Fluorescent-tagged sp2-iminosugars with potent β-glucosidase inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2010.09.003

New fluorescently-labelled sp²-iminosugars based on the 5N,6S-[N′-(4-aminobutyl)iminomethylidene]-6-thionojirimycin skeleton have been synthesized as photoprobes to monitor glycosidase binding. Dansyl, dapoxyl and coumarin fluorophores were app

Full text of DOI:10.1016/j.bmc.2010.09.003

Bioorganic & Medicinal Chemistry 18 (2010) 7439–7445
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorescent-tagged sp2-iminosugars with potent b-glucosidase
inhibitory activity
Matilde Aguilar-Moncayo a, M. Isabel García-Moreno a, Arnold E. Stütz b, José M. García Fernández c,
a,
Tanja M. Wrodnigg b, , Carmen Ortiz Mellet
⇑
⇑
a Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 553, E-41012 Sevilla, Spain
b Glycogroup, Institut für Organische Chemie der Technischen Universität Graz, Stremayrgasse 16, A-8010 Graz, Austria
c Instituto de Investigaciones Químicas, CSIC—Universidad de Sevilla, Américo Vespucio 49, Isla de la Cartuja, E-41092 Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
New fluorescently-labelled sp2-iminosugars based on the 5N,6S-[N0-(4-aminobutyl)iminomethylidene]-
6-thionojirimycin skeleton have been synthesized as photoprobes to monitor glycosidase binding.
Dansyl, dapoxyl and coumarin fluorophores were appended to the terminal amino group at the
N0-substituent by either sulfonamide or amide bridging reaction. All the conjugates behaved as strong
(low micromolar to nanomolar) and selective inhibitors of b-glucosidases (almonds and bovine liver)
and naringinase, in agreement with the inhibition pattern previously encountered for related
iso(thio)urea-type bicyclic sp2-iminosugars. The presence of the fluorescent probe allows real-time and
continuous monitoring of b-glucosidase inhibition by fluorescence resonance energy transfer (FRET), tak-
ing advantage of the intrinsic tryptophan-associated fluorescence of the protein.
Article history:
Received 29 May 2010
Revised 30 August 2010
Accepted 1 September 2010
Available online 7 September 2010
Keywords:
Glycosidase inhibitors
Fluorescence resonance energy transfer
(FRET)
Ó 2010 Elsevier Ltd. All rights reserved.
Iminosugars
Photolabeling
1. Introduction
the incorporation of substituents that provide additional interac-
tions with protein regions at the vicinity of the active site.8 For in-
Iminosugar glycosidase inhibitors such as 1-deoxynojirimycin
(1; DNJ) and 2,5-dideoxy-2,5-imino- -mannitol (2; DMDP) are
stance, the cyclic carbamate nojirimycin derivative 3 was a potent
and selective inhibitor of the neutral -glucosidase II of the endo-
D
a
important tools in glycobiology, helping both the unravelling of
the glycosidases action mechanism and the development of new
pharmaceuticals,1 for example, in the treatment of diabetes type II
symptoms2 and in hereditary enzyme deficiency diseases.3 How-
ever, most representatives of this family of alkaloids exhibit broad
inhibitory profiles within a given configurational substrate pattern,
plasmic reticulum (yeast), being inactive against b-glucosidases.
Derivatives of 3 incorporating N-substituents at the pseudoanomer-
ic (C-1) position have shown significant antiproliferative activity in
human breast carcinoma cells.9 In stark contrast to this, the N0-octyl
isothiourea analogue 4 was a selective inhibitor of b-glucosidases,
including the human lysosomal b-glucocerebrosidase (b-Glu) and
shows great promise as a chemical chaperone for the treatment of
Gaucher disease.10 The chaperone activity was assumed to rely on
the specific binding of 4 to the active site of misfolded b-Glu mutants
at the endoplasmic reticulum thereby facilitating trafficking to the
lysosome,11 although no direct proof could be obtained. In order to
investigate the mechanisms involved in cell internalization and
organelle distribution more deeply, the development of fluores-
cent-tagged sp2-iminosugars seemed very appealing (Fig. 1).
Previous work has shown that the introduction of fluorescent
probes such as dansyl residues onto lateral chains in classical
iminosugars, like 1 and 2, results in compounds that may exhibit
similar or even enhanced glycosidase inhibitory activity (e.g., 5
and 6).12,13 The requisite for these channels is that the presence
of a lipophilic substituent at the functionalized position in the
iminosugar core is tolerated, which is the case for the nitrogen
atom in 1 or the primary positions in 2. The utility of the fluores-
cently-labelled iminosugar inhibitors for the construction of
inhibiting simultaneously several a- and b-glycosidases, which rep-
resents a serious limitation for clinical applications.4 With the aim of
improving the enzyme selectivity we developed a new family of gly-
comimetics in which the endocyclic sp3-hybridized amine-type
nitrogen, characteristic for iminosugars, has been replaced by a
pseudoamide-type nitrogen atom having a high sp2-hybridisation
character (sp2-iminosugars).5 These compounds feature an extre-
mely strong anomeric effect that results in very high chemical and
configurational stabilities even for reducing derivatives, which is
notably different from the case of classical iminosugars.6,7 Most
remarkably, the affinity towards
a- or b-glycosidases as well as
towards specific isoenzymes within a series can be finely tuned by
⇑
Corresponding authors. Tel.: +43 316 873 8744; fax: +43 316 873 8740
(T.M.W.); tel.: +34 954559806; fax: +34 954624960 (C.O.M.).
E-mail addresses: t.wrodnigg@tugraz.at (T.M. Wrodnigg), mellet@us.es
(C.O. Mellet).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.003

7440
M. Aguilar-Moncayo et al. / Bioorg. Med. Chem. 18 (2010) 7439–7445
OH
H
N
R
NR
HO
HO
HO
OH
HO
1, R = H
5, R =
HO
2, R = OH
6, R =
O
O
O
O
S
S
N
H
N
5
N
H
N
H
N
5
O
N
O
S
7
N
N
HO
HO
HO
HO
HO
HO
OH
OH
3
4
Figure 1. Structures of DNJ (1), DMDP (2), the bicyclic nojirimycin analogues 3 and
4 and the fluorescent-tagged DNJ and DMDP derivatives 5 and 6.
sensors to detect glycosidase binding14 and as chemical chaper-
ones15 has been demonstrated, illustrating the potential of the
approach in glycobiology. Taking into consideration that the
hydrophobic chain in the sp2-iminosugar 4 is critical for the inhibi-
tion of b-glucosidases, it was conceived that related derivatives
having appended fluorescent moieties at this region of the
molecule might maintain the biological activity profile while
incorporating interesting chemo-optical properties. To test this
hypothesis we have now synthesized dansyl-, coumarin- and
dapoxyl-labelled analogues of 4 and evaluated their inhibitory
activity against a panel of commercial glycosidases. The feasibility
of fluorescence resonance energy transfer (FRET)16 as a protocol for
the real-time and continuous monitoring of glycosidase inhibition
by fluorescently-labelled sp2-iminosugars is also demonstrated.
Scheme 1. Reagents and conditions: (a) (1) SCN(CH2)4NHBoc, Et3N, pyridine, rt,
18 h; (2) TsOH, (1:1) CH2Cl2–MeOH, rt, 2 h, 70% (global); (b) MsCl, pyridine,
ꢀ20 °C?rt, 7 h, 78%; (c) (1) 90% TFA–H2O, rt, 30 min; (2) Amberlite IRA-68 (OHꢀ);
(3) HCl 1 N, 82%; (d) ClSO2R, DMF, Et3N, 0 °C, 4 h, 54–98%; (e) RCOOH, TBTU, DMF,
Et3N, rt, 1 h, 54–56%.
2. Results and discussion
2.1. Synthesis
ceived as the key pivotal intermediate for the preparation of the
title compounds.
The synthesis of 10 started from known 5-amino-5-deoxy-6-O-
In our molecular design of fluorescent sp2-iminosugar glycosi-
dase inhibitors we took advantage of the information previously
obtained from X-ray and thermodynamic studies of 4 and other
structurally related bicyclic nojirimycin glycomimetics in complex
with the b-glucosidase from Thermotoga maritime (TmGH1),17 an
enzyme that belongs to family GH1 in the CaZy classification.18
The octyl chain at the exocyclic nitrogen in the enzyme–inhibitor
complex was found to occupy a hydrophobic pocket at the en-
trance of the active site in all cases, keeping substantial mobility.
A similar situation was encountered for a related sp2-iminosugar
in complex with recombinant human b-glucocerebrosidase.19 It
was then inferred that structural modifications at this portion of
the molecule would not affect the extensive hydrogen bond net-
work involving the hydroxyl groups and would be well tolerated
as far as its hydrophobic nature is conserved. Shortening the alkyl
chain from octyl to butyl was considered to guarantee the neces-
sary flexibility for an optimal induced fit at the active site of b-glu-
cosidases without unduly extending the length of the substituent
after appending the fluorophore, which could result in steric
clashes or insufficient solubility in aqueous media. Coupling of
commercially available activated fluorescent probes to an amine-
functionalized scaffold was selected as the conjugation method
of choice since it represents one of the most efficient and popular
strategies for the preparation of fluorescent-tagged conjugates.20
Moreover, this approach is well-suited for convergent schemes in
which the fluorescent moiety is incorporated at the very last step.
Following these considerations the 5N,6S-(40-aminobutyliminome-
thylidene)-6-thionojirimycin derivative 10 (Scheme 1) was con-
tetrahydropyranyl-1,2-O-isopropylidene-
a
-D
-glucofuranose
7.21
The Boc-protectedbutylamino spacer was first incorporated by thio-
urea coupling reaction of 7 with 4-(tert-butoxycarbonylamino)butyl
isothiocyanate. Selective hydrolysis of the tetrahydropyranyl group
was achieved by treatment of the crude adduct with p-toluenesul-
fonic acid to give the vic-hydroxythiourea 8 (70% yield). Activation
of the primary hydroxyl by methanesulfonylation (mesylation) with
mesyl chloride proceeded with spontaneous nucleophilic displace-
mentof theresulting mesylate ester by the thiocarbonylsulfuratom,
affording the 2-aminothiazoline pseudo-C-nucleoside derivative 9.
Further rearrangement of the furanose ring into a piperidine deriv-
ative relies in the ability of the nitrogen atom of the cyclic isothiou-
rea functionality to participate in intramolecular nucleophilic
addition reactions to the masked aldehyde group of the monosac-
charide in the open chain form. Thus, simultaneous acid-promoted
hydrolysis of the acetal and Boc protecting groups in 9 using TFA–
water followed by elimination of the acid by coevaporation with
water and final neutralization with basic ion-exchange resin led to
the required sp2-iminosugar, characterized as the corresponding
hydrochloride salt 10, in 82% yield (Scheme 1).
For the final assembly of the target fluorescently-tagged bicyclic
nojirimycin derivatives two different types of coupling reactions
were considered, namely sulfonamide and amide coupling. Reac-
tion of 10 with dansyl and dapoxyl chloride in the presence of
Et3N afforded the corresponding sulfonamide conjugates 11 and
12 in 98% and 54% yield, respectively. The synthesis of the
amide-linked conjugates 13 and 14, was performed in 53–56%
yield by reaction of 10 with 6-dansylaminohexanoic acid10 and

M. Aguilar-Moncayo et al. / Bioorg. Med. Chem. 18 (2010) 7439–7445
7441
7-diethylaminocoumarin-3-carboxylic acid using TBTU as coupling
reagent (Scheme 1).
ence of the hydrophobic substituent results in unfavourable
contacts with amino acids at the vicinity of the catalytic site in
The 1H NMR spectra of the final compounds showed signals in
the aromatic region compatible with the presence of the fluores-
cent tags in the molecules. The coupling constant values between
vicinal protons at the piperidine ring were in agreement with the
4C1 conformation and the axial orientation of the pseudoanomeric
hydroxyl group (J1,2 = 3.4–3.5 Hz) fitting the anomeric effect, a dis-
tinctive feature of sp2-iminosugars. In the 13C NMR spectra, the
high field shift of the pseudoanomeric carbon resonance (76.3–
77.2 ppm) further confirmed the aminoketalic bicyclic structure.
the case of a-glucosidases. The situation is probably very similar
for compounds 10–14, leading to high b-anomeric selectivity.
As expected from the configurational pattern of the assayed
compounds, analogous to that of D-glucose, no or week inhibition
of enzymes acting on substrates other than glucosides was nor-
mally observed. The coumarin derivative 14 is an exception to this
rule, with significant inhibitory potency towards b-mannosidase
(Ki = 11 lM). In general, the presence of the aromatic fluorescent
moiety resulted in an increased selectivity towards almond b-glu-
cosidase as compared with the bovine cytosolic enzyme. The
ensemble of results stresses the utmost importance of pseudoagly-
conic interactions in the binding affinity of glycomimetics to differ-
ent glycosidases and the potential they offer for modulation of the
inhibition potency and selectivity.
2.2. Inhibitory activities
The fluorescent sp2-iminosugars 11–14 were assayed against a
panel of selected commercial glycosidases including
(yeast), b-glucosidase (almonds), b-glucosidase (bovine liver,
cytosolic), -mannosidase (Jack bean), b-mannosidase (Helix poma-
tia), trehalase (pig kidney), amyloglucosidase (Aspergillus niger),
naringinase (b-glucosidase/ -rhamnosidase; Penicillium decum-
bens),22,23
-galactosidase (green coffee beans) and isomaltase
a-glucosidase
a
2.3. Characterization of glycosidase binding by fluorescent
resonance energy transfer (FRET)
a
a
The above results demonstrate that the incorporation of fluores-
cent tags at the N0-substituent in the 5N,6S-(N0-alkyliminomethy-
lidene)-6-thionojirimycin framework is compatible with powerful
and selective inhibitory activity towards b-glucosidases. In princi-
ple, this allows the employment of fluorescence spectroscopy
methods for conducting binding studies. To check this possibility,
the dansyl conjugate 11 was chosen on the basis of its compara-
tively lower synthetic cost and favourable characteristics as chem-
ical chaperone for different mutant human lysosomal b-Glu related
to Gaucher disease (data not shown). The fluorescence spectrum of
(yeast). The non-fluorescent precursor 10 and the reference com-
pound 4 were also included in our study for comparative purposes
(Table 1). According to previous results on related sp2-iminosugars,
the presence of the N0-substituent results in a higher affinity for
b- as compared to
ally-oriented pseudoanomeric hydroxyl group that is stereocomple-
mentary to the aglycon in the natural -glucosides. Thus, no or weak
inhibition of yeast -glucosidase, isomaltase, trehalase or amyloglu-
a-glucosidases, in spite of the presence of an axi-
a
a
cosidase was observed in all cases. In contrast, 11–14 behaved as
strong competitive inhibitors of both almond and bovine (cytosolic)
b-glucosidases, the inhibition potency being about 2 orders of mag-
almond b-glucosidase (1.25 lM) was first recorded at 37 °C in
phosphate buffer (pH 7.3) for a wavelength range between 320
and 520 nm, showing the characteristic emission for the first ex-
cited singlet state of tryptophan at 339 nm. The dansyl fluorophore
is an appropriate energy acceptor for this emission; consequently,
binding of the dansyl labelled inhibitor 11 to the active site of the
enzyme suppresses the tryptophan-related protein fluorescence
highly efficiently. In a typical titration experiment, the addition
of aliquots of a solution of the dansyl labelled inhibitor 11
nitude higher for the first enzyme (Ki = 0.36–0.55
lM vs Ki = 29–
32 M). The presence of the fluorophores notably increased the
l
activity as compared to the precursor derivative 10, but was much
less influenced by the nature of the linking functionality, amide or
sulfonamide. A strong inhibition of the b-glucosidase activity of
naringinase was also detected (Ki = 0.072–1.2
It has been previously observed for compound 4 that although
the -anomer, was the only species detected by NMR in water
lM).
a
(50 lM) to a solution of b-glucosidase, while maintaining constant
solution, as determined by the anomeric effect, mutarotation
may occur in the presence of b-glucosidase. Thus, 4 was encoun-
tered in the b-orientation in the active site of TmGH1, thereby
matching the enzyme anomeric specificity.17 It has been also ar-
gued that compound 4 and other derivatives sharing the bicyclic
isothiourea skeleton, which are protonated at pH 7.0–7.3 to an ex-
tend of at least 50% (pKa 7.0–7.7), might interact preferentially
with a doubly deprotonated catalytic apparatus of the b-glucosi-
dases.17 Moreover, molecular modelling indicated that the pres-
the concentration of the latter, resulted in a gradual decrease of the
intensity of the band at 339 nm and the corresponding increase in
the emission band for the dansyl group at 515 nm (Fig. 2). Any of
these parameters can be used to build the corresponding binding
isotherm. Least-square fitting of the experimental points was com-
patible with a 1:1 binding stoichiometry, discarding contributions
from unspecific adsorption of the fluorescent moiety at the protein
surface. A dissociation constant (Kd) of 0.20 0.03
obtained from the binding curve, in good agreement with the Ki va-
lue (0.36 M) determined from competition experiments using p-
nitrophenyl b- -glucopyranoside as substrate. A virtually identical
Kd value, was obtained by a reverse titration experiment using a
constant concentration of 11 (1.25 M) and increasing concentra-
lM (Fig. 3) was
l
D
Table 1
Ki values (
Enzyme
-Glucosidase (baker’s yeast)
l
M) for 4, 10, 11–14 against several glycosidasesa
l
4
10
11
12
13
14
tions of almond b-glucosidase (see Supplementary data).
a
nib
ni
246
714
451
313
b-Glucosidase (almond), pH 7.3
b-Glucosidase (bovine liver)
0.76 12.8 0.36 0.55 0.50 0.45
3.7
ni
nd
13.4 116
nd ni
ni
ni
ni
29
ni
387
293
ni
23
32
32
199
11
28.2
167
3. Summary and conclusions
a-Mannosidase (Jack beans)
235
522
ni
407
186
179
ni
b-Mannosidase (Helix pomatia)
Trehalase (pig kidney)
Amyloglucosidase (Aspergillus
niger)
Naringinase (Penicillium
decumbens)
The results here collected illustrate the suitability of the syn-
thetic strategy disclosed for bicyclic sp2-iminosugars having a
5N,6S-(N0-alkyliminomethylidene)-6-thionojirimycin skeleton for
the preparation of fluorescent-tagged glycosidase inhibitors
through a convergent conjugation scheme that implies sulfon-
amide or amide-coupling using commercially available fluorescent
reagents. In general, the use of a tetramethylene spacer has been
shown to preserve the configurational selectivity of the fluorescent
ni
0.23 1.8
0.21 1.2
0.18 0.072
a
Inhibition was competitive in all cases. No inhibition was observed for any
compound at 2 mM concentration on, green coffee bean
isomaltase.
a-galactosidase or yeast
b
ni, no inhibition observed at 2 mM concentration of the inhibitor.

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M. Aguilar-Moncayo et al. / Bioorg. Med. Chem. 18 (2010) 7439–7445
were measured with a JASCO P-2000 polarimeter, using a sodium
[11] (μM)
lamp (k = 589 nm) at 22 °C in 1 cm or 1 dm tubes. NMR experi-
ments were performed at 300 (75.5) and 500 (125.7) MHz using
Bruker DMX300 and DRX500, and Varian INOVA 500 spectrome-
ters. 1-D TOCSY as well as 2-D COSY and HMQC experiments were
carried out to assist in signal assignment. In the FABMS spectra, the
primary beam consisted of Xe atoms with maximum energy of
8 keV. The samples were dissolved in m-nitrobenzyl alcohol or thi-
oglycerol as the matrices and the positive ions were separated and
accelerated over a potential of 7 keV. NaI was added as cationizing
agent. Thin-layer chromatography was performed on E. Merck pre-
coated TLC plates, silica gel 30F-245, with visualization by UV light
and by caring with 10% H2SO4 or 0.2% w/v cerium(IV) suphate-5%
ammonium molybdate in 2 M H2SO4 or 0.1% ninhydrin in EtOH.
Column chromatography was performed on Chromagel (SdS silica
200
100
0
0
0.17
0.33
0.66
0.98
1.30
1.92
2.53
3.12
4.54
5.88
325
350
375
400
425
λ (nm)
450
475
500
60 AC.C 70–200 lm). Elemental analyses were performed at the
Servicio de Microanálisis del Instituto de Investigaciones Químicas
Figure 2. Fluorescence emission spectra of almond b-glucosidase (1.25 lM) in the
de Sevilla, Spain. Compound 7 was prepared by hydrogenation of
5-azido-5-deoxy-1,2-O-isopropylidene-6-O-tetrahydropyranyl-a-
presence of increasing concentrations of the dansyl conjugate 11. The band at
339 nm corresponds to the tryptophan-associated fluorescence, which is quenched
by the dansyl fluorophore following inhibitor–enzyme complex formation, whereas
the band at 515 nm is ascribed to the dansyl-associated emission.
D
-glucofuranose (see hereinafter), obtained at its turn from com-
mercial 6,3-
D-glucuronolactone, following the procedure already
reported.21
0
-20
-40
4.1.1. Inhibition studies
Inhibition constant (Ki) values were determined by spectropho-
tometrically measuring the residual hydrolytic activities of the gly-
cosidases against the respective o- (for b-glucosidase from bovine
liver) or p-nitrophenyl
sidases) or
a0-trehalose (for trehalase) in the presence of com-
pounds 4, 10–14. Each assay was performed in phosphate buffer
or phosphate–citrate buffer (for - or b-mannosidase and amylo-
a- or b-D-glycopyranoside (for other glyco-
Kd = 0.20 0.03
-60
a,
a
-80
glucosidase) at the optimal pH for the enzymes. The reactions were
initiated by addition of enzyme to a solution of the substrate in the
absence or presence of various concentrations of inhibitor. The
mixture was incubated for 10–30 min at 37 °C or 55 °C (for amylo-
glucosidase) and the reaction was quenched by addition of 1 M
Na2CO3 or a solution of GLC-Trinder (Sigma, for trehalase). Reac-
tion times were appropriate to obtain 10–20% conversion of the
substrate in order to achieve linear rates. The absorbance of the
resulting mixture was determined at 405 nm or 505 nm (for treha-
lase). Approximate values of Ki were determined using a fixed con-
centration of substrate (around the KM value for the different
glycosidases) and various concentrations of inhibitor. Full Ki deter-
minations and enzyme inhibition mode were determined from the
slope of Lineweaver–Burk plots and double reciprocal analysis.
Representative examples of the Lineweaver–Burk plots, with typi-
cal profile for competitive inhibition mode, are shown in Supple-
mentary data.
-100
0
1
2
3
4
5
6
[11] (μM)
Figure 3. Binding isotherm plot for titration of almond b-glucosidase (1.25
with the fluorescent sp2-iminosugar inhibitor 11.
lM)
conjugates as determined by the hydroxylation pattern at the
piperidine ring, complementary of that of -glucose, as well as
the anomeric b- versus -glucosidase selectivity. A notable in-
D
a
crease in the selectivity towards almond b-glucosidase was ob-
served when considering the pair of b-glucosidase isoenzymes
from almonds and bovine liver (cytosolic) in comparison with
the N0-octyl derivative 4, which must be ascribed to the presence
of the aromatic rings of the fluorophores. The chemo-optical prop-
erties of the new compounds allows direct determination of the
binding parameters to glycosidases by exploiting the transfer of
energy between the first excited singlet state of tryptophan groups
of the protein and the fluorophore moiety of the inhibitor, as illus-
trated for the particular case of the dansyl conjugate 11 and the en-
zyme from almonds. Most interestingly, the incorporation of the
fluorescent tag is compatible with strong binding to normal b-glu-
cocerebrosidase as well as Gaucher disease-associated mutants
and allows investigation of important aspects related to the use
of sp2-iminosugars as chemical chaperones such as cellular uptake
pathways and intracellular distribution. Results in that direction
will published in due curse.
4.1.2. Binding studies by fluorescence spectroscopy
Quenching of the intrinsic tryptophan fluorescence of b-glucosi-
dase from almonds by titration with 11 was studied by fluores-
cence resonance energy transfer (FRET) using a F-2500 Hitachi
spectrofluorophotometer. Fluorescence spectra were recorded in
conventional 1-cm quartz cuvettes at 37 0.1 °C between 320
and 520 nm, using 2.5 mm excitation and emission slits. Excitation
wavelength (kex) was 280 nm. In a typical titration experiment a
1.25 lM stock solution of the enzyme was prepared in 0.05 M
potassium phosphate buffer, pH 7.3, a 1.5 mL aliquot was trans-
ferred to the titration cuvette and the initial fluorescence spectra
4. Experimental
was recorded. Then a solution of inhibitor 11 (50 lM) in the stock
solution of enzyme was prepared and portion-wise added to the
4.1. General methods
quartz cell via microsyringe. The additions were continued until
no change in fluorescence intensity were observed ([11] ꢁ 6
lM).
Reagents and solvents were purchased from commercial
sources and used without further purification. Optical rotations
A spectrum was recorded after each addition and the fluorescence
intensity at 339 nm (tryptophan fluorescence decrease) and

M. Aguilar-Moncayo et al. / Bioorg. Med. Chem. 18 (2010) 7439–7445
7443
515 nm (dansyl fluorescence increase) obtained at 10–15 different
enzyme–inhibitor ratios were then plotted against inhibitor con-
centration. Iterative least-squares fitting of the experimental bind-
ing isotherm to a 1:1 binding model allowed estimation of Kd
within a 15% estimated error.24 Dissociation constant was also
determined by reversed fluorescence titration of b-glucosidase
from almonds with 11 at constant concentration. In this case the
solution was extracted with CH2Cl2 (3 ꢂ 20 mL). The combined ex-
tracts were washed with iced saturated aqueous NaHCO3 (25 mL),
dried (MgSO4), filtered, and concentrated. The resulting residue
was purified by column chromatography using 20:1?10:1
CH2Cl2–MeOH as eluent to obtain 9 (384 mg, 78%). [
a]
D ꢀ7.3 (c 1.0
in CH2Cl2); Rf 0.46 (7:1 CH2Cl2–MeOH); mmax 3372, 2934, 1696,
1521, 1367, 1251, 1166, 1075 cmꢀ1; dH (300 MHz, CDCl3) 5.93 (d,
1H, J1,2 = 3.6 Hz, H-1), 4.79 (br s, 1H, NH), 4.54 (d, 1H, H-2), 4.42
(m, 1H, H-5), 4.25 (br s, 2H, OH, NH), 4.23 (d, 1H, J3,4 = 2.5 Hz, H-3),
4.03 (dd, 1H, J4,5 = 8.4 Hz, H-4), 3.51 (dd, 1H, J6a,6b = 10.9 Hz,
J5,6a = 7.4 Hz, H-6a), 3.39 (dd, 1H, J5,6b = 4.9 Hz, H-6b), 3.24 (m, 2H,
CH2N), 3.11 (m, 2H, CH2NHCO), 1.53 (m, 4H, CH2), 1.48, 1.30 (2 s,
6H, CMe2), 1.33 (s, 9H, CMe3); dC (75.5 MHz, CDCl3) 164.6 (CN),
156.1 (CO), 111.5 (CMe2), 105.1 (C-1), 85.2 (C-2), 81.6 (C-4), 79.3
(CMe3), 74.9 (C-3), 69.8 (C-5), 44.9 (CH2N), 40.0 (CH2NHCO), 37.4
(C-6), 28.4 (CMe3), 27.3, 26.8 (CH2), 26.8, 26.1 (CMe2); m/z (FAB)
454 ([M+Na]+, 50%), 432 (100). (Found: C, 52.66; H, 7.65; N, 9.48;
S, 7.19. C19H33N3O6S requires C, 52.88; H, 7.71; N, 9.74; S, 7.43).
emission was recorded between 400 and 600 nm. A 1.0 lM stock
solution of sp2-iminosugar 11 in 0.05 M potassium phosphate buf-
fer, pH 7.3, was prepared and the initial spectrum was recorded.
The enzyme was dissolved in the stock solution (containing the
inhibitor) at a final concentration of 5
the cuvette under stirring until no changes in fluorescence inten-
M). Fluorescence intensity at
lM, and added stepwise to
sity were observed ([enzyme] ꢁ1.9
l
515 nm was measured after each addition and data points were
plotted versus enzyme concentration. Least-square fitting to a
1:1 binding model furnished a Kd value that did not significantly
differ from the direct titration procedure.24
4.2. 5-[N0-(4-tert-Butoxycarbonylaminobutyl)thioureido]-5-
4.4. 5-N,6-S-[N0-(4-Amino)butyliminomethylidene]-6-
deoxy-1,2-O-isopropylidene-
a
-D
-glucofuranose (8)
thionojirimycin hydrochloride (10)
A solution of 5-azido-5-deoxy-1,2-O-isopropylidene-6-O-tetra-
hydropyranyl-
-glucofuranose21 (700 mg, 2.13 mmol) in MeOH
The corresponding 2-amino-2-thiazoline precursor 9 (340 mg,
0.79 mmol) was treated with TFA–H2O (9:1, 3.5 mL) for 30 min, con-
centrated under reduced pressure, coevaporated several times with
water, neutralized with Amberlite IRA-68 (OHꢀ) ion-exchange resin,
and subjected to column chromatography using 10:1:1?6:3:1
CH3CN–H2O–NH4OH as eluent to obtain the isothiourea 10 as the
a-D
(12 mL) was hydrogenated at atmospheric pressure for 1 h using
10% Pd/C (234 mg) as catalyst. The suspension was filtered through
Celite and concentrated to give 5-amino-5-deoxy-1,2-O-isopropyl-
idene-6-O-tetrahydropyranyl-a-D
-glucofuranose21 (7) as a hygro-
scopic material that was used in the next step without
purification. To a solution of 7 thus obtained in pyridine (12 mL),
Et3N (1.6 mL, 11.7 mmol) and 4-(tert-butoxycarbonylamino)butyl
isothiocyanate (433 mg, 2 mmol) were added and the mixture
was stirred at room temperature for 18 h. Then, the solvent was re-
moved under reduced pressure and the residue coevaporated sev-
eral times with toluene. The syrup was dissolved in CH2Cl2–MeOH
(1:1, 42 mL) and p-toluenesulfonic acid (69 mg, 0.16 mmol) was
added. The reaction mixture was stirred for 2 h at room tempera-
ture, and then diluted with CH2Cl2 (15 mL), washed with saturated
aqueous NaHCO3 (2 ꢂ 15 mL), dried (MgSO4), filtered, and concen-
trated. The resulting residue was purified by column chromatogra-
phy using 30:1?15:1 CH2Cl2–MeOH as eluent to obtain 8 (629 mg,
corresponding hydrochloride (258 mg, 82%).
integration); [
D ꢀ11.3 (c 1.0 in H2O); Rf 0.22 (6:3:1 CH3CN–H2O–
NH4OH). anomer: dH (500 MHz, D2O) 5.61 (d, 1H, J1,2 = 3.7 Hz, H-
1), 4.32 (m, 1H, H-5), 3.76 (m, 2H, H-3 , H-6a), 3.62 (dd, 1H,
a–b ratio 1:0.1 (H-1
a]
a
a
J2,3 = 9.5 Hz, H-2), 3.56 (t, 1H, J3,4 = J4,5 = 9.6 Hz, H-4), 3.46 (m, 3H,
H-6b, CH2N), 3.00 (t, 2H, 3JH,H = 7.1 Hz, CH2NH2), 1.73 (m, 4H, CH2);
dC (125.7 MHz, D2O) 175.7 (CN), 79.0 (C-1), 75.7 (C-4), 74.2 (C-3),
73.1 (C-2), 66.0 (C-5), 50.6 (CH2N), 41.5 (CH2NH2), 33.9 (C-6), 27.7,
26.5 (CH2). b anomer: dH (500 MHz, D2O) 5.02 (d, 1H, J1,2 = 8.2 Hz,
H-1), 4.08 (m, 1H, H-5), 3.76 (m, 1H, H-3), 3.70 (dd, 1H,
J6a,6b = 11.6 Hz, J5,6a = 7.6 Hz, H-6a), 3.60 (m, 1H, H-2), 3.56 (m, 1H,
H-4), 3.46 (m, 3H, H-6b, CH2N), 3.00 (m, 2H, CH2NH2), 1.73 (m, 4H,
CH2); dC (125.7 MHz, D2O) 175.7 (CN), 87.4 (C-1), 77.1 (C-4), 75.8
(C-3), 72.8 (C-2), 69.1 (C-5), 50.5 (CH2N), 41.5 (CH2NH2), 32.5 (C-
6), 27.9, 26.5 (CH2); m/z (FAB) 314 ([M+NaꢀHCl]+, 40%), 292 (90).
(Found: C, 39.95; H, 6.47; N, 12.49; S, 9.41. C11H22ClN3O4S requires
C, 40.30; H, 6.76; N, 12.82; S, 9.78).
70%). [a] +45.5 (c 1.0 in CH2Cl2); Rf 0.33 (15:1 CH2Cl2–MeOH);
D
mmax cmꢀ1 3342, 2933, 1682, 1549, 1367, 1254, 1165, 1075; kmax
(CH2Cl2) nm 247 (emM 12.3); dH (300 MHz, CDCl3, 313 K) dY 6.96
(br s, 1H, N0H), 6.72 (br d, 1H, JNH,5 = 7.8 Hz, NH), 5.92 (d, 1H,
J1,2 = 3.6 Hz, H-1), 5.07 (br s, 1H, OH), 4.82 (br s, 1H, NH), 4.58 (d,
1H, H-2), 4.54 (m, 1H, H-5), 4.19 (d, 1H, J3,4 = 1.9 Hz, H-3), 4.09
(dd, 1H, J4,5 = 9.8 Hz, H-4), 4.03 (dd, 1H, J6a,6b = 11.3 Hz,
J5,6a = 3.1 Hz, H-6a), 3.80 (dd, 1H, J5,6b = 3.0 Hz, H-6b), 3.47 (m,
2H, CH2NHCS), 3.11 (m, 2H, CH2NHCO), 1.60 (m, 4H, CH2), 1.49,
1.31 (2 s, 6H, CMe2), 1.49 (s, 9H, CMe3); dC (75.5 MHz, CDCl3,
313 K) d 181.8 (CS), 156.7 (CO), 111.6 (CMe2), 104.9 (C-1), 84.7
(C-2), 79.8 (CMe3), 79.7 (C-4), 73.8 (C-3), 62.4 (C-6), 53.7 (C-5),
44.1 (CH2NHCS), 40.2 (CH2NHCO), 28.4 (CMe3), 27.4 (CH2), 26.7,
26.0 (CMe2), 25.7 (CH2); m/z (FAB) 472 ([M+Na]+, 100%), 450 (15).
(Found: C, 50.78; H, 7.93; N, 9.28; S, 6.96. C19H35N3O7S requires
C, 50.76; H, 7.85; N, 9.35, S, 7.13).
4.5. 5-N,6-S-[N0-(4-Dansylamino)butyliminomethylidene]-6-
thionojirimycin (11)
To a solution of 10 (68 mg, 0.21 mmol) in anhydrous DMF
(15 mL) at 0 °C under Ar, triethylamine (64 lL, 1 equiv) and 5-dim-
ethylaminonaphtalene-1-sulfonyl chloride (61.8 mg, 1.1 equiv)
were added. The reaction mixture was stirred for 4 h and the sol-
vent was removed under reduce pressure. The resulting residue
was purified by column chromatography using 90:10:1?60:10:1
CH2Cl2–MeOH–H2O to obtain 11 (107 mg, 98%). [
a
]
ꢀ7.4 (c 0.7
D
in MeOH); Rf 0.63 (40:10:1 CH2Cl2–MeOH–H2O); dH (500 MHz,
CD3CN) 8.52 (d, 1H, dansyl), 8.24 (d, 1H, dansyl), 8.15 (d, 1H, dan-
syl), 7.60 (m, 2H, dansyl), 7.28 (d, 1H, dansyl), 5.48 (d, 1H,
J1,2 = 3.4 Hz, H-1), 3.79 (m, 1H, H-5), 3.62 (t, 1H, J2,3 = J3,4 = 9.5 Hz,
H-3), 3.46 (dd, 1H, J6a,6b = 11.2 Hz, J5,6a = 4.4 Hz, H-6a), 3.40 (dd,
1H, H-2), 3.30 (t, 1H, J4,5 = 9.3 Hz, H-4), 3.10 (dd, 1H,
4.3. (4R)-2-(4-tert-Butoxycarbonylamino)butylamino-4-[(40R)-
10,20-O-isopropylidene-b- -threofuranos-40-yl]-2-thiazoline (9)
L
To a solution of the corresponding thioureido derivative 8
(514 mg, 1.14 mmol) in anhydrous pyridine (17 mL) at ꢀ20 °C under
3
J5,6b = 7.3 Hz, H-6b), 3.06 (t, 2H, JH,H = 10.5 Hz, CH2N), 3.00 (m,
Ar, methanesulfonic chloride (110
lL, 1.42 mmol, 1.2 equiv) was
2H, CH2NH), 2.83 (br s, 6H, dansyl), 1.44 (m, 2H, CH2), 1.34 (m,
2H, CH2); dC (125.7 MHz, CD3CN) 163.1 (CN), 152.0, 135.4, 130.2–
129.5, 129.2, 128.4, 123.7, 119.1, 115.5 (dansyl), 76.3 (C-1), 74.3
added. The reaction mixture was stirred for 7 h and allowed to warm
to room temperature. Then, ice-water (30 mL) was added and the

7444
M. Aguilar-Moncayo et al. / Bioorg. Med. Chem. 18 (2010) 7439–7445
(C-4), 72.8 (C-3), 71.5 (C-6), 61.1 (C-5), 54.5 (CH2N), 44.9 (dansyl),
42.5 (CH2NH), 31.2 (C-6), 26.8, 26.7 (CH2); m/z (FAB) 547.1661
[M+Na]+. C23H32N4O6NaS2 requires 547.1661. (Found: C, 52.34; H,
6.10; N, 10.47; S, 11.89. C23H32N4O6S2 requires C, 52.65; H, 6.15;
N, 10.68; S, 12.22).
119.2 (C-90), 115.4 (C-70), 76.5 (C-1), 74.3 (C-4), 73.0 (C-3), 71.6 (C-
2), 61.6 (C-5), 52.0 (CH2N), 44.9 (CH3N), 42.7, 35.6 (CH2NH), 35.7
(C-6), 28.9, 26.9, 26.6, 25.8, 25.0 (CH2). m/z (HRFAB) 660.2503
[M+Na]+. C29H43N5O7NaS2 requires 660.2502. (Found: C, 53.39; H,
6.67; N, 10.71; S, 9.72. C29H43N5O7S2 requires C, 54.61; H, 6.80; N,
10.98; S, 10.05).
4.6. 5-N,6-S-[N0-(4-Dapoxylsulfonylamino)butyliminomethylid-
ene]-6-thionojirimycin (12)
4.8. 5-N,6-S-[N0-[4-(70-Diethylaminocoumarin-30-ylcarbox-
amido)butyl]iminomethylidene]-6-thionojirimycin (14)
To a solution of 10 (32.2 mg, 0.11 mmol) in anhydrous DMF
(10 mL) at 0 °C under Ar, triethylamine (15
l
L, 1 equiv) and dap-
To a solution of 10 (50.2 mg, 0.17 mmol) in anhydrous DMF
oxylsulfonyl chloride (39.9 mg, 1 equiv) were added. The reaction
mixture was stirred for 4 h and the solvent was removed under re-
duce pressure. The resulting residue was purified by column chro-
matography using 100:10:0.5?70:10:0.5 CH2Cl2–MeOH–NH4OH
(14 mL), 7-diethylaminocoumarin-3-carboxylic acid (45.1 mg,
1 equiv), TBTU (55.4 mg, 1 equiv) and triethylamine (24 lL, 1 equiv)
were added. The reaction wasstirred for 1 h, the solvent was removed
under reduce pressure, and the residue was purified by column chro-
matography using 100:10:0.5?70:10:0.5 CH2Cl2–MeOH–NH4OH as
as eluent to obtain 12 (37 mg, 54%). [
a
]
ꢀ14.7 (c 0.3 in MeOH);
D
Rf 0.40 (40:10:1 CH2Cl2–MeOH–NH4OH); dH (500 MHz, CD3CN)
eluent to obtain 14 (36.6 mg, 53%). [a D
]
ꢀ6.4 (c 0.6 in MeOH); Rf
8.20 (d, 2H, J2 ,3 = 8.8 Hz, H-20), 7.90 (d, 2H, H-30), 7.65 (d, 2H,
0.62 (70:10:0.5 CH2Cl2–MeOH–NH4OH); dH (500 MHz, MeOD) 8.50
0
0
J9 ,10 = 8.0 Hz, H-90), 7.61 (s, 1H, H-70), 6.80 (d, 2H, H-100), 5.57
(br s, 1H, OH), 5.31 (d, 1H, J1,2 = 3.4 Hz, H-1), 5.18, 4.78, 4.68 (3
br s, 3H, OH), 3.47 (m, 1H, H-5), 3.43 (m, 1H, H-3), 3.27 (dd, 1H,
J6a,6b = 10.7 Hz, J5,6a = 6.4 Hz, H-6a), 3.12 (dd, 1H, J2,3 = 9.3 Hz, H-
2), 3.01 (t, 1H, J4,5 = 9.3 Hz, H-4), 2.96 (s, 6H, CH3N), 2.95 (m, 2H,
(br s, 1H, H-40), 7.44 (d, 1H, J8 ,9 = 8.8 Hz, H-90), 6.71 (d, 1H, H-80),
6.45 (br s, 1H, H-60), 5,50 (d, 1H, J1,2 = 3.5 Hz, H-1), 4.39 (m, 1H, H-
5), 3.62 (t, 1H, J2,3 = J3,4 = 9.3 Hz, H-3), 3.53 (m, 1H, H-6a), 3.42 (m,
2H, CH2CH3), 3.35(m, 4H, H-2, H-4, CH2N), 3.28 (m, 3H, H-6b, CH2NH),
0
0
0
0
3
3.11 (m, 2H, CH2CH3), 1.62 (m, 4H, CH2), 1.21 (t, 3H JH,H = 7.3 Hz,
3
3
CH2N), 2.87 (dd, 1H, J5,6b = 8.8 Hz, H-6b), 2.76 (t, 2H, JH,H = 6.4 Hz,
CH3), 1.13 (t, 3H, JH,H = 6.8 Hz, CH3); dC (125.7 MHz, MeOD) 163.5
CH2N), 1.40 (m, 4H, CH2); dC (125.7 MHz, CD3CN) 168.6 (CN), 158.2
(CN), 157.3 (CN), 153.4 (C-60), 151.2 (C-110), 142.0 (C-10), 130.8 (C-
40), 128.1 (C-20), 126.8 (C-30), 126.2 (C-70), 122.1 (C-90), 115.3 (C-80),
112.8 (C-100), 77.2 (C-1), 75.1 (C-4), 73.9 (C-3), 72.5 (C-2), 59.6 (C-
5), 54.4 (CH2N), 43.2 (CH3N), 40.8 (CH2NH), 31.0 (C-1), 28.7, 27.7
(CH2). m/z (HRFAB) 640.1909 [M+Na]+. C28H35N5O7NaS2 requires
640.1876.
(CN), 163.5 (CO ester), 162.9 (CO amide), 158.0 (C-30), 153.5 (C-100),
148.1 (C-70), 131.5 (C-40), 110.5–96.1 (C-80, C-90, C-60, C-50), 77.2 (C-
1), 74.3 (C-4), 73.0 (C-3), 71.8 (C-2), 62.5 (C-5), 52.0 (CH2N), 44.8
(CH2CH3), 38.8 (CH2NH), 31.2 (C-6), 26.8, 26.7 (CH2), 11.5, 8.0 (CH3);
m/z (HRFAB) 557.2042 [M+Na]+. C25H34N4O7NaS requires 557.2046.
(Found: C, 55.91; H, 6.28; N, 10.22; S, 5.64. C25H34N4O7S requires C,
56.16; H, 6.41; N, 10.48; S, 6.00.
4.7. 5-N,6-S-[N0-[4-(6-Dansylaminohexanamido)butyl]imi-
nomethylidene]-6-thionojirimycin (13)
Acknowledgements
The Spanish Ministerio de Ciencia e Innovación (contract
numbers CTQ2006-15515-CO2-01, CTQ2009-14551-C02-01 and
CTQ2007-61180/PPQ; cofinanced with the Fondo Europeo de
Desarrollo Regional FEDER), the Spanish Ministerio de Educación
(joint Austrian-Spanish action number HU2007-0006), the Funda-
ción Ramón Areces, and the Junta de Andalucía are thanked for
funding. M.A.-M. is a FPU doctoral fellow holder.
To a solution of dansyl chloride (742 mg, 2.75 mmol) in CH2Cl2
(25 mL), triethylamine (0.8 mL, 2.1 equiv) and methyl 6-aminohex-
anoate hydrochloride (500 mg, 1.0 equiv) were added and the mix-
ture was stirred at room temperature for 18 h. The mixture was
partitioned between CH2Cl2 and 6% aqueous HCl and the organic
layer was washed with saturated aqueous NaHCO3 until neutral
pH, dried (MgSO4), filtered, and concentrated under reduced pres-
sure. The residue was purified by column chromatography using
cyclohexane?1:3 EtOAc–cyclohexane. To a 10% solution of the
resulting residue in dioxane–H2O 1:1 (v/v), aqueous NaOH (10%)
was added dropwise until pH 10 was reached and the mixture was
stirred at room temperature for 24 h. The solution was neutralised
with Amberlite IRA-120 (H+) ion-exchange resin, filtered, and con-
centrated under reduced pressure to give the free acid10 (81%). To
a 10% solution of this compound in anhydrous DMF, compound 10,
TBTU, and triethylamine (1 equiv) were added and the reaction mix-
ture was stirred at room temperature for 1 h. The solvent was re-
moved under reduce pressure and the residue was purified by
column chromatography using 100:10:0.5?50:10:0.5 CH2Cl2–
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.09.003.
References and notes
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ꢀ1.7 (c
D
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0
J8 ,9 = 8.8 Hz, H-90), 8.16 (d, 1H, J2 ,3 = 7.3 Hz, H-20), 7.60 (m, 2H, H-
0
0
0
0
30, H-80), 7.27 (d, 1H, J7 ,8 = 7.8 Hz, H-70), 5.51 (d, 1H, J1,2 = 3.4 Hz,
H-1), 3.88 (m, 1H, H-5), 3.60 (t, 1H, J2,3 = J3,4 = 9.3 Hz, H-3), 3.52
(dd, 1H, J6a,6b = 10.7 Hz, J5,6a = 7.1 Hz, H-6a), 3.40 (dd, 1H, H-2),
3.31 (t, 1H, J4,5 = 9.8 Hz, H-4), 3.23 (m, 3H, H-6b, CH2N), 3.13 (m,
2H, CH2NH), 2.80 (m, 10H, CH2NH, CH3), 1.58 (m, 2H, CH2), 1.49
(m, 2H, CH2), 1.33 (m, 4H, CH2), 1.13 (m, 2H, CH2); dC (125.7 MHz,
CD3CN) 162.4 (CN), 160.0 (CO), 152.1 (C-60), 135.7 (C-10), 130.2–
129.6 (C-40, C-50, C-100), 129.2 (C-20), 128.3 (C-80), 123.7 (C-30),
0
0

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