Exploring a multivalent approach to α-L-fucosidase inhibition

Article abstract of DOI:10.1002/ejoc.201300878

To probe the utility of a multivalent approach for fucosidase inhibition, a series of di- and tri-valent imino sugars based on L-fuco-configured 1,4-imino- and 1,4-bis(imino)-cyclitol epitopes has been synthesized and analyzed for fucosidase inhibition with the best trivalent species yielding a modest improvement in binding constant. Structural analysis of a representative pair of mono- and tri-valent imino sugars has been performed on a bacterial fucosidase, BtFuc2970. The 3D structures show binding of the imino-cyclitol in the ³E conformation, consistent with the known pathway for fucosidase action. Di- and tri-valent imino sugars based on L-fuco-configured 1,4-imino- and 1,4-bis(imino)-cyclitol moieties present α-L-fucosidase inhibitory activities in the μM and nM range. Copyright

Full text of DOI:10.1002/ejoc.201300878

FULL PAPER
DOI: 10.1002/ejoc.201300878
Exploring a Multivalent Approach to α- -Fucosidase Inhibition
L
Elena Moreno-Clavijo,[a] Ana T. Carmona,[a] Antonio J. Moreno-Vargas,[a] Lidia Molina,[a]
Daniel W. Wright,[b] Gideon J. Davies,[b] and Inmaculada Robina*[a]
Dedicated to Professor Pierre Vogel on the occasion of his 70th birthday
Keywords: Inhibitors / Enzymes / Carbohydrates / Imino sugars / Glycoconjugates / Structure–activity relationships
To probe the utility of a multivalent approach for fucosidase
inhibition, a series of di- and tri-valent imino sugars based
representative pair of mono- and tri-valent imino sugars has
been performed on a bacterial fucosidase, BtFuc2970. The
3D structures show binding of the imino-cyclitol in the 3E
conformation, consistent with the known pathway for fucosi-
dase action.
on
L-fuco-configured 1,4-imino- and 1,4-bis(imino)-cyclitol
epitopes has been synthesized and analyzed for fucosidase
inhibition with the best trivalent species yielding a modest
improvement in binding constant. Structural analysis of a
been found in human seminal plasma and in the mem-
branes of human sperm cells and facilitate sperm transport
and sperm-egg interactions.[10]
Introduction
Fucose is a 6-deoxyhexose commonly incorporated into
glycoconjugates, by either the direct attachment to proteins
or lipids, or attachment to N-linked glycans. Such glycocon-
jugates have roles in a number of physiological activities,
such as oncogenesis,[1] the blood coagulation cascade and
clot dissolution,[2] antigenic determination[3] and host-mi-
crobial interactions.[4] α-l-Fucosidases (EC 3.2.1.51) cata-
lyze the hydrolytic cleavage of fucose residues located at the
non-reducing end of glycoconjugates and, like other glycos-
idases, also catalyze glycosylation reactions. The accumu-
lation of glycoconjugates containing fucose, due to the ab-
sence or deficiency of α-l-fucosidases, induces the recogni-
tion of the α-l-fucose moieties by specific lectins that leads
to the neurovisceral disorder known as fucosidosis.[5] In
mammals, these enzymes, which may be lysosomally com-
partmentalised (FUCA1), or secreted (FUCA2), are impli-
cated in several pathological events. An abnormal α-l-fucos-
idase distribution, both extracellular and intracellular, is
found in inflammatory responses,[4a] cancer[6] and cystic fi-
brosis.[7] Human α-l-fucosidases are of substantial interest
as diagnostic markers of early colorectal[1b,6b] and hepato-
cellular cancers,[6a,8] and as modulators of metastasis in
breast cancer cells.[9] Furthermore, α-l-fucosidases have
The inhibition of α-l-fucosidases may be clinically im-
portant for a variety of reasons. α-l-Fucosidase may be a
target for small molecule chaperone therapy for fucos-
idosis.[11] This approach has been applied successfully to
other lysosomal storage diseases.[12,13] Helicobacter pylori
infection has been shown to have a correlation with in-
creased expression of α-l-fucosidase in the stomach. The
introduction of inhibitory compounds against α-l-fucosid-
ase has been shown to reduce H. pylori virulence in vitro.[14]
Because l-fucose and/or l-fucose-containing molecules
have been shown to inhibit sperm–egg interactions in hu-
mans, specific α-l-fucosidase inhibitors are expected to be
powerful tools in elucidating the biological role of α-l-
fucosidase in spermiogenesis and sperm maturation.[15]
For these reasons, a number of structural studies of α-l-
fucosidases have been reported in the last decade. In this
context, the focus has been on CAZY (www.cazy.org[16]
)
family GH29 fucosidases into which the relevant mamma-
lian enzymes are classified on the basis of amino-acid se-
quence similarities. In 2004 a small angle X-ray scattering
model of the GH29 α-l-fucosidase from Sulfolobus Solfat-
aricus was proposed.[17] Since then, crystallographic struc-
tures have been determined for α-l-fucosidases from
Thermotoga maritima,[18] Bifidobacterium bifidum[19] and
Bacteroides thetaiotaomicron.[20] All these structures allow,
to differing extents, the study of enzyme inhibition by small
molecule sugar mimics providing important information
about the mechanism of action and chemical topography of
the active site of the enzyme. Thus, crystal structures of a
number of complexes of α-l-fucosidases from both T. mari-
[a] Department of Organic Chemistry, Faculty of Chemistry,
University of Seville,
1, C/ Prof. García González, 41012 Seville, Spain
E-mail: robina@us.es
http://grupo.us.es/gqbya/es/inicio/
[b] Structural Biology Laboratory, Department of Chemistry,
University of York,
Heslington, York YO10 5DD, UK
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300878.
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Exploring a Multivalent Approach to α-l-Fucosidase Inhibition
tima and Bacteroides thetaiotaomicron with inhibitors dis- creased binding through avidity, there may be virtue in en-
playing both pyranose and iminocyclitol configurations zyme inhibitors wherein multiple copies of the inhibitory
have been reported.[18–21]
warhead are coupled reflecting the slower off-rate of the
In the last several years efforts have been devoted to the multivalent glycomimetic moiety relative to its monovalent
synthesis of α-l-fucosidase inhibitors based on monosac- counterparts. Several examples of multivalent effects on gly-
charides reaching the nano- and pico-molar range.[22] Re- cosidases have been recently reported (i.e. for amyloglucos-
cently, fucosidase-targeted ligands have shown strong anti- idases and α-mannosidases,[28] for α- and β-glucosidases[29]
proliferative effects on MDA-MB-231 cancer cells.[23] These and β-galactosidases).[30] The concept of multivalency for a
types of compounds have been shown to be useful for the glycosidase of therapeutic interest has also been applied in
treatment of liver disorders and liver tumors,[24] such as the development of chaperones for the treatment of
hepatocellular carcinoma and also for treatment and diag- Gaucher’s disease.[31] Herein, we explore such a multivalent
nosis of H. pylori infection.[25]
approach on α-l-fucosidase inhibition for the first time. We
Our group is striving to find new ways to extend these describe the synthesis and biological evaluation of short
successful studies with “monovalent” sugar mimics to and long-tethered di- and trivalent derivatives (1–4, Fig-
multivalent systems. In carbohydrate binding to receptors, ure 2) incorporating different imino sugars based on fuco-
multivalency effects may increase the affinity of sugar for a configured 1,4-imino- and 1,4-bis(imino)- moieties; these
given target as well as potentially improving their solubility compounds display α-l-fucosidase inhibitory activities in
in aqueous media.[26] The recognition of clustered sugars the μm and nm range. The 3D structures of both monomer
by proteins has been broadly studied in the case of lectins; and multivalent forms of one compound class have been
however, the effect of multivalency on glycosidase inhibi- studied using the Bacteroides thetaiotaomicron GH29 α-l-
tion has been only scarcely probed. It is generally believed fucosidase (BtFuc2970) as a target system providing insight
that multivalency may provide an enhancement to binding into both the conformational basis for enzyme inhibition
affinity in enzymatic systems by one of two processes.[26] and a consideration of different models for multivalency on
One process involves the simultaneous binding or chelation this system.
of multiple (sub)ligands to different binding sites of a pro-
tein – the classical avidity mechanism with polyvalent recep-
Results and Discussions
tors and multivalent ligands. The second process, named
the “statistical rebinding” or “proximity effect”, reflects the
increased propensity for ligand rebinding when two relevant
coupled ligands are in close proximity (Figure 1).[27] Thus,
even in the absence of appropriately situated sites for in-
Synthesis
The design of di- and trivalent-scaffolded imino sugars
of interest is based on the inhibitory properties towards α-
l-fucosidase of monovalent 1,4-imino- and 1,4-bis(imino)-
cyclitols 5–9. Compounds 7 and 8 are prepared for the first
time herein and the others were previously prepared by our
research group: 5,[32] 6[33] and 9,[34] (Figure 3). Biphenyl de-
rivatives 5 and 6 are valid reference compounds for the
comparison with dimer 1, when attaching another imino
sugar moiety to the biphenyl moiety. In a similar way, benz-
Figure 1. Multivalent ligand binding via a statistical rebinding
mechanism.
Figure 2. Structures for short-tethered bivalent glycomimetics 1 and 2, trivalent glycomimetics 3 and long-tethered trivalent glycomimetic
4.
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ylamino derivatives 7, 8 and 9 are monovalent compounds cleavage and oxidation gave carboxylic acid 12.[32a] Peptide
for the comparison with dimer 2 and trimers of type 3 and coupling with benzylamine using PyBOP as the coupling
4. For this purpose, the preparation of new pyrrolidines 7 agent and DIPEA as base, gave protected amide 13. Subse-
and 8 was accomplished (Scheme 1). The synthesis was car- quent standard deprotection of 13 afforded pyrrolidine 7 in
ried out starting from diol 10 that was easily obtained from good yield. Amine 8 was obtained from N-Boc-protected
d-mannose diacetonide as previously reported by us. Glycol diol 11 after glycol cleavage, reductive amination of the al-
dehyde with benzylamine and final acid hydrolysis.
For the syntheses of short-tethered bivalent glycomimet-
ics 1 and 2 and trivalent glycomimetics 3, commercial di-
carboxylic acid 15, m-xylylenediamine 16 or synthetic tri-
amine 17[35] were used as templates (Scheme 2). Standard
amide coupling (PyBOP as coupling agent and DIPEA as
base) between 15 and aminomethyl-imino sugar 14[33b] fol-
lowed by standard deprotection gave 1 in 43% yield. Simi-
larly, reaction of imino sugar acid derivative 12,[32a] and
amines 16 and 17 and subsequent deprotection gave dimer
2 and trimer 3a, respectively, in moderate to good yields.
Reaction of 1,4-imino-furan carboxylic acid 19[34] with tri-
amine 17 and deprotection, equally gave trimer 3b in 46%
yield. For the synthesis of long-tethered trivalent glycomi-
metic 4, compound 20[36] was used as a C-3 symmetric tem-
plate. Thus, after removal of the Boc groups in 20, the cou-
pling reaction with carboxylic acid 12 was carried out under
standard amide coupling conditions. Final deprotection of
the corresponding adduct gave 4 in good yield (Scheme 2).
Figure 3. Monovalent α-l-fucosidase inhibitors.
3D Structural Analysis of a Representative Mono/Trivalent
Inhibitor Pair
In order to probe the structural basis for GH29 fucosid-
ase inhibition by the iminocyclitols, crystal structures of
BtFuc2970 liganded with compounds 3a and 7 were deter-
mined, both at a resolution of ≈ 1.7 Å (see Supporting In-
formation, Table S1). Compounds 3a and
7 inhibit
BtFuc2970 with Ki values of 0.7Ϯ0.02 and 4.7Ϯ0.30 μm,
respectively (see Supporting Information, Figure S1). Elec-
tron density for the iminocyclitol ring for each inhibitor is
clear and unambiguous. Each adopts an essentially iden-
tical 3E envelope conformation as recently observed for
other five-membered iminocyclitols on this enzyme.[37] This
3
conformation reflects the putative H4 transition-state for
the enzyme-catalyzed reaction (discussed, for example, in
Refs. 18, 20 & 37, Figure 4). Beyond the core cyclitol, the
conformations observed for the amide and beyond are both
dynamic (indeed occasionally disordered) and dependent
on crystal packing environment resulting in a large spread
of observed conformations (see Supporting Information,
Figure S2). Representations of inhibitors 3a and 7 lying in
their respective crystallographic active sites are provided in
Figure S3. The BtFuc2970 crystal form used conveys two
independent observations of ligand binding, reflecting the
two independent molecules in the crystallographic asym-
metric unit. For monovalent compound 7, clear unambigu-
ous density is observed for one independent observation
(Figure 4, a) (that with the most interactions with a crystal-
packing neighboring molecule) whereas a more disordered
“aglycon” is observed in the second molecule in the asym-
Scheme 1.
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Exploring a Multivalent Approach to α-l-Fucosidase Inhibition
Scheme 2.
metric unit. Indeed in this latter case, with no packing con- observed for 3a beyond the phenyl ring reflecting a highly
straints, the “aglycon” is completely disordered beyond the disordered trimer (and the absence of appropriately placed
first methylene carbon pendant to the amide unit (see Sup- active sites for avidity effects). That the different degrees
porting Information, Figure S2). In contrast, but likely re- of order observed reflects adventitious interactions provides
flecting its larger steric bulk, the trimeric counterpart 3a is encouragement and valuable insight for those working on
better ordered in the less tightly packed protein molecule inhibitor design as well as providing a rationale for the dif-
(Figure 4, b). Despite this freedom, no electron density is ferent Ki values reported on different enzyme targets. In
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light of these data the full range of inhibitors was tested for Table 1. Inhibitory activities of mono- and multi-valent imino
sugars towards bovine kidney α-l-fucosidase.
fucosidase inhibition on the mammalian enzyme.
Compound
Monomers
% Inhibition at 1 mm, IC50 and Ki in μm
optimal pH = 6, 37 °C.[a],[b]
6[c]
7
85%, (IC50 = 8.6 μm), Ki = 1.0 μm
99%, (IC50 = 15 μm), Ki = 2.1 μm
89%, (IC50 = 100 μm), Ki = 11.7 μm
91%, (IC50 = 38 μm), Ki = 3.8 μm
8
9[d]
Dimers
Trimers
1
2
98%, (IC50 = 12.8 μm), Ki = 1.3 μm
99%, (IC50 = 6.5 μm), Ki = 4.0 μm
3a
3b
4
99%, (IC50 = 1.6 μm), Ki = 0.3 μm
98%, (IC50 = 17 μm), Ki = 2.1 μm
96%, (IC50 = 3.8 μm), Ki = 0.4 μm
[a] For measurement conditions, see ref. 38. [b] Competitive mode
of inhibition for given Ki. [c] Percentage of inhibition was deter-
mined at 0.1 mm and was previously reported.[33b] [d] A Ki of 2.2 μm
was previously determined against human placental fucosidase.[34]
inhibitor than the monovalent analogue 7 (Ki = 2.1 μm) of
both bovine kidney and Bacteroides enzymes. Furthermore,
bivalent inhibitor 2 was less potent an inhibitor than both
its monovalent parent 7 and trivalent offspring 3a. An in-
crease in the length of the spacers in the scaffold (3a vs. 4),
did not improve the inhibitory activity; both compounds
displayed practically equivalent activities.
Figure 4. (a) and (b) observed electron density (Fo – Fc maps, prior
to the incorporation of ligand in refinement) contoured at (a) 5 σ
(equates to 0.3 electrons/Å3, compound 7) and (b) 3 σ (equates to
0.15 electrons/Å3, compound 3a) bound to BtFuc2970. (c) Interac-
tions made between the iminocyclitol core of compounds 3a and 7
and residues at or near the active site of BtFuc2970.
Conclusions
We have provided an efficient method for the preparation
and biological evaluation of five di/tri-valent imino sugars
by coupling different aromatic templates to imino sugar
precursors. It is worth noting that amide 7 is a six-fold more
potent inhibitor than amine 8. This observation provides
justification for the use of amide linkages to attach mono-
valent imino sugars to their corresponding templates. Al-
Biological Evaluation of Glycomimetics
Multivalent glycomimetics 1–4 and monovalent imino though much more research is clearly needed to understand
sugars 7 and 8 were analyzed for their inhibitory activities multivalency and its utility in the area of glycosidase inhibi-
against a panel of twelve commercially available glycosid- tion, the synthetic approach reported is certainly suitable
ases.[38] Table 1 shows the results for inhibition analysis for the development of such multivalent glycosidase inhibi-
against α-l-fucosidase from bovine kidney, in comparison tors. As expected, most derivatives proved to be specific in-
with previous results of imino sugars 6 and 9 (inhibition hibitors of α-l-fucosidases with inhibition constants in the
data for other glycosidases are summarized in the Support- μm range. Data on enhanced inhibition are less convincing.
ing Information). Most of the new compounds are specific Only trivalent imino sugar 3a proved to be a more potent
inhibitors of α-l-fucosidase; no significant inhibition was inhibitor of α-l-fucosidases than its monovalent; the tri-
observed for any of the other enzymes assayed (α-galactos- valent inhibitor was seven-fold more active than its mono-
idases from coffee beans, β-galactosidases from Escherichia valent analogue 7 against both bovine kidney fucosidase
coli and Aspergillus oryzae, α-glucosidases from yeast and and the Bacteroides enzyme. The 3D structures of mamma-
from rice, amyloglucosidases from Aspergillus niger, β-glu- lian GH29 enzymes are not known, but it is unlikely from
cosidases from almonds, α-mannosidases from Jack beans, any of the reported 3D structures of bacterial homologs
β-mannosidases from snail, β-xylosidases from Aspergillus that the multi-valent inhibitors could span different active
niger, and β-N-acetylglucosaminidases from Jack beans).
centres and gain the massive affinity increase through avid-
The monovalent compounds, along with all new di- and ity effects that is possible in lectin-like systems. Any increase
trivalent glycomimetics inhibited α-l-fucosidases in the low observed may reflect subtle differences in (de)solvation and
μm range. Upon comparing each dimeric and trimeric in- entropic effects or may indeed represent a statistical rebind-
hibitor with its corresponding monovalent analogue (1 vs. ing phenomenon that is more likely in trimeric over di- or
6, 2 vs. 7, 3a vs. 7, 3b vs. 9, 4 vs. 7), enhanced inhibition monomeric compounds. Across the series, the ΔΔG values
was only found to occur in the case of trivalent imino sugar for binding cover just a small range. However, given the
3a (Ki = 0.3 μm). Compound 3a is a seven-fold more potent structural data which highlight the role of adventitious in-
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Exploring a Multivalent Approach to α-l-Fucosidase Inhibition
40:1) affording 13 (98.6 mg, 0.232 mmol, 95%). [α]2D4 = +28.0 (c =
0.89 in CH2Cl2). 1H NMR (300 MHz, [D6]DMSO, 363 K): δ =
8.58–8.48 (m, 1 H, CONH), 7.34–7.25 (m, 10 H, H-arom.), 5.06
(d, 2JH,HЈ = 12.7 Hz, 1 H, CH2 of Cbz), 4.97 (d, 1 H, CH2 of Cbz),
4.68 (t, J4,5 = J4,3 = 6.0 Hz, 1 H, 4-H), 4.59 (d, 1 H, 3-H), 4.43 (s,
1 H, 2-H), 4.29 (dd, 2J1Јa,1Јb = 15.2, J1Јa, NH = 5.8 Hz, 1 H, 1Ј-Ha),
4.22 (dd, J1Јb, NH = 6.0 Hz, 1 H, 1Ј-Hb), 4.08 (q, J5,Me-5 = 6.3 Hz,
teractions beyond the imino-cyclitol core, there is clearly
room to consider expansion of our current inventory of fu-
cosidase inhibitors so as to generate therapeutically signifi-
cant and specific inhibitors.
Experimental Section
1 H, 5-H), 1.43, 1.30 (2s, 3H each, C(CH3)2), 1.34 (d, JMe-5,5
=
6.6 Hz, 3 H, Me-5) ppm. 13C NMR (75.4 MHz, [D6]DMSO,
363 K): δ = 169.6 (CONH), 154.6 (C=O of Cbz), 138.5, 136.2 (Cq-
arom.), 127.7, 127.6, 127.1, 126.9, 126.8, 126.2 (C-arom.), 110.5
(C(CH3)2), 81.2, 80.1 (C-3, C-4), 66.5 (C-2), 65.5 (CH2 of Cbz),
57.3 (C-5), 42.0 (C-1Ј), 25.6, 24.5 (C(CH3)2), 14.5 (Me-5) ppm. IR:
General Methods: Optical rotations were measured with a 1.0 cm
or 1.0 dm tube with a Jasco P-2000 spectropolarimeter. 1H and 13
C
NMR spectra were recorded with a Bruker AV300, AMX300 and
AV500 for solutions in D2O, CD3OD and [D6]DMSO. All assign-
ments were confirmed by two-dimensional NMR experiments
(COSY and HSQC). Infrared spectra were recorded with a Jasco
FTIR-410 spectrophotometer. Mass spectra (CI, LSI and ESI)
were recorded using Micromass AutoSpeQ and QTRAP spectrom-
eters. The LSI was performed using thioglycerol as the matrix. TLC
was performed on silica gel HF254 (Merck), with detection by UV
light charring with H2SO4, vanillin, ninhydrin or with Pancaldi rea-
gent [(NH4)6MoO4, Ce(SO4)2, H2SO4, H2O]. Silica gel 60 (Merck,
230 mesh) was used for preparative chromatography.
ν = 3344, 2983, 2936, 1708, 1653, 1539, 1397, 1310, 1233, 1213,
˜
1027, 875 cm–1. CIMS: m/z (%)= 425 (10) [M + H]+. CIHRMS m/z
found 425.2062, calcd. for C24H29N2O5 [M + H]+: 425.2076.
(2R,3S,4R,5S)-2-Benzylcarbamoyl-5-methylpyrrolidine-3,4-diol (7):
A solution of 13 (66 mg, 0.156 mmol) in HCl (1 m)/THF, 1:1
(3.6 mL) was stirred overnight at room temp. The solvent was then
evaporated and the resulting residue was purified by column
chromatography (CH2Cl2/MeOH, 20:1). The obtained product
(58.7 mg, 0.153 mmol) was dissolved in MeOH (3 mL) and hydro-
genated with Pd/C (10%) as catalyst. After 1.5 h, the catalyst was
removed by filtration through celite and the solution concentrated.
The residue was purified by column chromatography (CH2Cl2/
MeOH, 10:1) affording 7 (34.4 mg, 0.137 mmol, 88 %, 2 steps).
Glycosidase Inhibition Assays: Glycosidases and the corresponding
p-nitrophenyl-O-glycoside substrates for inhibition assays were pur-
chased from Sigma–Aldrich. The experiments were performed es-
sentially as described previously.[38] Briefly, 0.01–0.5 units/mL of
enzyme and inhibitor were pre-incubated for 5 min at room temp.,
and the reaction started by addition of the substrate, buffered to
the optimal pH of the enzyme. After 20 min of incubation at 37 °C,
the reaction was stopped by addition of sodium borate buffer pH
9.8. The p-nitrophenolate formed was measured by visible absorp-
tion spectroscopy at 405 nm.
1
[α]2D4 = +18.3 (c = 0.89 in MeOH). H NMR (300 MHz, CD3OD):
2
δ = 7.31–7.20 (m, 5 H, H-arom.), 4.44 (d, JH,H = 15.2 Hz, 1 H,
CH-Ph), 4.39 (d, 1 H, CH-Ph), 4.15 (dd, J3,2 = 7.4, J3,4 = 4.2 Hz,
1 H, 3-H), 3.85–3.82 (m, 1 H, 4-H), 3.58 (d, 1 H, 2-H), 3.23 (qd,
J5,Me-5 = 6.6, J5,4 = 3.2 Hz, 1 H, 5-H), 1.16 (d, 3 H, Me-5) ppm.
13C NMR (75.4 MHz, CD3OD): δ = 176.0 (CONH), 139.9 (Cq-
arom.), 129.6, 128.5, 128.2 (C-arom.), 79.2 (C-3), 75.9 (C-4), 66.2
(C-2), 57.2 (C-5), 43.9 (CH2-Ph), 15.0 (Me-5) ppm. CIMS: m/z (%)
= 251 [(100) [M + H]+]. CIHRMS m/z found 251.1289, calcd. for
C13H19N2O3 [M + H]+: 251.1396.
(2S,3S,4R,5S)-N-(tert-Butoxycarbonyl)-2(1Ј,2Ј-dihydroxyethyl)-
3,4-O-isopropylidene-5-methylpyrrolidine-3,4-diol (11): To a solu-
tion of N-(Benzyloxycarbonyl-2(1Ј,2Ј-dihydroxyethyl)-3,4-O-iso-
propylidene-5-methylpyrrolidine-3,4-diol 10[32a] (86.3 mg,
0.246 mmol) in MeOH (2.5 mL), Pd/C (10%) and (Boc)2O (81 mg,
0.37 mmol) were added. The mixture was hydrogenated for 48 h.
After filtration through celite, the filtrate was purified by column
chromatography (CH2Cl2/MeOH, 30:1) to give 11 (54.5 mg,
0.172 mmol, 70%). [α]2D4 = +76.4 (c = 1.3 in MeOH). 1H NMR
(300 MHz, [D6]DMSO, 363 K): δ = 4.73 (d, J3,4 = 6.3 Hz, 1 H, 3-
H), 4.57 (br. s, 1 H, OH), 4.56 (t, J4,5 = 6.2 Hz, 1 H, 4-H), 4.16
(br. s, 1 H, OH), 3.99 (d, J2,1Ј = 4.5 Hz, 1 H, 2-H), 3.89–3.81 (m,
(2S,3S,4R,5S)-2-Benzylaminomethyl-5-methylpyrrolidine-3,4-diol
Hydrochloride (8): A solution of NaIO4 (440.6 mg, 2.06 mmol) in
water (4 mL) was added dropwise to a solution of 11 (326.5 mg,
1.03 mmol) in THF (4 mL) cooled to 0 °C. After stirring for 1 h at
room temp., THF was evaporated and the residue dissolved in
CH2Cl2 and washed successively with water, saturated NaHCO3
(aq.) and brine. The organic phase was dried, filtered and concen-
trated. To a solution of the corresponding aldehyde in dry 1,2-
dichloroethane (9 mL), benzylamine (337 μL, 3.09 mmol) and
NaBH(OAc)3 (343.3 mg, 1.54 mmol) were added. The reaction
mixture was stirred overnight at room temp. under N2. Then, satu-
rated NaHCO3(aq.) was added and the mixture extracted with Ac-
OEt, dried (Na2SO4), filtered and evaporated in vacuo. The residue
was purified by column chromatography (AcOEt/petroleum ether,
1:3) affording the corresponding amino pyrrolidine (214 mg,
0.569 mmol, 55%, 2 steps). A solution of the protected pyrrolidine
(75.2 mg, 0.2 mmol) in THF/HCl (1 m) (1:1, 5 mL) was stirred at
1 H, 1Ј-H), 3.78 (q, J5,Me = 6.5 Hz, 1 H, 5-H), 3.37 (dd, J2Јa,1Ј
=
4.5, 2J2Јa,2Јb = 11.2 Hz, 1 H, 2Ј-Ha), 3.27 (dd, J2Јb,1Ј = 6.3 Hz, 1 H,
2Ј-Hb), 1.42 (s, 9 H, (CH3)3C), 1.38 (s, 3 H, C(CH3)2), 1.28–1.26
(m, 6 H, C(CH3)2, Me-5) ppm. 13C NMR (75.4 MHz, [D6]DMSO,
363 K): δ = 153.7 (C=O of Boc), 109.5 ((CH3)2C), 80.3 (C-4), 79.6
(C-3), 78.2 ((CH3)3C), 70.6 (C-1Ј), 64.6 (C-2), 62.2 (C-2Ј), 56.7 (C-
5), 27.7 (CH3)3C), 25.5, 24.5 (C(CH3)2), 14.8 (Me-5). CIMS: m/z
(%) = 318 (20) [M + H]+, 218 (100) [M – Boc + H]+. CIHRMS
m/z found 318.1923, calcd. for C15H28NO6 [M + H]+: 318.1917.
(2R,3S,4R,5S)-N-Benzyloxycarbonyl-2-benzylcarbamoyl-3,4-O- room temp. for 3 h. The solvent was evaporated affording 8
1
isopropylidene-5-methylpyrrolidine-3,4-diol (13): Acid 12[32a] (82 mg, (56.5 mg, 0.2 mmol, quant.). [α]2D4 = –16.4 (c = 0.88 in MeOH). H
0.245 mmol) was dissolved in DMF and benzylamine (27 μL,
0.247 mmol), DIPEA (85 μL, 0.49 mmol) and PyBOP (130 mg,
NMR (300 MHz, CD3OD): δ = 7.62–7.58 (m, 2 H, H-arom.), 7.51–
7.45 (m, 3 H, H-arom.), 4.37 (d, J = 13.1 Hz, 1 H, CH2 of Bn),
2
0.249 mmol) were added. The mixture was stirred overnight at 4.33 (d, 1 H, CH2 of Bn), 4.24 (dd, J3,2 = 7.3, J3,4 = 4.8 Hz, 1 H,
room temp. Then, the solvent was evaporated and the residue dis-
solved in CH2Cl2 (10 mL) and washed with satd. aq. sol. of citric
acid (2ϫ 10 mL) and brine (10 mL). The organic phase was dried
(Na2SO4), filtered and evaporated in vacuo. The resulting crude
mixture was purified by column chromatography (CH2Cl2/MeOH,
3-H), 3.96 (t, J4,5 = 4.8 Hz, 1 H, 4-H), 3.86 (td, J2,1Јa = 7.4, J2,1Јb
2
= 5.9 Hz, 1 H, 2-H), 3.75–3.67 (m, 1 H, 5-H), 3.69 (dd, J1Јa,1Јb
=
13.6 Hz, 1 H, 1Ј-Ha), 3.60 (dd, 1 H, 1Ј-Hb), 1.49 (d, JMe,5 = 7.1 Hz,
3 H, Me-5) ppm. 13C NMR (75.4 MHz, CD3OD): δ = 131.9 (Cq-
arom.), 131.3, 131.0, 130.4 (C-arom.), 75.3 (C-4), 73.9 (C-3), 62.6
Eur. J. Org. Chem. 2013, 7328–7336
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7333

I. Robina et al.
FULL PAPER
(C-5), 59.9 (C-2), 53.1 (CH2 of Bn), 47.8 (C-1Ј), 16.1 (Me-5) ppm.
1,3,5-Tris((2R,3S,4R,5S)-N-benzyloxycarbonyl-2-carbonylamino-
methyl-3,4-O-isopropylidene-5-methylpyrrolidine-3,4-diol)benzene
IR: ν = 3389, 3164–3155, 3073, 2941, 2927, 2845–2610, 1536, 1457,
˜
1129, 1086, 1004, 787, 745, 692 cm–1. CIMS: m/z (%) = 237 [(43) (18): The acid 12[32a] (176 mg, 0.525 mmol) was dissolved in DMF
[M + H]+]. CIHRMS m/z found 237.1598, calcd. for C13H21N2O2
and 1,3,5-tris(aminomethyl)benzene trihydrochloride[35] (43.5 mg,
0.159 mmol), DIPEA (1.6 mL, 9.45 mmol) and PyBOP (278.6 mg,
0.535 mmol) were added. The mixture was stirred overnight at
room temp. Then, the solvent was evaporated and the residue dis-
solved in CH2Cl2 (30 mL) and washed with satd. aq. sol. of citric
acid (2ϫ 30 mL) and brine (30 mL). The organic phase was dried
(Na2SO4), filtered and evaporated in vacuo. The resulting crude
mixture was purified by column chromatography (CH2Cl2/MeOH,
30:1) affording 18 (117 mg, 0.105 mmol, 66%). [α]2D4 = –34.3 (c =
0.96 in CH2Cl2). 1H NMR (500 MHz, [D6]DMSO, 363 K): δ = 8.53
(t, JNH,1Јa = JNH,1Јb = 5.9 Hz, 3 H, CONH), 7.34–7.27 (m, 15 H,
H-arom.), 7.04 (s, 3 H, H-arom.), 5.02 (s, 6 H, CH2 of Cbz), 4.67
(t, J4,5 = J4,3 = 6.1 Hz, 3 H, 4-H), 4.59 (dd, J3,2 = 1.0 Hz, 3 H, 3-
H), 4.42 (br. s, 3 H, 2-H), 4.30 (dd, 2J1Јa,1Јb = 15.1 Hz, 3 H, 1Ј-Ha),
4.09–4.04 (m, 6 H, 1Ј-Hb, 5-H), 1.43, 1.30 (2s, 9H each, C(CH3)2),
1.32 (d, JMe-5,5 = 6.5 Hz, 9 H, Me-5) ppm. 13C NMR (125.7 MHz,
[D6]DMSO, 363 K): δ = 169.6 (CONH), 154.7 (CO of Cbz), 138.8,
136.3 (Cq-arom.), 127.8, 127.2, 126.9, 124.6 (C-arom.), 110.6
(C(CH3)2), 81.2 (C-3), 80.1 (C-4), 66.5 (C-2), 65.6 (CH2 of Cbz),
57.3 (C-5), 41.9 (C-1Ј), 25.7, 24.6 (C(CH3)2), 14.6 (Me-5) ppm. IR:
[M + H]+: 237.1603.
4,4Ј-Bis((3S,4S,5R,6S)-3-carbonylaminomethyl-6-methyl-hexa-
hydropyridazine-4,5-diol)biphenyl Dihydrochloride (1): To a solution
of 14[33b] (163 mg, 0.486 mmol) and commercial dicarboxylic acid
15 (59 mg, 0.243 mmol) in DMF (3 mL), DIPEA (166 μL,
0.953 mmol) and PyBOP (252 mg, 0.484 mmol) were added. The
mixture was stirred overnight at room temp. Then, the solvent was
evaporated and the residue dissolved in CH2Cl2 and washed with
satd. aq. sol. of citric acid and brine. The organic phase was dried
(Na2SO4), filtered and evaporated in vacuo. The crude product thus
obtained was treated with HCl (1 m)/THF, 1:1 (10 mL) and stirred
5 h at room temp. The solvent was then evaporated and the re-
sulting residue was purified by column chromatography (CH2Cl2/
MeOH, 20:1). The obtained product (83.4 mg, 0.105 mmol) was
dissolved in MeOH (15 mL), and Pd/C (10 %) and HCl (5 m,
100 μL) were added. The mixture was hydrogenated at 1 atm for
3 h, then diluted with MeOH, filtered through celite, and evapo-
rated, affording corresponding unprotected derivative 1 in 43%
overall yield. [α]2D5 = –20.9 (c = 0.6 in MeOH). 1H NMR (300 MHz,
D2O): δ = 7.79 (d, J = 8.3 Hz, 4 H, H-arom.), 7.68 (d, J = 8.3 Hz,
4 H, H-arom.), 4.04 (m, 2 H, 4-H or 5-H), 3.78 (dd, 2J1Јa,1Јb = 14.2,
J1Јa,3 = 2.5 Hz, 2 H, 1Ј-Ha), 3.69 (dd, J = 10.0, J = 2.8 Hz, 2 H,
ν = 2988, 2937, 1751–1587, 1454, 1405, 1352, 1306, 1209, 1140,
˜
1027, 867 697 cm–1. LSIMS: m/z (%) = 1139 [(5) [M + Na]+].
LSIHRMS m/z found 1139.4910, calcd. for C60H72N6O15Na [M +
Na]+: 1139.4953.
4-H or 5-H), 3.60–3.45 (m, 6 H, 1Ј-Hb, 3-H, 6-H), 1.31 (d, JMe,6
=
1,3,5-Tris((2R,3S,4R,5S)-2-carbonylaminomethyl-5-methylpyrrol-
idine-3,4-diol)benzene (3a): A solution of 18 (78.8 mg, 0.07 mmol)
in HCl (5 m)/THF, 1:1 (1.2 mL) was stirred overnight at room temp.
The solvent was then evaporated and the resulting residue was puri-
fied by column chromatography (CH2Cl2/MeOH, 10:1). The ob-
tained product (57.7 mg, 0.058 mmol) was dissolved in MeOH
(2 mL), and hydrogenated with Pd/C (10%) as catalyst. After 2 h
the catalyst was removed by filtration through celite and the solu-
tion concentrated affording 3a (19.5 mg, 0.033 mmol, 47 %, 2
steps). [α]2D4 = –15.4 (c = 0.99 in MeOH). 1H NMR (300 MHz,
6.8 Hz, 6 H, Me-6) ppm. 13C NMR (75.4 MHz, D2O): δ = 170.6
(CONH), 142.7, 132.4 (Cq-arom.), 127.8, 127.2 (C-arom.), 68.1,
67.9 (C-4, C-5), 57.1, 54.6 (C-6, C-3), 38.9 (C-1Ј), 12.6 (Me-6) ppm.
IR: ν = 3552–3046, 1635, 1550, 1532, 1492, 1308, 1165, 1115, 1024,
˜
1006, 840 cm–1. LSIHRMS m/z found 551.2587, calcd. for
C26H36N6O6Na [M + Na]+: 551.2594.
1,3-Bis((2R,3S,4R,5S)-2-carbonylaminomethyl-5-methyl-pyrrol-
idine-3,4-diol)benzene (2): Acid 12[32a] (150 mg, 0.448 mmol) was
dissolved in DMF and commercial m-xylylenediamine (23 μL,
0.174 mmol), DIPEA (122 μL, 0.71 mmol) and PyBOP (233 mg,
0.448 mmol) were added. The mixture was stirred overnight at
room temp. Then, the solvent was evaporated and the residue dis-
solved in CH2Cl2 (10 mL) and washed with satd. aq. sol. of citric
acid (2ϫ 10 mL) and brine (10 mL). The organic phase was dried
(Na2SO4), filtered and evaporated in vacuo. The resulting crude
product was purified by column chromatography (CH2Cl2/MeOH,
50:1) affording the corresponding protected diamide (134.3 mg,
0.174 mmol, quant.), whose solution (60 mg, 0.08 mmol) in HCl
(1 m)/THF, 1:1 (1.4 mL) was stirred overnight at room temp. The
solvent was then evaporated and the resulting residue was purified
by column chromatography (CH2Cl2/MeOH, 10:1). The obtained
product (35 mg, 0.05 mmol) was dissolved in MeOH (3 mL), and
hydrogenated with Pd/C (10%) as catalyst. After 2 h, the catalyst
was removed by filtration through celite and the solution concen-
2
CD3OD): δ = 7.13 (s, 3 H, H-arom.), 4.41 (d, J1Јa,1Јb = 15.6 Hz, 3
H, 1Ј-Ha), 4.36 (d, 3 H, 1Ј-Hb), 4.15 (dd, J3,2 = 7.5, J3,4 = 4.2 Hz,
3 H, 3-H), 3.86–3.83 (m, 3 H, 4-H), 3.58 (d, 3 H, 2-H), 3.24 (qd,
J5,Me-5 = 6.6, J5,4 = 3.2 Hz, 3 H, 5-H), 1.16 (d, 9 H, Me-5) ppm.
13C NMR (75.4 MHz, CD3OD): δ = 176.1 (CONH), 140.8 (Cq-
arom.), 126.2 (C-arom.), 76.1 (C-3), 75.9 (C-4), 66.1 (C-2), 57.1 (C-
5), 43.7 (C-1Ј), 15.1 (Me-5) ppm. LSIMS: m/z (%) = 617 [(10) [M
+ Na]+]. LSIHRMS m/z found 617.2911, calcd. for C27H42N6O9Na
[M + Na]+: 617.2911.
N,NЈ,NЈЈ-(1,3,5-Phenylenetris(methylene))tris-[5-((2R,3S,4R)-3,4-di-
hydroxypyrrolidin-2-yl)-2-methylfuran-3-carboxamide](3b): Acid
19[34b] (88.9 mg, 0.246 mmol) was dissolved in DMF and 1,3,5-tris-
(aminomethyl)benzene[35] (11.5 mg, 0.07 mmol), DIPEA (43 μL,
0.246 mmol) and PyBOP (143.6 mg, 0.27 mmol) were added. The
mixture was stirred overnight at room temp. Then, the solvent was
evaporated and the residue dissolved in AcOEt (50 mL) and
trated affording 2 (22.9 mg, 0.05 mmol, 64%, 2 steps). [α]2D5 = –15.6
1
(c = 0.43 in MeOH). H NMR (300 MHz, CD3OD): δ = 7.32–7.09 washed with HCl (1 m) (3ϫ 20 mL) and brine (30 mL). The organic
(m, 4 H, H-arom.), 4.41 (br. s, 4 H, CH2-Ph), 4.16 (dd, J3,2 = 7.5, phase was dried (Na2SO4), filtered and evaporated in vacuo. The
J3,4 = 4.2 Hz, 2 H, 3-H), 3.86–3.84 (m, 2 H, 4-H), 3.58 (d, 2 H, 2-
H), 3.25 (qd, J5,Me-5 = 6.6, J5,4 = 3.3 Hz, 2 H, 5-H), 1.22 (d, 6 H,
resulting crude mixture was purified by column chromatography
(CH2Cl2/MeOH, 10:1) affording the corresponding protected tri-
Me-5) ppm. 13C NMR (75.4 MHz, CD3OD): δ = 175.9 (CONH), amide (41.2 mg, 0.034 mmol, 49 %), whose solution (39.6 mg,
140.3 (Cq-arom.), 129.9, 129.2, 127.3 (C-arom.), 79.1 (C-3), 75.9 0.033 mmol) in MeOH (2 mL) was hydrogenated with Pd/C (10%)
(C-4), 66.1 (C-2), 57.2 (C-5), 43.8 (CH2-Ph), 15.0 (Me-5) ppm. IR:
as catalyst. After 1.5 h the catalyst was removed by filtration
through celite and the solution concentrated affording 3b (24.5 mg,
0.031 mmol, 94%). [α]2D5 = –68.6 (c = 0.23 in H2O). 1H NMR
(500 MHz, D2O): δ = 7.23 (s, 3 H, H-arom.), 6.83 (s, 3 H, H-furan),
ν = 3626–3105, 2920, 1650, 1541, 1347, 1269, 1123, 1283, 990,
˜
750 cm–1. CIMS: m/z (%) = 423 [(100) [M + H]+]. CIHRMS m/z
found 423.2238, calcd. for C20H31N4O6 [M + H]+: 423.2244.
7334
www.eurjoc.org
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 7328–7336

Exploring a Multivalent Approach to α-l-Fucosidase Inhibition
4.59–4.50 (m, 15 H, 2-H, 3-H, 4-H, 1Ј-Ha, 1Ј-Hb), 3.64 (dd, J5a,5b
= 12.9, J5a,4 = 4.5 Hz, 3 H, 5-Ha), 3.35 (dd, J5b,4 = 2.0 Hz, 3 H, 5-
Hb), 2.48 (s, 9 H, Me) ppm. 13C NMR (125.7 MHz, D2O): δ =
166.1 (C=O), 158.2, 146.1 (Cq-arom.), 139.2 (C-arom.), 124.0 (Cq-
arom.), 116.2, 109.8 (C-arom.), 74.1, 69.7, 56.7 (C-2, C-3, C-4),
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49.9 (C-5), 42.7 (C-1Ј), 13.0 (Me-5) ppm. IR: ν = 3596–300, 2923,
˜
1636, 1579, 1534, 1421, 1401, 1340, 1228, 1119–1066 cm–1. ESI MS:
m/z (%) = 793 [(64) [M + H]+], 815 [(37) [M + Na]+].
N1,N3,N5-Tris(2-(2-(2-((2R,3S,4R,5S)-3,4-dihydroxy-5-methyl-
pyrrolidine-2-carboxamido)ethoxy)ethoxy)ethyl)benzene-1,3,5-tri-
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ration afforded crude 21. To a solution of 21 (0.063 mmol) and 12
(69.6 mg, 0.208 mmol) in DMF (3 mL), DIPEA (137 μL,
0.794 mmol) and PyBOP (110 mg, 0.212 mmol) were added. The
mixture was stirred overnight at room temp. Then, the solvent was
evaporated and the residue dissolved in CH2Cl2 and washed with
satd. aq. sol. of citric acid and brine. The organic phase was dried
(Na2SO4), filtered and evaporated in vacuo. The resulting crude
reaction was purified by column chromatography (CH2Cl2/MeOH,
30:1Ǟ15:1) affording the corresponding protected triamide
(68.6 mg, 70%). This derivative (62.0 mg, 0.040 mmol) was stirred
in HCl (5 m)/THF, 1:1 (2 mL) at room temp. for 5 h. Solvent was
then evaporated and the residue was purified by column
chromatography (CH2Cl2/MeOH, 10:1Ǟ6:1). A solution of the ob-
tained product (45.7 mg, 0.032 mmol) in MeOH (5 mL) was hydro-
genated with Pd/C (10%) as catalyst. After 4 h the catalyst was
removed by filtration through celite and the solution concentrated
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1
affording 4 in quantitative yield. H NMR (300 MHz, CD3OD): δ
= 8.46 (s, 3 H, H-arom.), 4.13 (dd, J = 7.5, J = 4.1 Hz, 3 H, 3-H),
3.85–3.82 (m, 3 H, 4-H), 3.71–3.55 (m, 33 H, 2-H, CH2), 3.41–3.37
(m, 6 H, CH2), 3.22 (qd, J5,Me-5 = 6.6, J5,4 = 3.0 Hz, 3 H, 5-H),
1.17 (d, 9 H, Me-5) ppm. 13C NMR (75.4 MHz, CD3OD): δ =
168.7 (CONH), 136.6 (Cq-arom.), 130.1 (C-arom.), 78.8 (C-3), 75.5
(C-4), 71.4, 70.5, 65.5 (C-2, CH2), 57.6 (C-5), 41.1, 40.2 (CH2),
[11]
[12]
14.4 (Me-5) ppm. IR: ν = 3655–3080, 1645, 1541, 1449–1437, 1292,
˜
1122–1093, 995 cm–1. LSIMS: m/z (%) = 1052 [(5) [M + Na]+].
LSIHRMS m/z found 1052.5142, calcd. for C45H75N9O18Na [M +
Na]+: 1052.5128.
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Supporting Information (see footnote on the first page of this arti-
1
cle): Supplemental crystallographic data, H- and 13C-NMR spec-
[15]
tra for all new compounds and complete glycosidase inhibition
data. PDB files, and observed structure factor data for complexes
3a and 7 bound to BtFuc2970, have been deposited in the PDB
with accession codes 2JL1 and 2JL2, respectively. See DOI:
10.1039/b000000x/.
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Acknowledgments
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M. Nagae, A. Tsuchiya, T. Katayama, K. Yamamoto, S. Wak-
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The authors thank the Ministerio de Ciencia e Innovación of Spain
(MICINN) (CTQ2008-01565/BQU and CTQ2012-31247 and FPU
fellowship to EMC), the European Commision [FP7, number
HEALTH-F2-2011-256986] and the Junta de Andalucía (FQM
345) for financial support. D. W. W. thanks the Biotechnology and
Biological Sciences Research Council (BBSRC) for a PhD student-
ship. X-ray diffraction facilities in York were provided, in part, by
the Wellcome Trust (grant 077371). G. J. D. and D. W. W. thank the
staff of the Diamond Light Source and the European Synchrotron
Radiation Facility for provision of synchrotron data collection fa-
cilities.
A. L. van Bueren, A. Ardevol, J. Fayers-Kerr, B. Luo, Y. M.
Zhang, M. Sollogoub, Y. Bleriot, C. Rovira, G. J. Davies, J.
Am. Chem. Soc. 2010, 132, 1804.
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347; Angew. Chem. Int. Ed. 2010, 49, 337.
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Received: June 14, 2013
Published Online: September 19, 2013
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