Bodipy-VAD-Fmk, a useful tool to study yeast peptide N-glycanase activity

Article abstract of DOI:10.1039/b711531h

In this paper the development of a fluorescent activity based probe, Bodipy-VAD-Fmk, for visualization of yeast peptide N-glycanase is described. The activity based probe is used to assess the efficacy of known and new chitobiose-based electrophilic traps to bind yeast peptide N-glycanase. The Royal Society of Chemistry.

Full text of DOI:10.1039/b711531h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Bodipy-VAD-Fmk, a useful tool to study yeast peptide N-glycanase activity†
Martin D. Witte, Carlos V. Descals, Sebastiaan V. P. de Lavoir, Bogdan I. Florea, Gijsbert A. van der Marel* and
Herman S. Overkleeft*
Received 27th July 2007, Accepted 13th August 2007
First published as an Advance Article on the web 29th August 2007
DOI: 10.1039/b711531h
In this paper the development of a fluorescent activity based probe, Bodipy-VAD-Fmk, for
visualization of yeast peptide N-glycanase is described. The activity based probe is used to assess the
efficacy of known and new chitobiose-based electrophilic traps to bind yeast peptide N-glycanase.
Introduction
The majority of proteins in eukaryotes are synthesized by mem-
brane bound ribosomes on the endoplasmic reticulum (ER). The
newly synthesized proteins are inserted into the lumen of the ER
via a specialized structure referred to as the translocon. In a co-
translational process, oligosaccharyl transferase may glycosylate
asparagine residues within the Asn-X-Ser/Thr (X cannot be
Pro) consensus sequence.1 The resulting Glc3Man9GlcNHAc2
glycan is deglycosylated to GlcMan9GlcNHAc2 which is then
recognized by calnexin and calrectulin. These chaperones retain
glycoproteins in the ER until they are folded properly. Properly
folded proteins progress through the ER and Golgi. A series of
deglycosylation/glycosylation events transform the high mannose
N-glycan into complex-type N-glycans. These glycans in turn help
in guiding the glycoprotein to its final destination, such as the cell
surface or the endocytic pathway.
The Man8GlcNAc2 glycan of misfolded N-linked glycoproteins
is recognized by EDEM, a lectin-like protein of the endoplasmic
reticulum associated degradation pathway, resulting in dislocation
of misfolded proteins from the ER to the cytosol. Upon arrival
the misfolded proteins are ubiquitinated and are then directed to
the proteasome for degradation.2
Fig. 1 Target compounds.
In this pathway, the amidase peptide N-glycanase (PNGase) is
responsible for deglycosylation of most N-glycosylated proteins.3
PNGase contains the characteristic catalytic Cys, His, Asp triad
responsible for the cleavage of the b-aspartyl–glucosamine bond.
PNGase can associate with the proteasome as well as with ER-
currently in use entails the deglycosylation of RNase B by
PNGase, followed by SDS-PAGE and visualization of diges-
tion products with Coomassie blue. Before use as a substrate,
commercially available RNase B requires further purification
bound proteins, suggesting that PNGase has multiple modes
and denaturation.6 Our group recently published an activity-
of action.4 For fundamental studies on the role of PNGase
based fluorescent proteasome probe, which enables direct in-
gel detection of proteasome activity.7 We reasoned that the
in vivo, specific inhibitors can be beneficial. However, only few
such inhibitors are available to date and little is known about
their efficacy and selectivity. PNGase activity can be blocked by
either commercially available broad spectrum caspase inhibitor
availability of a fluorescent analogue of 2, such as Bodipy TMR-
Ahx-Val-Ala-Asp(OMe)-fluoromethyl ketone 1 (b-VAD-Fmk),8
would allow straightforward screening of potential irreversible N-
glycanase inhibitors in an analogous fashion. We here report the
validity of this reasoning by the application of b-VAD-Fmk 1 in
the identification of the two new chitobiose-based N-glycanase
Z-VAD(OMe)-Fmk 2 or haloacetamidyl chitobiose inhibitor 3
(Fig. 1).5
In the search for new inhibitors, a sensitive and straightforward
assay would be of great use. The N-glycanase activity assay
inhibitors, 4 and 5 (Fig. 1).
Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University,
P.O. Box 9502, 2300 RA Leiden, The Netherlands. E-mail: marel_g@
Results and discussion
chem.leidenuniv.nl, h.s.overkleeft@chem.leidenuniv.nl; Fax: 31 71 527 4307;
Tel: 31 71 527 4342
† Electronic supplementary information (ESI) available: All general pro-
cedures, the synthesis and characterization of all new compounds, and
biological data. See DOI: 10.1039/b711531h
The synthesis of b-VAD-Fmk 1 is depicted in Scheme 1. Boc-
Asp-OBn 6 was converted to the fluoromethyl ketone via a
modified procedure of the patented method by Palmer.9 Hence,
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Fluoromethyl ketone 5 was synthesized as follows (Scheme 3).
C-glycoside 17, prepared according to the literature procedure,11
was treated with acidic methanol, followed by reaction with
benzaldehyde dimethylacetal to give after benzylation of the 3-OH
protected C-glycoside 18. Reaction of the ethylene moiety in 18
with mCPBA afforded the desired epoxide 19 as a diastereomeric
mixture. Regioselective opening of the epoxide in 19 with tetra-
butylammonium dihydrogen trifluoride furnished fluorohydrin 20.
Acetylation or benzoylation of the resulting alcohol followed
by opening of the benzylidene to the C6 position afforded
acceptors 23 and 24, respectively. Condensation of donor 11 with
either acceptor 23 or 24 by treatment with Ph2SO–Tf2O gave
disaccharides 25 and 26. Deprotection of the amine functionalities
in 25 and 26, ensuing acetylation of the resulting free amines and
O-deacetylation furnished fluorohydrin 27. During the removal
of the phthaloyl protective groups of benzoylated disaccharide
26, O to N benzoyl migration was observed. This problem was
however overcome by synthesizing acetyl protected 25. Oxidation
of 27 afforded fully protected fluoromethyl ketone 28. Global
deprotection gave inhibitor 5 in a reasonable yield.
Scheme 1 Reagents and conditions: (a) (i) MeI, K2CO3, DMF; (ii) 10%
Pd/C, H2, EtOAc, quant.; (b) (i) CDI, THF, 1 h; (ii) monobenzyl
fluoromalonate magnesium enolate, THF; (iii) 10% Pd/C, H2, EtOAc,
68%; (c) (i) 4 M HCl–dioxane, 45 min; (ii) 9, HCTU, DiPEA, DMF 68%;
(d) (i) TFA–H2O (95 : 5, v/v), 30 min; (ii) Bodipy TMR-OSu, DiPEA,
16 h, 41%.
Boc-Asp-OBn 6 was converted to the methyl ester, and ensuing
reduction of the benzyl ester gave Boc-Asp(OMe)OH 7. Treat-
ment of the resulting acid with 1,1ꢀ-carbodiimidazole followed
by reaction with the magnesium enolate of the monobenzyl-
fluoromalonate and hydrogenation gave fluoromethylketone 8. Re-
moval of the Boc protective group in 8 and condensation of the re-
sulting free amine with peptide 9 (see ESI†) was followed by N-Boc
deprotection and treatment with Bodipy TMR-OSu to give target
compound 1. b-VAD-Fmk 1 was obtained as a diastereomeric mix-
ture due to epimerization of the alanine a-carbon during the block
coupling.
With b-VAD-Fmk 1 in hand, our attention was focused on the
synthesis of reference compound 3 and epoxysuccinate inhibitor
4 (Scheme 2). Known donor 11 was condensed with acceptor
12 under the agency of Ph2SO–Tf2O giving disaccharide 13.10
Removal of the phthaloyl group and subsequent acetylation
afforded protected chitobiose 14. Reduction of the azido group
followed by coupling with either chloroacetic anhydride or epoxy-
succinate monoethylester afforded the protected inhibitors 15 and
16. Removal of protective groups furnished epoxide inhibitor 4.
In the case of inhibitor 3 partial reduction of the chloroacetamide
moiety was observed.
With probe 1 and inhibitors 2–5 in hand, we focused our
attention on their biological evaluation. First, the capability of
b-VAD-Fmk 1 to label PNGase was examined. To this end,
purified recombinant yeast peptide N-glycanase (YPng1) was
incubated with various concentrations of 1 for 1 h. Direct in-
gel visualization with a fluorescent scanner revealed that probe 1
labeled yeast peptide N-glycanase in a concentration-dependent
manner. From this image (Fig. 2(A)) it can be seen that the
saturation of the enzyme is reached at a concentration between 1
and 5 lM. Moreover, heat inactivated enzyme was not labeled by
b-VAD-Fmk 1 suggesting that probe 1 labeled YPng1 specifically
(Fig. 2(A), (B)). The sensitivity was tested by labeling a serial
dilution of YPng1 with 1 (0.5 lM). The labeling proved to be
very sensitive since as little as 0.9 ng of purified YPng1 could
be detected (Fig. 2(C)). The specificity of b-VAD-Fmk 1 was
further investigated by incubating the probe with an Escherichia
coli (E. coli) cell extract expressing YPng1. Exclusive labeling of
YPng1 by probe 1 was observed by in-gel visualization. We next
investigated the binding site of 1. The reported X-ray structure
of YPng1 co-crystallized with Z-VAD-Fmk 2 showed that Z-
VAD-Fmk 2 binds to the catalytic Cys residue 191 of the active
site of YPng1.12 It is therefore reasonable to assume that 1,
Scheme 2 Reagents and conditions: (a) 12, Ph2SO, TTBP, Tf2O, DCM, −60 to 0 ◦C, 85%; (b) (i) (H2NCH2)2–n-BuOH (1 : 10), 90 ◦C; (ii) Ac2O, pyr, 81%;
(c) (i) Lindlar’s cat. H2, DMF; (ii) (ClCH2CO)2O, Et3N, DMF; (iii) MeOH, p-TsOH, 16 h, 47%; (d) (i) Lindlar’s cat. H2; (ii) epoxysuccinate monoethylester,
HCTU, Et3N, DMF, 39%; (e) 5% TFA–DCM, 70%; (f) 20% Pd(OH)2/C, H2, MeOH 3: 14%, 4: 67%.
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just as lead-compound 2 binds to the same site. To verify this,
catalytically inactive YPng(C191A) was expressed in E. coli.
Incubation of YPng(C191A) with 1 followed by visualization
revealed, as expected, no significant labeling of YPng(C191A)
(Fig. 2(D), (E)).
enzyme. Next, unreacted active sites were labeled by treatment
with 1 for 30 min. Even after preincubation, fluoromethyl ketone
5 inhibits YPng1 in the high micromolar range (Fig. 2 (G)).
Conclusions
Having assessed the active-site dependent labeling-ability of 1,
its application in the identification of the inhibitory potential of
irreversible inhibitors was investigated. A solution of YPng1 and
BSA was incubated with serial dilutions of the known active-
site binding inhibitors 2 and 3, followed by incubation with
1.13 Remarkably, labeling of YPng1 was still observed at 1 mM
inhibitor concentration when the experiment was conducted in
the presence of 5 mM DTT. In addition, non-specific labeling
of BSA was detected, suggesting that reduction of disulfide
bonds by DTT caused non-specific labeling by b-VAD-Fmk 1
(Fig. 2(F)). Therefore a similar competition assay was conducted
without DTT. Non-specific labeling was minimized allowing us
determine the concentration at which 50% of enzyme activity
was inhibited (apparent IC50) by quantification of the data with
imaging software. The apparent IC50 of 2 was determined at 22
In summary, we have described the synthesis of b-VAD-Fmk 1 and
have demonstrated its ability to covalently bind to the active site
Cys191 of recombinant yeast peptide N-glycanase. b-VAD-Fmk
1 can now be used in an enzyme inhibitory assay that allows the
rapid identification of potential YPng1 inhibitors as we validated
for two known inhibitors (2 and 3) and two new chitobiose-based
inhibitors (4 and 5). Current research is focused on the design of
new inhibitors, both with respect to nature of the electrophilic trap
and the length of the oligosaccharide position.
Experimental
All general experimental details, the synthesis of peptide 9,
monobenzyl fluoromalonate magnesium enolate, disaccharides
13 and 14 and acceptors 23 and 24, the silver stained gels of
YPng labeling in E. coli cell extracts and inhibitor-dose-response
curves are available in the ESI.† TTBP (tert-butylpyrimidine),
Bodipy TMR-OSu, tetrabutylammonium dihydrogen trifluoride
(TBA·H2F3) and epoxisuccinate monoethyl ester were synthesized
as described.7,14
5 lM and the IC50 of haloacetamide 3 was 1.6
0.5 lM. We
are aware that our assay is not ideally suited for an accurate
determination of IC50 values and is not suitable at all for the
establishment of ki values. Such detailed kinetic studies are
executed best by making use of a substrate-based assay such as
those reported by Ito and Ploegh.5,6 We believe, however, that
our assay is highly useful for the rapid identification of potential
YPng1 inhibitors, and to give a first impression of its potency.
In order to further illustrate this point, the inhibitory activity
of potential chitobiose inhibitors 4 and 5 was examined via the
competition assay. The apparent IC50 of epoxysuccinate 4 proved
to be 1.6 0.5 lM. To our surprise, fluoromethyl ketone 5 proved
to be a poor inhibitor of YPng1. Disaccharide 5 was preincubated
with YPng1 for 2 h allowing 5 to react with the active site of the
N-tert-Butoxycarbonylaspartyl(OMe) fluoromethyl ketone (8)
Boc-Asp-OBn 6 (1.6 g, 5 mmol) was treated with iodomethane
(0.622 mL, 10 mmol, 2 equiv.) and K2CO3 (0.69 g, 5 mmol) in
DMF. After 16 h stirring, the reaction was diluted with EtOAc,
washed with 1 M HCl, NaHCO3 (sat. aq.), brine, dried (MgSO4)
and concentrated in vacuo. The resulting Boc-Asp(OMe)OBn
Fig. 2 (A) Fluorescent in-gel detection of YPng1 by labeling with the indicated concentration of 1. “PS” represents the molecular marker (prestained
marker purchased from Fermentas). “M” represents the dual color marker. “—” represents heat-inactivated YPng1, incubated with 50 lM 1 for 2 h at
37 ◦C. (B) Silver stained gel of labeled YPng1. (C) Determination of the sensitivity of probe 1 by incubating 1 (0.5 lM) with various amounts of YPng1.
(D) Labeling of catalytically inactive YPng(C191A) (1 mg mL−1) with a serial dilution of 1 in E. coli cell extracts. (E) Labeling of YPng1 (1 mg mL−1
)
with 1 in E. coli cell extracts. (F) Non-specific labeling of BSA by b-VAD-Fmk 1 in the presence of 5 mM DTT. (G) Competition assay. The indicated
amount of inhibitors 2–4 in the presence of 1 (0.5 lM) was incubated with YPng1 (100 ng) and BSA (9 lg) in PBS (20 mM sodium phosphate, 150 mM
NaCl, pH = 7.2) for 1 h. Fluoromethyl ketone 5 was preincubated with YPng1 for 2 h followed by incubation with 1 for 30 min.
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was dissolved in EtOAc before being reduced under the agency
of palladium (10%) on charcoal under H2 atmosphere. TLC-
analysis showed complete reduction of the benzyl ester after 3 h.
Filtration and ensuing concentration afforded Boc-Asp(OMe)OH
7 (1.36 g, 5 mmol) which was co-evaporated with toluene and
dissolved in THF (25 mL). The solution was cooled to 0 ◦C,
carbodiimidazole (851 mg, 5.25 mmol) was added and the reaction
was stirred for 1 h at 0 ◦C. The mixture was cooled to −20 ◦C
before monobenzyl fluoromalonate magnesium enolate (see ESI†)
(6.25 mmol, 1.5 equiv.) was added dropwise. After 45 min stirring
at −20 ◦C and 3.5 h additional stirring at room temperature, the
reaction was poured into 1 M HCl, extracted with EtOAc, washed
with NaHCO3 (sat. aq.), brine, dried (Na2SO4) and concentrated
in vacuo. The residue was redissolved in EtOAc, a catalytic amount
of activated palladium (10%) on charcoal was added and the
mixture was stirred overnight under H2 atmosphere. Filtration
over Celite, concentration under reduced pressure followed by
silica gel column chromatography (Tol → 5% EtOAc–Tol) gave
250 × 10.00 mm, particle size is 5); linear gradient 45 → 48.5%
in 3 cv. Silica gel chromatography (CHCl3 → 3% MeOH–CHCl3)
1
gave both diastereomers. Diastereomer 1: H NMR (600 MHz,
CDCl3) d/ppm 7.97–7.77 (m, 2H), 7.69–7.44 (m, 2H), 7.01–6.93
(m, 2H), 6.61–6.47 (m, 1H), 6.11–5.94 (m, 1H), 5.23–4.76 (m, 2H),
4.47–4.40 (m, 1H), 3.86 (s, 1H), 3.66 (s, 1H), 3.31–3.19 (m, 1H),
3.14–3.05 (m, 1H), 2.98–2.67 (m, 4H), 2.53 (s, 3H), 2.33–2.26 (m,
2H), 2.21 (s, 1H), 2.17–1.93 (m, 4H), 1.49–1.08 (m, 1H), 1.00–0.78
1
(m, 9H). Diastereomer 2: H NMR (600 MHz, CDCl3) d/ppm
7.90–7.84 (m, 2H), 7.14–7.05 (m, 1H), 6.98–6.94 (m, 2H), 6.61–
6.52 (m, 1H), 6.16–5.96 (m, 1H), 5.16 (dd, J = 46.2, 16.0 Hz, 1H),
5.02 (dd, J = 47.0, 16.3 Hz, 1H), 4.89–4.84 (m, 1H), 4.46–4.36 (m,
1H), 4.14 (td, J = 15.0, 6.9, 6.9 Hz, 1H), 3.86 (s, 1H), 3.68 (s, 3H),
3.36–3.22 (m, 1H), 3.14–2.66 (m, 5H), 2.55 (s, 3H), 2.36–2.24 (m,
2H), 2.22 (s, 1H), 2.14–1.96 (m, 3H), 1.47–1.07 (m, 1H), 1.01–0.69
(m, 9H). FT–IR: vmax(neat)/cm−1 3267.9, 2940.0, 1602.3, 1526.8,
1461.8, 1435.9, 1293.8, 1255.0, 1232.7, 1199.7, 1176.9, 1135.2,
1056.5, 995.8, 942.1, 836.6, 799.1, 721.9, 662.6, 609.8.
1
title compound 8 in 68% yield (0.893 g, 3.4 mmol). H NMR
(600 MHz, CDCl3) d/ppm 5.54 (d, J = 7.9 Hz, 1H, NH), 5.23
(dd, J = 47.0, 16.4 Hz, 1H, CH2F), 5.12 (dd, J = 47.3, 16.5 Hz,
1H, CH2F), 4.63 (td, J = 9.2, 5.2, 5.2 Hz, 1H, H-a), 3.70 (s, 3H,
CH3 OMe), 3.08 (dd, J = 17.3, 4.1 Hz, 1H), 2.84 (dd, J = 17.3,
4.5 Hz, 1H, H-b), 1.46 (s, 9H, CH3 tBu). 13C NMR (150 MHz,
N-(O-(2-Acetamido-3-O-benzyl-2-deoxy-b-D-glucopyranosyl)-
(1→4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-
glucopyranosyl)chloroacetamide (15)
Azide 14 (225 mg, 0.3 mmol) was dissolved in DMF (2 mL),
Lindlar’s catalyst (50 mg) was added and the solution was
stirred overnight under H2 atmosphere. Subsequently, the mixture
was purged with argon gas after which chloroacetic anhydride
(77 mg, 0.45 mmol, 1.5 equiv.) and Et3N (71 lL, 0.51 mmol,
1.7 equiv.) were added. The solution was stirred overnight, filtered,
concentrated in vacuo and redissolved in MeOH (2 mL). p-
Toluenesulfonic acid (6 mg, 30 lmol) was added. TLC analysis
showed complete conversion to a polar product after overnight
stirring. The reaction was quenched with Et3N (0.1 mL), concen-
trated and applied to silica gel chromatography (CH2Cl2 → 2%
MeOH–CH2Cl2) affording title compound 15 in 47% (109 mg,
=
CDCl3) d/ppm 203.17 (d, J = 16.4 Hz, C O ketone), 171.69
=
=
(C O COOtBu), 155.19 (C O Boc), 84.14 (d, J = 183.4 Hz,
CH2F), 80.76 (Cq tBu), 53.49 (CH-a), 52.21 (CH3 OMe), 35.36
23
(CH2-b), 28.20 (CH3 tBu). [a]D −5.6 (c = 1.26, CHCl3). FT-
IR: vmax(neat)/cm−1 3362.2, 2980.0, 1706.0, 1505.8, 1438.7, 1393.8,
1367.6, 1247.6, 1159.8, 1055.4, 1000.0, 861.3, 780.3, 631.8. HRMS:
(M − Boc + H+) calc. for C6H11FNO3 164.07175, found 164.07166.
Bodipy-Ahx-Val-Ala-Asp(OMe) fluoromethyl ketone (1)
Fluoromethyl ketone 8 (40 mg, 0.15 mmol, 1.25 equiv.) was
dissolved in 4 M HCl in dioxane (2 mL). After 45 min, TLC
analysis showed complete conversion to a very polar product.
The solution was concentrated in vacuo, co-evaporated thrice with
toluene and dissolved in DMF (2 mL) before Boc-Ahx-Val-Ala-
OH 9 (48 mg, 0.12 mmol), HCTU (62 mg, 0.15 mmol, 1.25 equiv.)
and diisopropylethylamine (52 lL, 0.30 mmol, 2.5 equiv.) were
added. LC/MS analysis showed complete consumption after 2 h.
The reaction was diluted with CH2Cl2, washed with NaHCO3
(sat. aq.), 1 M HCl, brine, dried (Na2SO4) and concentrated.
Purification by silica gel chromatography (CH2Cl2 → 1% MeOH–
CH2Cl2) gave peptide 10 (68%, 45 mg, 82 lmol). LC/MS: Rt
7.10 min; linear gradient 10 → 90% B in 13.5 min; m/z = 547.2
(M + H)+, 447.2 (M − Boc + H)+. Intermediate 10 was dissolved in
TFA–H2O (2 mL, 95 : 5 v/v), stirred for 30 min and concentrated
under reduced pressure. Residual traces of TFA were removed
by co-evaporation with toluene. Subsequently, the free amine
was dissolved in DMF, treated with Bodipy-TMR-OSu (41 mg,
82 lmol, 1 equiv.) and diisopropylethylamine (36 lL, 0.2 mmol,
2.5 equiv.) and stirred overnight. The solution was diluted with
CH2Cl2, washed with 1 M HCl, dried (Na2SO4) and concentrated.
RP-HPLC yielded Bodipy TMR-Ahx-Val-Ala-Asp(OMe)-Fmk 1
as a diastereomeric mixture (41%, 28.3 mg, 34 lmol). LC/MS: Rt
8.15 min; linear gradient 10 → 90% B in 13.5 min; m/z = 827.4
(M + H)+, 807.3 (M − F)+. RP-HPLC: Rt 2.8 cv (column volume =
1
0.14 mmol). H NMR (500 MHz, MeOD) d/ppm 7.41–7.23 (m,
15H, H arom), 5.06 (d, J = 11.6, CH2 Bn), 5.02 (d, J = 10.4 Hz,
1H, H-1) 4.90 (d, J = 11.5 Hz, 1H, CH2 Bn), 4.72–4.58 (m, 5H,
4 × CH2 Bn, H-1ꢀ), 4.12 (t, J = 9.2, 9.2 Hz, 1H, H-4), 4.02 (d,
J = 6.3 Hz, 1H, CH2Cl), 3.99 (t, J = 10.0, 10.0 Hz, 1H, H-2),
3.83–3.75 (m, 4H, H-2ꢀ, H-6, H-6aꢀ), 3.64 (dd, J = 9.9, 9.1 Hz, 1H,
H-3), 3.56–3.46 (m, 2H, H-3ꢀ, H-5), 3.42 (dd, J = 9.7, 8.8 Hz, 1H,
H-6bꢀ), 3.21 (ddd, J = 9.5, 7.1, 2.1 Hz, 1H, H-5ꢀ), 1.90 (s, 3H, CH3
NHAc), 1.90 (s, 3H, CH3 NHAc). 13C NMR (125 MHz, MeOD)
=
=
=
d/ppm 173.75 (C O), 173.52 (C O), 169.85 (C O), 140.29 (Cq
Bn), 139.67 (Cq Bn), 139.51 (Cq Bn), 129.64–128.57 (CH arom),
101.14 (C-1ꢀ), 83.87 (C-3ꢀ), 83.00 (C-3), 80.48 (C-1), 78.72 (C-5ꢀ),
78.35 (C-5), 77.00 (CH2 Bn), 75.83 (CH2 Bn), 75.75 (C-4), 74.36
(CH2 Bn), 72.67 (C-4ꢀ), 69.40 (C-6), 62.96 (C-6ꢀ), 57.30 (C-2ꢀ), 54.92
(C-2), 43.10 (CH2Cl), 23.17 (CH3 NHAc), 22.82 (CH3 NHAc). FT-
IR: vmax(neat)/cm−1 3277.8, 1651.8, 1557.8, 1455.5, 1372.5, 1312.7,
23
1050.9, 737.6, 696.0. [a]D −2.4 (c = 0.74, MeOH). HRMS: (M +
H+) calc. for C39H49ClN3O11 770.30501, found 770.30547.
N-(O-(2-Acetamido-2-deoxy-b-D-glucopyranosyl)-(1→4)-2-
acetamido-2-deoxy-b-D-glucopyranosyl)chloroacetamide (3)
Partially deprotected chloroacetamide 15 (30 mg, 39 lmol) was
dissolved in MeOH (1 mL). 20% Pd(OH)2 on activated charcoal
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(20 mg) was added. The resulting suspension was stirred under H2
atmosphere. After 4 h, the mixture was filtered and concentrated in
vacuo. The crude product was acetylated by treatment with acetic
anhydride (1 mL) and pyridine (2 mL). After stirring overnight,
the reaction was concentrated and purified by silica gel chro-
matography (CH2Cl2 → 2% MeOH–CH2Cl2). Fully acetylated
haloacetamide was deprotected by stirring it under the agency of
30% NaOMe in MeOH (0.1 mL) in methanol (1 mL) until TLC
showed complete conversion. Neutralization by amberlite IR-120
H+ followed by concentration and purification over HW-40 gel
filtration (1% AcOH–H2O) afforded title compound 3 as a white
solid (14%, 2.69 mg, 5.4 lmol). 1H NMR (600 MHz, D2O) d/ppm
5.09 (d, J = 9.7 Hz, 1H, H-1), 4.60 (d, J = 8.5 Hz, 1H, H-1ꢀ), 4.15
(d, J = 14.3 Hz, 1H, CH2Cl), 4.11 (d, J = 14.3 Hz, 1H, CH2Cl),
3.96–3.44 (m, 12H), 2.07 (s, 3H, CH3 NHAc), 2.00 (s, 3H, CH3
DMF). HRMS: (M + H+) calc. for C50H58N3O14 924.39133, found
924.39219.
(2S,3S)-3-N-(O-(2-Acetamido-2-deoxy-b-D-glucopyranosyl)-
(1→4)-2-acetamido-2-deoxy-b-D-glucopyranosylcarbamoyl)-
oxirane-2-carboxylic acid ethyl ester (4)
Epoxide 16 (27 mg, 29.2 lmol) was suspended in CH2Cl2 (1 ml),
cooled to 0 ◦C and treated with TFA (50 ll) and H2O (5 ll). After
45 min stirring at 0 ◦C, the reaction was quenched with NaHCO3
(sat. aq.) followed by extraction. The organic layer was dried
(MgSO4) and concentrated. Silica gel chromatography purification
(CH2Cl2 → 2% MeOH–CH2Cl2) afforded the partially protected
epoxide (68%, 17 mg, 20.3 lmol). The remaining benzyl groups
were removed by dissolving the epoxide in EtOH, followed by
the addition of 20% Pd(OH)2 on activated charcoal (cat.) and
stirring under H2 atmosphere for 16 h. The solution was filtered,
concentrated and applied to HW-40 gel filtration (1% AcOH–H2O)
furnishing epoxide inhibitor 4 as a white solid (67% over two steps,
11.06 mg, 19.6 lmol). 1H NMR (600 MHz, D2O) d/ppm 5.09 (d,
J = 9.6 Hz, 1H, H-1), 4.59 (d, J = 8.4 Hz, 1H, H-1ꢀ), 4.28 (q,
J = 7.1, 7.1, 7.1 Hz, 2H, CH2 OEt), 3.96–3.43 (m, 14H), 2.06
(s, 3H, CH3 NHAc), 2.01 (s, 3H, CH3 NHAc), 1.29 (t, J = 7.2,
7.2 Hz, 1H, CH3 OEt). 13C NMR (150 MHz, D2O) d/ppm 174.26
NHAc). 13C NMR (150 MHz, D2O) d/ppm 174.31 (C O), 174.06
=
ꢀ
=
=
(C O), 169.91 (C O), 100.83 (C-1 ), 78.21, 78.19 (C-1), 75.77,
75.33, 72.85, 71.98, 69.12, 59.95 (C-6 or C-6ꢀ), 59.31 (C-6 or C-
6ꢀ), 55.01 (C-2ꢀ), 53.12 (C-2), 41.46 (CH2Cl), 21.53 (CH3 NHAc),
21.35 (CH3 NHAc).
(2S,3S)-3-N-(O-(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-b-D-glucopyranosyl)-(1→4)-2-acetamido-3,6-di-O-benzyl-
2-deoxy-b-D-glucopyranosylcarbamoyl)oxirane-2-carboxylic acid
ethyl ester (16)
=
=
=
=
(C O), 174.06 (C O), 168.31 (C O), 167.98 (C O), 100.83 (C-
1ꢀ), 78.16, 77.85 (C-1), 75.79, 75.33, 72.87, 72.85, 71.92, 69.12,
62.90 (CH2 OEt), 59.95 (C-6 or C-6ꢀ), 59.30 (C-6 or C-6ꢀ), 55.01,
53.19 (C-2), 52.72 (C-2ꢀ), 52.05, 21.53 (CH3 NHAc), 21.38 (CH3
NHAc), 12.58 (CH3 OEt). FT-IR: vmax(neat)/cm−1 3275.0, 1737.5,
1651.0, 1539.8, 1410.3, 1374.5, 1309.5, 1205.3, 1159.8, 1023.7,
Azide 14 (225 mg, 0.3 mmol) was dissolved in DMF (2 mL),
Lindlar’s catalyst (50 mg) was added and the solution was stirred
overnight under H2 atmosphere. Subsequently, the mixture was
purged with argon gas after which epoxisuccinate monoethyl ester
(115 mg, 0.72 mmol, 2.4 equiv.), HCTU (323 mg, 0.78 mmol,
2.6 equiv.), Et3N (0.216 mL, 1.56 mmol, 5.2 equiv.) were added.
After stirring overnight, the reaction was concentrated in vacuo,
diluted with CH2Cl2, washed with aqueous 1 M HCl, NaHCO3
(sat. aq.) and brine, dried (Na2SO4) and evaporate to dryness. Silica
gel chromatography (CH2Cl2 → 2% MeOH–CH2Cl2) furnished
title compound 16 in 39% (110 mg, 0.119 mmol). 1H NMR
(500 MHz, DMF) d/ppm 8.72 (d, J = 9.0 Hz, 1H, NH), 8.24
(d, J = 8.7 Hz, 1H, NH), 8.21 (d, J = 8.9 Hz, 1H, NH), 7.52–
7.24 (m, 20H, H arom), 5.73 (s, 1H, CHPh), 5.12 (t, J = 9.3,
9.3 Hz, 1H), 5.00 (d, J = 11.1 Hz, 1H, CH2 Bn), 4.92 (d, J =
7.6 Hz, 1H, H-1ꢀ), 4.84 (d, J = 11.9 Hz, 1H, CH2 Bn), 4.72 (d,
J = 11.0 Hz, 1H, CH2 Bn), 4.70 (d, J = 11.8 Hz, 1H, CH2 Bn),
4.67 (d, J = 11.8 Hz, 1H, CH2 Bn), 4.62 (d, J = 11.8 Hz, 1H, CH2
Bn), 4.26–4.19 (m, 2H, CH2 OEt), 4.11 (dd, J = 10.2, 5.0 Hz, 1H,
H-6a), 4.01 (t, J = 9.0, 9.0 Hz, 1H), 3.97–3.90 (m, 3H), 3.88–3.83
(m, 2H), 3.82–3.77 (m, 2H), 3.74 (d, J = 1.8 Hz, 1H), 3.57–3.52
(m, 1H), 3.35–3.27 (m, 3H), 1.98 (s, 3H, CH3 NHAc), 1.90 (s,
3H, CH3 NHAc), 1.25 (t, J = 7.1, 7.1 Hz, 3H, CH3 OEt). 13C
943.9, 896.9. [a]D +19.2 (c = 0.24, H2O). HRMS: (M + H+)
23
calc. for C22H36N3O14 566.21918, found 566.21904.
(2R/S)-3-C-(O-(3-O-Benzyl-4,6-O-benzylidene-2-deoxy-2-
phthalimido-b-D-glucopyranosyl)-(1→4)-3,6-di-O-benzyl-2-deoxy-
2-phthalimido-b-D-glucopyranosyl)-1-fluoro-2-acetoxypropane (25)
Before being dissolved in freshly distilled CH2Cl2, known donor
11 (454 mg, 0.78 mmol, 1.2 equiv.), diphenyl sulfoxide (174 mg,
0.86 mmol, 1.3 equiv.) and TTBP (487 mg, 1.96 mmol, 3.0 equiv.)
were co-evaporated thrice with toluene. Subsequently, activated
˚
4A MS were added and the reaction mixture was cooled to
◦
−60 C. Tf2O (145 lL, 0.86 mmol, 1.3 equiv.) was added. After
10 min pre-activation, acceptor 23 (427 mg, 0.65 mmol) was
added, followed by stirring at −60 ◦C for 1 h. The temperature
was slowly raised to 0 ◦C over a period of 4 h, the reaction
was quenched by addition of Et3N, diluted with EtOAc, washed
with NaHCO3 (sat. aq.), brine, dried and concentrate in vacuo.
Column chromatography (Tol → 10% EtOAc–Tol) furnished title
compound 26 as a colorless oil (97%, 668 mg, 0.63 mmol) which
was directly used for the next reaction. 1H NMR (600 MHz,
CDCl3) d/ppm 8.03–6.79 (m, 28H, H arom), 5.53–5.50 (m, 1H,
CHPh), 5.39–5.34 (m, 1H, H-1ꢀꢀ), 5.22–5.00 (m, 1H, H-2ꢀꢀ), 4.85–
4.76 (m, 2H, CH2 Bn), 4.57–4.10 (m, 11H), 4.02–3.91 (m, 2H),
3.76–3.68 (m, 1H), 3.57–3.34 (m, 3H), 3.32–3.18 (m, 2H), 1.96–
1.85 (m, 3H, CH3 OAc), 1.71–1.51 (m, 2H, H-1ꢀ). 13C NMR
=
=
NMR (150 MHz, DMSO) d/ppm 169.48 (C O), 169.32 (C O),
=
=
166.78 (C O), 165.42 (C O), 138.92 (Cq Bn or Ph), 138.63 (Cq
Bn or Ph), 138.44 (Cq Bn or Ph), 137.47 (Cq Bn or Ph), 128.67–
125.87 (CH arom), 100.40 (C-1ꢀ), 99.93 (CHPh), 80.85, 80.68,
78.31, 76.01, 75.10, 73.36 (CH2 Bn), 73.15 (CH2 Bn), 71.81 (CH2
Bn), 68.22 (C-6 or C-6ꢀ), 67.68 (C-6 or C-6ꢀ), 65.50, 61.49 (CH2
OEt), 53.24, 52.67, 51.13, 22.88 (CH3 NHAc), 22.67 (CH3 NHAc),
13.79 (CH3 OEt). FT-IR: vmax(neat)/cm−1 3277.7, 1651.9, 1538.3,
1455.4, 1371.6, 1205.0, 1069.8, 735.8, 694.4. [a]D23 +11.2 (c = 0.66,
=
=
(150 MHz, CDCl3) d/ppm 170.31 (C O Ac), 170.09 (C O Ac),
=
=
=
167.86 (C O phth), 167.73 (C O phth), 167.70 (C O phth),
=
167.61 (C O phth), 138.53 (Cq Bn or Ph), 138.45 (Cq Bn or Ph),
3694 | Org. Biomol. Chem., 2007, 5, 3690–3697
This journal is The Royal Society of Chemistry 2007
©

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138.22 (Cq Bn or Ph), 138.04 (Cq Bn or Ph), 137.85 (Cq Bn or Ph),
137.30 (Cq Bn or Ph), 137.28 (Cq Bn or Ph), 133.86 (CH phth),
133.78 (CH phth), 131.40 (Cq phth), 128.93–126.01 (CH arom),
123.35 (CH phth), 123.25 (CH phth), 101.13 (CHPh), 97.58 (C-
1ꢀꢀ), 97.39 (C-1ꢀꢀ), 83.97 (d, J = 173.7 Hz, C-3ꢀ), 83.14, 83.13, 83.09
(d, J = 172.6 Hz, C-3ꢀ), 78.25, 78.16, 77.27, 77.22, 77.16, 77.01,
76.79, 76.18, 75.93, 74.43, 74.40, 74.35, 74.31, 74.04, 72.61, 72.59,
71.58, 71.33, 69.97 (d, J = 19.4 Hz, C-2ꢀ), 68.85 (d, J = 19.0 Hz,
C-2ꢀ), 68.70, 68.68, 68.17, 68.08, 65.69, 65.61, 56.46, 55.82, 55.67,
32.40 (d, J = 6.2 Hz, C-1ꢀ), 31.50 (d, J = 6.7 Hz, C-1ꢀ), 20.87 (CH3
OAc), 20.83 (CH3 OAc). FT-IR: vmax(neat)/cm−1 2873.8, 1774.7,
1739.9, 1710.2, 1612.1, 1496.1, 1468.1, 1454.0, 1383.9, 1233.5,
1173.0, 1144.7, 1070.8, 1027.4, 997.4, 967.8, 874.0, 794.3, 736.0,
719.7, 695.8, 661.5. HRMS: (M + Na+) calc. for C61H57FN2O14Na
1083.36860, found 1083.36897.
2% MeOH–CH2Cl2) furnished benzoyl migrated compound 30
(95 mg, 0.105 mmol, 23%, Scheme 3) and title compound 27
(211 mg, 0.250 mmol, 56%).
Starting from 25: disaccharide 25 (286 mg, 0.27 mmol) was
converted to fluorohydrin 27 as depicted above. After silica gel
purification (CH2Cl2 → 2% MeOH–CH2Cl2) title compound 27
1
was obtained as a white solid (67%, 152 mg, 0.180 mmol). H
NMR (600 MHz, DMSO) d/ppm 8.11 (d, J = 8.4 Hz, 1H, NH),
7.96–7.93 (m, 1H, NH), 7.43–7.23 (m, 20H, H arom), 5.67 (s,
1H, CHPh), 5.04–4.92 (m, 1H), 4.86–4.82 (m, 1H, CH2 Bn),
4.75–4.71 (m, 2H, CH2 Bn, H-1ꢀꢀ), 4.65–4.54 (m, 4H, CH2 Bn),
4.43–4.15 (m, 2H, H-3ꢀ), 4.06–4.00 (m, 1H), 3.94–3.81 (m, 1H),
3.79–3.29 (m, 12H), 3.19–3.08 (m, 1H), 1.88–1.79 (m, 6H, CH3
NHAc), 1.54–1.21 (m, 2H, H-1ꢀ). 13C NMR (150 MHz, DMSO)
=
=
=
d/ppm 169.31 (C O NHAc), 169.28 (C O NHAc), 169.16 (C O
NHAc), 139.29 (Cq Bn or Ph), 139.27 (Cq Bn or Ph), 138.77 (Cq
Bn or Ph), 138.74 (Cq Bn or Ph), 138.70 (Cq Bn or Ph), 137.60
(Cq Bn or Ph), 128.77–125.99 (CH arom), 100.91 (C-1ꢀꢀ), 100.07
(CHPh), 87.29 (d, J = 168.4 Hz, C-3ꢀ), 86.50 (d, J = 167.1 Hz,
C-3ꢀ), 80.95, 78.13, 78.02, 76.32, 74.72, 73.29, 73.26, 71.96, 71.86,
68.82, 67.80, 66.55 (d, J = 18.6 Hz, C-2ꢀ), 65.59, 65.49 (d, J =
18.9 Hz, C-2ꢀ), 54.17, 54.06, 35.01 (d, J = 7.8 Hz, C-1ꢀ), 34.85 (d,
J = 6.5 Hz, C-1ꢀ), 22.98 (CH3 NHAc), 22.92 (CH3 NHAc). FT–
IR: vmax(neat)/cm−1 3275.4, 2870.2, 1652.0, 1538.6, 1453.7, 1371.7,
1319.7, 1072.8, 1011.3, 746.9, 694.4, 668.1, 615.9. HRMS: (M +
H+) calc. for C47H56FN2O11 843.38627, found 843.38699.
(2R/S)-3-C-(O-(3-O-Benzyl-4,6-O-benzylidene-2-deoxy-2-
phthalimido-b-D-glucopyranosyl)-(1→4)-3,6-di-O-benzyl-2-deoxy-
2-phthalimido-b-D-glucopyranosyl)-1-fluoro-2-benzoyloxypropane
(26)
Acceptor 24 (574 mg, 0.88 mmol) was condensed with donor
11 as described for disaccharide 25. Purification by silica gel
chromatography (Tol → 7.5% EtOAc–Tol) gave disaccharide 26
in 51% (500 mg, 0.45 mmol) as a colorless oil.
(2R/S)-3-C-(O-(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-b-D-glucopyranosyl)-(1→4)-2-acetamido-3,6-di-O-benzyl-
2-deoxy-b-D-glucopyranosyl)-1-fluoro-2-hydroxypropane (27)
3-C-(O-(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-b-
D-glucopyranosyl)-(1→4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
b-D-glucopyranosyl)-1-fluoro-2-propanone (28)
Starting from 26: Disaccharide 26 (500 mg, 0.45 mmol) was
dissolved in n-BuOH–ethylenediamine (10 : 1 v/v, 11 mL), before
being stirred at 90 ◦C for 8 h. The mixture was cooled to rt,
concentrated in vacuo, co-evaporated with toluene and used for
the next reaction. The mixture was suspended in pyridine (5 mL),
Fluorohydrin 27 (211 mg, 0.250 mmol) was dissolved in CH2Cl2
(5 mL) before being reacted with Dess–Martin periodinane
(415 mg, 0.98 mmol, 3 equiv.). After 5 h, TLC analysis showed
complete consumption of the starting material and the reaction
was quenched with 10% aq. NaHCO3 (10 mL) and 2 M aq.
NaS2O3 (10 mL). The layers were separated, the organic layer
was dried (MgSO4) and concentrated. Purification over silica gel
column chromatography (CH2Cl2 → 2% MeOH–CH2Cl2) gave
fluoromethyl ketone 28 (162 mg, 0.193 mmol) in 77% yield as
a white solid. 1H NMR (600 MHz, DMSO) d/ppm 8.08 (d,
◦
cooled to 0 C, treated with Ac2O (3 mL) and stirred overnight.
After being quenched with MeOH, the mixture was concentrated
in vacuo, redissolved in dichloromethane, washed with 1 M HCl,
dried (MgSO4) and concentrated. The resulting off-white powder
was dissolved in MeOH (2 mL), reacted with 30% NaOMe in
MeOH (0.2 mL) for 1 h, after which it was neutralized with AcOH
and concentrated. Silica gel column chromatography (CH2Cl2 →
Scheme 3 Reagents and conditions: (a) (i) Amberlite H+, MeOH, reflux, 16 h, quant; (ii) PhCH(OMe)2, pTsOH, MeCN, 91%; (b) BnBr, NaH, TBAI,
DMF, 6 h, 68%; (c) mCPBA, CH2Cl2, reflux, 4 h, 88%; (d) TBA·H2F3, Tol, microwave, 180 ◦C, 20 min, 84%; (e) Ac2O, pyr, 96%; (f) BzCl, DMAP, pyr,
92%; (g) TES, TfOH, DCM, −78 ◦C, 45 min, 79–85%; (h) 11, Ph2SO, Tf2O, TTBP, DCM, −60 to 0 ◦C, 25: 97% 26: 51%; (i) (i) (H2NCH2)2–n-BuOH
(1 : 10), 90 ◦C; (ii) Ac2O, pyr; (iii) NaOMe, MeOH, starting from 25: 67%, starting from 26: 56%; (j) Dess–Martin periodinane, DCM, 77%; (k) 5%
TFA–DCM, H2O, 0 ◦C, 87%; (l) 20% Pd(OH)2/C, H2, MeOH, 45%.
This journal is
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©

View Article Online
1
J = 8.4 Hz, 1H, NH), 7.97 (d, J = 8.8 Hz, 1H, NH), 7.42–7.24
(m, 20H, H arom), 5.67 (s, 1H, CHPh), 5.06 (ddd, J =46.8, 30.1,
16.7 Hz, 2H, H-3ꢀ), 4.83 (d, J = 11.0 Hz, 1H, CH2 Bn), 4.74–4.69
(m, 2H, CH2 Bn, H-1ꢀꢀ), 4.61–4.52 (m, 4H, CH2 Bn), 4.03 (dd, J =
10.0, 4.7 Hz, 1H), 3.80–3.58 (m, 8H), 3.55 (t, J = 10.1, 10.1 Hz,
1H), 3.50 (t, J = 9.1, 9.1 Hz, 1H), 3.36 (dd, J = 9.2, 4.5 Hz,
1H), 3.13 (dt, J = 9.6, 9.4, 5.6 Hz, 1H), 1.84 (s, 3H, CH3 NHAc),
5 (16 mg, 33.2 lmol, 45%) was obtained as a white solid. H
NMR (600 MHz, D2O) d/ppm 5.08 (d, J = 46.5 Hz, 1H, H-3ꢀ),
4.57 (d, J = 8.5 Hz, 1H, H-1ꢀꢀ), 3.96–3.41 (m, 13H), 2.77–2.68
(m, 2H), 2.06 (s, 3H, CH3 NHAc), 2.00 (s, 3H, CH3 NHAc).
13C NMR (150 MHz, D2O) d/ppm 207.24 (d, J = 15.3 Hz, C-
ꢀ
ꢀꢀ
=
=
2 ), 174.57 (C O NHAc), 174.53 (C O NHAc), 101.41 (C-1 ),
85.35 (d, J = 180.1 Hz, C-3ꢀ), 79.45, 78.03, 75.88, 73.52, 73.42,
69.66, 60.49 (C-6 or C-6ꢀꢀ), 60.11 (C-6 or C-6ꢀꢀ), 55.56 (C-2ꢀꢀ),
54.41 (C-2), 40.09 (C-2ꢀ), 22.09 (CH3 NHAc), 22.04 (CH3 NHAc).
FT-IR: vmax(neat)/cm−1 3270.1, 2872.1, 1732.6, 1652.1, 1558.1,
1406.9, 1372.9, 1306.3, 1204.1, 1162.8, 1105.6, 1077.5, 1025.8,
1.80 (s, 3H, CH3 NHAc). 13C NMR (150 MHz, DMSO) d/ppm
ꢀ
=
=
203.14 (d, J = 14.8 Hz, C-2 ), 169.31 (C O NHAc), 169.20 (C O
NHAc), 139.05 (Cq Bn), 138.64 (Cq Bn), 138.56 (Cq Bn), 137.48
(Cq Bn), 128.66–125.86 (CH arom), 100.71 (C-1ꢀꢀ), 99.91 (CHPh),
85.12 (d, J = 179.8 Hz, C-3ꢀ), 81.46, 80.81, 78.17, 75.94, 74.13,
73.26 (CH2 Bn), 73.12 (CH2 Bn), 71.72 (CH2 Bn), 68.51 (C-6 or
C-6ꢀ), 67.67 (C-6 or C-6ꢀ), 65.47, 55.33 (C-2 or C-2ꢀ), 53.63 (C-2 or
C-2ꢀ), 40.71 (C-1ꢀ), 22.86 (CH3 NHAc), 22.72 (CH3 NHAc). FT-
IR: vmax(neat)/cm−1 3274.2, 2863.9, 1727.9, 1657.8, 1651.9, 1557.8,
1497.1, 1454.4, 1371.4, 1319.4, 1174.3, 1085.8, 1028.1, 1016.2,
23
948.3, 893.7. [a]D −11.1 (c = 0.36, H2O). HRMS: (M + H+)
calc. for C19H32FN2O11 483.19846, found 483.19824.
Expression of YPng1 and YPng(C191A)
BL21/DE3 E. coli transformed with either pET28a-YPng1 or
pET28a-YPng(C191A) were cultured overnight at 37 ◦C in 50 mL
LB media containing kanamycin (50 lg mL−1). The culture was
transferred to 450 mL fresh LB media and cultured at 37 ◦C until
OD600 = 0.8. Subsequently, IPTG (5 mL, 0.1 M) was added and the
cells were further incubated for 3 h at 37 ◦C. Next, the cells were
centrifuged at 6000 rpm for 20 min at 4 ◦C. The resulting pellet was
resuspended in 20 mM Tris (pH = 8), 100 mM NaCl, 5% glycerol
and 1% Triton X-100. The resulting suspension was incubate on ice
for 30 min followed by 20 min sonication (90% maximum power,
5 s pulse and 3 s wait), the solution was centrifuged at 12000 g
(10 min at 4 ◦C) affording the cell extracts.
23
960.2, 917.5, 733.9, 694.1. [a]D −5 (c = 0.28, DMF). HRMS:
(M + H+) calc. for C47H54FN2O11 841.37062, found 841.37119.
3-C-(O-(2-Acetamido-3-O-benzyl-2-deoxy-b-D-glucopyranosyl)-
(1→4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-
glucopyranosyl)-1-fluoro-2-propanone (29)
The benzylidene of fully protected 28 (152 mg, 0.181 mmol)
was removed as described in the first step of the synthesis
of disaccharide 4. Silica gel column chromatography (CH2Cl2
→ 5% MeOH–CH2Cl2) furnished title compound 29 (118 mg,
1
0.157 mmol) in 87% as a white powder. H NMR (500 MHz,
MeOD) d/ppm 7.37–7.22 (m, 15H, H arom), 5.00 (d, J = 10.9 Hz,
1H, CH2 Bn), 4.89 (d, J = 47.2 Hz, 1H, H-3ꢀ), 4.88 (d, J = 11.5 Hz,
1H, CH2 Bn), 4.66–4.53 (m, 6H), 4.00 (t, J = 9.2, 9.2 Hz, 1H),
3.86–3.65 (m, 7H), 3.53 (dd, J = 9.6, 9.0 Hz, 1H), 3.50–3.44 (m,
2H), 3.43–3.38 (m, 2H), 3.20 (ddd, J = 9.5, 7.2, 2.1 Hz, 1H),
2.70 (ddd, J = 16.2, 8.9, 2.1 Hz, 1H, H-1aꢀ), 2.58–2.52 (m, 1H,
H-1bꢀ), 1.89 (s, 3H, CH3 NHAc), 1.87 (s, 3H, CH3 NHAc). 13C
Labeling of purified YPNGase with bodipy probe 1
Purified recombinant yeast peptide N-glycanase (4.7 mg mL−1)
was diluted with 20 mM sodium-phosphate buffer (pH = 7.2),
150 mM NaCl, 5 mM DTT to a final enzyme concentration
of 11.1 ng lL−1. To assess the labeling properties of Bodipy
TMR-Ahx-Val-Ala-Asp(OMe)-Fmk 1, 9 lL of enzyme solution
(11.1 ng lL−1) was labeled for 2 h at 37 ◦C with increasing
concentrations of b-VAD-Fmk 1 (1 lL). The reaction mixture
was quenched by the addition of 4 × SDS-PAGE sample buffer
(5 lL), boiled for 3 min and separated on 10% SDS-PAGE. In
gel-visualization of protein labeling was directly performed in the
wet gel slabs by using the Cy3/Tamra settings (kex 532, kem 560)
on a Thyphoon Variable Mode Imager (Amersham Biosciences).
Afterwards the protein amount was quantified by silverstaining.
Non-specific labeling of YPng1 was evaluated: by treating heat
inactivated YPng1 (9 lL, 11.1 ng lL−1 boiled with 1% SDS for
3 min) with b-VAD-Fmk 1 (50 lM) or by treating a solution of
9 lL YPng1 (100 ng) and BSA (9 lg) with probe 1 (0.5 lM) in the
presence or in the absence of 5 mM DTT. To assess the minimal
amount of YPng1 which could be labeled and visualized with b-
VAD-Fmk 1, a serial dilution of YPng1 was incubated with 0.5 lM
1 for 1 h after which direct in gel-visualization was performed.
NMR (150 MHz, DMSO) d/ppm 203.20 (d, J = 14.9 Hz, C-
ꢀ
=
=
2 ), 169.32 (C O NHAc), 169.12 (C O NHAc), 139.25 (Cq Bn),
139.17 (Cq Bn), 138.54 (Cq Bn), 128.14–126.95 (CH arom), 99.99
(C-1ꢀꢀ), 85.15 (d, J = 179.9 Hz, C-3ꢀ), 82.37, 81.77, 78.27, 76.78,
75.23, 74.22, 73.26 (CH2 Bn), 72.85 (CH2 Bn), 71.86 (CH2 Bn),
70.09, 68.76 (C-6), 60.80 (C-6ꢀ), 55.00 (C-2 or C-2ꢀ), 53.55 (C-2
or C-2ꢀ), 40.80 (C-1ꢀ), 22.91 (CH3 NHAc), 22.73 (CH3 NHAc).
FT-IR: vmax(neat)/cm−1 3292.1, 1733.0, 1652.0, 1549.7, 1455.2,
1372.2, 1317.1, 1104.3, 1073.7, 1054.8, 1024.1, 741.9, 695.9, 634.0,
602.0. [a]D +23.3 (c = 0.42, MeOH). HRMS: (M + H+) calc. for
23
C40H50FN2O11 753.33931, found 753.33980.
3-C-(O-(2-Acetamido-2-deoxy-b-D-glucopyranosyl)-(1→4)-2-
acetamido-2-deoxy-b-D-glucopyranosyl)-1-fluoro-2-propanone (5)
Partly deprotected fluoromethyl ketone 29 (59 mg, 78.5 lmol)
was dissolved in MeOH (1 mL), followed by the addition of 20%
Pd(OH)2 on activate charcoal (15 mg). The reaction mixture was
stirred under H2 atmosphere for 8 h, after which TLC-analysis
showed complete conversion. Argon gas was bubbled through,
the solution was filtered over Celite concentrated and purified
over a HW-40 gel filtration (1% AcOH–H2O). Title compound
Labeling of YPng1 and C191A with b-VAD-Fmk 1
E. coli cell extracts (1 mg mL−1) overexpressing either YPng1 or
C191A were incubated with a serial dilution of b-VAD-Fmk 1 for
1 h. After denaturation, resolving on 10% SDS-PAGE, labeling
3696 | Org. Biomol. Chem., 2007, 5, 3690–3697
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was visualized as described for purified YPng1. The total protein
amount was quantified using silver staining (see ESI†).
3 C. Hirsch, D. Blom and H. L. Ploegh, EMBO J., 2003, 22, 1036; T.
Suzuki, H. Park, N. Hollingsworth, R. Sternglanz and W. J. Lennarz,
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4 S. Katiyar, S. Joshi and W. J. Lennarz, Mol. Biol. Cell, 2005, 16, 4584;
G. Li, G. Zhao, X. Zhou, H. Schindelin and W. J. Lennarz, Proc. Natl.
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Schindelin and W. J. Lennarz, Proc. Natl. Acad. Sci. U. S. A., 2006,
103, 8348.
5 S. Misaghi, M. E. Pacold, D. Blom, H. L. Ploegh and G. A. Korbel,
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W. J. Lennarz, H. Schindelin, N. Taniguchi, K. Totani, I. Matsuo and
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Competition experiments
For competition experiments, 9 lL of YPng1 (11.1 ng lL−1) in
reaction buffer (1 lg lL−1 BSA, 20 mM sodium-phosphate buffer
(pH = 7.2), 150 mM NaCl) was incubated with 1 lL of inhibitors
2–4 and Bodipy TMR-Ahx-Val-Ala-Asp(OMe)-Fmk 1 (0.5 lM)
for 1 h. The reaction was quenched by the addition of 4 × SDS-
PAGE sample buffer (5 lL) and boiling for 3 min. The samples
were analyzed by SDS-PAGE as described above. Data was
quantified with ImageQuant and analyzed with Graphpad Prism
(see ESI†). To analyze the inhibitory potential of fluoromethyl
ketone 5, 1 lL of 5 was preincubated with 9 lL of YPng1
(11.1 ng lL−1) in reaction buffer for 2 h followed by labeling with
b-VAD-Fmk 1 (0.5 lM) for 30 min. The reaction was quenched
and analyzed as described above.
6 C. Hirsch, S. Misaghi, D. Blom, M. E. Pacold and H. L. Ploegh, EMBO
Rep., 2004, 5, 201; T. Suzuki, Methods, 2005, 35, 360.
7 M. Verdoes, B. I. Florea, V. Menendez-Benito, C. J. Maynard, M. D.
Witte, W. A. van der Linden, A. M. van den Nieuwendijk, T.
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Marel, N. P. Dantuma and H. S. Overkleeft, Chem. Biol., 2006, 13,
1217.
8 Several fluorescent VAD-Fmk derivatives (Fam-VAD-Fmk, FITC-
VAD-Fmk, SR-VAD-Fmk) have been reported as caspase activity
probes. See: WO Pat., WO03058194, 2003; J. Grabarek, B. Adbelt,
L. Du and Z. Darzynkiewicz, Exp. Cell Res., 2002, 278, 61.
9 J. T. Palmer, US Pat., 5101068, 1992.
10 W. Bro¨der and H. Kunz, Carbohydr. Res., 1993, 249, 221; T. Ogawa, S.
Nakabayashi and K. Sasajima, Carbohydr. Res., 1981, 95, 308; J. D. C.
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11 B. A. Roe, C. G. Boojamra, J. L. Griggs and C. R. Bertozzi, J. Org.
Chem., 1996, 61, 6442.
Acknowledgements
YPng1 and active site mutant YPng(C191A) were provided by
S. Misaghi and H. L. Ploegh, Whitehead Institute for Biomedical
Research. This work was supported by the Netherlands Orga-
nization for Scientific Research (NWO) and the Netherlands
Proteomic Centre.
12 J.-H. Lee, J. M. Choi, C. Lee, K. J. Yi and Y. Cho, Proc. Natl. Acad.
Sci. U. S. A., 2005, 102, 9144.
13 BSA was added to the competition experiment to increase reproducibil-
ity: A. F. Kisselev and A. L. Goldberg, Methods Enzymol., 2005, 398,
364.
Notes and references
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1 R. E. Dempski, Jr. and B. Imperiali, Curr. Opin. Chem. Biol., 2002, 6,
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2 R. Y. Hampton, Curr. Opin. Cell Biol., 2002, 14, 476; R. G. Spiro, Cell.
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The Royal Society of Chemistry 2007
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