Understanding multivalent effects in glycosidase inhibition using: C -glycoside click clusters as molecular probes

Article abstract of DOI:10.1039/c6nj01311b

The synthesis of the first examples of multivalent C-glycosides based on C₆₀-fullerene or β-cyclodextrin cores by way of Cu(i)-catalyzed azide-alkyne cycloadditions is reported. These compounds were designed as molecular probes to understand the mechanisms underlying the outstanding multivalent effects observed in glycosidase inhibition. The inhibition results obtained support a multivalent-binding model based on two scenarios both involving nonspecific interactions and varying by the presence or the absence of active site specific interactions. The magnitude of the multivalent effect obtained depends on the identity of the glycosidase involved and more specifically on the accessibility of its catalytic active site. Large inhibitory multivalent effects can be obtained when both glycosidase active sites and non-catalytic sites at the protein surface are involved in binding events. On the other hand, nonspecific interactions alone are not sufficient to achieve relative affinity enhancements exceeding a simple statistical effect (i.e., a relative inhibition potency not better than one on a valence-corrected basis).

Full text of DOI:10.1039/c6nj01311b

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DOI: 10.1039/C6NJ01311B
New Journal of Chemistry
ARTICLE
Understanding multivalent effects in glycosidase inhibition using
C-glycoside click clusters as molecular probes
Fabien Stauffert,a Anne Bodlenner,a Thi Minh Nguyet Trinh,b M. Isabel García Moreno,c Carmen
Ortiz Mellet,c Jean-François Nierengarten,*b and Philippe Compain*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
The synthesis of the first examples of multivalent C-glycosides based on C60-fullerene or β-cyclodextrin cores by way of
Cu(I)-catalyzed azide-alkyne cycloadditions is reported. These compounds were designed as molecular probes to
understand the mechanisms underlying the outstanding multivalent effects observed in glycosidase inhibition. The
inhibition results obtained support a multivalent-binding model based on two scenarios both involving nonspecific
interactions and varying by the presence or the absence of active site specific interactions. The magnitude of the
multivalent effect obtained depends on the identity of the glycosidase involved and more specifically on the accessibility of
its catalytic active site. Large inhibitory multivalent effects can be obtained when both glycosidase active site and non-
catalytic sites at the protein surface are involved in binding events. On the other hand, nonspecific interactions alone are
not sufficient to achieve relative affinity enhancements exceeding a simple statistical effect (i. e., a relative inhibition
potency not better than one on a valence-corrected basis).
www.rsc.org/
biological medium and to form ammonium cations that
Introduction
interact strongly with
a nucleophilic catalytic residue,
commonly a carboxylate, in the enzyme active site.8 In sharp
contrast, lectins, an important class of proteins playing
numerous roles in biological recognition phenomena, appear
to be ideal candidates for multivalent design as they usually
display accessible, multiple carbohydrate-binding pockets.9 In
such systems, multivalent interactions are used by Nature as a
way to increase affinity and modulate selectivity since
monovalent carbohydrate ligands typically bind only weakly to
lectin receptors. The bridging of several adjacent binding sites
by multidentate ligands, i. e. the so-called chelate effect, is
indeed at the basis of the largest multivalent effects known to
date.9,10 For proteins with one binding site, as anticipated for
most enzymes, statistical rebinding effect, which is due to high
local ligand concentration, is believed to be one of the major
binding modes at play.10f In combination with chelation or
The recent discovery of multivalent glycosidase inhibitors
showing outstanding binding enhancements up to five orders
of magnitude over the corresponding monovalent ligands
challenges and stimulates (bio)chemist imagination.1-4 First,
because these enzymes which powerfully catalyse glycosidic
cleavage in carbohydrates and glycoconjugates, do not exhibit
the typical structural features required for multivalent design.
Glycosidases indeed display generally a single, buried catalytic
site5 and bind to their monovalent substrates with high affinity
and selectivity. The main approach to reversible glycosidase
inhibitors has been consequently based on natural substrate
mimetics, such as iminosugars in which the endocyclic oxygen
atom of the parent glycosides is replaced by a nitrogen
atom.6,7 The high affinity of iminosugars for glycosidases is
commonly explained by their ability to become protonated in
non-chelation
mechanisms,
additional
aggregation
phenomena have also to be considered, further complicating
the rationale behind the observed multivalent effects. It is
worth noting that systems that do not allow chelation
mechanisms typically show moderate multivalent effects and
require ligands with very high valency (>ca 100-150) to reach
significant affinity enhancements.10f Considering these points,
how to explain that the multivalent effects observed in
glycosidase inhibition with neoglycoclusters displaying up to 36
iminosugar ligands2,3 rival those encountered for
carbohydrate-lectin interactions? To answer to this
fundamental question, the molecular basis of the rather
counter-intuitive inhibitory multivalent effect has been
explored independently by several groups using different
a.Laboratoire de Synthèse Organique et Molécules Bioactives (SYBIO), Université de
Strasbourg/CNRS (UMR 7509), Ecole Européenne de Chimie, Polymères et
Matériaux (ECPM), 25 rue Becquerel, 67087 Strasbourg Cedex 2, France.
E-mail:philippe.compain@unistra.fr
b.Laboratoire de Chimie des Matériaux Moléculaires
Université de Strasbourg et CNRS (UMR 7509)
Ecole Européenne de Chimie, Polymères et Matériaux
25 rue Becquerel, 67087 Strasbourg Cedex 2, France.
E-mail: nierengarten@unistra.fr
c. Departamento de Química Orgánica
Facultad de Química
Universidad de Sevilla
Profesor García González 1, E-41012, Sevilla, Spain.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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strategies3,11,12a,13,14 and complementary techniques including and specific interactions with a more accessible VcieawtaArltyictliecOsnilitnee
isothermal titration calorimetry,12a mass spectrometry,3 would be simultaneously involved in binding events leading to
DOI: 10.1039/C6NJ01311B
electron microscopy imaging3 and atomic force microscopy.11 strong multivalent effects via chelation mechanisms. The
Beyond fundamental interest in understanding this new multivalent-binding model presented above is thus based on
phenomena, these studies are also stimulated by the two scenarios involving either just interactions at non-catalytic
therapeutic relevance of glycosidase inhibitors6,7,15 and by the sites or the concerted occurrence of both active site and non-
first promising applications of multivalent effect to cellular catalytic site-interactions. Whereas iminosugar glycomimetics
glycosidases for correcting protein folding defects.16 Recently, are ideally suited to bind at the catalytic site of glycosidases, it
the dual lectin- and glycosidase binding abilities of designed remains uncertain whether or not the interactions with the
C60-based sp2-iminosugar clusters17 were exploited to probe non-catalytic sites involved in multivalent enzyme inhibition
the involvement of non-catalytic sites and active site are governed by similar principles. To explore this hypothesis,
interactions in multivalent binding events. Competitive lectin- we have now designed neoglycoclusters as molecular probes
glycosidase assays which were performed in the presence or in which the iminosugar-based specific active-site-directed
the absence of a potent monovalent competitive inhibitor, inhibitory epitopes (inhitope) were replaced by C-glycoside
suggested two different multivalent binding modes depending motifs, with a much weaker affinity for the catalytic site. Here,
on the nature of the glycosidase involved. For glycosidases we describe the full details of our study from C-glycoside
known to possess relatively deep catalytic sites, the latter cluster synthesis to the contrasted inhibition results obtained
would be only marginally involved in multivalent binding whether the glycosidase active-site is supposed to be involved
events, leading to poor response to multivalent ligand or not in the multivalent binding events.
presentation. Most of the stabilizing interactions in the
enzyme-inhibitor complexes would arise from non-catalytic
Results and discussion
sites located at the protein surface and the inhibition would be
only due to the blockage of the catalytic site entrance. This
mechanistic model has been demonstrated by competitive
assays14 for yeast maltase which belongs to the glycosyl
hydrolase family GH13. It could be applied to other
glycosidases of the GH13 family or known to have buried
active sites such as GH1 or GH27 glycosyl hydrolases. Jack bean
α-mannosidase (JBα-man), which belongs to the glycosyl
hydrolase GH38 family is the glycosidase being the most
sensitive to multivalent presentation known to date. Despite
the fact that the 3-D crystallographic structure of JBα-man is
currently unknown, it is believed, by analogy with other
members of the GH38 family, that this enzyme possesses an
open active site cleft with several sugar-binding subsites. Due
to this structural feature, interactions with non-catalytic sites
Neoglycocluster design. The mechanistic probes we wanted to
construct needed first to be close analogues of iminosugar
click clusters such as
1 which are known to display significant
inhibitory multivalent effects (Figure 1).1,2 Secondly, these
neoglycoclusters have to be based on inhitopes that should be
hydrolytically stable18 and structurally as close as possible as
iminosugars without displaying their selectivity towards
glycosidase active-sites. To achieve this objective, we selected
a C-glycosidic motif. The absence of a basic nitrogen atom was
indeed expected to suppress key electrostatic interactions
with nucleophilic catalytic residues and thus inhitope affinity
with minimal structural modifications.
R
R
R
N
N
R
R
R
O
O
HO
N
R
R
O
O
N
( )5
O
HO
HO
O
O
O
O
HO
R
O
O
O
O
O
HO
O
O
HO
4a
OH
O
O
R
R
R
O
O
O
O
R
O
N
O
N
N
R1=
R2=
R
O
O
O
O
O
O
HO
O
N
O
O
( )5
HO
HO
OH
O
HO
HO
R
R
O
R
O
O
O
O
R
OH
O
HO
HO
O
O
OH
O
O
O
O
O
O
N
N
O
R
HO
N
O
O
R
O
HO
R
R
N
R
R3=
O
( )5
AcO
AcO
AcO
R
R
R
O
1b R = R1
2b R = R2
1a R = R1
2a R = R2
N
N
AcO
3
R = R3
( )5
Fig. 1 Mono- and multivalent glycomimetics.
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DOI: 10.1039/C6NJ01311B
[60]Fullerene (C60) and
β-cyclodextrin (β-CD) were selected C7 byproducts. Under these conditions, 1,4-BQ is believed to
since these platforms have led, in previous studies, to clusters oxidize ruthenium hydrides resulting from the decomposition
displaying large multivalent effects with relative inhibition of the metathesis catalyst and which are responsible for the
potency on molar basis (rp/n) up to 620-fold.1,2
olefin isomerization side-reactions.22,23 The selective one-step
removal of the benzyl protecting groups and reduction of the
Neoglycocluster synthesis. Based on these design criteria, we double bond in
targeted multivalent C-glycosides differing by the nature of with H2/ Pd-C. The resulting crude tetrol product obtained was
their central core (Figure 1). As shown in Scheme 1, the directly converted to the corresponding tetraacetate.
synthesis began with the preparation of the azide-armed C- Compound was thus obtained in 68% yield from (two
glycosides and 10 from methyl tetra-O-benzyl- steps). The displacement of the tosyl group with sodium azide
glucopyranoside ( ). Following the protocol of Hosomi and was achieved in 91% yield in DMF to provide the desired
Sakurai,19 compound
was reacted with allyltrimethylsilane in protected substrate for azide-alkyne cycloaddition coupling
the presence of TMSOTf. This efficient process afforded -C- with the
-CD-based scaffold 1224 (Scheme 2). Compound 10
allyl glycoside 6a20 in 73% yield after purification on silica gel, the fully deprotected analogue of
, prepared for the Cu(I)-
7 was performed by catalytic hydrogenolysis
2
8
7
9
D-
5
5
9
α
β
,
9
the minor
β
-epimer 6b being obtained in 7% yield.
catalyzed azide-alkyne cycloaddition (CuAAC) with fullerene
hexa-adduct 13, was obtained using anion exchange Amberlite
IRA-400 (OH-) resin. To assess any possible multivalent effect,
C-glycoside
9 was reacted with ethynylcyclopropane in the
presence of CuSO4•5H2O and sodium ascorbate to yield, after
deprotection, the corresponding adduct 4b used as
monovalent control. It can be noted that a cyclopropyl residue
was used for 4b and not a n-propyl one as in the case of 4a
.
Actually, the reaction between and 1-pentyne was
9
attempted but it led to side-products12b arising from prior
aerobic copper-catalyzed oxygenation at the propargylic
methylene group. We found that this problem could be
avoided by using the cyclopropylated alkyne as starting
material for the preparation of the C-glycoside model
compound. Having in hands the key azide-armed C-glycosides
9
and 10, we then performed the final steps of the cluster
synthesis (Schemes 2 and 3).
O
O
O
O
O
O
HO
O
O
HO
OH
O
O
O
O
Scheme 1 Synthesis of azide-armed C-glycosides 9-10 and of the
monovalent model 4b
O
O
O
O
OH
O
O
HO
Our objective was then to perform a cross-metathesis reaction
to complete the construction of the linker and functionalize
the terminal position with a tosyl group. Unfortunately, our
first attempts towards this goal were unsuccessful. The
O
O
OH
O
O
OH
O
O
O
O
O
reaction of 4-penten-1-yl tosylate21 with
α-C-allyl glycoside 6a
O
12
in the presence of Hoveyda-Grubbs II catalyst led to an
inseparable mixture of the expected product along with the
two corresponding analogues bearing a C5 or a C7 alkyl spacer
as judged by NMR and MS analysis. This product distribution
resulted from olefin isomerization/migration in the reactants
during the cross-metathesis step.22 After optimization,
AcO
AcO
AcO
O
CuSO4.5H2O
sodium asc.
AcO
DMF/H2O, 80°C (MW)
N3
( )5
9
83%
3
Amberlite IRA400 (OH-)
MeOH/H2O, rt
compound 7 was obtained in 68% yield using an additive to the
cross-metathesis ruthenium catalyst (Table S1 in the
supporting information). Specifically, treatment of 6a and 4-
penten-1-yl tosylate with Grubbs II catalyst (15 mol%) in the
presence of 1,4-benzoquinone (1,4-BQ, 20 mol%) at room
temperature was found to prevent the formation of the C5 and
93%
2b
Scheme 2 Synthesis of C-glycoside cluster 2b
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9 and heptakis(2,6-di- fullerene derivatives, it was not the case here VaienwdArtIicClePO-AnlEinSe
Microwave-assisted click conjugation of
O-propargyl)
tetradecavalent C-glycoside
β
-cyclodextrin 1224 led to the desired analysis revealed 19000 ppm of coppeDrOaI:t10t.h10is39s/Cta6gNeJ.01T31h1iBs
3
in 83% yield (Scheme 2). Copper prompted us to further filter the compound over a QuadraSilTM
residues were removed at this stage following a simple Mercaptopropyl column. Compound 2a was finally isolated in
protocol based on the filtration of the crude product on a pad 34% yield and ICP-AES analysis revealed a low level of residual
of silica gel using ammonia as a complexing agent in the eluent copper (994 ppm, i.e. a maximal concentration of 35 µM
before purification by flash chromatography. This purification during inhibition assays).27 The structure of 2a was confirmed
process have previously demonstrated its efficiency for the by its T-symmetry deduced from the 13C NMR spectrum
synthesis of iminosugar click clusters leading to low content of recorded in DMSO-d6 as well as by mass spectrometry.
residual copper ions (60 ppm) as measured by inductively
coupled plasma atomic emission spectroscopy (ICP-AES).3 Biological results. The inhibitory properties of C-glycosides
Subsequent O-deacetylation of adduct
3 using anion exchange clusters 2 as well as the monovalent control 4b were assayed
Amberlite IRA-400 (OH-) resin afforded the expected cluster 2b towards a panel of commercial glycosidases, including JBα-
in high yield. Compound 2b showed good solubility in water. man and yeast maltase (Table 1). As their corresponding
For the preparation of 2a, the fullerene scaffold 1325 had to be analogues in the iminosugar series,1,2 the mono- and
directly functionalized with the unprotected C-glycoside as the multivalent C-glycosides were found to display no inhibition
presence of the malonic esters on the C60 core is not towards
compatible with the reaction conditions used for the iminosugar clusters
deacetylation of a protected analogue. The click reaction differed on their behavior against
between 10 and 13 was performed in a ternary solvent system almonds) and -galactosidase (from green coffee beans):
β
-mannosidase and
and C-glycoside clusters
-glucosidase (from
β
-galactosidase. In contrast,
1
2
drastically
β
α
(CH2Cl2/DMSO/H2O) allowing the solubilisation of all the whereas 1a and 1b displayed moderate to good inhibitory
reagents but also of the final product. Importantly, no properties, compounds 2a and 2b showed no inhibition at
precipitation of partially clicked intermediates occurred during concentrations up to 2 mM. In the case of yeast maltase the
the course of the reaction thus allowing the complete inhibition profile were very similar irrespective of the C-
functionalization of the scaffold.26
glycoside or iminosugar nature of the inhitope. The 12-valent
fullerene clusters 1a and 2a were at the limit of exhibiting
formal multivalent effects, with relative inhibition potencies
relative to the monovalent references 4a and 4b, on a valence-
corrected basis (rp/n) close to one. The inhibitory efficiency
was significantly decreased for the
clusters 1b and 2b. Considering that
-galactosidase (green coffee beans) and yeast maltase
β
-CD-based 14-valent
β
-glucosidase (almonds),
α
possess deep catalytic sites, a priori not accessible for
multivalent conjugates, the above results can be interpreted in
terms of the relative affinity of the different constructs for the
non-catalytic sites involved in multivalent enzyme inhibition
and their ability to block access of the substrate to the
catalytic site. In the case of
galactosidase, the presence of the iminosugar motif in the
clusters is essential for binding, both fullerene and -CD
β-glucosidase (almonds) and α-
β
architectures being equally efficient. In the case of maltase,
however, the iminosugar and the C-glycoside inhitope are
equally efficient, but the enzyme was very sensitive to the
topology of the conjugates, the fullerene conjugates affording
much stronger inhibition as compared to the
β-CD
counterparts. The inhibitory performance of glycomimetic
clusters towards JBα-man, an enzyme for which the active site
and non-catalytic sites are simultaneously involved in
multivalent enzyme inhibition,14 was found to be strongly
dependent on the nature of the inhitope. As observed with
yeast maltase, both monovalent models 4a and 4b produced
weak inhibition. Yet, replacing the iminosugar inhitope by the
C-glycoside motif had a drastic impact on the inhibition
properties. Strong multivalent effects were indeed produced
Scheme 3 Synthesis of C-glycoside cluster 2a
At the end of the reaction, the product was precipitated with
methanol and dried. The work-up and purification process
proved difficult because of solubility problems and copper
contamination leading to modest yields of the desired product.
Purification of 2a was first achieved by gel permeation
chromatography (Sephadex G-25, H2O). Although this
purification protocol was typically sufficient to remove the
remaining amount of copper catalyst for related glycosylated
for the multivalent iminosugars
1 with rp/n up to 178, whereas
no to modest relative affinity enhancement (rp/n ~ 0.5) were
observed with
2
, the corresponding C-glycoside analogues. The
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higher differences in JBα-man inhibition potencies for form the C-glycoside derivative 4b was a two-fold better
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multivalent than for monovalent derivatives in the iminosugar inhibitor that the iminosugar derivative 4DaO,I: 1a0f.t1e03r9m/Cu6lNtiJv0a1l3e1n1Bt
and C-glycoside series suggest that interactions at non- presentation the iminosugar turned to be a much better
catalytic sites contribute significantly to the binding affinity of inhitope, the
β-CD conjugate 1b becoming about 10-fold more
the multivalent conjugates and that the iminosugar motif is efficient that the corresponding C-glycoside analogue 2b
.
better suited for such binding mode. As for maltase, a notable Amyloglucosidase, a GH15 enzyme, also possesses a relative
influence of the scaffold architecture in the inhibition deep glycone (-1) binding site, but the aglycone (+1) binding
performance was additionally observed, fullerene conjugates site has been shown to be relatively accessible28 and,
being much more efficient than
β
-CD derivatives.
β
-
interestingly, to bind 1-deoxynojirimycin.29 This probably
Glucosidase from bovine liver and amyloglucosidase from explains the strong inhibition properties of the multivalent
Aspergillus niger offer further examples of inhitope-dependent derivatives 1a and 1b, similar to that of the monovalent
and scaffold-dependent multivalent enzyme inhibition. As the reference 4a, in spite of the expected shift in the binding
almond enzyme, bovine liver β-glucosidase belongs to the GH1 mode. A preference of the β-CD architecture was also
family and the catalytic site is not expected to participate in observed in this case.
binding to multivalent conjugates. Whereas in monovalent
Table 1. Glycosidase inhibitory activities (Ki, μM) for monovalent derivative (4) as well as multivalent glycomimetics (1-2)a,b
Iminosugar derivatives
C-Glycoside derivatives
Enzyme
Mono DNJ
12-valent DNJ
14-valent DNJ
Mono
C-glycoside 4b
12-valent
C-glycoside 2a
14-valent
C-glycoside 2b
4a1,2
1a1
1b2
α-glucosidase
Yeast maltase
152
0.71
18 (0.7)b
0.69
270
0.2
217±40
160±32
19±3 (0.9)b
NIa
378±29
Amyloglucosidasec
NIa
β-glucosidase
Almonds
Bovine liver
11
482
95
247
111
NIa
282±45
NIa
NIa
NIa
201±10
25 (1.4)b
α-galactosidase
Green coffee bean
NI
NIa
322
NIa
84
NIa
28
NIa
NIa
NIa
NIa
NIa
NIa
NIa
NIa
β-galactosidase
E. coli
α-mannosidase
Jack Bean
0.15 (178)b
NIa
0.5 (46)b
NIa
524±35
NIa
79±10 (0.5)b
NIa
β-mannosidase
Helix pomatia
NIa
[a] NI: no inhibition detected at
2
mM. [b] Whenever relevant, rp/n values are given in bracket: rp
=
Ki (monovalent
reference)/Ki(glycocluster), n = number of inhitope units. [c] From A. Niger.
with the corresponding monovalent model questionable.
Instead, comparison of analogous multivalent structures let
evaluate the efficiency of different inhitope motifs and
topologies in promoting inhibition of glycosidases. Indeed. The
present body of work revealed that enzymes sensitive to
multivalent enzyme inhibition can exhibit marked differences
in their response to different inhitopes as well as to different
architectural presentations. It is expected that the implications
of these findings will be exploited in the design of potent
multivalent glycosidase inhibitors.
Conclusions
In conclusion, we have synthesized unprecedented multivalent
C-glycosides based on C60-fullerene or
β-cyclodextrin cores.
These compounds were designed as mechanistic probes to
understand the molecular basis of strong inhibition of
glycosidases by multivalent constructs. The inhibition results
further confirmed the hypothesis that large inhibitory
multivalent effects can be obtained when both glycosidase
active site and non-catalytic sites are involved in binding
events, as it is the case for JBα-man. For glycosyl hydrolases
with less accessible active sites, multivalent presentation of
Experimental Section
inhibitory motifs may lead to cancellation, conservation or Solvents were reagent grade and further dried when
enhancement of the inhibition capabilities. In the last two necessary. Dichloromethane (CH2Cl2) was distilled over CaH2
cases, binding of the multivalent conjugate occurs at non- under argon. Pyridine was distilled over KOH under argon and
catalytic sites of the enzyme, which makes a direct comparison stored over KOH. Dry acetonitrile and dry DMF (both over
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molecular sieves) were purchased from commercial vendors 11.0 Hz, 1H; CH2Ph), 5.10 (dd, J = 10.0, 1.5 Hz, 1H; HB-9), 5.14
View Article Online
and used as such. 4-Penten-1-yl tosylate,21 1224 and 1325 were (dd, J = 17.1, 1.5 Hz, 1H; HA-9), 5.85 (ddt,DJO=I:1170..110,3190/C.06NaJn0d13711.0B
prepared according to previously reported procedures. All Hz, 1H; H-8), 7.12-7.18 (m, 2H; HAr), 7.26-7.39 (m, 18H; HAr);
reactions were performed in standard glassware and 13C-NMR (100 MHz, CDCl3) δ 30.0 (C-7), 69.1 (C-6), 71.3 (C-5),
microwave reactions were carried out using Biotage 73.2 (CH2Ph), 73.6 (CH2Ph), 73.9 (C-1), 75.2 (CH2Ph), 75.6
microwave reactor vials and Initiator microwave synthesizer. (CH2Ph), 78.3 (C-4), 80.2 (C-2), 82.6 (C-3), 117.0 (C-9), 127.7,
The reactions were monitored by Thin Layer Chromatography 127.8, 127.9, 127.96, 128.01, 128.07, 128.12, 128.47, 128.53,
(TLC) on aluminium sheets coated with silica gel 60 F254 128.6 (20×CHAr), 134.9 (C-8), 138.2, 138.3, 138.4, 138.9 (4×CAr).
purchased from Merck KGaA. Visualization was accomplished
with UV light (at 254 nm) and exposure to TLC stains,
2,3,4,6-Tetra-O-benzyl-1-C-[6-(tosyloxy)hex-2-en-1-yl]-α-D-
phosphomolybdic acid or potassium permanganate, followed
by heating. Flash column chromatographies were carried out
on silica gel 60 (230-400 mesh, 40-63 µm) purchased from
Merck KGaA. 1H and 13C NMR experiments were carried out at
298K on either a Bruker Avance 300 MHz or a Bruker Avance III
HD 400 MHz spectrometer. The chemical shifts are reported as
δ values in parts per million (ppm) relative to residual solvent
signals used as an internal reference. The exponents “A” or “B”
will be used for diastereotopic protons, “A” is assigned to the
proton with the lowest chemical shift and “B” to the proton
with the highest chemical shift. Assignments of 1H and 13C
signals were made by DEPT, 1H-1H COSY, HSQC and HMBC
experiments. For convenience the assignation of 1H and 13C for
all the molecules were based on the same numbering (see
Supporting information). Optical rotations were measured at
589 nm (sodium lamp) and 20 °C on either a Perkin-Elmer 341
polarimeter or an Anton Paar MCP 200 polarimeter with a path
length of 1 dm. The concentration (c) is indicated in gram per
deciliter. Infrared (IR) spectra were recorded neat on a Perkin-
Elmer Spectrum Two FT-IR spectrometer. High-resolution
(HRMS) electrospray ionization-time-of-flight (ESI-TOF) mass
glucopyranose 7. An oven-dried Schlenk tube was charged with 6a
(52 mg, 0.0921 mmol), 4-penten-1-yl tosylate (120 mg, 0.4993
mmol, 5.4 eq.), 1,4-benzoquinone (2 mg, 0.0185 mmol, 0.2 eq.) and
Grubbs II catalyst (7.7 mg, 0.0091 mmol, 0.1 eq.). The Schlenk tube
was evacuated under vacuum and backfilled with argon (three
cycles). Dry and degassed CH2Cl2 (1.2 mL) was added and the
reaction mixture was stirred at rt under argon for 6 h. Additional
Grubbs 2nd generation catalyst (3.9 mg, 0.0046 mmol, 0.05 eq.) was
added to the reaction mixture, followed by additional stirring for 64
h. The reaction mixture was concentrated under reduced pressure.
The crude residue was purified by flash column chromatography
(EtOAc/petroleum ether, 15:85 to 20:80; solid deposition) to afford
7 (49 mg, 68%) as a mixture of E/Z isomers (colorless oil).
Rf 0.34 (EtOAc/petroleum ether, 20:80); IR (neat) 1175, 1360 cm-1;
1H-NMR (400 MHz, CDCl3) δ 1.62-1.72 (m, 2H; CH2-11), 1.96-2.08
(m, 2H; CH2-10), 2.31-2.46 (m, 5H; CH2-7 and CH3), 3.53-3.81 (m, 6H;
H-2, H-3, H-4, H-5 and CH2-6), 3.96-4.06 (m, 3H; H-1 and CH2-12),
4.43-4.50 (m, 2H; CH2Ph), 4.57-4.64 (m, 2H; CH2Ph), 4.65-4.71 (m,
1H; CH2Ph), 4.77-4.83 (m, 2H; CH2Ph), 4.90-4.96 (m, 1H; CH2Ph),
5.30-5.45 (m, 2H; H-8 and H-9), 7.10-7.15 (m, 2H; HAr), 7.22-7.36 (m,
20H; HAr), 7.76-7.80 (m, 2H; HAr); 13C-NMR (100 MHz, CDCl3) δ 21.8
(CH3), 28.5, 28.6, 28.7 (C-7, C-10, C-11), 69.2 (C-6), 70.1 (C-12), 71.2
(C-5), 73.2 (CH2Ph), 73.6 (CH2Ph), 74.0 (C-1), 75.2 (CH2Ph), 75.6
(CH2Ph), 78.3 (C-4), 80.3 (C-2), 82.5 (C-3), 127.6, 127.7, 127.8,
127.88, 127.93, 128.01, 128.03, 128.05, 128.12, 128.47, 128.54,
128.6 (C-8, C-9, 24×CHAr), 134.9 (C-8), 133.4, 138.2, 138.3, 138.4,
138.9, 144.8 (6×CAr); HRMS (ESI) m/z 799.3279 ([M+Na]+, calcd for
C47H52O8SNa: 799.3275).
spectra were recorded on
a Bruker micrOTOF® mass
spectrometer. Residual copper ions traces were measured on
an Inductively Coupled Plasma Optical Emission Spectrometer
(Varian 720-ES) at 324.754 nm.
α
-1-
At rt, allyltrimethylsilane (1.75 mL, 11.01 mmol, 3 eq.) was
added to a solution of (2.01 g, 3.62 mmol) in dry MeCN (20
C-Allyl-2,3,4,6-tetra-O-benzyl-D-glucopyranose (6a).
5
mL) and the solution was stirred for 30 min. The solution was
then cooled to 0 °C and TMSOTf (0.66 mL, 3.65 mmol, 1 eq.)
was added dropwise. The reaction mixture was allowed to
warm to rt and stirred for 19 h. Et2O was added and the
organic layer was successively washed with sat. aqueous
NaHCO3, water and brine, dried over Na2SO4, filtered and
concentrated under reduced pressure. The crude residue was
purified by flash column chromatography (Et2O/petroleum
ether, 20:80) to afford 6a (1.49 g, 73%) and 6b (150 mg, 7%) as
white solids. The analytical data of 6a were in complete
agreement with those reported in the literature.20
Rf 0.42 (Et2O/petroleum ether, 20:80); 1H-NMR (400 MHz,
CDCl3) δ 2.46-2.58 (m, 2H; CH2-7), 3.61-3.86 (m, 6H; H-2, H-3,
H-4, H-5 and CH2-6), 4.16 (ddd, J = 10.2, 5.4, 4.8 Hz, 1H; H-1),
4.50 (d, J = 12.2 Hz, 1H; CH2Ph), 4.50 (d, J = 10.6 Hz, 1H;
CH2Ph), 4.65 (d, J = 12.2 Hz, 1H; CH2Ph), 4.66 (d, J = 11.6 Hz,
1H; CH2Ph), 4.72 (d, J = 11.6 Hz, 1H; CH2Ph), 4.83 (d, J = 11.0
Hz, 1H; CH2Ph), 4.84 (d, J = 10.6 Hz, 1H; CH2Ph), 4.96 (d, J =
2,3,4,6-Tetra-O-acetyl-1-C-[6-(tosyloxy)hexyl]-α-D-glucopyranose
8. To a solution of 7 (144 mg, 0.1853 mmol) in THF (4 mL) and
MeOH (2 mL) were added 1M aqueous HCl (0.25 mL) and Pd/C (20
mg, 0.0188 mmol, 10% Pd on C, 0.1 eq.). The flask was evacuated
under vacuum and backfilled with argon (four cycles), then
evacuated under vacuum and backfilled with H2 (four cycles). The
reaction mixture was stirred under an atmosphere of H2 (balloon) at
rt for 4 h. The reaction mixture was then filtered through a pad of
Celite, previously washed first with 1M aqueous HCl (at least 250
mL) and then with water. The catalyst was washed with MeOH, and
the filtrate was concentrated under reduced pressure. The crude
residue was dissolved in a mixture of dry pyridine (4 mL) and Ac2O
(4 mL), then DMAP (4.5 mg, 0.0368 mmol, 0.2 eq.) was added to the
solution. The reaction mixture was stirred at rt for 5 h. Solvents
were evaporated under reduced pressure and the crude residue
was purified by flash column chromatography (EtOAc/petroleum
ether, 30:70) to afford 8 (74 mg, 68%) as a colorless oil.
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 7 of 11
New Journal of Chemistry
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ARTICLE
ꢀ ꢂꢄꢅ
Rf 0.43 (EtOAc/petroleum ether, 40:60); ꢁ
+ 40 (c 1.0, CHCl3); IR 2), 74.3 (C-5), 75.3 (C-3), 77.2 (C-1); HRMS (ESI) m/z 312.1556
ꢃ
View Article Online
+
(neat) 1175, 1365, 1744 cm-1; 1H-NMR (400 MHz, CDCl3) δ 1.22-1.48 ([M+Na] , calcd for C12H23N3O5Na : 312.1530).
DOI: 10.1039/C6NJ01311B
(m, 7H; HA-7, CH2-8, CH2-9 and CH2-10), 1.59-1.68 (m, 2H; CH2-11),
1.69-1.80 (m, 1H; HB-7), 2.02 (s, 3H; C(O)CH3), 2.03 (s, 3H; C(O)CH3), 2,3,4,6-Tetra-O-acetyl-1-C-[6-(4-cyclopropyl-1H-1,2,3-triazol-1-
2.04 (s, 3H; C(O)CH3), 2.07 (s, 3H; C(O)CH3), 2.45 (s, 3H; CH3), 3.79 yl)hexyl]-α-D-glucopyranose 11. To a solution of 9 (29 mg, 0.0634
(ddd, J = 9.4, 5.3 and 2.3 Hz, 1H; H-5), 4.01 (t, J = 6.4 Hz, 2H; CH2- mmol) and ethynylcyclopropane (0.02 mL, 0.2363 mmol, 3.7 eq.) in
12), 4.07 (dd, J = 12.1, 2.3 Hz, 1H; HA-6), 4.09-4.16 (m, 1H; H-1), 4.22 DMF (1.7 mL) in a microwave vial was added a yellow suspension of
(dd, J = 12.1, 5.3 Hz, 1H; HB-6), 4.96 (dd, J = 9.4, 8.8 Hz, 1H; H-4), CuSO4·5H2O (2 mg, 0.0080 mmol, 0.13 eq.) and sodium ascorbate (3
5.05 (dd, J = 9.5, 5.7 Hz, 1H; H-2), 5.30 (dd, J = 9.5, 8.8 Hz, 1H; H-3), mg, 0.0151 mmol, 0.24 eq.) in water (0.45 mL). The resulting
7.35 (d, J = 8.1 Hz, 2H; HAr), 7.78 (d, J = 8.1 Hz, 2H; HAr); 13C-NMR suspension was stirred and heated under microwave irradiation at
(100 MHz, CDCl3) δ 20.7 (C(O)CH3), 20.75 (2×C(O)CH3), 20.77 80 °C for 30 min. Additional ethynylcyclopropane (0.02 mL, 0.2363
(C(O)CH3), 21.7 (CH3), 24.8, 25.2, 25.4, 28.6, 28.8 (C-7, C-8, C-9, C- mmol, 3.7 eq.) was added and the reaction mixture was stirred and
10, C-11), 62.5 (C-6), 68.6 (C-5), 69.0 (C-4), 70.48, 70.52 (C-2, C-3, C- heated under microwave irradiation at 80 °C for another 30 min.
12), 72.5 (C-1), 127.9 (2×CHAr), 129.9 (2×CHAr), 133.2 (CAr), 144.8 The reaction mixture was diluted with water and extracted with
(CAr), 169.6, 169.7, 170.2, 170.7 (4×C(O)CH3); HRMS (ESI) m/z EtOAc (3×). The combined organic extracts were washed with water
609.1897 ([M+Na]+, calcd for C27H38O12SNa: 609.1976).
(3×), dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was diluted with mixture of
9. MeCN/H2O/NH4OH (10:1:1) and filtered through a pad of silica gel,
a
2,3,4,6-Tetra-O-acetyl-1-C-(6-azidohexyl)-α- -glucopyranose
D
NaN3 (41 mg, 0.6307 mmol, 4.6 eq.) was added to a solution of 8 using the same mixture as eluent. Copper salts precipitated as a
(80 mg, 0.1364 mmol) in dry DMF (2 mL), and the reaction mixture blue powder and remained at the top of the silica gel pad. The
was stirred at rt under argon for 17 h. The solvent was evaporated filtrate was concentrated under reduced pressure and the crude
under reduced pressure and the residue was diluted with EtOAc residue was purified by flash column chromatography
and washed with water (2×) and brine. The organic layer was dried (EtOAc/petroleum ether, 70:30) to afford 11 (30 mg, 90%) as a
over Na2SO4, filtered and concentrated under reduced pressure. colorless solid.
ꢀ ꢂꢄꢅ
The crude residue was purified by flash column chromatography Rf 0.36 (EtOAc/petroleum ether, 70:30); ꢁ
+ 54 (c 1.0, CHCl3); IR
ꢃ
(EtOAc/petroleum ether, 30:70) to afford 9 (57 mg, 91%) as a (neat) 1746 cm-1; 1H-NMR (400 MHz, CDCl3) δ 0.78-0.96 (m, 4H;
colorless solid.
CH2-16 and CH2-17), 1.18-1.49 (m, 7H; HA-7, CH2-8, CH2-9 and CH2-
+ 63 (c 2.0, CHCl3); IR 10), 1.67-1.80 (m, 1H; HB-7), 1.81-1.96 (m, 3H; CH2-11 and H-15),
ꢀ ꢂꢄꢅ
Rf 0.30 (EtOAc/petroleum ether, 30:70);
ꢁ
ꢃ
1
(neat) 1743, 2096 cm-1; H-NMR (400 MHz, CDCl3) δ 1.22-1.50 (m, 2.01 (s, 6H; 2×C(O)CH3), 2.03 (s, 3H; C(O)CH3), 2.05 (s, 3H; C(O)CH3),
7H; HA-7, CH2-8, CH2-9 and CH2-10), 1.53-1.63 (m, 2H; CH2-11), 1.70- 3.73-3.81 (m, 1H; H-5), 4.02-4.09 (m, 1H; HA-6), 4.09-4.16 (m, 1H; H-
1.81 (m, 1H; HB-7), 2.00 (s, 3H; C(O)CH3), 2.01 (s, 3H; C(O)CH3), 2.02 1), 4.17-4.31 (m, 3H; HB-6 and CH2-12), 4.91-4.99 (m, 1H; H-4), 5.00-
(s, 3H; C(O)CH3), 2.06 (s, 3H; C(O)CH3), 3.25 (t, J = 6.8 Hz, 2H; CH2- 5.08 (m, 1H; H-2), 5.24-5.32 (dd, J = 9.2 Hz, 1H; H-3), 7.20 (s, 1H; H-
12), 3.78 (ddd, J = 9.4, 5.3 and 2.4 Hz, 1H; H-5), 4.05 (dd, J = 12.1, 13); 13C-NMR (100 MHz, CDCl3) δ 6.8 (C-15), 7.8 (C-16, C-17), 20.76,
2.4 Hz, 1H; HA-6), 4.10-4.17 (m, 1H; H-1), 4.21 (dd, J = 12.1, 5.3 Hz, 20.82, 20.9 (4×C(O)CH3), 24.9, 25.3, 26.6, 28.8 (C-7, C-8, C-9, C-10),
1H; HB-6), 4.95 (dd, J = 9.4, 8.9 Hz, 1H; H-4), 5.04 (dd, J = 9.5, 5.8 Hz, 30.3 (C-11), 50.1 (C-12), 62.5 (C-6), 68.7 (C-5), 69.0 (C-4), 70.5, 70.6
1H; H-2), 5.29 (dd, J = 9.5, 8.9 Hz, 1H; H-3); 13C-NMR (100 MHz, (C-2, C-3), 72.6 (C-1), 119.5 (C-13), 150.3 (C-14), 169.6, 169.8, 170.3,
CDCl3) δ 20.7, 20.8 (4×C(O)CH3), 24.9, 25.2, 26.8, 28.8, 28.9 (C-7, C- 170.7 (4×C(O)CH3); HRMS (ESI) m/z 524.2628 ([M+H]+, calcd for
8, C-9, C-10, C-11), 51.4 (C-12), 62.5 (C-6), 68.7 (C-5), 69.0 (C-4), C25H38N3O9 : 524.2603).
70.5 (C-3), 70.6 (C-2), 72.6 (C-1), 169.6, 169.7, 170.2, 170.7
(4×C(O)CH3); HRMS (ESI) m/z 480.1976 ([M+Na]+, calcd for α-1-C-[6-(4-Cyclopropyl-1H-1,2,3-triazol-1-yl)hexyl]-
D-
C20H31N3O9Na : 480.1953).
glucopyranose 4b. To a solution of 11 (28 mg, 0.0535 mmol) in
MeOH (3 mL) was added Amberlite IRA400 (OH-) resin (1.0 g). The
-glucopyranose 10. To a solution of 9 (130 mixture was stirred using a rotavapor at rt for 16 h. The resin was
α-1-C-(6-Azidohexyl)-
D
mg, 0.2842 mmol) in a mixture of MeOH (7 mL) and H2O (7 mL) was then filtered, rinsed with MeOH and the filtrate was concentrated
added Amberlite IRA400 (OH-) resin (3.5 g). The mixture was stirred under reduced pressure. The crude residue was purified by flash
using a rotavapor at rt for 18 h. The resin was then filtered, rinsed column chromatography (CH2Cl2/MeOH, 90:10 to 85:15) to afford
with MeOH and the filtrate was concentrated under reduced 4b (17 mg, 89%) as a colorless oil.
ꢀ ꢂꢄꢅ
pressure. The crude residue was purified by flash column Rf 0.20 (CH2Cl2/MeOH, 90:10); ꢁ
+ 53 (c 1.0, MeOH); IR (neat)
ꢃ
1
chromatography (CH2Cl2/MeOH, 90:10) to afford 10 (70 mg, 85%) as 3340 cm-1; H-NMR (400 MHz, CD3OD) δ 0.72-0.79 (m, 2H; CH2-16
a colorless oil.
Rf 0.28 (CH2Cl2/MeOH, 90:10); ꢁ
or CH2-17), 0.91-1.00 (m, 2H; CH2-16 or CH2-17), 1.24-1.45 (m, 5H;
+ 69 (c 2.0, MeOH); IR (neat) HA-8, CH2-9 and CH2-10), 1.47-1.71 (m, 3H; CH2-7 and HB-8), 1.83-
ꢀ ꢂꢄꢅ
ꢃ
2096, 3353 cm-1; 1H-NMR (400 MHz, CD3OD) δ 1.26-1.48 (m, 5H; 1.99 (m, 3H; CH2-11 and H-15), 3.22 (dd, J = 9.5, 8.5 Hz, 1H; H-4),
HA-7, CH2-9 and CH2-10), 1.49-1.75 (m, 5H; HB-7, CH2-8 and CH2-11), 3.34-3.41 (m, 1H; H-5), 3.50 (dd, J = 9.4, 8.5 Hz, 1H; H-3), 3.57 (dd, J
3.21-3.34 (m, 3H; H-4 and CH2-12), 3.35-3.43 (m, 1H; H-5), 3.50-3.68 = 9.4, 5.6 Hz, 1H; H-2), 3.61 (dd, J = 11.5, 5.6 Hz, 1H; HA-6), 3.77 (dd,
(m, 3H; H-2, H-3 and HA-6), 3.75-3.82 (m, 1H; HB-6), 3.84-3.92 (m, J = 11.5, 2.3 Hz, 1H; HB-6), 3.86 (ddd, J = 10.3, 5.6 and 3.9 Hz, 1H; H-
1H; H-1); 13C-NMR (100 MHz, CD3OD) δ 25.2, 26.5, 27.8, 29.8, 30.0 1), 4.32 (t, J = 7.0 Hz, 2H; CH2-12), 7.67 (s, 1H; H-13); 13C-NMR (100
(C-7, C-8, C-9, C-10, C-11), 52.4 (C-12), 63.1 (C-6), 72.4 (C-4), 73.1 (C- MHz, CD3OD) δ 7.3 (C-15), 8.2 (C-16, C-17), 25.3 (C-7), 26.4 (C-8),
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Page 8 of 11
ARTICLE
Journal Name
27.4 (C-10), 29.8 (C-9), 31.2 (C-11), 51.2 (C-12), 63.2 (C-6), 72.5 (C- 68.6 (C-6'), 70.32, 70.35 (C-4, C-5'), 71.4 (C-2), 72.4 (C-V5ie),w7A2rt.ic8le(CO-n3lin')e,
DOI: 10.1039/C6NJ01311B
4), 73.1 (C-2), 74.4 (C-5), 75.3 (C-3), 77.2 (C-1), 121.8 (C-13), 151.4 73.5 (C-3), 75.7 (C-1), 79.4 (C-2'), 81.9 (C-4'), 100.4 (C-1'), 124.6,
(C-14); HRMS (ESI) m/z 356.2200 ([M+H]+, calcd for C17H30N3O5
:
125.2 (C-9', C-12'), 143.4, 143.9 (C-8', C-11'); HRMS (ESI) m/z
1430.4807 ([M+4H]4+, calcd for C252H424N42O105: 1430.4799).
356.2180).
Compound 3. To a solution of heptakis(2,6-di-O-propargyl)cyclo Compound 2a. A mixture of 13 (37.3 mg, 0.018 mmol), 4b (68 mg,
maltoheptaose 12 (13 mg, 0.0078 mmol) and 9 (55 mg, 0.1202 0.235 mmol), CuSO4.5H2O (1.3 mg, 52 µmol) and sodium ascorbate
mmol, 15.4 eq.) in DMF (1.2 mL) in a microwave vial was added a (3.5 mg, 0.018 mmol) in CH2Cl2/H2O/DMSO (0.6 : 0.4 : 0.4 mL) was
yellow suspension of CuSO4·5H2O (3 mg, 0.0120 mmol, 1.5 eq.) and stirred at 30°C under argon for 3 days. MeOH (10 mL) was added.
sodium ascorbate (5 mg, 0.0252 mmol, 3.2 eq.) in water (0.3 mL). The orange-brown precipitate was filtered and washed with MeOH
The resulting suspension was stirred and heated under microwave and acetone. Two successive gel permeation chromatographies
irradiation at 80 °C for 30 min. The reaction mixture was diluted (Sephadex G-25, H2O) followed by filtration over a QuadraSilTM
with water and extracted with EtOAc (3×10 mL). The combined Mercaptopropyl column gave 2a (33 mg, 34 %) as an orange-brown
organic extracts were washed with water, dried over Na2SO4, glassy product. IR (neat) 3340 (br, OH), 1738 (C=O) cm-1; UV/Vis
filtered and concentrated under reduced pressure. The residue was (H2O/DMSO 10:0.2) λmax(ε) = 235 (179900), 273 (sh, 66400), 337
1
diluted with a mixture of MeCN/H2O/NH4OH (15:0.5:0.5) and (sh, 31700) nm; H NMR (400 MHz, D2O/DMSO-d6 4:1) δ 7.84 (s,
filtered through a pad of silica gel, using the same mixture as 12H), 4.80 (m, 36H), 4,42-4,02 (m, 60H), 3.66 (m, 12H), 3.17 (m,
eluent. Copper salts precipitated as a blue powder and remained at 12H), 3.01 (m, 12H), 2.62 (m, 24H), 2.05-1.57 (m, 60H), 1.54-1.03
the top of the silica gel pad. The filtrate was concentrated under (m, 84H); 13C NMR (100 MHz, DMSO-d6) δ 163.2, 146.2, 145.4,
reduced pressure and the crude residue was purified by flash 141.2, 122.5, 75.5, 74.2, 73.8, 71.9, 71.3, 69.2, 67.0, 61.9, 49.9,
column chromatography (CH2Cl2/MeOH, 98:2 to 95:5) to afford 3 46.0, 30.1, 28.8, 28.2, 26.4, 25.4, 24.3, 21.9; ES-TOF-MS m/z
(52 mg, 83%) as a pale yellow oil.
1889.04 ([M+3Na]3+, calcd for C282H360N36O84Na3: 1889.02).
ꢀ ꢂꢄꢅ
Rf 0.34 (CH2Cl2/MeOH, 96:4); ꢁ
+ 57 (c 2.0, CHCl3); IR (neat)
ꢃ
1744, 3424 cm-1; 1H-NMR (400 MHz, CDCl3) δ 1.19-1.51 (m, 98H; HA- Inhibition studies with commercial enzymes
7, CH2-8, CH2-9 and CH2-10), 1.68-1.81 (m, 14H; HB-7), 1.81-1.95 (m, Inhibition constant (Ki) values were determined by
28H; CH2-11), 1.95-2.07 (several s, 168H; 56×C(O)CH3), 3.28-3.54 spectrophotometrically measuring the residual hydrolytic activities
(m, 14H; H-2' and H-4'), 3.56-3.73 (m, 7H; H-5'), 3.73-3.83 (m, 14H; of the glycosidases against the respective p-nitrophenyl α- or β-
D-
H-5), 3.91 (dd, J = 8.9 Hz, 7H; H-3'), 4.04 (dt, J = 12.1, 2.3 Hz, 14H; glycopyranoside, o-nitrophenyl β- -galactopyranoside (for β-
D
HA-6), 4.08-4.15 (m, 14H; H-1), 4.21 (dd, J = 12.1, 4.9 Hz, 14H; HB-6), galactosidases) in the presence of the C-glycosides. Each a ssay was
4.30 (t, J = 7.3 Hz, 14H; CH2-12), 4.35 (t, J = 7.3 Hz, 14H; CH2-12), performed in phosphate buffer or phosphate-citrate buffer (for α-
4.42-4.68 (m, 14H; CH2-6'), 4.69-4.81 (m, 7H; H-1'), 4.84-5.02 (m, or β- mannosidase and amyloglucosidase) at the optimal pH of each
14H; CH2-7' or CH2-10'), 4.95 (dd, J = 9.1 Hz, 14H; H-4), 5.02 (dd, J = enzyme. The reactions were initiated by addition of enzyme to a
9.5, 5.7 Hz, 14H; H-2), 5.23-3.31 (m, 28H; H-3 and CH2-7' or CH2- solution of the substrate in the absence or presence of various
10'), 7.54-7.80 (m, 14H; H-9' and H-12'); 13C-NMR (100 MHz, CDCl3) concentrations of inhibitor. The mixture was incubated for 10-30
δ 20.7, 20.78, 20.82 (CH3), 25.0, 25.3, 26.7, 28.8 (C-7, C-8, C-9, C- min at 37 ºC or 55 ºC (for amyloglucosidase) and the reaction was
10), 30.4 (C-11), 50.26, 50.35 (C-12), 53.5 (C-7’or C-10’), 62.4 (C-6), quenched by addition of 1M Na2CO3. Reaction times were
64.9, 65.2 (C-6’ and C-7' or C-10'), 68.67 (C-5 and C-5’), 68.99 (C-4), appropriated to obtain 10-20% conversion of the substrate in order
70.52, 70.55 (C-2 and C-3), 72.6 (C-1), 73.4 (C-3'), 79.1 (C-2'), 83.0 to achieve linear rates. The absorbance of the resulting mixture was
(C-4'), 101.7 (C-1'), 122.9, 123.6 (C-9', C-12'), 144.1, 144.7 (C-8', C- determined at 405 nm. Approximate values of Ki were determined
11'), 169.6, 169.7, 170.2, 170.7 (C(O)CH3); HRMS (ESI) m/z using a fixed concentration of substrate (around the Km value for
2713.4838 ([M+3Na]3+, calcd for C364H532N42O161Na3: 2713.4844).
the different glycosidases) and various concentrations of inhibitor.
Full Ki determinations and enzyme inhibition mode were
Compound 2b. To a solution of 3 (50 mg, 0.0062 mmol) in a mixture determined from Dixon plots.
of MeOH (1 mL) and water (1 mL) was added Amberlite IRA400
(OH-) resin (2.20 g). The mixture was stirred using the rotavapor at
Acknowledgements
rt for 20 h. The resin was then filtered, rinsed with MeOH and
water, and the filtrate was concentrated under reduced pressure.
The solvents were evaporated under reduced pressure to afford 2b
(33 mg, 93%) as a pale yellow oil.
The authors are grateful to financial supports from the Institut
Universitaire de France (IUF), the CNRS (UMR 7509), the
University of Strasbourg and the International Centre for
Frontier Research in Chemistry (icFRC). FS thanks the French
Department of Research for a doctoral fellowship. The Spanish
Ministerio de Economía y Competitividad (MINECO, contract
numbers SAF2013-44021-R) and cofinancing from the
European Regional Development Funds (FEDER and FSE) are
also thanked.
ꢁ
+ 80 (c 1.0, H2O); IR (neat) 3369 cm-1; 1H-NMR (300 MHz,
ꢀ ꢂꢄꢅ
ꢃ
D2O) δ 1.10-1.94 (m, 140H; CH2-7, CH2-8, CH2-9, CH2-10 and CH2-
11), 3.30-3.99 (m, 140H; H-1, H-2, H-2’, H-3, H-3’, H-4, H-4’, H-5, H-
5’, CH2-6 and CH2-6’), 4.15-4.65 (m, 42H; CH2-7' or CH2-10', CH2-12),
4.80-5.04 (m, 21H; H-1', CH2-7' or CH2-10'), 7.92 (br s, 7H; H-9' or H-
12'), 8.09 (s, 7H; H-9' or H-12'); 13C-NMR (75.5 MHz, D2O) δ 23.4,
23.5, 24.4, 24.5, 25.55, 25.62, 27.8, 27.9, 29.5, 29.6 (C-7, C-8, C-9, C-
10, C-11), 50.2, 50.4 (C-12), 61.1, 61.2 (C-6), 63.4, 64.5 (C-7', C-10'),
8 | J. Name., 2012, 00, 1-3
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ARTICLE
Journal Name
29 E. M. S. Harris, A. E. Aleshin, L. M. Firsov, R. B. Honzatko,
Biochemistry, 1993, 32, 1618-1626.
View Article Online
DOI: 10.1039/C6NJ01311B
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Multivalent C-glycosides based on C60 or β-cyclodextrine cores were designed to probe the influence
DOI: 10.1039/C6NJ01311B
of inhitopes in glycosidase binding events.