMR (CDCl3): δ = 156.1
(C=O, Cbz), 138.6–136.8 (C Ar), 129.7–127.0 (CH=CH, CH Ar),
84.5, 82.1, 81.7, 80.7, 77.2, 76.5, 54.8, 53.4 (C-1,1Ј, C-2,2Ј, C-3,3Ј,
C-4,4Ј, C-5,5Ј), 73.4–72.8 (OCH2Ph), 72.2, 72.0 (C-6,6Ј), 67.4, 66.4
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Iminosugar-Based Trehalase Inhibitors by Cross-Metathesis Reactions
(CH2 Cbz), 31.0, 30.0 (CH2-CH=CH) ppm. MS (TOF): m/z =
1367.66 [M + H]+; found 1367.00. C88H90N2O12 (1367.69): calcd.
C 77.28, H 6.63, N 2.05; found C 77.34, H 6.62, N 2.05.
40 H, ArH), 5.54–5.24 (m, 2 H, CH=CH), 5.23–4.94 (m, 4 H, CH2
Cbz), 4.69–4.21 (m, 12 H, OCH2Ph), 4.20–3.40 (m, 12 H, 1-H, 2-
H, 3-H, 4-H, 5-H), 1.29–1.24 (m, 4 H, CH2-CH=CH) ppm. 13C
NMR (CDCl3): δ = 156.3 (C=O, Cbz), 138.5–136.9 (C Ar), 128.7–
127.8 (CH=CH, CH Ar), 82.9, 76.7, 62.6, 61.3 (C-1, C-2, C-3, C-
4), 73.3, 71.4 (OCH2Ph), 68.9 (C-5), 67.1 (CH2 Cbz), 29.9 (CH2-
CH=CH) ppm. C72H74N2O10 (1127.38): calcd. C 76.71, H 6.62, N
2.48; found C 76.67, H 6.63, N 2.48.
Protected Heterodimer 14: Flash column chromatography (toluene/
1
EtOAc, 98:2). H NMR (CDCl3): δ = 7.33–7.08 (m, 45 H, ArH),
5.48 (m, 1 H, CH=CH), 5.35 (m, 1 H, CH=CH), 5.02 (s, 2 H, CH2
Cbz), 4.84 (d, J = 10.9 Hz, 1 H, OCH2Ph), 4.75–4.70 (m, 2 H,
OCH2Ph), 4.60–4.19 (m, 13 H, OCH2Ph), 4.11 (m, 1 H, 1-H), 3.94
(m, 1 H, 1Ј-H), 3.87–3.43 (m, 12 H, 2-H, 3-H, 4-H, 5-H, 6a-H, 6b-
H), 2.50 (m, 1 H, CH2-CH=CH), 2.37 (m, 1 H, CH2-CH=CH),
2.23 (m, 2 H, CH2-CH=CH) ppm. 13C NMR (CDCl3): δ = 156.2
(C=O Cbz), 139.0–136.7 (C Ar), 130.3–127.5 (CH=CH, CH Ar),
82.7, 82.6, 81.7, 80.8, 80.3, 78.3, 74.3, 71.3, 54.8, 53.3 (C-1,1Ј, C-
2,2Ј, C-3,3Ј, C-4,4Ј, C-5,5Ј), 75.6, 75.2, 73.7, 73.1, 73.0, 72.8, 72.3,
72.0 (OCH2Ph), 69.1, 67.5 (C-6,6Ј), 60.7 (CH2 Cbz), 30.0, 28.8
(CH2-CH=CH) ppm. MS (TOF): m/z = 1234.60 [M + H]+; found
1234.80. C80H83NO11 (1234.54): calcd. C 77.83, H 6.78, N 1.13;
found C 77.79, H 6.77, N 1.14.
Pyrrolidine 25: Allyl ammine 32 (575 mg, 1.3 mmol) was dissolved
in water (5 mL) and NaHCO3 (218 mg, 2.6 mmol) was added. The
suspension was stirred until complete dissolution of the salt, then
dioxane (6 mL) was added. The resulting mixture was cooled to
0 °C with an ice bath and CbzCl (186 μL, 1.3 mmol) was added
dropwise. After 10 min, the ice bath was removed, and the reaction
mixture was stirred for 20 h at room temperature. After addition
of EtOAc (27 mL) and water (8 mL), the aqueous layer was sepa-
rated and washed with EtOAc (3ϫ10 mL); the combined organic
extracts were washed with 1 m HCl (2ϫ10 mL) and brine and dried
with Na2SO4. The solvent was evaporated, and the residue was
purified by flash column chromatography (petroleum ether/EtOAc,
2:1) to afford pure 25 (590 mg, 1.02 mmol, 79%) as a colorless oil.
1H NMR (CDCl3): δ = 7.51–7.10 (m, 20 H, ArH), 5.73–5.29 (m, 1
H, CH2CH=CH2), 5.18–5.11 (m, 2 H, CH2 Cbz), 5.05–4.82 (m, 2
H, CH2CH=CH2), 4.53–4.40 (m, 6 H, OCH2Ph), 4.20–4.01 (m, 3
H, 1-H, 2-H, 4-H) 3.79 (t, J = 4.2 Hz, 1 H, 3-H), 3.72–3.46 (m, 2 H,
5-H), 2.50–2.32 (m, 2 H, CH2CH=CH2) ppm. 13C NMR (50 MHz,
CDCl3): δ = 151.1 (C=O Cbz), 137.9–136.3 (C Ar), 133.8
(CH2CH=CH2), 128.1–127.0 (C Ar), 117.2 (CH2CH=CH2), 78.4
(C-3), 77.4 (C-2), 72.9, 71.3, 71.0 (OCH2Ph), 67.8 (C-5), 66.6 (CH2
Cbz), 61.5–60.43 (C-1, C-4), 36.4–35.9 (CH2CH=CH2) ppm. MS
(ESI): m/z (%) = 600.28 (79) [M + Na]+. C37H39NO5 (577.72):
calcd. C 76.92, H 6.80, N 2.42; found C 76.85, H 6.81, N 2.42.
1
Homodimer 18: H NMR (D2O): δ = 4.19–3.05 (m, 12 H, 1-H, 2-
H, 3-H, 4-H, 5-H), 1.88–1.58 (m, 4 H, CH-CH2-CH2), 1.48–1.27
(m, 4 H, CH2-CH2-CH2-CH2) ppm. 13C NMR (D2O): δ = 79.1
73.7 (C-2, C-3), 64.9, 64.0 (C-1, C-4), 60.6 (C-5), 27.4 (CH-CH2-
CH2), 26.5 (CH2-CH2-CH2-CH2) ppm. C14H28N2O6 (320.39):
calcd. C 52.48, H 8.81, N 8.74; found C 52.41, H 8.80, N 8.75.
1
Homodimer 19: H NMR (D2O): δ = 4.30–3.17 (m, 12 H, 1-H, 2-
H, 3-H, 4-H, 5-H), 1.80–1.49 (m, 4 H, CH-CH2-CH2), 1.47–1.26
(m, 4 H, CH2-CH2-CH2-CH2) ppm. 13C NMR (D2O): δ = 75.8,
73.0 (C-2, C-3), 65.2, 62.9 (C-1, C-4), 59.9 (C-5), 32.2 (CH-CH2-
CH2), 27.8 (CH2-CH2-CH2-CH2) ppm. C14H28N2O6 (320.39):
calcd. C 52.48, H 8.81, N 8.74; found C 52.43, H 8.80, N 8.73.
1
Heterodimer 20: H NMR (D2O): δ = 4.17–3.19 (m, 12 H, 1-H, 2-
H, 3-H, 4-H, 5-H), 1.82–1.50 (m, 4 H, CH-CH2-CH2), 1.48–1.27
(m, 4 H, CH2-CH2-CH2-CH2) ppm. 13C NMR (D2O): δ = 80.7,
76.9, 75.8, 73.0 (C-2, C-2Ј, C-3, C-3Ј), 64.9, 64.9 63.9, 63.9 (C-1,
C-1Ј, C-4, C-4Ј), 60.6, 59.9 (C-5, C-5Ј), 32.2, 32.2 (CH-CH2-CH2),
27.7, 27.7 (CH2-CH2-CH2-CH2) ppm. C14H28N2O6 (320.39): calcd.
C 52.48, H 8.81, N 8.74; found C 52.52, H 8.82, N 8.74.
Homodimer 26: 1H NMR (D2O): δ = 3.90–3.75 (m, 2 H, 1-H), 3.66
(dd, J = 12.2, 1.6 Hz, 2 H, 6a-H), 3.59–3.41 (m, 6 H, 2-H, 5-H,
6b-H), 3.41–3.30 (m, 2 H, 3-H), 3.16 (t, J = 9.2 Hz, 2 H, 4-H),
1.63–1.50 (m, 2 H, CH2), 1.49–1.37 (m, 2 H, CH2), 1.36–1.26 (m,
2 H, CH2), 1.25–1.17 (m, 2 H, CH2) ppm. 13C NMR (D2O): δ =
78.4, 76.0, 75.0, 74.0, 73.1 (C-1, C-2, C-3, C-4, C-5), 63.9 (C-6),
26.9, 26.1 (CH2) ppm. MS (TOF): m/z = 383.19 [M + H]+; found
383.17. C16H30O10 (382.41): calcd. C 50.52, H 7.91; found C 50.47,
H 7.92.
Protected Homodimer 21: Flash column chromatography (petro-
leum ether/EtOAc, 87.5:12.5). 1H NMR (CDCl3): δ = 7.48–6.97
(m, 40 H, ArH), 5.39–5.25 (m, 2 H, CH=CH), 5.23–4.98 (m, 4 H,
CH2 Cbz), 4.69–4.21 (m, 12 H, OCH2Ph), 4.21–3.40 (m, 12 H, 1-
H, 2-H, 3-H, 4-H, 5-H), 1.26 (t, J = 7.1 Hz, 4 H, CH2-CH=CH)
ppm. 13C NMR (CDCl3): δ = 154.4 (C=O, Cbz), 138.7–136.7 (C
Ar), 129.9 (CH=CH), 128.7–127.6 (CH Ar), 83.6, 82.7, 64.5, 63.0
(C-1, C-2, C-3, C-4), 73.2, 71.2 (OCH2Ph), 68.9, 68.0 (C-5,5Ј), 67.1
(CH2 Cbz), 29.9 (CH2-CH=CH) ppm. MS (TOF): m/z = 1127.54
[M + H]+; found 1127.20. C72H74N2O10 (1127.38): calcd. C 76.71,
H 6.62, N 2.48; found C 76.75, H 6.61, N 2.48.
Protected Homodimer 27: Flash column chromatography (petro-
leum ether/EtOAc, 85:15). 1H NMR (CDCl3): δ = 7.43–7.18 (m,
40 H, ArH), 5.55 (br. d, J = 21.9 Hz, 2 H, CH=CH), 4.94 (d, J =
11.0 Hz, 2 H, OCH2Ph), 4.85–4.78 (m, 4 H, OCH2Ph), 4.70–4.61
(m, 6 H, OCH2Ph), 4.52–4.41 (m, 4 H, OCH2Ph), 4.10 (m, 2 H, 1-
H), 3.88–3.51 (m, 12 H, 2-H, 3-H, 4-H, 5-H, 6a-H, 6b-H), 2.47
(m,4 H, CH2-CH=CH) ppm. 13C NMR (CDCl3): δ = 139.0, 138.5,
138.4, 138.2 (C Ar), 128.7–127.8 (CH=CH, CH Ar), 82.6, 80.3,
78.2, 74.2, 71.3 (C-1, C-2, C-3, C-4, C-5), 75.7, 75.3, 73.7, 73.2
(OCH2Ph), 69.0 (C-6), 29.9, 28.8 (CH2-CH=CH) ppm. MS (TOF):
m/z = 1101.55 [M + H]+; found 1101.60. C72H76O10 (1101.39):
calcd. C 78.52, H 6.96; found C 78.59, H 6.94.
Protected Homodimer 22: Flash column chromatography (petro-
leum ether/EtOAc, 80:20). 1H NMR (CDCl3): δ = 7.36–7.11 (m,
40 H, ArH), 5.51–5.25 (m, 2 H, CH=CH), 5.17–5.03 (m, 4 H, CH2
Cbz), 4.58–4.22 (m, 12 H, OCH2Ph), 4.21–3.41 (m, 12 H, 1-H, 2-
H, 3-H, 4-H, 5-H), 1.25 (t, J = 7.1 Hz, 4 H, CH2-CH=CH) ppm.
13C NMR (CDCl3): δ = 155.9 (C=O, Cbz), 138.5–136.9 (C Ar),
128.7–127.7 (CH=CH, CH Ar), 77.6, 76.6, 61.9, 61.3 (C-1, C-2, C-
3, C-4), 73.3, 71.7 (OCH2Ph), 68.3 (C-5), 67.1 (CH2 Cbz), 30.0
(CH2-CH=CH) ppm. MS (TOF): m/z = 1127.54 [M + H]+; found
1127.30. C72H74N2O10 (1127.38): calcd. C 76.71, H 6.62, N 2.48;
found C 76.77, H 6.63, N 2.48.
Pyrrolidine 31: To a cooled (0 °C) solution of nitrone 30 (960 mg,
2.3 mmol) in anhydrous THF (30 mL) was dropwise added allyl-
magnesium bromide (1.0 m in diethyl ether, 6.9 mL, 6.9 mmol). Af-
ter stirring for 3 h at 0 °C, the reaction was quenched with satu-
rated aqueous NH4Cl (20 mL). The reaction mixture was diluted
with diethyl ether (25 mL), the organic layer was separated, and
the aqueous layer was extracted with diethyl ether (2ϫ20 mL). The
combined organic extracts were washed with brine (20 mL), dried
with Na2SO4, filtered, and concentrated under reduced pressure to
Protected Heterodimer 23: Flash column chromatography (petro-
leum ether/EtOAc, 85:15). 1H NMR (CDCl3): δ = 7.56–7.00 (m,
Eur. J. Org. Chem. 2011, 3995–4000
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3999

D. Bini, M. Forcella, L. Cipolla, P. Fusi, C. Matassini, F. Cardona
FULL PAPER
afford crude 31 (1.05 g, quantitative), which was employed in the
following step without further purification. A sample of crude
product was purified by flash column chromatography (petroleum
ether/EtOAc, 3:1) to give pure 31 as a white solid. M.p. 63–65 °C.
1H NMR (CDCl3): δ = 7.32–7.29 (m, 15 H, ArH), 5.94–5.83 (m, 1
H, CH2CH=CH2), 5.08–5.00 (m, 2 H, CH2CH=CH2), 4.58–4.51
(m, 5 H, OCH2Ph), 4.43 (d, J = 11.7 Hz, 1 H, OCH2Ph), 3.72 (t,
J = 5.4 Hz, 1 H, 3-H), 3.56 (dd, J = 5.3, 2.3 Hz, 2 H, 5-H), 3.47
(dd, J = 7.3, 5.8 Hz, 1 H, 2-H), 3.39–3.30 (m, 2 H, 1-H, 4-H), 2.46–
2.32 (m, 2 H, CH2CH=CH2) ppm. 13C NMR (CDCl3): δ = 138.2,
138.1, 138.0 (C Ar), 135.5 (CH=CH2), 128.4–127.7 (C Ar), 116.7
(CH=CH2), 77.3 (C-2), 75.4 (C-3), 73.3 (C-4), 71.9, 71.7, 71.4
(OCH2Ph), 70.1 (C-1), 69.7 (C-5), 36.0 (CH2CH=CH2) ppm. MS
(ESI): m/z (%) = 482.42 (100) [M + Na]+. C29H33NO4 (459.58):
calcd. C 75.79, H 7.24, N 3.05; found C 75.69, H 7.21, N 3.07.
Firenze, Italy, for financial support, and Ministero dell’Università
e della Ricerca (MIUR) under project PRIN 2008.
[1] A. D. Elbein, Y. T. Pan, I. Pastuszak, D. Carroll, Glycobiology
2003, 13, 17R–27R.
[2] J. C. Arguelles, Arch. Microbiol. 2000, 174, 217–224.
[3] N. Asano, Glycobiology 2003, 13, 93R–104R.
[4] a) N. Asano, M. Takeuchi, Y. Kameda, K. Matsui, Y. Kono,
J. Antibiot. 1990, 43, 722–726; b) R. P. Gibson, T. M. Gloster,
S. Roberts, R. A. J. Warren, I. Storch de Gracia, Á. García,
J. L. Chiara, G. J. Davies, Angew. Chem. Int. Ed. 2007, 46,
4115–4119.
[5] S. V. Kyosseva, Z. N. Kyossev, A. D. Elbein, Arch. Biochem.
Biophys. 1995, 316, 821–826.
[6] 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,
N. Asano, Tetrahedron: Asymmetry 2003, 14, 325–331.
[7] F. Cardona, C. Parmeggiani, E. Faggi, C. Bonaccini, P. Grat-
teri, L. Sim, T. M. Gloster, S. Roberts, G. J. Davies, D. R. Rose,
A. Goti, Chem. Eur. J. 2009, 15, 1627–1636.
[8] F. Cardona, A. Goti, C. Parmeggiani, P. Parenti, M. Forcella,
P. Fusi, L. Cipolla, S. Roberts, G. J. Davies, T. M. Gloster,
Chem. Commun. 2010, 46, 2629–2631.
[9] a) L. Cipolla, B. La Ferla, M. Gregori, Comb. Chem. High
Throughput Screening 2006, 9, 571–582; b) I. Robina, P. Vogel,
Synthesis 2005, 675–702; c) P. Merino, I. Delso, E. Marca, T.
Tejero, R. Matute, Curr. Chem. Biol. 2009, 3, 253–271.
[10] P. Compain, O. R. Martin (Eds.), Iminosugars: From Synthesis
to Therapeutic Applications, Wiley-VCH, Weinheim, 2007.
[11] N. Asano, A. Kato, K. Matsui, Eur. J. Biochem. 1996, 240,
692–698.
[12] M. Forcella, F. Cardona, A. Goti, C. Parmeggiani, L. Cipolla,
M. Gregori, R. Schirone, P. Fusi, P. Parenti, Glycobiology 2010,
20, 1186–1195.
[13] See, for example: a) D. V. Jarikote, P. V. Murphy, Eur. J. Org.
Chem. 2010, 4959–4970; b) R. H. Grubbs, Tetrahedron 2004,
60, 7117–7140.
[14] L. Cipolla, L. Lay, F. Nicotra, C. Pangrazio, L. Panza, Tetrahe-
dron 1995, 51, 4679–4690.
[15] L. Cipolla, M. Reis Fernandes, M. Gregori, C. Airoldi, F. Nic-
otra, Carbohydr. Res. 2007, 342, 1813–1830.
Pyrrolidine 32: A solution of hydroxylamine 31 (923 mg, 2.0 mmol)
in acetic acid (10 mL) and water (10 mL) was treated with Zn pow-
der (2.6 g, 40.0 mmol). The resulting mixture was stirred at room
temperature for 40 min, diluted with water (50 mL), and treated
with Na2CO3 (9.3 g) until bubbling of CO2 stopped. The reaction
mixture was extracted with DCM (3ϫ50 mL), and the combined
organic extracts were washed with 3 m NaOH (50 mL) and brine,
dried (Na2SO4), and evaporated under reduced pressure to give
pure ammine 32 (704 mg, 1.6 mmol, 80%) as a colorless oil, which
did not need further purification. An analytically pure sample was
obtained after purification by flash column chromatography
(CHCl3/Et2O, 2:1). 1H NMR (CDCl3): δ = 7.36–7.24 (m, 15 H,
ArH), 5.78 (ddt, J = 17.1, 10.1, 7.0 Hz, 1 H, CH2CH=CH2), 5.08–
5.01 (m, 2 H, CH2CH=CH2), 4.60–4.45 (m, 6 H, OCH2Ph), 3.74
(t, J = 5.3 Hz, 1 H, 3-H), 3.51–3.40 (m, 4 H, 2-H, 4-H, 5-H), 3.35–
3.30 (m, 1 H, 1-H), 2.41–2.35 (m, 1 H, CH2CH=CH2), 2.08 (td, J
= 14.2, 6.9 Hz, 1 H, CH2CH=CH2) ppm. 13C NMR (CDCl3): δ =
138.3–138.2 (C Ar), 135.2 (CH=CH2), 128.3–127.6 (C Ar), 117.1
(CH2CH=CH2), 81.3 (C-2), 78.2 (C-3), 73.2, 71.8, 71.6 (OCH2Ph),
71.1 (C-5), 61.6 (C-4), 60.6 (C-1), 38.4 (CH2CH=CH2) ppm. MS
(ESI): m/z (%) = 444.42 (100) [M + H]+. C29H33NO3 (443.58):
calcd. C 78.52, H 7.50, N 3.16; found C 78.59, H 7.48, N 3.15.
[16] a) Y.-J. Hu, R. Roy, Tetrahedron Lett. 1999, 40, 3305–3308; b)
G. Godin, P. Compain, O. R. Martin, Org. Lett. 2003, 5, 3269–
3272; c) A. J. Vernall, A. D. Abell, Aldrichimica Acta 2003, 36,
93–105.
Enzyme Assay: Trehalase activity was measured through a coupled
assay with glucose-6-phosphate dehydrogenase and hexokinase ac-
cording to Wegener et al.[23] All enzyme assays were performed in
triplicates at 30 °C by using sample volumes varying from 5 to
20 μL in 1 mL test and using a Cary3 UV/Vis Spectrophotometer.
Enzyme activities were analyzed by Cary Win UV application soft-
ware for Windows XP. The specific activity (Umg–1) was expressed
as μmolmin–1 mgprotein–1. Values were expressed as mean ϮS.E.
of replicated.
[17] a) R. Roy, S. K. Das, Chem. Commun. 2000, 519–529; b) A.
Dondoni, P. P. Giovannini, A. Marra, J. Chem. Soc. Perkin
Trans. 1 2001, 2380–2388.
[18] A. Dondoni, P. P. Giovannini, D. Perrone, J. Org. Chem. 2005,
70, 5508–5518.
[19] F. Cardona, E. Faggi, F. Liguori, M. Cacciarini, A. Goti, Tetra-
hedron Lett. 2003, 44, 2315–2318.
[20] I. Delso, T. Tejero, A. Goti, P. Merino, Tetrahedron 2010, 66,
Supporting Information (see footnote on the first page of this arti-
cle): Details of the enzyme assays and selected copies of the NMR
spectra.
1220–1227.
[21] F. Sladojevich, A. Trabocchi, A. Guarna, Org. Biomol. Chem.
2008, 6, 3328–3333.
[22] E.-L. Tsou, Y.-T. Yeh, P.-H. Liang, W.-C. Cheng, Tetrahedron
2009, 65, 93–100.
[23] G. Wegener, V. Schiedel, P. Schlöder, O. Ando, J. Exp. Biol.
2003, 206, 1233–120.
Acknowledgments
We gratefully acknowledge Consorzio CINMPIS, University of
Milano-Bicocca under FAR 2009, and Ente Cassa di Risparmio di
Received: April 5, 2011
Published Online: June 1, 2011
4000
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3995–4000