Nanomolar affinity, iminosugar-based chemical probes for specific labeling of lysosomal glucocerebrosidase

Article abstract of DOI:10.1016/j.bmc.2009.10.060

Three different photoprobes were synthesized to label β-glucosidases; one probe was based on glucose, two probes on the iminosugar deoxynojirimycin. The affinity of the probes for three different β-glucosidases was determined. Furthermore, their labeling efficiencies, binding specificities through competition with deoxynojirimycin, and binding specificities in the presence of cell lysate, were evaluated. Especially one showed very high affinity towards non-lysosomal glucoceramidase (IC₅₀ = 20 nM).

Full text of DOI:10.1016/j.bmc.2009.10.060

Bioorganic & Medicinal Chemistry 18 (2010) 267–273
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Nanomolar affinity, iminosugar-based chemical probes for specific labeling
of lysosomal glucocerebrosidase
Monique van Scherpenzeel a, Richard J. B. H. N. van den Berg b, Wilma E. Donker-Koopman c,
Rob M. J. Liskamp a, Johannes M. F. G. Aerts c, Herman S. Overkleeft b, Roland J. Pieters a,
*
a Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80082, 3508 TB Utrecht, The Netherlands
b Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
c Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Three different photoprobes were synthesized to label b-glucosidases; one probe was based on glucose,
two probes on the iminosugar deoxynojirimycin. The affinity of the probes for three different b-glucosi-
dases was determined. Furthermore, their labeling efficiencies, binding specificities through competition
with deoxynojirimycin, and binding specificities in the presence of cell lysate, were evaluated. Especially
one showed very high affinity towards non-lysosomal glucoceramidase (IC50 = 20 nM).
Ó 2009 Elsevier Ltd. All rights reserved.
Received 4 September 2009
Revised 22 October 2009
Accepted 29 October 2009
Available online 31 October 2009
Keywords:
Glucosidases
Proteomics probes
Photolabeling
1. Introduction
substrate or inhibitor of the enzyme can potentially be a very use-
ful tool. Romaniouk et al. synthesized the first photoaffinity deriv-
The glucosidases are a large class of hydrolases that occur in
plants, fungi, mammals and microorganisms. The glucosidases cat-
alyze the hydrolysis of glycosidic bonds, and are therefore involved
in many important processes such as the biosynthesis and
degradation of glycoproteins.1,2 In mammalian systems, two
well-studied types of b-glucosidases are the cytosolic and lyso-
somal b-glucosidases.3 Cytosolic b-glucosidases are mainly present
in the liver, the kidneys and the intestine. Gopolan et al. suggested
that cytosolic b-glucosidases play a key role in the detoxification of
plant b-glucosides,4 which seems to correspond to the significant
quantities of this enzyme present in especially the organs contain-
ing detoxification enzymes, namely the liver and the intestine.
The Lysosomal b-glucosidase cleaves the substrate glucosylcer-
amide (glucocerebroside) into ceramide and glucose. Therefore, the
enzyme is also called lysosomal glucocerebrosidase or glucosylce-
ramidase (E.C. 3.2.1.45).3 Lysosomal b-glucosidase is present in
most tissues and cell types with various levels of activity. The en-
zyme is partially defective in Gaucher disease patients. The disor-
der is characterized by the accumulation of glucosylceramide in
tissue macrophages (Gaucher cells).5,6
ative of the iminosugar deoxynojirimycin.7 With this [125I]-labeled
probe, it was possible to identify the peptide that is part the active
site of the protein glucosidase I. The group of Withers8,9 synthe-
sized a probe to profile retaining b-endoglycosidases in complex
proteomes. Their probe consisted of a mechanism-based inactiva-
tor of the enzyme. A number of peptides were identified, and they
all proved to be derived from proteins belonging to the family of
retaining glycanases. In a different approach, a fluorescent dansyl
moiety was coupled to 1-amino-1,2,5-trideoxy-2,5-imino-D-man-
nitol by Hermetter et al.10 with two powerful b-glucosidase probes
as a result. In one compound the dansyl group was coupled directly
to the sugar while the other probe contained a spacer in between
the sugar and the fluorescent group. Both compounds were highly
potent inhibitors of Agrobacterium sp. b-glucosidase, with Ki-values
of 2 nM. With one probe it was possible to fluorescently label b-
glucosidase selectively in a mixture with three other related pro-
teins and visualize the labeled enzyme on a native PAGE.10 Within
this context we here describe our efforts in the synthesis of chem-
ical probes containing a photolabel (benzophenone and acetophe-
none) for glucosidase capture. An alkyne moiety is part of the
probe to enable the attachment via ‘click’ chemistry of a reporter
tag, such as a fluorescein-azide for visualization in a gel. As the rec-
ognition moiety glucose and 1-deoxynojirimycin were used which
exhibit greatly different inhibitory potencies for the enzymes tar-
geted in our studies: lysosomal glucocerebrosidase (GBA), non-
To study glycosidases and capture them selectively in the pres-
ence of complex protein mixtures, a chemical probe, based on a
* Corresponding author. Tel.: +31 30 2536944; fax: +31 30 2536655.
E-mail address: r.j.pieters@uu.nl (R.J. Pieters).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.060

268
M. van Scherpenzeel et al. / Bioorg. Med. Chem. 18 (2010) 267–273
Scheme 1. Synthesis of glucose-benzophenone (4). Reagents and conditions: (a) propargyl bromide, K2CO3, acetone, 16 h, 31%; (b) Ac2O, NaOAc,
D, 30 min, quant.; (c)
NH2CH2CH2NH2 (1.2 equiv), AcOH (1.4 equiv), THF, 18 h, 71%; (d) Cl3CCN (2.5 equiv), DBU (0.2 equiv), CH2Cl2, 0 °C to rt, 4 h, 89%; (e) TMSOTf (0.2 equiv), CH2Cl2, ꢀ20 °C to rt,
16 h; (f) 3, NaOMe, MeOH, 10 min, 23% over two steps.
lysosomal glucoceramidase (GBA2), and almond b-glucosidase. The
abilities of the probes to specifically capture the enzymes in the
presence of a human cell lysate were evaluated.
acetophenone derivative 1112 was successfully coupled using
EDC/NHS. After a final preparative HPLC purification benzophe-
none probe 8 and acetophenone probe 9 were obtained in 13%
and 39% isolated yield, respectively (Scheme 2).
2. Results and discussion
2.2. Evaluation of the probes
2.1. Syntheses
2.2.1. Enzyme inhibition experiments
The probes were tested for their ability to inhibit three different
b-glucosidases: lysosomal glucocerebrosidase (CerezymeÒ, recom-
binant GBA, CeredaseÒ, human placental GBA), non-lysosomal
glucoceramidase (GBA2), and almond b-glucosidase. The assays
for the first two enzymes were based on a competition of the probe
with the substrate 4-methylumbelliferyl (4-MU) b-glucoside. The
4-MU part is cleaved from b-glucose by the enzyme, and its
amount can be measured with a fluorometer. The inhibitory po-
tency is expressed as the inhibitor concentration resulting in 50%
inhibition (IC50-values).14 The assay for the third enzyme is based
on a competition of the probe with the substrate para-nitrophenol
b-glucoside (PNPG), whose liberated PNP group was quantified by
UV spectroscopy at 405 nm. Measurements were performed with
different substrate concentrations, from which a Ki-value was
2.1.1. Synthesis of the glucose-based probe (4)
D-Glucose was acetylated and converted to imidate-donor 1
according to known procedures.11 Commercially available 4,40-
dihydroxybenzophenone was mono-alkylated with propargyl bro-
mide and an excess of base to give 2 (Scheme 1). Subsequently, the
photolabel was coupled to the sugar under standard coupling condi-
tions (cat. TMSOTf in dry CH2Cl2).11 Deprotection of the sugar using
Zemplen’s method yielded the glucose-based chemical probe 4.
2.1.2. Synthesis of iminosugar-based probes
The synthesis of the two iminosugar-based probes started with
the alkylation of the hydrochloride salt of 1-deoxynojirimycin 513
by bromide 6. After removal of the Boc-group with TFA, the puri-
fied OSu ester 1012 was used to prepare 8. For the synthesis of 9
OH
OH
OH
H
N
NHR
NHBoc
N
a
c, d
N
HO
HO
HO
HO
HO
HO
OH
O
OH
OH
5
7
6
8: R =
b
O
Br
NH3+Br-
Br
NHBoc
O
O
9: R =
O
O
O
O
OH
O
O
O
N
O
O
O
10
O
11
Scheme 2. Synthesis of the iminosugar-based probes. Reagents and conditions: (a) 6, K2CO3, DMF, 85 °C, 18 h; (b) Boc2O, dioxane/H2O/NaOH, 1 h, 0 °C, 91%; (c) TFA/CH2Cl2
1:1 (v/v), 1 h, 0 °C; (d) for 8:10, DMF, 16 h, 0 °C to rt, 13%; for 9:11, EDC, NHS, DMF, 16 h, 0 °C to rt, 39%.

M. van Scherpenzeel et al. / Bioorg. Med. Chem. 18 (2010) 267–273
269
determined. Results of our measurements are shown in Table 1.
The affinity of DNM 5 and probe 4 for almond b-glucosidase are gi-
ven for comparison. Especially the affinities of 8 and 9 for the
membrane-bound glucosidase GBA2 were very high (IC50 20 and
75 nM, respectively). The best inhibitor thus far described in the
literature has a IC50-value of 1.7 nM,14 which is only a factor of
10 lower than the value obtained for compound 8. For a monosac-
charide-protein interaction this is exceptional. The other Ki-values
given in Table 1 are all in the low micromolar or high nanomolar
range, so both 8 and 9 bind with high affinities.
bands from the gel, trypsin digestion and MALDI-TOF analysis were
performed. In this fashion the glucose-binding proteins tryptopha-
nase,
a-galactosidase, elongation factor Tu, outer membrane pro-
tein and OmpF porin were identified with high confidence (100%
C.I.) according to Mascot (see Supplementary data).19 Especially
OmpF is an interesting result, because this protein is important
in nutrient (including glucose) regulation.20 Since this protein is
a membrane protein, it is often lost in common 2D-gel MS identi-
fication techniques.21,24
Figure 2B shows an important control experiment, which is the
profile of benzophenone-labeled proteins. From this experiment,
we can conclude that the influence of the photolabel is dominant
in the chemical probe; there are only small differences in the gel
profile between the glucose-probe 4 and benzophenone 2.
An explanation is probably the large size of the photolabel com-
pared to the rest of the probe, as well as its hydrophobicity. Carbo-
hydrate–protein interactions are usually weak,25 which makes
these probes more of a ‘water-soluble photolabel’ rather than a tar-
geted probe. Despite this result, some glucose specific labeled pro-
teins were obtained by subtracting the benzophenone-labeled
proteins from the glucose-probe labeled proteins using suitable
software.26
2.2.2. Labeling of b-glucosidase with (4)
Probe 4 was evaluated first on its ability to label b-glucosidase.
A general procedure, developed by Speers and Cravatt15 that was
also previously used in our group was followed.16–18 During incu-
bation of the enzyme with 4 for 30 min, the sample was irradiated
with UV light. Fluorescein-N3 was ‘clicked’ on overnight and the
samples were resolved on SDS–PAGE. The results are shown in Fig-
ure 1. The probe was able to label the enzyme. It turned out that
the fluorescence intensity depends on several factors such as the
concentration of the probe, the amount of enzyme, the reaction
time of the click reaction, the irradiation time and the extensive-
ness of washing the gel (which lowers the background) before fluo-
rescence scanning. Clearly, UV light irradiation is necessary to
fluorescently label the protein.
2.2.4. Labeling of lysomal glucocerebrosidase with the
iminosugar-based probes (8) and (9)
Lysosomal glucocerebrosidase was used to test the difference in
labeling efficiency of the probes. The iminosugar probes 8 and 9
were compared with the glucose-probe 4. Concentration ranges
2.2.3. Profiling sugar binding and processing proteins with 4
In a second experiment we turned to cell lysates, to find out if it
was possible to profile sugar binding proteins and sugar processing
were made of the probes, from 10 down to 0.1
shown in Figure 3A. It turned out that clear labeling could only
be observed at the 10 M probe concentration. At this concentra-
lM. The result is
enzymes (e.g., glycosidases, glycosyltransferases) in such
a
complex mixture. The result is shown in Figure 2A, in which the
fluorescence scan and the silver stain of one gel are compared.
It is clear that only a small percentage of all the proteins present
were labeled with the probe. After excision of the fluorescent
l
tion, there is a large difference in efficiency between the com-
pounds. Probe 8 labeled the best, and the labeling by probe 9
was only negligible. The difference in affinity of both compounds
for this enzyme was about a factor 5 (Table 1), which probably
influences the labeling efficiency. However, the difference in reac-
tivity between acetophenone and benzophenone is likely of greater
influence, considering their absorptions in near UV.12 Since com-
pounds 4 and 8 differ roughly a factor of 100 in their binding con-
stant (Table 1), the difference in labeling efficiency between these
compounds is most likely a matter of affinity.
Table 1
Affinity measurements of the probes on three different enzymes
a
a
Compds GBA IC50
M)
GBA2 IC50
M)
Almond b-glucosidase Kia
(lM)
(
l
(
l
9
8
5
4
1.65 ( 0.15)
0.35 ( 0.05)
506c
0.075 ( 0.03)
0.02 ( 0.005)
28.80c
6.4 ( 0.1)
1.3 ( 0.1)
167 ( 1)
To test if 4, 8 and 9 bind the enzyme specifically in the binding
pocket, a competition experiment with 5 was performed. The re-
sult is shown in Figure 3B.
n.d.
n.d.
588 ( 133)b
a
Values are means of two experiments, standard deviation is given in paren-
In this experiment, the labeling efficiency of 8 is again higher
than the efficiency of 9. For all molecules it is clear that they com-
pete with 5: the fluorescent signal decreases with an increase in
concentration of 5. From this picture we can conclude that both
probes 8 and 9 bind specifically in the binding pocket of lysosomal
glucocerebrosidase, and this seems to be the case also for glucose-
probe 4.
theses (n.d. = not determined).
b
Probe 4 can be considered as an inhibitor even though it is a slowly converting
substrate.
c
Values from Ref. 12 are given for comparison.
2.2.5. Specificity of probe 8 for labeling GBA in the presence of
human cell lysate
The final experiment was set up to find out if probe 8 was spe-
cific for GBA in the presence of other proteins. The chosen probe
concentration was relatively low, to avoid non-specific interac-
tions. However, since the labeling efficiency of this compound is
not that high, the samples had to contain a considerable amount
(300 ng) of the enzyme. A concentration range of a human cell
lysate was prepared up to 3
are shown in Figure 4.
lg of total proteins/sample. The results
Probe 8 seems to be highly specific for GBA, as we can conclude
from the fluorescence picture. Surprisingly, the fluorescent signal
does not decrease with an increasing amount of other proteins
present, which is in contrast to another highly selective probe to
Figure 1. Almond b-glucosidase is labeled by probe 4. Without light irradiation, no
covalent bond is formed between the probe and the protein, and no fluorescent
labeling occurs.

270
M. van Scherpenzeel et al. / Bioorg. Med. Chem. 18 (2010) 267–273
Figure 2. Profiling glucose-binding proteins in E. Coli cell lysate. (A) E. coli lysate spiked with different amounts of b-glucosidase, was incubated with 40 lM of 4. A fluorescent
picture (lanes 1 and 2) and silver stain (lanes 3 and 4) of the same gel are shown. (B) Comparison between the fluorescent patterns of 4 (lane 5) and 2 (lane 6), which showed
only minor differences. The boxes indicate the position of b-glucosidase.
the galectin-3 probe binds in a surface groove benefiting from elec-
trostatic interactions with arginines.28 Therefore, the galectin-3
probe is probably more likely to capture additional proteins than
probe 8.
3. Conclusions
We started our project with the easy to synthesize glucose-
probe 4. A disadvantage of this probe is that it is a substrate of
the enzyme as well, which causes cleavage of the benzophenone
moiety and subsequent non-specific labeling. Furthermore, it
Figure 3. Labeling CeredaseÒ with different probe concentrations (A), or with
increasing concentrations of deoxynojirimycin (DNM, 5) (B). (A) Each lane contains
100 ng of enzyme. Concentrations are from left to right for each probe: 10, 1 and
turned out that the relatively small sugar had only small influence
on binding specificity, when it was coupled to a large, bulky,
hydrophobic photolabel. We could have changed to a less hydro-
phobic photolabel, such as a diazirine moiety, however this label
is reported to possess lower labeling efficiency, and a higher reac-
tivity towards nucleophiles and the solvent, than aryl ketones.29,30
We instead opted to switch to the high affinity iminosugar DNM,
and we were able to couple our benzophenone photolabel, as well
as the less bulky and hydrophobic acetophenone photo affinity la-
bel. The prepared DNM-based labels were tested for their affinity
towards three different glycosidases, and were compared with
the glucose-probe 4. Especially probe 8 showed a very high inhib-
itory potency with an IC50 of 20 nM for the non-lysosomal glucoce-
ramidase (GBA2) assay. The probes were tested for their ability to
label different b-glucosidases, and their binding specificity through
competition experiments with DNM. From those experiments, it
was concluded that all the probes bound the enzyme specifically
in its binding pocket. Although in general, labeling efficiencies
were low, lysosomal glucocerebrosidase could be labeled specifi-
cally in the presence of a complex protein mixture. The labeling
efficiency can possibly be improved by coupling the photolabel di-
rectly, or with a shorter spacer, to the iminosugar. However, a pos-
sible negative influence of this bulky substituent on binding
specificity cannot be excluded. Nevertheless, the chemical probes
presented here do label three different b-glucosidases specifically,
and two of them bind with exceptionally high affinity. Further-
0.1
concentrations are 5
prepared: from left to right for all probes 0, 10, 100 and 500
l
M. (B) The same amount of CeredaseÒ (200 ng) is present in all samples. Probe
l
M in all samples. From DNM 5 a concentration range was
lM.
Figure 4. Labeling of Ceredase by probe 8 (3 lM) in the presence of a human cell
lysate. In all of the lanes, 300 ng of CeredaseÒ is present. The amount of total protein
per lane increases from left to right: 0, 0.3, 0.7, 1.3 and 2.7 per lane. Lanes 1–5:
fluorescent picture; lanes 6–10: silver stain of lanes 1–5.
label galectin-3.12 It is likely that 8 binds the enzyme in a deep
pocket in accordance with a recent crystal structure.27 In contrast

M. van Scherpenzeel et al. / Bioorg. Med. Chem. 18 (2010) 267–273
271
more, the experiments clearly show that creating a successful
4.2.3. Glucose-probe (4)
probe is a multidimensional challenge involving issues of lipophil-
icity, affinity and photoreactivity.
To a solution of compound 3 in MeOH (5 mL) was added a few
drops of NaOMe (30% w/v in MeOH) and the mixture was stirred
for 30 min. The mixture was neutralized with Dowex H+-resin, fil-
tered and concentrated in vacuo to give 4 in 23% yield over two
steps as a white solid. 1H NMR (CD3OD): d = 3.02 (s, 1H, C„CH),
3.37- 3.56 (m, 3H, H-3, H-2, H-6a), 3.72 (dd, 1H, J = 5.8, 17.3 Hz,
H-5, H-6b), 3.92 (ꢂd, 2H, J = 11.8 Hz, H-4), 4.86 (s, 2H, OCH2),
5.06 (d, 1H, J = 6.9 Hz, H-1), 7.12 (d, 2H, J = 8.8 Hz), 7.22 (d, 2H,
J = 8.8 Hz), 7.76 (d, 2H, J = 5.5 Hz), 7.79 (d, 2H, J = 5.2 Hz); 13C
NMR: d = 52.8, 58.5, 67.3, 70.8, 73.9, 74.3, 97.7, 111.7, 113.2,
128.2, 129.1, 158.5, 158.7, 192.6; HR-ESI-MS calcd for C22H22O8
437.1213 [M+Na]+, found 437.1192.
4. Experimental
4.1. General methods
All reagents were purchased from commercial sources and
were used without further purification. The compound 1-deoxy-
nojirimycin 5 was prepared according to the reported protocol.13
Dowex 50X8 (H+ form, 20–50 mesh) was purchased from Sigma.
Analytical thin layer chromatography (TLC) was performed on
Merck pre-coated Silica Gel 60 F254 (0.25 mm) plates. Spots
were visualized with UV light, H2SO4, or ninhydrin. Column
chromatography was carried out using Merck Kieselgel 60 (40–
63 mm). 1H NMR and 13C NMR were obtained on a Varian
300 MHz spectrometer. Chemical shifts are given in ppm with
respect to internal TMS for 1H NMR. Two-dimensional 1H–1H
correlation and total correlation spectroscopy (COSY and TOCSY)
and 1H–13C correlated heteronuclear single quantum coherence
(HSQC) NMR spectra (500 MHz) were recorded at 300 K with a
Varian Unity INOVA 500 spectrometer. Low resolution ESI-MS
experiments were performed on a Shimadzu LC–MS QP8000
system. Exact masses were measured by nanoelectrospray
time-of-flight mass spectrometry on a Micromass LCToF mass
spectrometer at a resolution of 5000 fwhm. Gold-coated capillar-
4.2.4. 3-Bromo-propyl-1-NHBoc (6)
The compound 3-bromopropylamine (500 mg, 2.28 mmol) and
Boc2O (548 mg, 2.51 mmol) were dissolved in dioxane/H2O (10/
5 mL). 5 mL 1 M NaOH-solution in H2O was added and the mixture
was stirred for 1½ h. The reaction mixture was diluted with 50 mL
of EtOAc and 20 mL of H2O. The organic layer was washed once
with 1 N KHSO4, dried with Na2SO4, filtered and concentrated in
vacuo. Compound 6 was obtained as a colorless oil in 91% yield
(516 mg, 2.17 mmol). Rf (hexane/EtOAc, 4:1 v/v) = 0.43; 1H NMR
(300 MHz, CDCl3): d = 1.45 (s, 9H, 3 ꢁ CH3), 2.05 (quintet, 2H,
J = 6.6 Hz, CH2CH2CH2), 3.27 (quartet, 2H, J = 6.3 Hz, CH2NH), 3.44
(t, 2H, J = 6.6 Hz, CH2Br), 4.77 (br s, 1H, NH); 13C NMR (75 MHz,
CDCl3): d = 28.5 (3 ꢁ CH3), 30.9 (CH2Br), 32.9 (CH2CH2CH2), 39.1
(CH2NH), 79.5 (C(CH3)3), 156.1 (C@O).
ies were loaded with 1 lL of sample (concentration 20 lM)
dissolved in a 1:1 (v/v) mixture of CH3CN–H2O. NaI or poly(eth-
ylene glycol) (PEG) was added as internal standard. The capillary
voltage was set between 1100 and 1350 V, and the cone voltage
was set at 30 V.
4.2.5. N-(Propylamide-benzophenone)-1-deoxynojirimycin (8)
Compound 5 (17 mg, 0.1 mmol) was reacted with 6 (36 mg,
0.15 mmol) in DMF (0.5 mL) with K2CO3 (42 mg, 0.3 mmol) at
85 °C for 18 h. Reaction mixture was filtered and concentrated in
vacuo. The crude product was dissolved in 1 mL MeOH, cooled to
0 °C, and 1 mL TFA and a droplet H2O were added. The mixture
was stirred for 1 h at 0 °C, and evaporated to dryness. The crude
solid was dissolved in dry DMF (2 mL) and benzophenone-succi-
ninmidyl ester 10 (63 mg, 0.17 mmol) and DiPEA (0.1 mL,
0.6 mmol) were added. The mixture was stirred for 18 h, concen-
trated in vacuo and 8 was obtained after preparative HPLC and
lyophilization in 13% isolated yield (34 mg) as colorless oil. 1H
NMR (500 MHz, CD3OD): d = 2.02 (m, 2H, CH2CH2NH), 2.91–3.01
(m, 3H, H-5, H-1ax, C„CH), 3.24 (ꢂs, 2H, CH2Nimino), 3.31 (t, 1H,
J = 9.5 Hz, H-3), 3.41–3.56 (m, 4H, H-4, H-1eq, CH2NHC@O), 3.63
(m, 1H, H-2), 3.86, 4.05 (2 ꢁ d, 2H, J = 12 Hz, H-6ab), 4.78 (s, 2H,
OCH2), 7.06 (d, 2H, J = 9.0 Hz, CHar), 7.75 (t, 4H, J = 8 Hz, CHar),
7.90 (d, 2H, J = 8.0 Hz, CHar); 13C NMR (125 MHz, CD3OD):
d = 33.4 (CH2CH2NH), 36.1 (CH2NHC@O), 47.3 (CH2Nimino), 52.9
(C-1, C-6), 54.9 (OCH2), 65.7 (C-5, C-2), 66.9 (C-4), 76.2 (C-3),
75.4 (C„CH), 77.0 (C„CH), 113.6, 126.4, 128.6 (6 ꢁ Car), 129.1
(Car, etherC@O), 131.5 (2 ꢁ Car), 136.1 (CarC(@O)NH), 140.0
(Car, amideC@O), 161.2 (CarOCH2C„CH), 167.6 (C(@O)NH), 195.7
4.2. General procedures
4.2.1. 4-Hydroxy-40-(prop-2-ynyloxy)benzophenone (2)
4,40-Dihydroxybenzophenone (1.00 g, 4.67 mmol), was dis-
solved in acetone (30 mL) and K2CO3 (600 mg, 4.34 mmol) was
added. Propargylbromide (80% w/v solution in toluene, 500 lL,
3.61 mmol) was slowly added to the solution and the mixture
was stirred for 16 h. The mixture was purified by silica gel column
chromatography (hexane/EtOAc, 5:1, v/v). The product was ob-
tained as a white solid (362 mg, 31% yield). 1H NMR (CD3OD):
d = 3.02 (s, 1H), 4.84 (s, 2H), 4.92 (br s, 1H), 6.89 (d, 2H,
J = 8.3 Hz), 7.11 (d, 2H, J = 8.8 Hz), 7.69 (d, 2H, J = 8.3 Hz), 7.75 (d,
2H, J = 8.3 Hz); 13C NMR (CDCl3): d = 55.6, 76.2, 78.0, 114.9, 129.1,
131.6, 132.0, 132.6, 161.3, 162.2, 194.2.
4.2.2. Acetylated glucose-probe (3)
The two compounds 1 (258 mg, 0.60 mmol) and 2 (75 mg,
0.30 mmol) were dissolved in dry CH2Cl2 (10 mL) and stirred at -
(C@O). HRMS (m/z): calcd for C26H31N2O7 [M+H]+ 483.2131;
þ
20 °C. Trimethylsilyl triflate (13 lL, 0.06 mmol) was added and
the mixture was stirred for 16 h allowing the mixture to warm to
rt The mixture was neutralized with Et3N, concentrated in vacuo
and purified by silica gel column chromatography (hexane/EtOAc,
5:1, v/v) to give 3 as a white solid (41 mg, 0.070 mmol). 1H NMR
(CD3OD): d = 2.00, 2.01, 2.05 (3 ꢁ s, 12H), 3.03 (s, 1H), 4.18 (d,
2H, J = 11.7 Hz, H-6ab), 4.33 (dd, 1H, J = 5.3 Hz, 8.8 Hz, H-4), 4.86
(s, 2H, OCH2), 5.15 (t, 1H, J = 9.8 Hz, H-5), 5.23 (t, 1H, J = 8.8 Hz,
H-2), 5.42 (t, 1H, J = 9.5 Hz, H-3), 5.52 (d, 1H, J = 7.8 Hz, H-1),
7.12 (d, 2H, J = 8.3 Hz), 7.15 (d, 2H, J = 8.3 Hz), 7.76 (d, 2H,
J = 7 Hz), 7.77 (d, 2H, J = 7 Hz); 13C NMR (CDCl3): d = 20.6–20.7
(4 ꢁ COCH3) 55.8, 61.8, 68.1, 71.0, 72.1, 72.5, 76.2, 77.7, 98.2,
114.4, 116.0, 131.0, 132.0, 132.2, 132.9, 159.6, 160.9, 169.4,
170.2, 170.6, 194.2.
found 483.2114.
4.2.6. N-(Propylamide-acetophenone)-1-deoxynojirimycin (9)
Compound 5 (33 mg, 0.2 mmol) was reacted with 6 (71 mg,
0.3 mmol) in 1 mL DMF with K2CO3 (83 mg, 0.6 mmol) at 85 °C
for 18 h. Reaction mixture was filtered and concentrated in vacuo.
The crude product was dissolved in 2 mL of MeOH, cooled to 0 °C,
and 2 mL of TFA and a drop of H2O were added. The mixture was
stirred for 1 h at 0 °C, and evaporated to dryness. The crude solid
was dissolved in dry DMF (4 mL) and acetophenone derivative 11
(139 mg, 0.6 mmol), EDC (115 mg, 0.6 mmol), NHS (69 mg,
0.6 mmol) and DiPEA (0.2 mL, 1.2 mmol) were added. The mixture
was stirred for 18 h, concentrated in vacuo and 9 was obtained

272
M. van Scherpenzeel et al. / Bioorg. Med. Chem. 18 (2010) 267–273
after preparative HPLC and lyophilization in 39% isolated yield
(34 mg) as yellow oil. 1H NMR (500 MHz, DMSO): d = 1.79 (ꢂbr s,
2H, CH2CH2NH), 2.45 (t, 2H, J = 6.5 Hz, CH2C(@O)NH), 2.50 (s, 2H,
CH2Nimino), 2.90 (quartet, 1H, J = 10.5 Hz, H-1ax), 2.97 (t, 1H, H-5),
3.12 (ꢂd, 2H, J = 5.5 Hz, CH2NHC@O), 3.21 (m, 4H, CH2C(@O)Car,
H-1eq, H-3), 3.33 (m, 1H, H-4), 3.56 (br s, 1H, H-2), 3.63 (s, 1H,
C„CH), 3.75, 3.90 (2 ꢁ d, 2H, J = 11 Hz, H-6ab), 4.91 (s, 2H,
HC„CCH2O), 7.09 (d, 2H, J = 9.0 Hz, CHar), 7.97 (d, 2H, J = 8.5 Hz,
CHar), 8.03 (s, 1H, NH), 9.28 (br s, 1H, NHþimino); 13C NMR
(125 MHz, DMSO): 22.9 (CH2CH2NH), 29.3 (CH2C(@O)NH), 33.0
(CH2C(@O)Car), 35.7 (CH2NHC@O), 39.8 (CH2Nimino), 53.3 (C-1, C-
6), 55.7 (OCH2C„ CH), 65.0 (C-5), 66.1 (C-2), 67.1 (C-4), 76.2 (C-
3), 78.5 (C„CH), 114.4, 129.7 (4 ꢁ Car), 160.6 (CarOCH2C„CH),
171.4 (C(@O)NH), 198.5 (C@O). HRMS (m/z): calcd for
4.3.4. Labeling CeredaseÒ with different probe concentrations,
in competition with DNM (5), or in the presence of human cell
lysate
CeredaseÒ (1.5 or 2.3
l
L, 1 mg/mL,) was incubated and irradi-
ated for 30 min under a 366 nm UV lamp at 4 °C in 50 L samples
containing 10, 5, 3, 1, 0.5, 0.1 M of probe 4, 8 or 9 (stock solutions
in water), 0, 0.5, 5 or 25 L of DNM 5 (1 mM stock solution in
water), 0, 5, 10, 20 or 40 L HeLa cell lysate (0.5 mg/mL as deter-
l
l
l
l
mined by standard BCA protein concentration assay) in 50 mM
HEPES-buffer, pH 6.1. Samples were submitted to conjugation to
fluorescein-N316 and loaded onto a gel and visualized as described
for the labeling of almond b-glucosidase with different probe
concentrations.
C22H31N2O7 [M+H]+ 435.2131; found 435.2129.
4.4. Lysate preparation procedures
þ
4.3. General procedures to label b-glucosidase with a chemical
probe
4.4.1. Bacterial lysate preparation
Bacterial cells (BL21(DE3) from Novagen, OD600 = 2.2, 1.5 mL)
were pelleted by centrifugation at 5000 rpm for 10 min. The cells
4.3.1. Labeling study of b-glucosidase with (4)
The following components were incubated and irradiated for
30 min under a 366 nm UV lamp at 4 °C: 3 lL of a 1 mg/mL stock
were resuspended in 300 l
L of B-PerÒ Reagent (Pierce) and vor-
texed for 2 min. the homogeneous mixture was centrifuged at
13,000 rpm for 5 min. The supernatant was taken for the labeling
studies. Protein concentrations of the solutions were determined
by standard BCA protein concentration assay, using bovine serum
albumin as a standard.
solution of b-glucosidase (from almonds, Sigma) and 4
lL of a
1 mM stock solution of 4 in 5% DMSO in water, in 100
l
L final
volume of 50 mM HEPES-buffer, 150 mM NaCl, pH 7.4. The control
sample without UV treatment was protected from daylight with alu-
minium foil. After photoincubation, 10
50 mM stock solution in water), 10 L of CuSO4 (50 mM stock solu-
tion in water) and 1 equiv, with respect to probe 4, of fluorescein-
lL TCEP (freshly prepared
4.4.2. HeLa cell lysate preparation
2.5 ꢁ 105 cells were washed twice with PBS and pelleted by
centrifugation at 1400 rpm for 5 min. The cells were resuspended
l
14
in 100 l
L of B-PerÒ Reagent (Pierce) and vortexed for 1 min. the
N3 (40
the volume was adjusted to 200
from15). The samples were gently shaken for 1 h at rt and denatured
by the addition of 100 L sample buffer (10% SDS, 40% glycerol and
2% DTT solution) and subsequent incubation at 95 °C for 5 min. From
the total volume of 300 L, 20 L was loaded onto a 12% Tris–HCl gel
l
L of a 100
l
M stock solution in water) were added and
lL (click conditions adapted
homogeneous mixture was centrifuged at 12,000 rpm for 5 min.
The supernatant was taken for the labeling studies. Protein con-
centrations of the solutions were determined by standard BCA
protein concentration assay, using bovine serum albumin as a
standard.
l
l
l
for SDS–PAGE. After extensive washing of the running gel (1 h in
water) to eliminate excess dye reagent, fluorescence was detected
with a Typhoon fluorescence scanner and the gel was subsequently
silver stained (BioRad Silver staining plus kit).
4.5. Affinity determination procedures
4.5.1. Determination of IC50-values of probes 8 and 9 for
inhibition of the enzymes lysomal glucocerebrosidase (GBA)
and non-lysomal glucoceramidase (GBA2)
4.3.2. Labeling studies with probe (4) (and (2) as control) in
bacterial lysate
Experiments were performed as described in the literature.14
Bacterial lysate (40
protein assay), 6, 1.5 or 0.75
mond b-glucosidase, and 4 (Fig. 4A) or 10
stock solution of probe 4 (or 2) in 5% DMSO in water, in 50 mM
HEPES-buffer, 150 mM NaCl, pH 7.4 (total volume 100 L), were
l
L, 1 mg/mL as determined by standard BCA
L of a 0.5 mg/mL stock solution of al-
L (Fig. 4B) of a 1 mM
l
4.5.2. Determination of Ki-values of probes 5, 8 and 9 for
inhibition of almond b-glucosidase
The activity of almond b-glucosidase was determined with 0.2–
5 mM p-nitrophenol-b-D-glucopyranoside (PNPG) as a substrate in
0.1 M acetate-buffer pH 5.0. The amount of liberated p-nitrophenol
was measured at 405 nm after 15 min incubation at 37 °C. Ki-val-
ues were determined by variation of inhibitor concentrations (I)
and substrate concentrations (PNPG, 0.2–5 mM). The assay was
l
l
14
photoactivated and submitted to conjugation to fluorescein-N3
and loaded onto a gel and visualized as described above.
4.3.3. Labeling almond b-glucosidase with different probe
concentrations
b-Glucosidase (1.5
incubated and irradiated for 30 min under a 366 nm UV lamp at
4 °C in 50 L samples containing 10, 5, 1, 0.5, 0.1 M of probe 4, 8
or 9 (stock solutions in water) in 50 mM HEPES-buffer, 150 mM
NaCl, pH 7.4. After photoincubation, 2 L TCEP (freshly prepared
50 mMstock solutionin water), 2 L of CuSO4 (50 mM stock solution
in water) and 15 L of fluorescein-N3 (100
water) were added and the volume was adjusted to 100
conditions adapted from13). The samples were gently shaken for
16 h at 4 °C and denatured by the addition of 50 L sample buffer
(10% SDS, 40% glycerol and 2% DTT solution) and subsequent incuba-
tion at 95 °C for 5 min. From the total volume of 150 L, 20 L was
l
L of a 1 mg/mL stock solution, Sigma) was
performed in 96-wells format; samples of 150
in each well, consisting of 50 L PNPG-solution (dilutions from a
freshly prepared 20 mM stock in water) and 100 L inhibitor in
acetate-buffer pH 5.0. The reaction was started by adding 50
b-glucosidase (in 0.2% BSA in 0.01 M Na2HPO4, pH 7.0). The
Michaelis constant KM and Ki-values were determined from the ob-
tained curves using the equation:31,32
lL were prepared
l
l
l
l
l
L
l
l
16
l
l
M stock solution in
L (click
ꢀ
ꢁ
l
1
V
1
KM
½Iꢃ
1
¼
þ
1 þ
ꢄ
:
Vmax Vmax
Ki
½PNPGꢃ
l
l
l
Acknowledgements
loaded onto a 12% Tris–HCl gel for SDS–PAGE. After extensive wash-
ing of the running gel (1 h in water) to eliminate excess dye reagent,
fluorescence was detected with a Typhoon fluorescence scanner.
M.v.S. was supported by the Netherlands Proteomics Center
(NPC).

M. van Scherpenzeel et al. / Bioorg. Med. Chem. 18 (2010) 267–273
273
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