Towards dynamic drug design: Identification and optimization of β-galactosidase inhibitors from a dynamic hemithioacetal system

Article abstract of DOI:10.1002/cbic.201000158

A discovery strategy relying on the identification of fragments through resolution of a constitutional dynamic system, coupled to subsequent static ligand design and optimization, is demonstrated. The strategic design and synthesis of the best molecular fragments identified from a dynamic hemithioacetal system into static ligand structures yielded a range of β-galactosidase inhibitors. Two series of structures mimicking the hemithioacetal motif were envisaged: thioglycosides and C-glycosides. Inhibition studies provided important structural information for the two groups, and 1-thiobenzyl-b-d-galactopyranoside demonstrated the best inhibitory effects.

Full text of DOI:10.1002/cbic.201000158

DOI: 10.1002/cbic.201000158
Towards Dynamic Drug Design: Identification and
Optimization of b-Galactosidase Inhibitors from a Dynamic
Hemithioacetal System
Rꢀmi Caraballo, Morakot Sakulsombat, and Olof Ramstrçm*[a]
A discovery strategy relying on the identification of fragments
through resolution of a constitutional dynamic system, cou-
pled to subsequent static ligand design and optimization, is
demonstrated. The strategic design and synthesis of the best
molecular fragments identified from a dynamic hemithioacetal
system into static ligand structures yielded a range of b-galac-
tosidase inhibitors. Two series of structures mimicking the
hemithioacetal motif were envisaged: thioglycosides and C-
glycosides. Inhibition studies provided important structural in-
formation for the two groups, and 1-thiobenzyl-b-d-galactopyr-
anoside demonstrated the best inhibitory effects.
Introduction
Emerging in the mid-1990s, fragment-based methods have
been increasingly adopted in drug-discovery processes, dem-
onstrated as promising, potent, and powerful alternatives to
more conventional methods.[1a–f] Thus, fragment-based drug
discovery (FBDD) is usually associated with facilitated handling
and smaller compound collections than other high-throughput
chemistry protocols.[1b,c,f] In addition, fragment-based methods
offer more efficient screening of the chemical space than other
techniques.[2a–b] The principle of FBDD relies on the successive
identification, chemical modification, optimization, and combi-
nation of small molecular fragments exhibiting low affinities
for a given biological target/receptor. As in conventional drug
design, these structural changes aim to increase the protein–
ligand binding affinity; this leads to the synthesis and discov-
ery of potent drug leads. However, several challenges of the
fragment-based method in drug discovery remain, especially in
the identification and linkage approaches of the different mo-
lecular fragments constituting the final lead.[1d] The recent and
increasingly adopted concept of constitutional dynamic
chemistry (CDC) offers a way to meet these challenges.[3a–f]
Starting from molecular fragments (building blocks), the rever-
sible formation of component associations through covalent or
noncovalent bonds has proven successful for the formation
and identification of biologically relevant ligands. Furthermore,
the capability of a dynamic system under specific conditions to
adapt and reorganize itself in the presence of a receptor can
lead to amplification of the best component associations,
which facilitates the identification process. Therefore, target-
driven compound screening combined with reversible self-as-
sembly properties renders the CDC concept a rapid and con-
venient approach for identification of potential leads in FBDD,
and, more generally, in the whole drug-discovery area.[1f,3e,4a–c]
However, the dynamic character of the system generally re-
strains the protein–ligand binding affinity. As a consequence,
the molecular fragments reversibly associated in a dynamic
system, generally do not fully exhibit their potential as ligands
or inhibitors.
A way to overcome this issue would be to irreversibly link
these component associations without influencing the ligand
structure and geometry. This strategy amounts to a dynamic
drug design (DDD) strategy that relies on the identification of
fragments through dynamic resolution, coupled to subsequent
static ligand design and optimization. To date, this approach
remains unexplored, except for examples of “frozen” dynamic
systems generated in order to facilitate the ligand characteriza-
tion.[5a–d] This challenge has thus been addressed in the present
study, in which we report on the use of a dynamic-system-
based drug-design strategy for the identification, synthesis,
and evaluation of enzyme inhibitors (Figure 1).
Results and Discussion
Recently, we successfully described the inhibitory properties of
a virtual dynamic hemithioacetal system against b-galactosi-
dase activity.[6] 1H STD NMR experiments, supported by inhibi-
tion studies demonstrated binding and inhibition differences
between the different species, respectively. Among the system
components, two molecular fragments (aldehydes), when suc-
cessively associated with a thiogalactose moiety, exhibited the
highest affinity for the enzyme. Therefore, despite their transi-
ent existence in aqueous solution, the component associations
that result from the addition of 1-thio-b-d-galactose to 4-pyri-
dine carboxaldehyde or isovaleraldehyde demonstrated signifi-
cant inhibition activities (Scheme 1A). However, the very low
[a] R. Caraballo, M. Sakulsombat, Prof. O. Ramstrçm
Department of Organic Chemistry, KTH–Royal Institute of Technology
Teknikringen 30, 10044 Stockholm (Sweden)
Fax: (+46)8-791-2333
E-mail: ramstrom@kth.se
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000158.
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Dynamic System-Based Drug Design
Figure 1. Concept of enzyme inhibitor synthesis designed from a dynamic
hemithioacetal system. Encircled arrows denote dynamic system.
concentration of the active inhibitors during the enzymatic ex-
periments, caused by a very fast dissociation (cleavage) rate of
these products, remains a shortcoming when efficient and
potent drug leads are targeted. Therefore, the design and syn-
thesis of b-galactosidase inhibitors, that are directly inspired
from the previously described dynamic hemithioacetal system
were envisaged (Scheme 1B, C).[6] The molecular fragments
necessary for the inhibition of the enzyme would be, in this
case, irreversibly connected through a scaffold mimic of the
hemithioacetal linkage.
Design of b-galactosidase inhibitors
A key feature of this approach relies on the design and synthe-
sis of irreversible hemithioacetal-like inhibitors. Thus, the struc-
tural features causing hemithioacetal dissociation provide es-
sential information for the design of kinetically stable inhibi-
tors. The reversibility of the hemithioacetal reaction originates
from the presence of vicinal sulfur (e.g., thioether) and hydroxy
groups. Therefore, the removal or replacement of one of these
functionalities would generally lead to the formation of a hem-
ithioacetal-like derivative without any reversibility properties.
In this context, derivatives of thioglycosides and C-glycosides
can be considered as synthetic equivalents of the hemithioace-
tal scaffold (Scheme 1B, C). The designed set of thioglycoside
inhibitors thus resembles the corresponding hemithioacetal
products owing to the presence of a sulfur atom at the glyco-
sidic position of the carbohydrate ring, however, without the
vicinal hydroxy group. The C-glycoside derivatives 1e and 2e,
on the other hand, mimic the dynamic inhibitors owing to the
presence of a hydroxy group analogous to the hemithioacetal
scaffold. The design of C-glycoside ketones (1d and 2d) would
provide further structural information regarding the enzyme
selectivity. In order to underline the expected enzyme–sub-
strate specificities, both carbohydrate series contained the ex-
Scheme 1. Library design: A) best b-galactosidase inhibitors from the dy-
namic hemithioacetal library, B) thioglycoside inhibitors, and C) C-glycoside
inhibitors.
pected b-galactosidase inhibitors (galactose derivatives) and
noninhibitors (glucose derivatives). Furthermore, the introduc-
tion of benzyl derivatives, absent from the dynamic system for
compatibility issues, into the library would support the impor-
tance of the constitution and properties of the aromatic-ring
fragment in terms of polarity, hydrogen bonding, and electron-
ic structure. Therefore, after preliminary results from the thio-
glycoside series, benzyl derivatives were primarily chosen for
the C-glycoside series.
Synthesis of the designed set of b-galactosidase inhibitors
b-Galactosidase is a retaining hydrolase. Thus, its function is
based on the specific hydrolysis of b-galactoside derivatives at
the O-glycosidic linkage; this results in the formation of free b-
galactose, and the corresponding alcohol.[7a,b] Thiogalactoside
derivatives, which have an increased resistance towards hydro-
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O. Ramstrçm et al.
lytic systems conferred by the presence of the sulfur atom,
usually provide potent b-galactosidase inhibitors.[8a–d] Previous
studies on this enzyme have demonstrated that hydrophobic
regions in the vicinity of the active site favor the binding of ar-
omatic aglycons over alkyl-chain derivatives.[8d] For these rea-
sons, the designed thioglycoside series, which embraces both
alkyl and aromatic molecular fragments, was expected to pro-
vide a broader panel of inhibitor potency and was therefore
synthesized first (Scheme 2A, B). Thioglycosides are readily ac-
and diketone structures as well as on the solvent.[11] Therefore,
only the most potent molecular-fragment combinations were
selected for the synthesis of the targeted C-glycoside deriva-
tives. In addition to providing the desired b-C-glycosides, the
Lubineau method offers the great advantage, in our case, of
directly yielding the desired b-C-glycoside ketones 1d and 2d
(Scheme 2C). b-C-Glycosides 1e and 2e were subsequently ob-
tained from 1d and 2d by reduction with NaBH4 in methanol.
Unfortunately, compounds 1e and 2e were isolated as insepa-
rable mixtures of diastereoiso-
mers (3:1 and 1:1, respectively).
Therefore, the inhibitory proper-
ties of the pure diastereoisomers
against b-galactosidase activity
could not be evaluated. Howev-
er, the use of a catalytic amount
of acetic acid during the reduc-
tion step of compound 1d con-
veniently afforded compound 1e
with the opposite diastereoiso-
meric composition (1:2). There-
fore, inhibition experiments with
the different mixtures of diaste-
reoisomers would provide an
indication as to the preference
of b-galactosidase.
1H NMR inhibition experiments
Subsequent 1H NMR inhibition
studies were conducted, and
each potential inhibitor was
evaluated for inhibitory activity
Scheme 2. Synthesis of the static library components. A) 4-Pyridyl thioglycoside derivatives: a) 4-(bromomethyl)-
pyridine hydrobromide, KOH, MeOH; B) thioglycoside derivatives: b) BF3.Et2O, RSH, CHCl3; c) NaOMe, MeOH; C) C-
glycoside derivatives: d) dibenzoylmethane, NaHCO3, EtOH/H2O, 908C; e) NaBH4, MeOH.
against b-galactosidase. To com-
pare both systems, the experi-
mental conditions and methods
were chosen to be similar to the
previously described dynamic
cessed in one or two steps by using different approaches.
Thus, the Lewis acid-mediated coupling method between per-
acetylated d-aldopyranose 5 or 6 and a thiol derivative was
used for the synthesis of the desired alkyl and aryl thioglycosi-
des.[9a,b] However, for the synthesis of compounds 1a and 2a, a
different strategy proved more suitable. In this case, 1-thio-b-
d-aldopyranose 3 or 4, which is easily available, was treated
with a bromopyridine derivative in the presence of a base to
generate the thioglycosides (Scheme 2A).
hemithioacetal system.[6] o-Nitrophenyl-b-galactopyranoside
(ONPG), a well-studied b-galactosidase substrate, was used as
substrate during the inhibition studies.[12] The hydrolysis rate of
1
ONPG was followed by H NMR spectroscopy in the presence
or absence of any potential inhibitor. The inhibition experi-
ments were performed with a 1:1 inhibitor/substrate ratio, and
the inhibitory effect was determined as the ratio of the t50
values corresponding to the time required for 50% ONPG hy-
drolysis. The results were highly conspicuous, and the analysis
of the thioglycoside series fully confirmed the enzyme selectiv-
ity. Indeed, solely the galactose-containing derivatives exhibit-
ed significant inhibition, whereas thioglucosides only provided
trace effects on the ONPG hydrolysis rate (Figure 2). Thus, the
inhibitory effect values for compounds 2a–c were estimated at
1.0, 1.1 and 1.3, respectively. In the thiogalactoside series, the
observed inhibitory effects were significantly higher than those
recorded during the previous dynamic hemithioacetal system
studies.[6] In the latter case, an inhibitor/substrate ratio of 5:1
was necessary before significant inhibition was observed.
Synthetic methods of forming C-glycosides, on the other
hand, are less explored and often lead to the formation of the
a-anomer derivatives. However, Lubineau and co-workers re-
cently developed
a straightforward route to b-C-glycosi-
des.[10a,b] With this method, b-C-glycosides are accessed in a
one-pot procedure, successively involving a Knoevenagel con-
densation between a b-diketone carbanion and a reducing car-
bohydrate structure, water elimination, an intramolecular 1,4-
addition, and a retro-Claisen aldolization.[10a] However, the reac-
tion efficiency proved highly dependent on the carbohydrate
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Dynamic System-Based Drug Design
UV inhibition experiments: IC50 and Ki determination
1
The H NMR results provided a good comparison between the
previously described component associations from the dynam-
ic system and their nondynamic synthetic equivalents. In addi-
tion, the results allowed an estimation of the different inhibitor
potencies from which several trends could be drawn. However,
in order to quantify the potencies of the designed inhibitors
against b-galactosidase activity, further investigations were car-
ried out. Thus, kinetic studies followed by UV–visible spectros-
copy on the galactose series were subsequently performed for
the experimental determination of the IC50 values and the in-
hibition constants (Ki; Table 1).[13]
Table 1. IC50 values and inhibition constant (Ki) of the galactosides series
of inhibitors at 258C and pH 7.4.
Figure 2. Inhibitory effect of the thiogalactoside and C-glycoside species.
[a] diastereoisomeric ratio 3:1, [b] diastereoisomeric ratio 1:2.
Galactoside
IC50 [mm]
Ki [mm]
1
2
3
4
5
6
1a
1b
1c
1d
1e[a]
1e[b]
70.9
315.0
18.4
864.8
253.4
176.0
17.6
78.2
4.6
214.6
62.9[c]
43.7[c]
Therefore, the latest results confirm the predicted enhance-
ment of the inhibitor potency from the static system.
The derivatives containing aromatic groups were, as expect-
ed, better inhibitors than the alkyl ones (Figure 2). The 4-pyrid-
yl-substituted thiogalactoside 1a and the alkyl derivative 1b,
respectively, analogous to the two most potent component as-
sociations of the dynamic system, severely decreased the rate
of ONPG hydrolysis (12.4 and 4.9 times, respectively). Com-
pound 1c has previously been reported as an efficient binder
and inhibitor of b-galactosidase.[8d] However, the effect on the
substitution of an endocyclic carbon from the aromatic ring
with a heteroatom remained to be investigated. Thus, com-
pound 1c exhibited the highest inhibitory effect of the panel
(32.9 times); this unambiguously demonstrates the importance
of the aromatic ring structure. Substitution of the benzyl
group with a more hydrophilic pyridine fragment considerably
decreased the inhibitory potency. For comparison, isopropyl-b-
d-1-thiogalactopyranoside (IPTG), a known b-galactosidase in-
hibitor, was also evaluated and an inhibitory effect of about
three times the normal ONPG hydrolysis rate was recorded.
This value, which is slightly lower than the one recorded for
compound 1b, is in complete agreement with the expected
trend, in which a galactoside bearing a longer alkyl chain pro-
vides a stronger b-galactosidase inhibitor.[8a,d]
[a] Diastereoisomeric ratio of 3:1. [b] Diastereoisomeric ratio of 1:2.
[c] Average.
As expected, the results demonstrated the same trend as
the 1H NMR studies, and therefore, the enzyme selectivity
proved analogous in aqueous buffer and under deuterated
buffer conditions. Thus, kinetic analysis of the thiogalactoside
aromatic compounds 1a and 1c, respectively, yielded the most
potent inhibitors with Ki values of 17.6 and 4.6 mm (Table 1, en-
tries 1 and 3). In the C-glycoside series, the ketone derivative
1d (entry 4) afforded the lowest inhibition character with a Ki
value of 0.2 mm. The different diastereomeric mixtures 1ea and
1eb (entries 5 and 6, average Ki values of 62.9 mm and 43.7 mm,
respectively) proved slightly better inhibitors than the thioga-
lactoside 1b (entry 2, Ki value of 78.2 mm).
Influence of the galactoside structure on the b-galactosidase
inhibition
The C-glycoside series also demonstrated the expected b-
galactosidase ligand specificity. As a consequence, the galac-
tose derivatives affected the ONPG hydrolysis rate while the
glucopyranosides demonstrated a weak or no inhibitory effect
(inhibitory effect value of 1.1 and 1.6 for compounds 2d and
2e, respectively). Interestingly, the ketone derivative 1d dem-
onstrated a very poor inhibitory effect (1.5) on the substrate
hydrolysis rate, in contrast to compound 1e, which proved to
be a more efficient inhibitor. Evaluation of the diastereoisomer-
ic mixtures of 1e revealed some differences as to the configu-
ration. Thus, compounds 1e[a] (3:1) and 1e[b] (1:2) decreased
the ONPG hydrolysis rate by approximately 4.9 and 7.4 times,
respectively. These results indicate a slight difference in inhibi-
tory effect for the different diastereoisomers.
1
The results of both the H NMR and UV–visible kinetic studies
confirmed the expected trends and provided new structural in-
formation from the designed set of inhibitors. b-Galactosidase
specificity and selectivity were clearly displayed and proved
highly dependent on the nature of three functional groups/
substituents (Figure 3). Firstly, when considering the glycosidic
linkage atom, a thioether linkage proved to be an efficient
connection between the galactose moiety and the other sub-
stituents. However, the C-galactoside ligands also demonstrat-
ed inhibitory properties, albeit to an apparently lower extent.
This difference could arise from hydrogen-bonding with the ty-
rosine (Tyr503) residue in the enzyme active site,[14b] the hydro-
gen bonding properties of the sulfur atom led to stronger in-
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O. Ramstrçm et al.
Conclusions
In conclusion, we have successfully demonstrated the extend-
ed concept of dynamic system-based drug design (DDD) and
its application in the design and synthesis of b-galactosidase
inhibitors. Based on the identification of fragment combina-
tions from the resolution of a dynamic hemithioacetal system,
a series of kinetically stable (static) compounds was designed
and probed for inhibitory activities. Mimicking the hemithioa-
cetal motif thus led to the synthesis of thioglycoside and C-gly-
coside derivatives in which the molecular fragments were irre-
versibly linked to the glycoside moieties. Inhibition studies re-
vealed the presence of good b-galactosidase inhibitors and
structural differences could be established. Thus, the enzyme
proved highly selective and aromatic thiogalactosides dis-
played the strongest inhibition character among the members
of the compound series. We believe that this approach is very
efficient, and its application in drug discovery could offer a
convenient, cheap and rapid means to potent lead discovery
and synthesis.
Figure 3. Schematic representation of the importance of the substituent for
the inhibition of b-galactosidase. (+) is for the substituents providing very
good inhibition, (=) is for the substituents providing good inhibition, and
(À) is for the substituents providing poor inhibition. (Figure made by using
PyMOL software).
teractions than those of the carbon atom of the C-glycoside
series. Thus, the nature of the glycosidic linkage constitutes
one of the important factors to consider for the design of b-
galactosidase ligands and inhibitors.
Experimental Section
Material and general methods: All commercial reagents and sol-
vents were used as received. b-Galactosidase from E. coli (EC
3.2.1.23, 151 UmgÀ1) was purchased from Fluka. Chemical reactions
were monitored with thin-layer chromatography by using precoat-
ed silica gel 60 (0.25 mm thickness) plates. Flash column chroma-
tography was performed on silica gel 60 (0.040–0.063 mm).
1H NMR and 13C NMR spectra were recorded on a Bruker Avance
400 spectrometer at 400 (100) MHz and/or Bruker Avance DMX
500 at 500 (125) MHz, respectively. d-Buffer solutions were pre-
pared from D3PO4 (85% w/w in D2O) and NaOD (40% w/w in D2O)
using an Ecoscan pH meter equipped with a Ross Combination pH
Semi Microelectrode. Phosphate buffered saline (PBS, pH 7.4) solu-
tions for UV/Vis experiments were prepared from preweighed
preparations (Sigma). UV-Vis measurements were performed on a
Perkin–Elmer, Lambda 759 UV/Vis spectrometer.
Further structural information is provided by the different
substituents at the carbon vicinal to the glycosidic position.
Considering a first substitution, the observed trend clearly un-
derlines a severe decrease in inhibitory potency when a phenyl
group is successively replaced by a pyridine derivative and an
isobutyl group; the latter group providing the lowest effect on
the ONPG hydrolysis rate. This reveals the importance and
nature of the aromatic group, in which replacement of a
benzyl fragment by a pyridine derivative still generates a fairly
good inhibitor, but of lower potency. This effect is to some
degree explained by the presence of hydrophobic regions at
the enzyme active site. A second substitution at the carbon vi-
cinal to the glycosidic position also proved important for the
inhibitory effect. Thus, the presence of a hydrogen atom at
this position yielded the best inhibitors, and in the C-galacto-
side series, the presence of a hydroxy group also resulted in
good inhibition. However, modification of the structure from a
hydroxy functionality to a ketone derivative completely im-
paired the inhibitory properties. These effects could imply the
presence of steric constraints in the active site, which can less
easily accommodate a keto-derivative than its reduced coun-
terpart. However, both structures are sterically likely to fit
inside the b-galactosidase active site, and therefore the hydro-
gen-bond-donor character of the reduced ligands is more
likely to play a major role in the ligand–enzyme binding mode.
Compound 1e possesses hydrogen-bond-donor character, in
contrast to ketone 1d, and could therefore be stabilized at the
enzyme active site by hydrogen bonding with negatively
charged amino acid residues involved in the glycolysis or in its
vicinity.[14a–d] b-Galactosidase selectivity was also demonstrated
and, among the hydroxy derivatives, one diastereoisomer ex-
hibited a slightly greater inhibitory effect.
Synthesis of 1-S-(4-methylpyridyl)-b-d-glycopyranoside: KOH
(21 mg, 0.37 mmol) was added to a mixture of 1-thio-b-d-glycopyr-
anose sodium salt 3 or 4 (80 mg, 0.37 mmol) and 4-(bromomethyl)-
pyridine (111 mg, 0.44 mmol) in MeOH (5 mL). After completion of
the reaction, the solvent was evaporated, and the crude product
was purified by flash column chromatography (eluent: EtOAc/
EtOH/H2O 3:1:0.1) to give the desired products as a light brown
powder (yield 69–77%).
1-S-(4-Methylpyridyl)-b-d-galactopyranoside
(1a):
1H NMR
(500 MHz, D2O): d=8.47 (dd, J=4.8, 1.6 Hz, 2H; H-Ar), 7.49 (dd, J=
4.8, 1.6 Hz, 2H; H-Ar), 4.24 (d, J=9.0 Hz, 1H; H-1), 4.05 (d, J=
13.9 Hz, 1H; H-7), 3.94 (d, J=3.5 Hz, 1H; H-6), 3.93 (d, J=13.9 Hz,
1H; H-7’), 3.66 (d, J=6.2 Hz, 2H; H-3, H-4), 3.52–3.60 (m, 3H; H-2,
H-6’, H-5); 13C NMR (125 MHz, D2O): d=149.5, 148.5, 124.8, 84.6,
78.9, 73.9, 69.5, 68.7, 60.9, 32.7; HRMS (ESI): (m/z): calcd for
C12H18N1O5S1: 288.0900 [M+H]+, found: 288.0902.
1-S-(4-Methylpyridyl)-b-d-glucopyranoside
(2a):
1H NMR
(500 MHz, D2O): d=8.48 (dd, J=4.6, 1.3 Hz, 2H; H-Ar), 7.48 (dd, J=
4.6, 1.3 Hz, 2H; H-Ar), 4.32 (d, J=9.7 Hz, 1H; H-1), 4.05 (d, J=
13.9 Hz, 1H; H-7), 3.93 (d, J=13.9 Hz, 1H; H-7’), 3.79 (dd, J=12.6,
2.3 Hz, 1H; H-6), 3.65 (dd, J=12.6, 5.7 Hz, 1H; H-6’), 3.28–3.42 (m,
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Dynamic System-Based Drug Design
4H; H-5, H-2, H-3, H-4); 13C NMR (125 MHz, D2O): d=149.4, 148.5,
124.8, 84.2, 79.7, 77.2, 77.1, 69.4, 60.7, 32.7; HRMS (ESI): (m/z): calcd
for C12H18NO5S: 288.0900 [M+H]+, found: 288.0903.
Synthesis
of
b-C-glycosides:[11]
Glycopyranose
(500 mg,
2.78 mmol) dibenzoylmethane (847 mg, 3.78 mmol) and NaHCO3
(350 mg, 4.17 mmol) were dissolved in EtOH/H2O (4:1, 5 mL) and
heated under reflux overnight. The reaction mixture was acidified
to pH 2 with an exchange acidic resin, and filtered. After removal
of the solvent, the dark solid crude product was dissolved in water
and extracted with diethyl ether. The combined organic layers
were dried over MgSO4, and filtered. The crude product was puri-
fied by flash column chromatography (eluent: EtOAc/MeOH 9:1) to
give the b-C-glycosides ketone 1d (yield 38%) and 2d (yield 64%).
b-C-Glycoside ketone 1d or 2d (250 mg, 0.89 mmol) was dissolved
in MeOH (15 mL), and NaBH4 (44.7 mg, 1.07 mmol) was carefully
added portionwise. The resulting mixture was evaporated and pu-
rified by flash column chromatography (eluent: EtOAc/MeOH 9:1)
to yield an inseparable mixture of diastereoisomer 1e (yield 78%)
and 2e (yield 86%).
Synthesis of 1-S-b-d-glycosides:[9a,b] 1,2,3,4,6-Penta-O-acetyl-b-d-
glycopyranose (500 mg, 1.28 mmol) and
a
thiol derivative
(1.54 mmol) were dissolved in dry CHCl3 (5 mL), and BF3·Et2O
(0.81 mL, 6.41 mmol) was added dropwise. The resulting mixture
was allowed to react for 15 min before addition of aqueous
NaHCO3 to quench the reaction. The combined organic layers were
successively washed with water and brine, dried over MgSO4, fil-
tered, and concentrated. The crude product was purified by
column chromatography on silica gel (eluent: hexane/EtOAc 3:1)
to give the 1-S-2,3,4,6-tetra-O-acetyl-b-d-glycopyranoside derivative
(yield 90–98%), which was directly dissolved in a solution of
NaOMe in MeOH (1.1 equiv, 15 mL). After completion of the reac-
tion, the resulting mixture was neutralized by exchanging acidic
resin, thereby quantitatively yielding the corresponding 1-S-b-d-
glycosides.
1
b-C-Galactoside alcohol (1e): Mixture of diastereoisomers. H NMR
(500 MHz, D2O): d=7.34–7.47 (m, 5H; 5H’, H-Ar, H’-Ar), 5.01 (dd,
J=9.9, 3.2 Hz, 1H; H-8), 4.99 (dd, J=9.5, 5.7 Hz, 1H’, H’-8), 3.96 (d,
J=3.2 Hz, 1H; H-4), 3.88 (d, J=3.1 Hz, 1H’, H’-4), 3.75 (dd, J=11.8,
8.0 Hz, 1H’, H’-6), 3.74 (dd, J=11.7, 8.0 Hz, 1H; H-6), 3.69 (dd, J=
10.3, 4.3 Hz, 1H’, H’-6’), 3.67 (dd, J=6.4, 4.3 Hz, 1H; H-6), 3.60–3.64
(m, 2H; H-3, H-5), 3.37–3.50 (m, 2H; 3H’, H-1, H-2, H’-2, H’-3, H’-5),
2.83 (dt, J=10.3, 2.1 Hz, 1H’, H’-1), 2.31 (ddd, J=15.2, 5.0, 2.0 Hz,
1H; H-7), 2.26 (ddd, J=13.6, 9.3, 2.1 Hz, 1H’, H’-7), 2.05 (ddd, J=
13.8, 10.2, 5.5 Hz, 1H’, H’-7’), 1.77–1.86 (m, 1H; H-7’); 13C NMR
(125 MHz): d=142.6, 128.7, 128.1, 126.6, 78.5, 77.3, 73.9, 71.6, 70.9,
69.0, 61.4, 39.5, and 144.0, 128.7, 127.8, 125.9, 75.5, 76.4, 71.0, 69.8,
69.1, 61.4, 40.2; HRMS (ESI): (m/z): calcd for C14H20O6Na: 307.1152
[M+Na]+, found: 307.1155.
1-S-Isopentyl-2,3,4,6-tetra-O-acetyl-b-d-galactopyranoside (7a):
1H NMR (500 MHz, CDCl3): d=5.4 (d, J=3.4 Hz, 1H; H-4), 5.21 (t,
J=10.0 Hz, 1H; H-2), 5.02 (dd, J=10.0, 3.4 Hz, 1H; H-3), 4.45 (d, J=
10.0 Hz, 1H; H-1), 4.13 (dd, J=11.3, 6.8 Hz, 1H; H-6), 4.08 (dd, J=
11.3, 6.8 Hz, 1H; H-6’), 3.91 (t, J=6.8 Hz, 1H; H-5), 2.61–2.73 (m,
2H; H-8), 2.12 (s, 3H; OAc), 2.04 (s, 3H; OAc), 2.01 (s, 3H; OAc),
1.95 (s, 3H; OAc), 1.60–1.70 (m, 1H; H-9), 1.48 (t, J=7.5 Hz, 1H; H-
7), 1.46 (t, J=7.5 Hz, 1H; H-7’), 0.88 (dd, J=6.7, 1.4 Hz, 6H; H-10);
13C NMR (125 MHz, CDCl3): d=170.3, 170.1, 170.0, 169.5, 84.0, 72.3,
71.9, 67.3, 67.2, 61.5, 38.5, 28.0, 27.2, 22.2, 22.0, 20.7, 20.6, 20.5;
HRMS (ESI): (m/z): calcd for C19H30O9SNa: 457.1503 [M+Na]+,
found: 457.1491.
b-C-Glucoside alcohol (2e): Mixture of diastereoisomers. 1H NMR
(500 MHz, D2O): d=7.33–7.48 (m, 5H; 5H’, H-Ar, H’-Ar), 5.00 (dd,
J=9.8, 3.5 Hz, 1H’, H’-8), 4.98 (dd, J=9.7, 5.7 Hz, 1H; H-8), 3.88 (dd,
J=15.0, 2.1 Hz, 1H; 1H’, H-6, H’-6), 3.67 (dd, J=10.0, 6.1 Hz, 1H;
1H’, H-6’, H’-6’), 3.53 (dt, J=9.7, 2.1 Hz, 1H; H-1), 3.47 (t, J=8.8 Hz,
1H; H-3), 3.30–3.41 (m, 2H; 1H’, H-4, H-5, H’-4), 3.25 (t, J=9.0 Hz,
1H’, H’-3), 3.21 (dd, J=9.0, 4.5 Hz, 1H’, H’-2), 3.13–3.21 (m, 1H; 1H’,
H-2, H-5), 2.89 (dt, J=9.9, 2.3 Hz, 1H’, H’-1), 2.22–2.34 (m, 1H; 1H’,
H-7, H’-7), 2.02 (ddd, J=14.0, 10.4, 5.5 Hz, 1H’, H’-7’), 1.78 (ddd, J=
14.5, 10.2, 3.5 Hz, 1H; H-7’); 13C NMR (125 MHz): d=142.5, 128.7,
128.1, 126.5, 79.5, 77.3, 76.8, 73.5, 69.9, 69.8, 61.0, 39.5 and 144.0,
128.8, 127.8, 125.9, 79.5, 77.4, 76.0, 73.6, 71.5, 70.1, 61.1, 40.1;
HRMS (ESI): (m/z): calcd for C14H20O6Na: 307.1152 [M+Na]+, found:
307.1155.
1-S-Isopentyl-2,3,4,6-tetra-O-acetyl-b-d-glucopyranoside
(8a):
1H NMR (500 MHz, CDCl3): d=5.22 (t, J=9.4 Hz, 1H; H-3), 5.08 (t,
J=9.8 Hz, 1H; H-4), 5.03 (t, J=9.8 Hz, 1H; H-2), 4.48 (d, J=10.0 Hz,
1H; H-1), 4.34 (dd, J=12.4, 5.0 Hz, 1H; H-6), 4.14 (dd, J=12.4,
2.2 Hz, 1H; H-6’), 3.70 (ddd, J=10.0, 5.0, 2.2 Hz, 1H; H-5), 2.62–2.74
(m, 2H; H-8), 2.07 (s, 3H; OAc), 2.05 (s, 3H; OAc), 2.02 (s, 3H; OAc),
2.00 (s, 3H; OAc), 1.62–1.72 (m, 1H; H-9), 1.44–1.52 (m, 2H; H-7),
0.90 (dd, J=6.7, 2.0 Hz, 6H; H-10); 13C NMR (125 MHz, CDCl3): d=
170.6, 170.2, 169.4, 169.4, 83.5, 75.9, 73.9, 69.9, 68.4, 62.2, 38.4,
27.9, 27.3, 22.2, 22.1, 20.7, 20.7, 20.6, 20.6; HRMS (ESI): (m/z): calcd
for C19H30O9SNa: 457.1503 [M+Na]+, found: 457.1491.
1-S-Isopentyl-b-d-galactopyranoside (1b): 1H NMR (500 MHz,
D2O): d=4.46 (d, J=9.7 Hz, 1H; H-1), 3.98 (d, J=3.5 Hz, 1H; H-4),
3.67–3.79 (m, 3H; H-5, H-6, H-6’), 3.64 (dd, J=9.5, 3.5 Hz, 1H; H-3),
3.55 (t, J=9.8 Hz, 1H; H-2), 2.70–2.84 (m, 2H; H-8), 1.61–1.72 (m,
1H; H-9), 1.53 (t, J=7.7 Hz, 1H; H-7), 1.52 (t, J=7.3 Hz, 1H; H-7’),
0.89 (dd, J=6.6, 2.9 Hz, 6H; H-10); 13C NMR (125 MHz, D2O): d=
85.8, 78.9, 74.0, 69.7, 68.8, 61.0, 38.4, 28.0, 26.8, 21.5, 21.4; HRMS
(ESI): (m/z): calcd for C11H22O5SNa: 289.1080 [M+Na]+, found:
289.1084.
General 1H NMR inhibition studies: Inhibitor (0.83 mm, 1 equiv)
was added to a phosphate buffer solution (pD 7.2, 0.2m, RT) con-
taining b-galactosidase (0.125 U). The solution was gently mixed,
and the volume was adjusted to 480 mL with buffer. After 5 min,
ONPG (0.83 mm) was introduced, and the kinetics were recorded
1
by H NMR spectroscopy. The formation of ONP (hydrolyzed ONPG)
was followed over time. The inhibitor solutions were prepared in
buffer, and the concentrations were calibrated with the substrate
(ONPG) solution.
1-S-Isopentyl-b-d-glucopyranoside (2b): 1H NMR (500 MHz, D2O):
d=4.50 (d, J=9.9 Hz, 1H; H-1), 3.88 (dd, J=12.3, 1.8 Hz, 1H; H-6),
3.69 (dd, J=12.3, 5.9 Hz, 1H; H-6’), 3.36–3.50 (m, 3H; H-3, H-4, H-
5), 3.30 (t, J=9.9 Hz, 1H; H-2), 2.69–2.82 (m, 2H; H-8), 1.60–1.72
(m, 1H; H-9), 1.52 (t, J=7.7 Hz, 1H; H-7), 1.51 (t, J=7.2 Hz, 1H; H-
7’), 0.88 (dd, J=6.6, 4.3 Hz, 6H; H-10); 13C NMR (125 MHz, D2O): d=
85.3, 79.8, 77.2, 72.3, 69.6, 60.9, 38.4, 28.0, 26.7, 21.5, 21.4; HRMS
(ESI): (m/z): calcd for C11H22O5SNa: 289.1080 [M+Na]+, found:
289.1083.
General UV/Vis inhibition studies: Different concentrations of in-
hibitor were added to
a phosphate-buffered saline solution
(pH 7.4, 0.01m, RT) containing b-galactosidase (0.48 mm). The solu-
tion (1.5 mL) was gently mixed. After 5 min, ONPG (44.3 mm) was
introduced and the kinetics were recorded by UV/Vis spectroscopy
at 420 nm. The formation of ONP was followed over time by using
a time-drive analysis method for the first 15 min. The inhibitor sol-
ChemBioChem 2010, 11, 1600 – 1606
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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O. Ramstrçm et al.
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This study was supported by the Swedish Research Council, the
Royal Institute of Technology and the European Commission
(contracts no. MRTN-CT-2005-019561 and MRTN-CT-2006-
035614).
Keywords: beta-galactosidase · dynamic chemistry · fragment-
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Received: March 10, 2010
Published online on June 11, 2010
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