Synthesis and biological evaluation of a chitobiose-based peptide VV-glycanase inhibitor library

Article abstract of DOI:10.1021/jo801906s

Peptide N-glycanase (PNGase), the enzyme responsible for the deglycosylation of N-linked glycoproteins, has an active site related to that of cysteine proteases. Chitiobiose was equipped with electrophilic traps often used in cysteine protease inhibitors,

Full text of DOI:10.1021/jo801906s

Synthesis and Biological Evaluation of a Chitobiose-Based Peptide
N-Glycanase Inhibitor Library
Martin D. Witte,† Danielle Horst,‡ Emmanuel J. H. J. Wiertz,‡,§
Gijsbert A. van der Marel,*,† and Herman S. Overkleeft*,†
Leiden Institute of Chemistry, Leiden UniVersity, P.O. Box 9502, 2300 RA Leiden, The Netherlands, and
Department of Medical Microbiology, Center of Infectious Diseases, Leiden UniVersity Medical Center,
P.O. Box 9600, 2300 RC Leiden, The Netherlands
marel_g@chem.leidenuniV.nl; h.s.oVerkleeft@chem.leidenuniV.nl
ReceiVed August 28, 2008
Peptide N-glycanase (PNGase), the enzyme responsible for the deglycosylation of N-linked glycoproteins,
has an active site related to that of cysteine proteases. Chitiobiose was equipped with electrophilic traps
often used in cysteine protease inhibitors, and the resulting compounds were evaluated as PNGase
inhibitors. We found that the electrophilic trap of the inhibitor has a great influence on the potency of the
compounds with the chloromethyl ketone inhibitor being the first potent C-glycoside-based PNGase
inhibitor.
Introduction
Some oligopeptides escape via the transporter associated with
antigen presentation (TAP) back to the ER to become part of
the major histocompatibilty complex class I (MHCI) and are
thus presented to the cell surface immune surveillance system.4
The importance of deglycosylation by PNGase in epitope
formation was demonstrated in the following. The epitope of
tyrosinase (369-377) is glycosylated at Asn(369). This N-glycan
is removed by PNGase, thereby converting asparagine to aspartic
acid prior to expression by MHC class 1 molecules.5 In addition
to this, it was shown that N-linked glycans near epitopes also
influence the epitope formation. Although fully glycosylated
glycoproteins can still be partially processed by the proteasome,
the amount of cleavage sites is restricted, thereby limiting the
formation of MHC class I peptides.6
Nascent proteins that are secreted into the endoplasmic
reticulum during ribosomal protein synthesis receive one or more
N-linked glycans in a cotranslational process. These N-glycans
assist in folding of the protein and serve as a signal molecule
for localization and functioning of the protein once the folding
process is completed successfully.1 Current estimations are that
about 10% of the thus-generated N-glycan proteins do not have
the appropriate fold, and these are subsequently degraded by
the endoplasmatic reticulum associated degradation pathway.2
During degradation, peptide N-glycanase (PNGase) is respon-
sible for the removal of N-linked glycans in the cytosol. The
carbohydrate core of the N-glycan binds to the C-terminal
domain of PNGase after which the glycosyl amide bond is
cleaved by the thiol residue of the catalytic cysteine, histidine,
aspartic acid triad.3 Deglycosylated proteins are then further
processed by the proteasome and downstream aminopeptidases.
The first small molecule found to inhibit PNGase is the
peptide fluoromethylketone ZVAD-Fmk. This inhibitor, identi-
fied from a generic compound library by Ploegh and co-workers,
is by no means specific for PNGase.7 Rather, the peptide-based
† Leiden University.
‡ Leiden University Medical Center.
(4) Antoniou, A. N.; Powis, S. J. Immunology 2008, 124, 1–12.
(5) Altrich-VanLith, M. L.; Ostankovitch, M.; Polefrone, J. M.; Mosse, C. A.;
Shabanowitz, J.; Hunt, D. F.; Engelhard, V. H. J. Immunol. 2006, 177, 5440–
5450.
(6) Kario, E.; Tirosh, B.; Ploegh, H. L.; Navon, A. J. Biol. Chem. 2008,
283, 244–254.
§ Current Address: Department of Medical Microbiology, University Medical
Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
(1) (a) Ruddock, L. W.; Molinari, M. J. Cell Sci. 2006, 119, 4373–4380. (b)
Spiro, R. G. Cell. Mol. Life Sci. 2004, 61, 1025–1041.
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2002, 277, 12953–12959.
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10.1021/jo801906s CCC: $40.75
Published on Web 12/10/2008
2009 American Chemical Society
J. Org. Chem. 2009, 74, 605–616 605

Witte et al.
thiomethyl ketones,15 (B) the oxiranes16 and aziridines,17 and
(C) Michael acceptors such as vinyl sulfones18 and R,ꢀ-
unsaturated esters.19 Based on these known cysteine protease
inhibitors, we synthesized a library of potential PNGase
inhibitors (Figure 1B) and evaluated their biological activity.
Results and Discussion
Synthesis of Acetamide Inhibitors. Potential acetamide
inhibitors 10-14 were synthesized as depicted in Scheme 1.
Known azide 420 was reduced by Lindlars’ catalyst, and
subsequent condensation of the resulting amine with acids 5-921
gave the fully protected inhibitors. Global deprotection was
accomplished by either hydrogenation in the presence of
Pd(OH)2 in trifluoroethanol(TFE)/MeOH (for 10, 12-14) or
selective removal of the benzylidene using 10% TFA in CH2Cl2
followed by hydrogenation in the presence Pd(OH)2 in MeOH
(for 11).
Martichonok et al. revealed that the potency of the aziridine
warhead depended on the pH, with pH 4 being the optimum
for such inhibitors.22 The pH dependency could be reduced by
N-acylation of the aziridine.23 Since cytosolic PNGase is active
at neutral pH, it is likely that compounds 13 and 14 will not be
very potent at this pH. Therefore, aziridines 15 and 16,
intermediates of the synthesis of 13 and 14, were benzoylated
under the agency of benzoyl chloride in pyridine/CH2Cl2
affording 17 and 18. Removal of the protective groups gave
acylated aziridine inhibitors 19 and 20 (Scheme 2).
The synthesis of thioacetamide 25 was first attempted
following the route detailed in Scheme 1. Although inhibitor
25 in its protected form was obtained in reasonable to good
yield, deprotection with Pd(OH)2 proved ineffective. We
reasoned that introduction of the thioether after global depro-
tection would not only avoid this problem (Scheme 3) but in
addition would provide us mesyl derivative 24 as a potential
inhibitor. Therefore, the azide function in 4 was reduced and
coupled with glycolic acid to furnish hydroxyacetamide 21. The
hydroxyl in 21 was mesylated under the agency of methane-
sulfonic anhydride (Ms2O) and Et3N in dioxane/DMF. During
synthesis, it appeared that mesylate 22 is extremely labile.
Conversion of mesylate 22 to the corresponding chloride 23
was observed by LC/MS when the reaction was performed in
solvents containing traces of chloride ions, such as CH2Cl2 and
after purification over silica column chromatography, probably
due to traces of CaCl2.24 Removal of the protective groups in
FIGURE 1. (A) Known inhibitors of PNGase. (B) Electrophilic traps
that are the subject of this paper.
inhibitor is a broad-spectrum caspase inhibitor and as such
widely used in studies on caspase activity and their role in
apoptosis events.8 Ito and co-workers were the first to address
the subject of selective PNGase inhibitors in their studies on
the rational design of chitobiose-based inhibitor 1 (Figure 1A).9
We recently synthesized chitobiose-based epoxysuccinate 2 and
fluoromethyl ketone 3 and revealed that these, too, inhibit the
enzyme.10 Interestingly, the nature of the electrophilic trap
appeared to have a large effect on the biological activity.
Whereas chitobiose derivatives 1 and 2 are potent inhibitors,
fluoromethyl ketone 3 is a poor inhibitor of PNGase. We
reasoned that chitobiose equipped with a variety of traps would
be helpful to get insight in the influence of the electrophilic
trap on the potency of the inhibitor. Since the active site of
PNGase shares great similarities to the active site of cysteine
proteases, the nature of the known cysteine protease inhibitors
provide a guidline for the design of these inhibitors.
The synthesis of selective and covalent inhibitors of cysteine
proteases has received considerable attention over the past
decades, and various electrophilic traps have been evaluated.11
Three of the major classes that are used as warheads nowadays
are as follows: (A) the activated methylketones and their
acetamide analogues such as halomethyl ketones,12 acyloxym-
ethyl ketones,13 diphenylphosphinyloxymethyl ketones,14 and
(8) (a) Clermont, F.; Gonzalez, N. S.; Communi, D.; Franken, S.; Dumont,
J. E.; Robaye, B. J. Cell. Biochem. 2008, 104, 1161–1171. (b) Vandenabeele,
P.; Vanden Berghe, T.; Festjens, N. Sci. STKE 2006, 358, pe44.
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H.; Taniguchi, N.; Totani, K.; Matsuo, I.; Ito, Y. J. Biol. Chem. 2006, 281, 22152–
22160. (b) Hagihara, S.; Miyazaki, A.; Matsuo, I.; Tatami, A.; Suzuki, T.; Ito,
Y. Glycobiology 2007, 17, 1070–1076.
(10) Witte, M. D.; Descals, C. V.; De Lavoir, S. V. P.; Florea, B. I.; Van
der Marel, G. A.; Overkleeft, H. S. Org. Biomol. Chem. 2007, 5, 3690–3697.
(11) Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. J. Chem. ReV. 2002,
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(12) (a) Rasnick, D. Anal. Biochem. 1985, 149, 461–465. (b) Rauber, P.;
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C. J.; McDowell, A.; Martin, L. S.; Lynas, J. F.; Vanderbroeck, K.; Walker, B.
Biochem. J. 2002, 366, 953–958.
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4429–4431. (b) Kato, D.; Boatright, K. M.; Berger, A. B.; Nazif, T.; Blum, G.;
Ryan, C.; Chehade, K. A. H.; Salvesen, G.; Bogyo, M. Nature Chem. Biol. 2005,
1, 33–38.
(15) Marquis, R. W.; Ru, Y.; Yamashita, D. S.; Oh, H.-J.; Yen, J.; Thompson,
S. K.; Carr, T. J.; Levy, M. A.; Tomaszek, T. A.; Ijames, C. F.; Smith, W. W.;
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1986, 29, 104–111. (b) Ettari, R.; Nizi, E.; Di Francesco, M. E.; Dude, M.-A.;
Pradel, G.; Vicˇ´ık, R.; Schirmeister, T.; Micale, N.; Grasso, S.; Zappala`, M. J. Med.
Chem. 2008, 51, 988–996.
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K.; Toole, B. P. Tetrahedron lett. 2001, 42, 3001–3004.
(21) For the synthesis of 5 and 7, see the Supporting Information. (a)
Synthesis of 6: Korn, A.; Rudolph-Bo¨hner, S.; Morodor, L. Tetrahedron 1994,
50, 8381–8392. (b) Synthesis of 8 and 9: Breuning, A.; Vicik, R.; Schirmeister,
T. Tetrahedron: Asymmetry 2003, 14, 3301–3312.
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606 J. Org. Chem. Vol. 74, No. 2, 2009

Chitobiose-Based Peptide N-Glycanase Inhibitor Library
SCHEME 1
SCHEME 2
SCHEME 3
crude 22 followed by HPLC purification prevented the formation
of chloroacetamide 23 giving 24 in 44% yield over three steps.
The mesylate in 24 was converted to the corresponding thioether
25 by reaction with thiophenol and K2CO3 in methanol.
Synthesis of C-Glycoside Inhibitors. The synthesis of the
Michael acceptors 33 and 38 commences with the synthesis of
alkene 31 (Scheme 4). Known 3-C-(3-O-benzyl-4,6-O-ben-
zylidene-2-deoxy-2-phthalimido-ꢀ-D-glucopyranosyl)-1-pro-
pene was transformed to acceptor 26 by reductive opening of
the benzylidine group.10 Ph2SO/Tf2O-mediated condensation of
donor 2725 with acceptor 26 gave disaccharide 29 in a low (45%)
yield.26 Furthermore, activation of donor 27 by treatment with
N-iodosuccinimide/TMSOTf did not result in productive cou-
pling. Therefore, acceptor 26 was reacted with known imidate
(24) Lai, G.; Tan, P.-Z.; Ghoshal, P. Synth. Commun. 2003, 33, 1722–1732.
J. Org. Chem. Vol. 74, No. 2, 2009 607

Witte et al.
SCHEME 4
SCHEME 5
28 giving disaccharide 29 in 97% yield.27 Removal of the
phthaloyl groups followed by acetylation of the resulting amine,
debenzylation under Birch conditions, and ensuing acetylation
of the hydroxyls furnished fully protected alkene 31. Vinyl ester
32 was synthesized by cross-metathesis (CM) of alkene 31 with
ethyl acrylate in the presence of Grubbs second-generation
catalyst as was previously described for monosaccharides.28
Upon ensuing deacetylation, the ethyl ester was transesterified
with methanol to give methyl vinyl ester 33.
Although alkene 31 was easily converted to vinyl ester 32
by reaction with Grubbs second-generation catalyst, this catalyst
was ineffective for the synthesis of electron-deficient vinyl
sulfone34(Scheme5).WethereforeswitchedtoHoveyda-Grubbs
second-generation catalyst, which is more reactive toward
electron-deficient alkenes.29 After 2 days of refluxing in the
presence of 20% of Hoveyda-Grubbs second-generation cata-
lyst, alkene 31 was converted to the corresponding vinyl sulfone
34 in 57%. Removal of the acetyl protective groups with MeOH/
H2O/Et3N resulted in a mixture of compounds. NMR analysis
showed that the double bond in R,ꢀ-unsaturated sulfone 38 was
migrated, forming ꢀ,γ-unsaturated sulfone 35.30 The migration
under alkaline conditions was proven by treating 34 with DBU
in CH2Cl2. After overnight stirring, complete migration of the
double bond was observed as judged by NMR analysis. To
prevent alkene migration during deprotection, disaccharide 31
was equipped with acid-labile protective groups (Scheme 5).
To this end, 31 was deacetylated under Zemplen conditions and
subsequently silylated with tert-butyldimethylsilyl trifluo-
romethanesulfonate in pyridine affording 36. Cross-metathesis
with methyl vinyl sulfone under the agency of Hoveyda-Grubbs
second-generation catalyst afforded fully protected vinyl sulfone
37 in 66% yield. Cleavage of the silyl ethers in 37 under acidic
conditions proceeded smoothly to give vinyl sulfone 38.
The various methyl ketone based inhibitors 44, 45, 47, and
48 were synthesized from general intermediate hydroxylmethyl
ketone 41 (Scheme 6). The synthesis of this key intermediate
started with the condensation of acceptor 26 with imidate 39
giving disaccharide 40 in excellent yield. Conversion of 40 to
41 was achieved in four successive steps: dihydroxylation of
the double bond in 40 with OsO4, removal of the phthaloyl group
by treatment with ethylenediamine (EDA), selective acetylation
of the resulting amines, and oxidation of the secondary alcohol
under the agency of dibutyltin oxide and NBS,31 giving key
intermediate 41 in 50% yield over four steps. Mesylation of
hydroxymethyl ketone 41 and in situ conversion to the corre-
(25) Structure 27:
Synthesis: De la Fuente, J. M.; Penade´s, S. Tetrahedron: Asymmetry 2002, 17,
1879–1888.
(26) Code´e, J. D. C.; Litjens, R. E. J. N.; den Heeten, R.; Overkleeft, H. S.;
van Boom, J. H.; van der Marel, G. A. Org. Lett. 2003, 5, 1519–1522.
(27) Kretzschmar, G.; Stahl, W. Tetrahedron 1998, 54, 6341–6358.
(28) Timmer, M. S. M.; Chumillas, M. V.; Donker-Koopman, W. E.; Aerts,
J. M. F. G.; Van der Marel, G. A.; Overkleeft, H. S.; Van Boom, J. H.
J. Carbohydr. Chem. 2005, 24, 335–351.
(29) (a) Graber, S. B.; Kingsbury, J. S.; Gray, B. L.; Hoveyda, A. H. J. Am.
Chem. Soc. 2000, 122, 8168–8179. (b) Michrowska, A.; Bieniek, M.; Kim, M.;
Klajn, R.; Grela, K. Tetrahedron 2003, 59, 4525–4531.
(30) Wang, J.-F.; Yang, X.-D.; Zhang, L.-R.; Yang, Z.-J.; Zhang, L.-H.
Tetrahedron 2004, 60, 8535–8546.
(31) Shiina, J.; Nishiyama, S. Tetrahedron 2003, 59, 6039–6044.
608 J. Org. Chem. Vol. 74, No. 2, 2009

Chitobiose-Based Peptide N-Glycanase Inhibitor Library
SCHEME 6
sponding acyloxymethyl and chloromethyl ketones using dim-
ethylbenzoic acid (DBA) or lithium chloride furnished 42 and
43. Global deprotection of 42 under reductive conditions
proceeded smoothly giving acyloxymethyl ketone 44 in 83%
yield. For the synthesis of chloromethyl ketone 45, however,
hydrogenolysis was not suitable. Due to reductive dehalogena-
tion, ketone 46 was the only product observed by LC/MS after
hydrogenolysis. The benzyl/benzylidene protective groups could,
however, be efficiently removed under the agency of FeCl3 in
CH2Cl2 furnishing chloromethyl ketone 45. Treatment of fully
deprotected 45 with diphenylphosphinic acid (DPP) and K2CO3
or thiophenol and K2CO3 afforded compounds 47 and 48.
Inhibition Studies. With the inhibitors in hand, we evaluated
their biological activity as described.10 Purified yeast peptide
N-glycanase was incubated with a serial dilution of the
chitobiose-based inhibitors followed by treatment with the
fluorescent active-site label, ꢀ-VAD-Fmk (0.5 µM).32 Although
the chitobiose-based inhibitors bind to the carbohydrate binding
subsite and ꢀ-VAD-Fmk binds in the peptide binding subsite,
both covalently modify the nulceophilic thiol of cysteine 191.
Therefore, an increasing concentration of a potent inhibitor will
result in a drop of ꢀ-VAD-Fmk binding which is evidenced by
reduced fluorescence. However, we realize that both the ꢀ-VAD-
Fmk and the chitobiose based inhibitors bind in a time-
dependent fashion, and therefore, IC50 values will have to be
interpreted as relative values. Furthermore, we cannot exclude
that weaker inhibitors may bind to the active site of yeast
PNGase and induce the binding of ꢀ-VAD-Fmk via an induced
fit. After quantification of the intensities of the bond, the relative
IC50 values were determined of both known inhibitor 1, as a
reference, and the newly synthesized inhibitors 10-14, 19, 20,
24, 25, 33, 38, 44, 45, 47 and 48. The apparent IC50 values
allowed us to compare the different inhibitors (Table 1).
The IC50 values of the synthesized inhibitors reveal several
trends. First, the nature of the leaving group of the acetamides
1, 10, 12, 24, and 25 and methyl ketones 3, 44, 45, 47, and 48
has a great influence on the inhibitory potential of the compound.
A good leaving group is required for strong irreversible
inhibition of purified yeast PNGase. Fluoromethyl ketone,
acyloxymethyl ketone, and diphenylphosphinyloxymethyl ke-
tone warheads, which were originally designed to react selec-
tively with the thiol of the catalytic triad and to be quiescent in
the presence of other bionucleophiles, were inefficient inhibitors
of yeast PNGase. The stereochemistry of the epoxide and
aziridine electrophilic trap is of great importance. Inhibitors with
the S,S-configuration (2 and 19) showed an approximately 100-
fold higher activity in comparison with the R,R-configured 11
and 20. In addition to this stereoselectivity, N-acylation of the
aziridine (8.77 µM for 19 compared to >1000 µM for 13) is
beneficial for the IC50 value at pH 7.4. Somewhat surprisingly,
the Michael acceptor containing compounds 33 and 38 did not
reveal inhibitory activity at all. The lack of activity might be
due to improper alignment of E-vinylsulfone 38 and E-vinylester
33 with the active site cysteine, thereby preventing nucleophilic
attack on the Michael acceptor. The corresponding Z-vinylsul-
fones and Z-vinylesters might align properly in the active site
of yeast PNGase and therefore might be inhibitors of PNGase.
To evaluate the inhibitory potential in a cell culture model,
epoxide 2 and its peracetylated analogue 49 were tested in a
model involving the deglycosylation and degradation of MHC
class I heavy chains in the context of a viral protein, US11.33
Human cytomegalovirus encodes two glycoproteins, US2 and
US11, each of which is sufficient to catalyze the dislocation of
MHC class I heavy chains (HCs) from the ER into the cytosol,
where they become ubiquitinated, deglycosylated, and degraded
by the proteasome.33,34 Here, U373 cells stably expressing US11
were used to monitor the effects of our compounds on HC
deglycosylation by PNGase. Control cells and US11-expressing
cells were metabolically labeled with [35S]-cysteine/methonine.
The cells were lysed in the presence of detergent, and the HCs
(32) Structureꢀ-VAD-Fmk:
(33) Wiertz, E. J. H. J.; Jones, T. R.; Sun, L.; Bogyo, M.; Geuze, H. J.;
Ploegh, H. L. Cell 1996, 84, 769–779.
(34) Wiertz, E. J.; Tortorella, D.; Bogyo, M.; Yu, J.; Mothes, W.; Jones,
T. R.; Rapoport, T. A.; Ploegh, H. L. Nature 1996, 384, 432–438.
J. Org. Chem. Vol. 74, No. 2, 2009 609

Witte et al.
TABLE 1. Apparent IC50 Values of the Inhibitors
were isolated by immunoprecipitation. Whereas the HCs were
readily detectable in control U373 cells (Figure 2, lane 1), the
HCs were completely degraded in U373 cells expressing US11
(lane 2). However, in the presence of the proteasome inhibitor
ZL3VS, a deglycosylated form of the HCs was observed (lane
3). Fully glycosylated HCs were detected when the cells were
incubated with the PNGase-inhibitor ZVAD-Fmk and ZL3VS
(lane 4). To test our compounds, US11 cells were preincubated
with epoxides 2 and 49 in the presence of ZL3VS, followed by
labeling of the proteins with [35S]-labeled cysteine and me-
thionine (lanes 5 and 6). In the presence of 100 µM of epoxides
2 and 49, the HCs were deglycosylated with similar efficiency
as in cells treated with ZL3VS alone (compare lanes 3, 5, and
6). We reasoned that the lack of activity of the inhibitors could
be caused by hydrolysis of the glycosidic bond in 2 and 49 by
hexosaminidase present inside the cell. To investigate this
possibility, the cells were treated with 2 and 49 in the presence
of ZL3VS and the broad spectrum hexosaminidase inhibitor
NGT 50. The addition of 50 had, however, no effect on the
state of glycosylation of the HCs (lanes 7 and 8). An explanation
for this fact could be that epoxides 2 and 49 cannot penetrate
the cell membrane. Alternatively, the epoxides may enter the
cell but the electrophilic group is hydrolyzed inside the cell. It
is also possible that the acetates in 49 are stable to esterases
present in the U373 cells.
FIGURE 2. Inhibition of human PNGase in U373 cells. U373 control
cells and US11-expressing U373 cells were incubated with the inhibitors
indicated (final concentrations: ZVAD-Fmk, 30 µM; 2 and 49, 100
µM; ZL3VS, 1.25 µM; and NGT, 50 µM) in starvation medium for 45
min, followed by metabolic labeling for 30 min at 37 °C with [35S]-
labeled cysteine and methionine, again in the presence of the compounds
indicated. The cells were lysed in the presence of NP40 lysis buffer,
and the MHC class I HCs were immunoprecipitated using the
monoclonal antibody HC10. The samples were separated by SDS-PAGE
and visualized by phosphoimaging. The presence of the single N-linked
glycan on the HCs is indicated (+ and - CHO).
In summary, a library of modified chitobiose derivatives as
potential PNGase inhibitors was synthesized. The IC50 value
of the newly synthesized inhibitors was determined by incubat-
ing them with purified yeast PNGase followed by visualizing
the remaining activity by activity-based enzyme profiling. The
results of this assay demonstrate that the nature of electrophilic
610 J. Org. Chem. Vol. 74, No. 2, 2009

Chitobiose-Based Peptide N-Glycanase Inhibitor Library
10.0, 8.8 Hz, 1H), 3.50-3.42 (m, 2H), 2.04 (s, 3H), 1.97 (s, 3H);
13C NMR (150 MHz, D2O) δ ppm 174.4, 174.1, 171.0, 170.9, 100.8,
79.6, 78.4, 78.2, 77.5, 75.7, 75.3, 72.8, 72.0, 69.1, 59.9, 59.3, 55.0,
53.1, 21.5, 21.3; FT-IR Vmax (neat)/cm-13280.8, 1661.4, 1651.8,
1562.8, 1557.7, 1538.4, 1435.8, 1373.4, 1319.7, 1200.7, 1103.2,
1043.1, 1012.4; LC/MS tR 1.28 min; linear gradient 0f20% B in
13.5 min; ESI/MS m/z ) 484.00 (M + H)+; HRMS (M + H+)
calcd for C18H31FN3O11 484.19371, found 484.19344; mp 203 °C
(start of decomposition).
trap has significant influence on the potency of the inhibitor.
To study the selectivity, the library will be tested for their
inhibitory activity of human chitinases35 and the inhibitors will
be converted to probes by the introduction of an azidoacetyl or
by equipping them with a fluorophore (e.g., BodipyFl). The
resulting probes may find use in the study of the selectivity of
the inhibitors in cell lysates as well as whole cells. Furthermore,
the fluorescent probes can be used to study their cell-perme-
ability by microscopy as was reported by Ito and co-workers.36
(2R,3R)-3-N-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-
(1f4)-2-acetamido-2-deoxy-ꢀ-D-glucopyranosylcarbamoyl)ox-
irane-2-carboxylic Acid Ethyl Ester (11). Azide 4 (100 mg, 0.124
mmol) was reduced, and the resulting amine was condensed with
(R,R) epoxysuccinate monoethyl ester as described in the general
procedure. Silica gel column chromatography (CH2Cl2f6% MeOH/
CH2Cl2) yielded the protected epoxide. The resulting epoxide was
deprotected according procedure 2. RP-HPLC purification (linear
gradient 3f10.5% B in 15 min) afforded epoxide 11 (27% over
three steps, 19.20 mg, 34 µmol): 1H NMR (400 MHz, D2O) δ ppm
5.01 (d, J ) 9.6 Hz, 1H), 4.51 (d, J ) 8.4 Hz, 1H), 4.21 (q, J )
7.2, 7.1, 7.1 Hz, 2H), 3.84 (d, J ) 12.1 Hz, 1H), 3.81 (dd, J )
10.0, 9.6 Hz, 1H), 3.75 (d, J ) 11.7 Hz, 1H), 3.72-3.35 (m, 11H),
1.98 (s, 3H), 1.93 (s, 3H), 1.21 (t, J ) 7.2, 7.2 Hz, 3H); 13C NMR
(150 MHz, D2O) δ ppm 174.9, 174.6, 168.8, 168.6, 101.4, 78.8,
78.4, 76.4, 75.9, 73.4, 72.6, 69.7, 63.5, 60.5, 59.9, 55.6, 53.7, 53.3,
53.2, 52.4, 29.6, 22.1, 22.0, 13.2; FT-IR Vmax (neat)/cm-1 3270.2,
1651.8, 1563.5, 1557.7, 1373.7, 1312.0, 1202.3, 1025.7. LC/MS
tR 6.02 min; linear gradient 0f20% B in 13.5 min; ESI/MS m/z )
566.00 (M + H)+; HRMS (M + H+) calcd for C22H36N3O14
566.21918, found 566.21894; mp 201 °C (start of decomposition).
N-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-(1f4)-2-ac-
etamido-2-deoxy-ꢀ-D-glucopyranosyl) 2,6-Dimethylbenzoyloxy-
acetamide (12). Disacharride 4 (78 mg, 98 µmol) was transformed
to the acyloxyamide as depicted in the general procedure. Purifica-
tion over silica gel chromatography (CH2Cl2f4% MeOH/CH2Cl2)
gave the protected acyloxyacetamide: LC/MS tR 10.76 min; linear
gradient 10f90% B in 13.5 min; ESI/MS m/z ) 972.27 (M +
H)+. Deprotection was performed as described in general procedure
1. Preparative RP-HPLC (linear gradient 0f50% B in 10 min)
afforded title compound 12 (17% over three steps, 10.18 mg, 16.6
µmol): 1H NMR (600 MHz, D2O) δ ppm 7.16 (d, J ) 7.7 Hz, 1H),
5.11 (d, J ) 9.7 Hz, 1H), 4.88 (d, J ) 15.1 Hz, 1H), 4.84 (d, J )
15.1 Hz, 1H), 4.57 (d, J ) 8.5 Hz, 1H), 3.90 (d, J ) 12.6 Hz, 1H),
3.86 (t, J ) 9.7, 9.7 Hz, 1H), 3.81 (d, J ) 11.8 Hz, 1H), 3.77-3.71
(m, 3H), 3.65 (t, J ) 9.5, 9.5 Hz, 1H), 3.63 (dd, J ) 12.6, 4.7 Hz,
1H), 3.60-3.57 (m, 1H), 3.55 (dd, J ) 10.0, 9.0 Hz, 1H), 3.50-3.42
(m, 2H), 2.29 (s, 1H), 2.04 (s, 3H), 1.96 (s, 3H); 13C NMR (150
MHz, D2O) δ ppm 174.2, 174.0, 170.7, 169.6, 135.0, 131.1, 130.0,
127.2, 100.8, 78.2, 77.7, 75.8, 75.3, 72.9, 72.1, 69.1, 62.6, 59.9,
59.3, 55.0, 53.2, 21.5, 21.4, 18.1; FT-IR Vmax (neat)/cm-13273.9,
1668.1, 1651.8, 1645.8, 1564.0, 1557.7, 1435.8, 1373.8, 1115.9,
1075.9; LC/MS tR 6.43 min; linear gradient 10f50% B in 13.5
min; ESI/MS m/z ) 614.00 (M + H)+. HRMS (M + H+) calcd
for C27H40N3O13 614.25556, found 614.25546; mp 228 °C (start of
decomposition).
Experimental Section
General Procedure for the Synthesis of Acetamide Inhibi-
tors. Disacharride 4 was dissolved in DMF (10 mL/mmol).
Lindlars’ catalyst was added, and the solution was stirred under
H2 atmosphere for 3 h. The solution was purged with argon after
which acid (3 equiv), 2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3-
tetramethylaminium hexafluorophosphate (HCTU) (3 equiv), and
DiPEA (6 equiv) were added. The resulting mixture was stirred
overnight, concentrated under reduced pressure, and purified over
silica gel chromatography (CH2Cl2fMeOH/CH2Cl2) to give the
fully protected inhibitors.
General Procedure 1 for Global Deprotection. The protected
inhibitors were directly deprotected. Hence, they were dissolved
in 2,2,2-trifluoroethanol/methanol (10 mL/mmol). Subsequently,
20% Pd(OH)2 on activated charcoal (20 mg) was added. The
reaction mixture was stirred under H2 atmosphere until TLC
revealed complete conversion to a very polar product. The mixture
was purged with argon, filtered, concentrated, and purified over
reversed-phase HPLC.
General Procedure 2 for Global Deprotection. The inhibitor
was dissolved in CH2Cl2 (2 mL) and cooled to 0 °C before
trifluoroacetic acid (100 µL) and H2O (10 µL) were added. The
mixture was stirred until TLC showed complete conversion to a
lower running product. The reaction was quenched with NaHCO3
(satd aq) and diluted with CH2Cl2. The organic layer was washed
with NaHCO3 (satd aq), dried over Na2SO4, and concentrated under
reduced pressure. The resulting disaccharide was redissolved in
MeOH (2 mL) and 20% Pd(OH)2 on activated charcoal (20 mg)
was added after which the reaction was stirred under H2 atmosphere
until TLC showed complete conversion. Subsequently, the reaction
was purged with argon, filtered, concentrated, and purified by
reversed-phase HPLC.
N-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-(1f4)-2-ac-
etamido-2-deoxy-ꢀ-D-glucopyranosyl) fluoroacetamide (10). Dis-
acharride 4 (76 mg, 94 µmol) was converted to the protected
fluoroacetamide as described in the general procedure. Silica gel
chromatography (CH2Cl2f4% MeOH/CH2Cl2) gave the protected
fluoroacetamide: LC/MS tR 9.43 min; linear gradient 10f90% B
in 13.5 min; ESI/MS m/z ) 842.27 (M + H)+. Deprotection
according to general procedure 1 followed by preparative RP-HPLC
(linear gradient 0f8% B in 10 min) furnished title compound 10
(20% over three steps, 9.14 mg, 18.9 µmol): 1H NMR (600 MHz,
D2O) δ ppm 5.12 (d, J ) 9.7 Hz, 1H), 4.89 (dd, J ) 46.2, 14.8
Hz, 1H), 4.87 (dd, J ) 46.2 Hz, 14.7 Hz, 1H), 4.58 (d, J ) 8.4 Hz,
1H), 3.90 (t, J ) 9.7, 9.7 Hz, 1H), 3.90 (dd, J ) 12.5, 1.2 Hz, 1H),
3.81 (d, J ) 11.7 Hz, 1H), 3.77 (dd, J ) 9.8, 9.1 Hz, 1H),
3.75-3.71 (m, 2H), 3.66 (t, J ) 8.3, 8.3 Hz, 1H), 3.63 (dd, J )
11.2, 3.4 Hz, 1H), 3.59 (dd, J ) 9.6, 3.5 Hz, 1H), 3.55 (dd, J )
(2S,3S)-3-N-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-
(1f4)-2-acetamido-2-deoxy-ꢀ-D-glucopyranosylcarbamoyl)aziri-
dine-2-carboxylic Acid Ethyl Ester (13). Protected aziridine 15
(50 mg, 62 µmol) was synthesized as described in the general
procedure. Silica gel chromatography (CH2Cl2f3% MeOH/CH2Cl2)
furnished protected aziridine 15 (72%, 41 mg, 44.4 µmol): LC/MS
tR 9.46 min; linear gradient 10f90% B in 13.5 min; ESI/MS m/z
) 923.27 (M + H)+. The resulting aziridine was converted to the
title compound as depicted in general procedure 1. Preparative RP-
HPLC (linear gradient 0f10% B in 14.5 min) furnished title
(35) (a) Fusetti, F.; von Moeller, H.; Houston, D.; Rozeboom, H. J.; Dijkstra,
B. W.; Boot, R. G.; Aerts, J. M. F. G.; van Aalten, D. M. F. J. Biol. Chem.
2002, 277, 25537–25544. (b) Boot, R. G.; Blommaart, E. F.; Swart, E.;
Ghauharali-van der Vlugt, K.; Bijl, N.; Moe, C.; Place, A.; Aerts, J. M. F. G.
J. Biol. Chem. 2001, 276, 6770–6778. (c) Aguilera, B.; Ghauharali-van der Vlugt,
K.; Helmond, M. T.J.; Out, J. M. M.; Donker-Koopman, W. E.; Groener, J. E. M.;
Boot, R. G.; Renkema, G. H.; van der Marel, G. A.; van Boom, J. H.; Overkleeft,
H. S.; Aerts, J. M. F. G. J. Biol. Chem. 2003, 278, 40911–40916.
(36) Hagihara, S.; Miyazaki, A.; Matsuo, I.; Tatami, A.; Suzuki, T.; Ito, Y.
Glycobiology 2007, 17, 1070–1076.
1
compound 13 (30% over three steps, 10.46 mg, 18.5 µmol): H
NMR (600 MHz, D2O) δ ppm 5.05 (d, J ) 9.8 Hz, 1H), 4.57 (d,
J ) 8.4 Hz, 1H), 4.21 (q, J ) 7.1, 7.1, 7.1 Hz, 2H), 3.89 (d, J )
11.0 Hz, 1H), 3.87 (dd, J ) 10.0, 9.5 Hz, 1H), 3.80 (d, J ) 11.4
J. Org. Chem. Vol. 74, No. 2, 2009 611

Witte et al.
Hz, 1H), 3.76-3.70 (m, 3H), 3.64 (dd, J ) 9.5, 8.8 Hz, 1H), 3.62
(dd, J ) 12.1, 4.7 Hz, 1H), 3.58-3.52 (m, 2H), 3.50-3.42 (m,
2H), 2.91 (s, 1H), 2.81 (s, 1H), 2.03 (s, 3H), 1.99 (s, 3H), 1.23 (t,
J ) 7.2, 7.2 Hz, 3H); 13C NMR (150 MHz, D2O) δ ppm 174.2,
174.0, 170.3, 170.0, 100.8, 78.2, 77.9, 75.7, 75.3, 72.8, 72.1, 69.1,
62.4, 59.9, 59.3, 55.0, 53.1, 35.9, 34.9, 21.5, 21.4, 12.6; FT-IR
Vmax (neat)/cm-1 3271.2, 1667.9, 1651.7, 1645.7, 1634.2, 1563.5,
1557.7, 1538.7, 1435.8, 1373.9, 1200.2, 1049.7; LC/MS tR 5.58
min; linear gradient 0f20% B in 13.5 min; ESI/MS m/z ) 565.20
(M + H)+; HRMS (M + H+) calcd for C22H37N4O13 565.23516,
found 565.23497; mp 177 °C (start of decomposition).
(2R,3R)-3-N-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-
(1f4)-2-acetamido-2-deoxy-ꢀ-D-glucopyranosylcarbamoyl)aziri-
dine-2-carboxylic Acid Ethyl Ester (14). Disacharride 4 (80 mg,
99 µmol) was transformed to 16 according the general procedure.
Subsequent purification by silica gel chromatography gave protected
16 (94%, 86 mg, 93 µmol): LC/MS tR 9.40 min; linear gradient
10f90% B in 13.5 min; ESI/MS m/z ) 923.33 (M + H)+. Fully
protected aziridine 16 was deprotected as given in general procedure
1. Ensuing preparative RP-HPLC (linear gradient 0f10% B in 14.5
min) furnished title compound 14 (27% over three steps, 15.47 mg,
27.4 µmol): 1H NMR (400 MHz, D2O) δ ppm 5.11 (d, J ) 9.8 Hz,
1H), 4.62 (d, J ) 8.3 Hz, 1H), 4.28 (q, J ) 7.2, 7.2, 7.1 Hz, 2H),
3.98-3.45 (m, 12H), 3.15 (s, 1H), 3.09 (s, 1H), 2.08 (s, 3H), 2.03
(s, 3H), 1.30 (t, J ) 7.1, 7.1 Hz, 3H); 13C NMR (100 MHz, D2O)
δ ppm 174.8, 174.7, 101.5, 78.9, 78.7, 76.4, 76.0, 73.5, 72.7, 69.8,
63.4, 60.6, 60.0, 55.7, 53.9, 36.7, 35.6, 22.2, 22.1, 13.3; FT-IR
Vmax (neat)/cm-1 3261.5, 1668.3, 1650.7, 1557.6, 1435.3, 1376.7,
1313.4, 1201.2, 1113.6, 1067.2, 1026.4; LC/MS tR 5.57 min; linear
gradient 0f20% B in 13.5 min; ESI/MS m/z ) 565.53 (M + H)+;
HRMS (M + H+) calcd for C22H37N4O13 565.23516, found
565.23500; mp 177 °C (start of decomposition).
mg, 109 µmol) was dissolved in CH2Cl2/pyridine (2 mL, 1/1 v/v)
before being cooled to 0 °C. Benzoyl chloride (25 µL, 0.22 mmol)
was added. An additional amount of benzoyl chloride (300 µL)
was added after 1 h of stirring, and the reaction was stirred until
TLC showed complete conversion of the starting material. The
solution was diluted with CH2Cl2, washed with NaHCO3 (satd aq)
and 10% citric acid, dried over Na2SO4, and concentrated. Purifica-
tion by silica gel chromatography (CH2Cl2f3% MeOH/CH2Cl2)
1
gave title compound 18 (48%, 52 mg, 51 µmol): H NMR (400
MHz, CDCl3/MeOD) δ ppm 7.91-7.88 (m, 2H), 7.53-7.21 (m,
23H), 5.47 (s, 1H), 4.84 (d, J ) 7.5 Hz, 1H), 4.82 (d, J ) 6.6 Hz,
1H), 4.82 (d, J ) 7.4 Hz, 1H), 4.65 (d, J ) 11.7 Hz, 1H), 4.61 (d,
J ) 7.2 Hz, 1H), 4.58 (d, J ) 6.8 Hz, 1H), 4.54 (d, J ) 8.3 Hz,
1H), 4.45 (d, J ) 11.7 Hz, 1H), 4.10 (dd, J ) 10.5, 5.0 Hz, 1H),
4.07-3.93 (m, 4H), 3.78 (t, J ) 8.7, 8.7 Hz, 1H), 3.65-3.57 (m,
4H), 3.56 (d, J ) 2.4 Hz, 1H), 3.55 (d, J ) 2.4 Hz, 1H), 3.50 (dd,
J ) 9.0, 8.5 Hz, 1H), 3.47-3.42 (m, 2H), 3.39 (d, J ) 10.3 Hz,
1H), 3.19 (td, J ) 14.6, 5.1, 5.1 Hz, 1H), 1.85 (s, 3H), 1.84 (s,
3H), 1.08 (t, J ) 7.1, 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3/
MeOD) δ ppm 138.3, 137.9, 137.5, 136.8, 132.8, 132.0, 128.6,
128.2, 128.1, 128.0, 127.9, 127.8, 127.8, 127.8, 127.7, 127.5, 127.3,
127.1, 125.6, 100.8, 100.1, 81.8, 80.1, 78.7, 77.5, 76.2, 74.8, 73.7,
73.1, 68.1, 67.5, 65.3, 62.0, 55.4, 52.7, 41.4, 40.8, 22.3, 21.9, 13.1;
FT-IR Vmax (neat)/cm-13277.1, 2873.3, 1739.0, 1656.9, 1651.7,
1562.9, 1557.7, 1538.4, 1452.0, 1370.9, 1318.1, 1202.2, 1174.1,
1069.2; LC/MS tR 9.75 min; linear gradient 10f90% B in 13.5
min; ESI/MS m/z ) 1027.5 (M + H)+; HRMS (M + H+) calcd
for C57H62N4O14 1027.43353, found 1027.43508; mp 252 °C (start
of decomposition).
1-Benzoyl-(2S,3S)-3-N-(O-(2-acetamido-2-deoxy-ꢀ-D-glucopy-
ranosyl)-(1f4)-2-acetamido-2-deoxy-ꢀ-D-glucopyranosylcar-
bamoyl)aziridine-2-carboxylic Acid Ethyl Ester (19). Fully
protected benzoylated aziridine 17 (42 mg, 41 µmol) was depro-
tected as described in general procedure 2. Semipreparative RP-
HPLC (linear gradient 20%f29.375% B in 12.5 min) furnished
title compound 19 (51%, 13.8 mg, 21 µmol): 1H NMR (400 MHz,
D2O) δ ppm 7.94 (d, J ) 7.8 Hz, 2H), 7.71 (t, J ) 7.4, 7.4 Hz,
1H), 7.57 (t, J ) 7.7, 7.7 Hz, 2H), 4.95 (d, J ) 9.7 Hz, 1H), 4.58
(d, J ) 8.4 Hz, 1H), 4.15 (q, J ) 7.1, 7.1, 7.1 Hz, 2H), 3.92 (d, J
) 12.2 Hz, 1H), 3.87 (t, J ) 10.0, 10.0 Hz, 1H), 3.81 (dd, J )
12.1, 1.3 Hz, 1H), 3.78 (d, J ) 2.3 Hz, 1H), 3.77-3.71 (m, 3H),
3.69 (d, J ) 2.2 Hz, 1H), 3.65 (dd, J ) 9.5, 8.7 Hz, 1H), 3.63 (d,
J ) 12.4 Hz, 1H), 3.60-3.42 (m, 4H), 2.06 (s, 3H), 1.98 (s, 3H),
1.15 (t, J ) 7.1, 7.1 Hz, 3H); 13C NMR (100 MHz, D2O) δ ppm
177.6, 174.7, 174.6, 167.8, 167.5, 134.3, 131.6, 129.2, 128.3, 101.4,
78.7, 78.5, 76.3, 75.9, 73.5, 72.6, 69.7, 63.6, 60.6, 59.9, 55.6, 53.6,
42.3, 41.3, 22.2, 22.1, 13.1; FT-IR Vmax (neat)/cm-1 3290.2, 1668.0,
1651.7, 1645.7, 1563.5, 1557.7, 1538.4, 1435.8, 1373.6, 1318.4,
1200.9, 1050.1. LC/MS tR 7.45 min; linear gradient 10f50% B in
13.5 min; ESI/MS m/z ) 669.07 (M + H)+; HRMS (M + H+)
calcd for C29H41N4O14 669.26138, found 669.26145; mp 150 °C
(start of decomposition).
1-Benzoyl-(2R,3R)-3-N-(O-(2-acetamido-2-deoxy-ꢀ-D-glucopy-
ranosyl)-(1f4)-2-acetamido-2-deoxy-ꢀ-D-glucopyranosylcar-
bamoyl)aziridine-2-carboxylic Acid Ethyl Ester (20). Benzoy-
lated 18 (52 mg, 51 µmol) was deprotected as described in general
procedure2.SemipreparativeRP-HPLC(lineargradient20%f29.375%
B in 12.5 min) gave title compound 20 (35%, 11.9 mg, 18 µmol):
1H NMR (400 MHz, D2O) δ ppm 7.95 (d, J ) 7.9 Hz, 2H), 7.71
(t, J ) 7.5, 7.5 Hz, 1H), 7.57 (t, J ) 7.8, 7.8 Hz, 2H), 4.98 (d, J
) 9.6 Hz, 1H), 4.59 (d, J ) 8.4 Hz, 1H), 4.15 (dq, J ) 7.1, 7.0,
7.0, 2.9 Hz, 2H), 3.92 (dd, J ) 12.3, 1.6 Hz, 1H), 3.86 (t, J ) 9.9,
9.9 Hz, 1H), 3.79-3.70 (m, 6H), 3.66 (dd, J ) 9.5, 8.9 Hz, 1H),
3.60 (dd, J ) 17.8, 5.8 Hz, 1H), 3.57 (dd, J ) 9.8, 8.9 Hz, 1H),
3.53-3.44 (m, 2H), 2.06 (s, 3H), 1.99 (s, 3H), 1.14 (t, J ) 7.1, 7.1
Hz, 3H); 13C NMR (100 MHz, D2O) δ ppm 177.4, 174.8, 174.6,
167.7, 167.4, 134.3, 131.4, 129.2, 128.3, 101.4, 78.7, 78.6, 76.2,
75.9, 73.5, 72.6, 69.7, 63.7, 60.6, 59.8, 55.6, 53.8, 42.2, 41.1, 22.1,
22.0, 13.1; FT-IR Vmax (neat)/cm-13273.5, 1661.5, 1651.7, 1645.7,
1-Benzoyl-(2S,3S)-3-N-(O-(2-acetamido-3-O-benzyl-4,6-O-ben-
zylidene-2-deoxy-ꢀ-D-glucopyranosyl)-(1f4)-2-acetamido-3,6-di-
O-benzyl-2-deoxy-ꢀ-D-glucopyranosylcarbamoyl)aziridine-2-
carboxylic Acid Ethyl Ester (17). Fully protected aziridine 15 (66
mg, 72 µmol) was dissolved in CH2Cl2/pyridine (2 mL, 1/1 v/v).
The solution was cooled to 0 °C before benzoyl chloride (17 µL,
0.143 mmol) was added. Additional benzoyl chloride (130 µL) was
added after 3 h of stirring. The reaction was stirred until TLC
showed complete consumption of starting material after which the
reaction was diluted with CH2Cl2 and washed with NaHCO3 (satd
aq) and 10% citric acid. The organic layer was dried over Na2SO4,
concentrated in vacuo, and purified by column chromatography
(CH2Cl2f3% MeOH/CH2Cl2) affording title compound 17 (57%,
1
42 mg, 41 µmol): H NMR (400 MHz, CDCl3/MeOD) δ ppm
7.93-7.90 (m, 2H), 7.57-7.51 (m, 1H), 7.47-7.19 (m, 22H), 5.47
(s, 1H), 4.87 (d, J ) 11.3 Hz, 1H), 4.85 (d, J ) 9.9 Hz, 1H), 4.82
(d, J ) 12.4 Hz, 1H), 4.65 (d, J ) 11.7 Hz, 1H), 4.62 (d, J ) 7.7
Hz, 1H), 4.49 (d, J ) 11.7 Hz, 1H), 4.10-3.92 (m, 5H), 3.79 (t, J
) 9.0, 9.0 Hz, 1H), 3.71-3.67 (m, 2H), 3.63 (dd, J ) 12.7, 9.2
Hz, 1H), 3.58 (d, J ) 2.4 Hz, 1H), 3.52 (dd, J ) 7.9, 7.3 Hz, 1H),
3.49 (d, J ) 2.4 Hz, 1H), 3.49-3.46 (m, 1H), 3.40 (t, J ) 10.3,
10.3 Hz, 1H), 3.19 (dt, J ) 9.8, 9.6, 4.9 Hz, 1H), 1.86 (s, 3H),
1.85 (s, 3H), 1.10 (t, J ) 7.1, 7.1 Hz, 3H); 13C NMR (100 MHz,
CDCl3/MeOD) δ ppm 175.5, 173.3, 172.5, 167.2, 167.1, 139.4,
139.0, 138.6, 137.9, 133.8, 133.2, 129.6, 129.2, 129.1, 129.0, 128.9,
128.83, 128.77, 128.62, 128.58, 128.4, 128.3, 128.1, 126.6, 101.8,
101.2, 82.8, 81.3, 79.7, 78.7, 77.3, 75.9, 74.9, 74.8, 74.0, 69.1, 68.7,
66.4, 63.0, 56.6, 53.9, 42.4, 42.0, 23.2, 23.0, 14.1; FT-IR Vmax (neat)/
cm-13276.4, 1738.9, 1668.0, 1651.7, 1645.7, 1562.9, 1557.6,
1452.1, 1371.8, 1317.9, 1204.3, 1174.6, 1069.9; LC/MS tR 10.38
min; linear gradient 10f90% B in 13.5 min; ESI/MS m/z )
1027.27 (M + H)+; HRMS (M + H+) calcd for C57H62N4O14
1027.43353, found 1027.43497; mp 250 °C (start of decomposition).
1-Benzoyl-(2R,3R)-3-N-(O-(2-acetamido-3-O-benzyl-4,6-O-
benzylidene-2-deoxy-ꢀ-D-glucopyranosyl)-(1f4)-2-acetamido-
3,6-di-O-benzyl-2-deoxy-ꢀ-D-glucopyranosylcarbamoyl)aziridine-
2-carboxylic Acid Ethyl Ester (18). Protected aziridine 16 (100
612 J. Org. Chem. Vol. 74, No. 2, 2009

Chitobiose-Based Peptide N-Glycanase Inhibitor Library
1563.0, 1557.7, 1373.7, 1305.4, 1199.9, 1117.0, 1023.9; LC/MS
tR 5.67 min; linear gradient 0f90% B in 13.5 min; ESI/MS m/z )
669.07 (M + H)+; HRMS (M + H+) calcd for C29H41N4O14
669.26138, found 669.26145; mp 164 °C (start of decomposition).
N-(O-(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-ꢀ-
D-glucopyranosyl)-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
ꢀ-D-glucopyranosyl)hydroxyacetamide (21). Glycolic acid (14 mg,
0.186 mmol) was condensed with azide 4 (100 mg, 0.124 mmol)
in the presence of HCTU (77 mg, 0.186 mmol) and DiPEA (65
µL, 0.371 mmol) as described in the general procedure. The
resulting hydroxyacetamide was purified (CH2Cl2f5% MeOH/
CH2Cl2) affording 21 (77%, 80 mg, 95 µmol) as a white solid: 1H
NMR (400 MHz, CDCl3/MeOD) δ ppm 8.01 (d, J ) 9.0 Hz, 1H),
7.56 (d, J ) 9.0 Hz, 1H), 7.52-7.21 (m, 20H), 6.62 (d, J ) 9.2
Hz, 1H), 5.50 (s, 1H), 4.91 (dd, J ) 9.4, 6.3 Hz, 1H), 4.86 (d, J )
12.4 Hz, 2H), 4.71 (d, J ) 11.7 Hz, 1H), 4.64 (d, J ) 12.0 Hz,
1H), 4.64 (d, J ) 11.4 Hz, 1H), 4.55 (d, J ) 8.3 Hz, 1H), 4.46 (d,
J ) 11.7 Hz, 1H), 4.16-4.11 (m, 1H), 4.09-4.00 (m, 2H), 3.99
(s, 2H), 3.83 (t, J ) 8.8, 8.8 Hz, 1H), 3.71-3.50 (m, 6H), 3.44 (t,
J ) 10.3, 10.3 Hz, 1H), 3.24 (dt, J ) 9.6, 9.5, 5.0 Hz, 1H), 1.87
(s, 6H); 13C NMR (100 MHz, CDCl3/MeOD) δ ppm 174.1, 172.2,
171.3, 138.4, 138.0, 137.6, 136.9, 128.6, 128.1, 127.9, 127.8, 127.8,
127.6, 127.4, 127.3, 127.1, 125.6, 100.8, 100.2, 81.8, 80.1, 78.4,
76.2, 74.9, 73.7, 73.6, 73.1, 68.1, 67.7, 65.3, 61.1, 55.5, 52.8, 22.3,
21.8; FT-IR Vmax (neat)/cm-1 3273.7, 1652.0, 1537.8, 1452.6,
1371.2, 1315.8, 1066.7; LC/MS tR 8.26 min; linear gradient
10f90% B in 13.5 min; ESI/MS m/z ) 840.4 (M + H)+; HRMS
(M + H+) calcd for C46H53N3O12 839.37020, found 839.37101; mp
159 °C (start of decomposition).
N-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-(1f4)-2-ac-
etamido-2-deoxy-ꢀ-D-glucopyranosyl)mesyloxyacetamide (24).
Hydroxyacetamide 21 (80 mg, 95 µmol) was dissolved in dioxane/
DMF (4/1, v/v, 1.5 mL). Ms2O (60 mg, 0.34 mmol) and Et3N (53
µL, 0.38 mmol) were added. After 2 h, an additional amount of
Ms2O (15 mg, 86 µmo l) and Et3N (26 µL, 0.188 mmol) was added.
The reaction was quenched after additional stirring with MeOH
and subsequently concentrated under reduced pressure: LC/MS tR
9.44 min; linear gradient 10f90% B in 13.5 min; ESI/MS m/z )
918.5 (M + H)+. Crude mesylate 22 was dissolved in MeOH/TFE.
p-Toluenesulfonic acid (25 mg) was added after which the reaction
was stirred at 50 °C. After 1 h, the solution was cooled to room
temperature, Pd(OH)2 on charcoal was added, and the mixture was
stirred under H2 atm. TLC showed complete conversion to a lower
running spot after 30 min. The mixture was filtered, concentrated
in vacuo and purfied by semipreparative RP-HPLC (linear gradient
0f6% B in 15.25 min) furnishing mesylate 24 (44%, 23.14 mg,
41.4 µmol): 1H NMR (600 MHz, D2O) δ ppm 5.11 (d, J ) 9.8 Hz,
1H), 4.58 (d, J ) 8.5 Hz, 1H), 3.91 (dd, J ) 11.8, 2.8 Hz, 1H),
3.90 (t, J ) 10.8, 10.8 Hz, 1H), 3.82 (dd, J ) 12.2, 1.4 Hz, 1H),
3.79-3.72 (m, 3H), 3.66 (dd, J ) 9.3, 8.8 Hz, 1H), 3.64 (dd, J )
12.1, 4.5 Hz, 1H), 3.59 (ddd, J ) 10.0, 4.6, 1.7 Hz, 1H), 3.55 (dd,
J ) 10.3, 8.6 Hz, 1H), 3.49 (ddd, J ) 10.2, 5.6, 1.7 Hz, 1H), 3.46
(dd, J ) 9.6, 8.7 Hz, 1H), 3.27 (s, 3H), 2.05 (s, 3H), 1.98 (s, 3H);
13C NMR (150 MHz, D2O) δ ppm 174.3, 174.1, 168.6, 100.8, 78.2,
77.7, 75.8, 75.3, 72.9, 72.1, 69.1, 65.8, 60.0, 59.3, 55.0, 53.0, 21.5,
21.4; FT-IR Vmax (neat)/cm-1 3269.8, 1681.6, 1651.4, 1544.9,
1431.7, 1351.6, 1201.0, 1165.4, 1108.7, 1041.5; LC/MS tR 2.77
min; linear gradient 0f20% B in 13.5 min; ESI/MS m/z ) 560.00
(M + H)+; HRMS (M + H+) calcd for C19H34N3O14S 560.17560,
found 560.17540; mp 181 °C (start of decomposition).
(m, 5H), 4.96 (d, J ) 9.7 Hz, 1H), 4.55 (d, J ) 8.4 Hz, 1H), 3.90
(dd, J ) 12.1, 1.5 Hz, 1H), 3.81 (t, J ) 9.3, 9.3 Hz, 1H), 3.80 (dd,
J ) 12.0, 1.5 Hz, 1H), 3.76-3.39 (m, 10H), 2.05 (s, 3H), 1.73 (s,
3H); 13C NMR (100 MHz, MeOD/D2O) δ ppm 175.3, 175.2, 173.8,
135.3, 130.4, 129.5, 128.0, 102.5, 80.0, 79.9, 77.3, 77.1, 74.6, 73.8,
70.9, 61.7, 61.0, 56.7, 54.5, 37.8, 23.1, 22.8; FT-IR Vmax (neat)/
cm-1 3272.1, 1651.1, 1539.7, 1435.8, 1373.7, 1310.0, 1026.9; LC/
MS tR 6.52 min; linear gradient 0f50% B in 13.5 min; ESI/MS
m/z ) 573.93 (M + H)+; HRMS (M + H+) calcd for C24H36N3O11S
574.20651, found 574.20644; mp 230 °C (start of decomposition).
3-C-(O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-glu-
copyranosyl)-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-ꢀ-
D-glucopyranosyl)propene (29). Imidate 28 (2.53 g, 4.36 mmol)
and acceptor 26 (1.54 g, 3 mmol) were coevaporated three times
with anhydrous Tol and dissolved in CH2Cl2 (5 mL/mmol) after
which 3 Å moleculare sieves (MS) were added. The mixture was
cooled to -30 °C and stirred for 30 min before TMSOTf (cat.)
was added. After 2 h, the reaction was quenched with Et3N (1 mL),
diluted with CH2Cl2, extracted with NaHCO3 (satd aq), dried
(Na2SO4), and concentrated. Purification by Sephadex LH-20
column (50 mmD × 1500 mmL) gave disaccharide 29 (97%, 2.719
g, 2.9 mmol) as a thick syrup: 1H NMR (400 MHz, CDCl3) δ ppm
7.96-7.84 (m, 2H), 7.79-7.71 (m, 3H), 7.69-7.62 (m, 2H), 7.57
(d, J ) 6.4 Hz, 1H), 7.39-7.15 (m, 5H), 7.06-6.96 (m, 2H),
6.87-6.82 (m, 3H), 5.81 (t, J ) 9.9, 9.9 Hz, 1H), 5.70-5.58 (m,
1H), 5.54 (d, J ) 8.3 Hz, 1H), 5.13 (t, J ) 9.6, 9.6 Hz, 1H),
4.90-4.76 (m, 3H), 4.59 (d, J ) 12.1 Hz, 1H), 4.53 (d, J ) 12.1
Hz, 1H), 4.46 (d, J ) 12.5 Hz, 1H), 4.34 (dd, J ) 10.4, 8.5 Hz,
1H), 4.26-4.18 (m, 3H), 4.06-4.00 (m, 2H), 3.95 (d, J ) 12.7
Hz, 1H), 3.56 (d, J ) 11.1 Hz, 1H), 3.53-3.47 (m, 1H), 3.45 (dd,
J ) 11.4, 3.0 Hz, 1H), 3.33-3.28 (m, 1H), 2.18-2.06 (m, 2H),
2.00 (s, 3H), 1.96 (s, 3H), 1.84 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ ppm 170.6, 170.0, 169.4, 168.0, 167.7, 138.7, 138.5,
134.4, 133.7, 133.6, 133.4, 131.5, 131.4, 128.7, 128.2, 127.8, 127.7,
127.4, 127.3, 126.9, 123.5, 123.2, 123.1, 116.9, 96.8, 78.3, 77.8,
76.4, 74.5, 74.2, 72.6, 71.4, 70.6, 68.8, 68.2, 61.5, 55.7, 55.3, 36.9;
FT-IR Vmax (neat)/cm-12945.5, 1747.8, 1713.8, 1469.3, 1455.3,
1385.6, 1222.8, 1081.2, 1036.1; HRMS (M + Na+) calcd for
C51H50N2O15Na 953.31034, found 953.31077.
3-C-(O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-ꢀ-D-glucopy-
ranosyl)-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-ꢀ-D-glu-
copyranosyl)propene (30). After coevaporation with toluene,
disaccharide 29 (3.35 g, 3.6 mmol) was dissolved in n-BuOH/
ethylenediamine (50 mL, 9/1 v/v), 4 Å MS were added, and the
solution was subsequently stirred overnight at 90 °C. The solution
was filtered, concentrated in vacuo, coevaporated with toluene (3×),
redissolved in pyridine (50 mL), and cooled to 0 °C before Ac2O
(30 mL) was added. The reaction mixture was stirred overnight,
after which TLC analysis showed complete conversion of the
starting material. Concentration in vacuo, redissolving in CH2Cl2,
extraction with 1 M HCl, drying (Na2SO4), and concentrating gave
the crude title compound 30. Purification by silica gel chromatog-
raphy (CH2Cl2f2% MeOH/CH2Cl2) furnished 30 (75%, 2.048 g,
2.71 mmol) as an off-white solid: 1H NMR (400 MHz, DMSO) δ
ppm 8.05 (d, J ) 9.2 Hz, 1H), 7.86 (d, J ) 9.3 Hz, 1H), 7.37-7.21
(m, 10H), 5.88-5.76 (m, 1H), 5.15-4.96 (m, 3H), 4.89-4.78 (m,
3H), 4.63-4.54 (m, 3H), 4.02 (dd, J ) 12.2, 4.0 Hz, 1H), 3.81 (d,
J ) 12.1 Hz, 1H), 3.79-3.67 (m, 3H), 3.66-3.58 (m, 2H), 3.51
(t, J ) 9.4, 9.4 Hz, 1H), 3.48-3.43 (m, 1H), 3.37-3.33 (m, 1H),
3.26 (m, 1H) 2.26 (dd, J ) 14.2, 7.6 Hz, 1H), 2.04 (td, J ) 14.4,
6.9, 6.9 Hz, 1H), 1.94 (s, 3H), 1.92 (s, 3H), 1.86 (s, 3H), 1.77 (s,
6H); 13C NMR (100 MHz, DMSO) δ ppm 169.8, 169.6, 169.4,
169.3, 169.1, 139.4, 138.8, 135.0, 128.2, 127.9, 127.2, 127.0, 126.9,
126.9, 116.6, 99.7, 82.2, 78.2, 77.6, 76.0, 72.7, 72.3, 71.9, 70.7,
68.8, 68.5, 61.7, 54.1, 53.6, 35.9, 22.9, 22.7, 20.4, 20.3; FT-IR
Vmax (neat)/cm-1 3297.9, 2940.5, 1740.4, 1652.0, 1557.8, 1538.9,
1455.7, 1373.3, 1227.3, 1118.2, 1045.5; LC/MS tR 7.96 min; linear
gradient 10f90% B in 13.5 min; ESI/MS m/z ) 755.00 (M +
N-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-(1f4)-2-ac-
etamido-2-deoxy-ꢀ-D-glucopyranosyl)thiophenylacetamide (25).
Mesylate 24 (44 mg, 78 µmol) was dissolved in MeOH (1 mL).
Thiophenol (16.5 µL, 0.16 mmol) and K2CO3 (22 mg, 0.16 mmol)
were added. The solution was stirred for 16 h before removal of
the volatiles and extraction with H2O/Et2O. The aqueous layer was
concentrated in vacuo and purified by RP-HPLC (linear gradient
10f30% B in 10 min) giving title compound 25 (43%, 19.12 mg,
1
33.3 µmol): H NMR (400 MHz, MeOD/D2O) δ ppm 7.43-7.22
J. Org. Chem. Vol. 74, No. 2, 2009 613

Witte et al.
H)+; HRMS (M + H+) calcd for C39H51N2O13 755.33857, found
755.33881; mp 224 °C.
4.59 (d, J ) 8.5 Hz, 1H), 3.92 (dd, J ) 12.5, 0.9 Hz, 1H), 3.83
(dd, J ) 12.0, 1.3 Hz, 1H), 3.78-3.72 (m, 5H), 3.71-3.53 (m,
6H), 3.53-3.43 (m, 3H), 2.56 (ddd, J ) 15.6, 6.3, 5.1 Hz, 1H),
3-C-(O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-ꢀ-D-glucopy-
ranosyl)-(1f4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-ꢀ-D-glu-
copyranosyl)propene (31). Disaccharide 30 (154 mg, 0.2 mmol)
was suspended in THF (5 mL) and added to a solution of NH3 (5
mL) and sodium (100 mg) at -78 °C. The mixture was stirred at
-78 °C for 2 h after which it was quenched with MeOH. The
temperature was raised to room temperature. Residual NH3 was
removed by a flow of argon. Subsequently, the solution was
neutralized with AcOH, concentrated, redissolved in pyridine/Ac2O,
and stirred overnight. The reaction mixture was concentrated in
vacuo, redissolved in CH2Cl2, extracted with 1 M HCl and NaHCO3
(satd aq), dried (Na2SO4), and concentrated under reduced pressure.
Silica gel column chromatography (CHCl3f2% MeOH/CHCl3)
afforded 31 (quant, 131 mg, 0.2 mmol) as a white solid: 1H NMR
(400 MHz, CDCl3/MeOD) δ ppm 5.84-5.72 (m, 1H), 5.25 (t, J )
9.9, 9.9 Hz, 1H), 5.05-4.94 (m, 4H), 4.69 (d, J ) 8.3 Hz, 1H),
4.40 (d, J ) 11.8 Hz, 1H), 4.39 (dd, J ) 12.3, 4.0 Hz, 1H), 4.04
(dd, J ) 11.8, 6.1 Hz, 1H), 3.99 (dd, J ) 12.4, 1.8 Hz, 1H), 3.82
(t, J ) 10.2, 10.2 Hz, 1H), 3.75-3.65 (m, 3H), 3.54-3.49 (m,
1H), 3.37 (ddd, J ) 10.3, 7.6, 3.0 Hz, 1H), 2.35-2.10 (m, 2H),
2.07 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.98 (s, 3H), 1.97 (s, 3H),
1.88 (s, 6H); 13C NMR (100 MHz, CDCl3/MeOD) δ ppm 172.7,
172.4, 172.0, 171.68, 171.65, 171.4, 170.6, 134.3, 117.5, 101.4,
78.2, 77.3, 77.1, 75.8, 73.1, 72.1, 69.2, 63.6, 62.5, 55.53, 55.45,
54.0, 36.6, 22.9, 22.8, 21.1, 21.0, 20.84, 20.77, 20.7; FT-IR Vmax
(neat)/cm-13293.9, 1739.7, 1699.9, 1660.9, 1652.0, 1538.1, 1435.8,
1373.5, 1223.9, 1136.9, 1042.6; LC/MS tR 5.58 min; linear gradient
10f90% B in 13.5 min; ESI/MS m/z ) 659.00 (M + H)+; HRMS
(M + H+) calcd for C29H43N2O15 659.26579, found 659.26579; mp
235 °C (start of decomposition).
(E)-Ethyl 4-C-(O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-ꢀ-
D-glucopyranosyl)-(1f4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-
ꢀ-D-glucopyranosyl)but-2-enoate (32). Allyl C-glycoside 31 (131
mg, 0.2 mmol) was coevaporated with toluene and dissolved in
CH2Cl2 (6 mL). Grubbs second-generation catalyst (8 mg) and ethyl
acrylate (92 µL, 1 mmol) were added. The reaction mixture was
refluxed overnight, concentrated, and applied to silica gel chroma-
tography (CH2Cl2f5% MeOH/CH2Cl2), furnishing title compound
32 as an off-white solid (quant, 146 mg, 0.2 mmol): 1H NMR (400
MHz, CDCl3) δ ppm 6.92 (dt, J ) 15.2, 6.7 Hz, 1H), 6.40 (d, J )
8.8 Hz, 1H), 6.15 (d, J ) 9.5 Hz, 1H), 5.88 (d, J ) 15.6 Hz, 1H),
5.27 (t, J ) 9.8, 9.8 Hz, 1H), 5.06 (t, J ) 9.6, 9.6 Hz, 1H), 4.99
(t, J ) 9.6, 9.6 Hz, 1H), 4.67 (d, J ) 8.2 Hz, 1H), 4.39 (dd, J )
12.5, 3.8 Hz, 1H), 4.36 (d, J ) 12.0 Hz, 1H), 4.22 (dd, J ) 12.1,
6.0 Hz, 1H), 4.17 (q, J ) 6.9 Hz, 2H), 4.06-3.98 (m, 2H), 3.81
(q, J ) 9.4, 9.1, 9.1 Hz, 1H), 3.74-3.64 (m, 2H), 3.57-3.52 (m,
1H), 3.36 (dt, J ) 10.2, 9.9, 2.9 Hz, 1H), 2.49 (dd, J ) 13.6, 6.8
Hz, 1H), 2.40 (td, J ) 15.6, 7.2, 7.2 Hz, 1H), 2.13 (s, 3H), 2.08 (s,
3H), 2.06 (s, 3H), 2.00 (s, 6H), 1.94 (s, 6H), 1.27 (t, J ) 7.1, 7.1
Hz, 3H); 13C NMR (100 MHz, CDCl3) δ ppm 171.3, 171.1, 170.7,
170.6, 170.5, 170.4, 169.4, 166.3, 144.2, 123.3, 101.1, 101.0, 77.7,
76.7, 76.5, 74.3, 72.4, 71.7, 68.3, 62.6, 61.9, 60.2, 54.9, 53.6, 34.4,
23.1, 20.9, 20.64, 20.57, 20.56, 14.2; FT-IR Vmax (neat)/cm-13286.0,
1738.6, 1656.7, 1651.8, 1562.7, 1538.0, 1435.7, 1373.1, 1221.9,
1179.5, 1032.5, 979.7, 905.9; LC/MS tR 5.43 min; linear gradient
10f90% B in 13.5 min; ESI/MS m/z ) 731.5(M + H)+; HRMS
(M + H+) calcd for C32H47N2O17 731.28692, found 731.28713; mp
225 °C.
2.48 (td, J ) 14.5, 6.6, 6.6 Hz, 1H), 2.07 (s, 3H), 1.98 (s, 3H); 13
C
NMR (125 MHz, D2O) δ ppm 174.6, 174.3, 169.2, 146.1, 122.0,
101.4, 79.7, 78.1, 76.5, 75.9, 73.8, 73.5, 69.7, 60.5, 60.3, 55.6, 54.5,
52.1, 34.4, 22.10, 22.07; FT-IR Vmax (neat)/cm-13268.9, 1627.8,
1562.6, 1557.7, 1436.0, 1377.2, 1304.3, 1199.8, 1079.8, 1044.4;
LC/MS tR 4.583 min; linear gradient 0f20% B in 13.5 min; ESI/
MS m/z ) 507.00 (M + H)+; HRMS (M + H+) calcd for
C21H35N2O12 507.21845, found 507.21818; mp 150 °C (start of
decomposition).
3-C-(O-(2-Acetamido-3,4,6-tri-O-tert-butyldimethylsilyl-2-
deoxy-ꢀ-D-glucopyranosyl)-(1f4)-2-acetamido-3,6-di-O-tert-bu-
tyldimethylsilyl-2-deoxy-ꢀ-D-glucopyranosyl)propene (36). Per-
acetylated 31 (255 mg, 387 µmol) was dissolved in MeOH (10
mL) before NaOMe in MeOH (3M, 0.3 mL) was added. TLC
analysis showed complete conversion to a very polar product after
2 h. The mixture was neutralized with Amberlite IR-120 H+,
filtered, concentrated, and redissolved in pyridine. The solution was
cooled, after which tert-butyldimethylsilyl triflate (TBSOTf) (0.533
mL, 2.32 mmol) was added. Additional amounts of TBSOTf (0.15
mL, 0.653 mmol) were added after 24 and 72 h. The reaction was
quenched by the addition of MeOH (1 mL) and concentrated under
reduced pressure. Residual amounts of pyridine were removed by
coevaporation with toluene. Silica gel column chromatography
(CH2Cl2 f 2% MeOH/CH2Cl2) afforded title compound 36 (64%,
251 mg, 0.246 mmol) as a white solid: 1H NMR (400 MHz, CDCl3)
δ ppm 6.25 (d, J ) 9.7 Hz, 1H), 5.90-5.79 (m, 1H), 5.77 (d, J )
10.3 Hz, 1H), 5.06 (dd, J ) 17.2, 1.6 Hz, 1H), 5.00 (dd, J ) 10.2,
1.8 Hz, 1H), 4.52 (d, J ) 6.9 Hz, 1H), 4.14-4.08 (m, 1H), 3.97
(d, J ) 4.2 Hz, 1H), 3.94 (dd, J ) 4.8, 2.7 Hz, 1H), 3.89 (dd, J )
9.3, 2.9 Hz, 1H), 3.80-3.63 (m, 7H), 3.51 (ddd, J ) 6.6, 6.0, 2.8
Hz, 1H), 3.09 (dt, J ) 8.6, 8.5, 3.6 Hz, 1H), 2.32 (ddd, J ) 10.3,
6.7, 3.2 Hz, 1H), 2.23 (td, J ) 14.9, 7.3, 7.3 Hz, 1H), 1.98 (s, 3H),
1.93 (s, 3H), 0.93 (s, 9H), 0.89 (s, 9H), 0.89 (s, 18H), 0.87 (s, 9H),
0.14 (s, 3H), 0.13 (s, 3H), 0.12 (s, 6H), 0.11 (s, 3H), 0.08 (s, 3H),
0.05 (s, 9H), 0.04 (s, 3H); 13C NMR (100 MHz, CDCl3) δ ppm
169.4, 168.8, 135.0, 116.4, 99.0, 81.2, 78.9, 77.0, 76.4, 73.9, 73.2,
70.0, 63.8, 63.3, 55.4, 54.3, 38.3, 25.94, 25.92, 25.87, 25.84, 25.77,
25.67, 23.4, 23.2, 18.3, 18.2, 17.82, 17.79, -4.0, -4.4, -4.6, -4.7,
-4.8, -5.1, -5.2, -5.4; FT-IR Vmax (neat)/cm-12930.3, 2857.5,
1651.9, 1558.6, 1473.7, 1374.0, 1308.3, 1252.1, 1102.6, 1005.9;
HRMS (M + H+) calcd for C49H102N2O10Si5 1019.64536, found
1019.64668; mp 193.2-194.7 °C.
(E)-3-C-(O-(2-Acetamido-3,4,6-tri-O-tert-butyldimethylsilyl-
2-deoxy-ꢀ-D-glucopyranosyl)-(1f4)-2-acetamido-3,6-di-O-tert-
butyldimethylsilyl-2-deoxy-ꢀ-D-glucopyranosyl)-1-methanesulfo-
nylpropene (37). Alkene 36 (240 mg, 0.235 mmol) was dissolved
in freshly distilled CH2Cl2 under argon atmosphere. Methyl
vinylsulfone (206 µL, 2.35 mmol) and Hoveyda-Grubbs second-
generation catalyst (15 mg, 23.5 µmol) were added. The reaction
was refluxed overnight before an additional amount of catalyst and
vinylsulfone was added. The resulting mixture was refluxed until
TLC analysis showed complete conversion to a more polar product.
Concentration under reduced pressure followed by purification over
silica gel chromatography (CH2Cl2f3% MeOH/CH2Cl2) afforded
1
vinyl sulfone 37 (66%, 171 mg, 0.156 mmol) as a brown oil: H
NMR (400 MHz, CDCl3) δ ppm 6.93 (td, J ) 14.3, 6.9, 6.9 Hz,
1H), 6.43 (d, J ) 14.9 Hz, 2H), 5.81 (d, J ) 10.2 Hz, 1H), 4.51
(d, J ) 6.8 Hz, 1H), 4.08 (dd, J ) 9.5, 7.3 Hz, 1H), 4.00-3.95
(m, 2H), 3.90 (dt, J ) 9.1, 9.0, 1.8 Hz, 1H), 3.82 (dd, J ) 4.4, 2.8
Hz, 1H), 3.78-3.63 (m, 6H), 3.54 (dt, J ) 6.5, 6.2, 1.9 Hz, 1H),
3.22 (dt, J ) 8.7, 8.6, 3.4 Hz, 1H), 2.92 (s, 3H), 2.54 (ddd, J )
15.1, 7.2, 2.9 Hz, 1H), 2.45 (dd, J ) 15.3, 7.6 Hz, 1H), 2.00 (s,
3H), 1.95 (s, 3H), 0.94 (s, 9H), 0.89 (s, 18H), 0.89 (s, 9H), 0.87 (s,
9H), 0.13 (s, 6H), 0.12 (s, 6H), 0.11 (s, 3H), 0.08 (s, 3H), 0.05 (s,
12H); 13C NMR (100 MHz, CDCl3) δ ppm 169.8, 169.1, 144.7,
131.1, 98.9, 81.3, 78.9, 76.3, 75.4, 73.6, 72.7, 70.0, 63.8, 63.2, 55.7,
(E)-Methyl 4-C-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyrano-
syl)-(1f4)-2-acetamido-2-deoxy-ꢀ-D-glucopyranosyl)but-2-
enoate (33). Vinyl ester 32 (73 mg, 0.1 mmol) was dissolved in
methanol/H2O (1/1 v/v, 4 mL). Et3N (1 mL) was added, and the
resulting mixture was stirred for 48 h. Concentration under reduced
pressure followed by preparative RP-HPLC purification (linear
gradient 0f40% B in 10 min) gave title compound 33 (12%, 6.01
mg, 11.9 µmol) as a white solid: 1H NMR (500 MHz, D2O) δ ppm
7.01 (td, J ) 14.6, 7.0, 7.0 Hz, 1H), 5.95 (d, J ) 15.8 Hz, 1H),
614 J. Org. Chem. Vol. 74, No. 2, 2009

Chitobiose-Based Peptide N-Glycanase Inhibitor Library
54.2, 42.8, 36.2, 26.0, 25.9, 25.83, 25.79, 23.5, 23.2, 18.4, 18.23,
18.19, 17.9, -4.3, -4.4, -4.6, -4.66, -4.73, -5.0, -5.2, -5.3,
-5.4; FT-IR: Vmax (neat)/cm-12931.6, 2857.8, 1667.3, 1469.6,
residual traces of n-BuOH/ethylenediamine. The resulting amine
was dissolved in MeOH (50 mL) and cooled to 0 °C before Ac2O
(5 mL) and Et3N (7 mL) were added. The reaction was stirred
overnight and crystallized. The diol was dissolved in CHCl3/MeOH
(30 mL, 1/1 v/v). Dibutyltin oxide (0.883 g, 3.5 mmol) was added,
and the reaction was refluxed for 2 h. The resulting clear solution
was concentrated and dissolved in CHCl3 (20 mL), and N-
bromosuccimide (0.627 g, 3.52 mmol) was added. After 2 h of
stirring, Celite was added, and the reaction mixture was concentrated
in vacuo. Purification by silica gel chromatography (CH2Cl2f6%
MeOH/CH2Cl2) afforded hydroxymethyl ketone 41 (50% over four
1302.1, 1253.1, 1092.8; HRMS (M
+
H+) calcd for
C50H104N2O12SSi5 1097.62291, found 1097.62394.
(E)-3-C-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-(1f4)-
2-acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-1-methanesulfonyl-
propene (38). Vinyl sulfone 37 (89 mg, 74.7 µmol) was cooled to
0 °C before being dissolved in trifluoroacetic acid (2 mL) in the
presence of H2O (200 µL). The reaction was allowed to come to
room temperature. LC/MS analysis showed complete conversion
after 4 h. The solution was diluted with Tol/H2O and concentrated
under reduced pressure. The resulting solids were redissolved in
H2O, washed with EtOAc (3×), and concentrated. Semipreparative
reversed-phase HPLC purification (linear gradient in 0f6% B in
14.00 min) afforded deprotected vinyl sulfone 38 (78%, 30.63 mg,
1
steps, 1.35 g, 1.6 mmol): H NMR (400 MHz, CDCl3/MeOD) δ
ppm 7.56-7.18 (m, 20H), 5.49 (s, 1H), 4.86 (d, J ) 11.9 Hz, 1H),
4.83 (d, J ) 11.1 Hz, 1H), 4.67 (d, J ) 10.4 Hz, 1H), 4.64 (d, J )
10.5 Hz, 1H), 4.62 (d, J ) 11.5 Hz, 1H), 4.52 (d, J ) 8.3 Hz, 1H),
4.45 (d, J ) 11.7 Hz, 1H), 4.21 (s, 2H), 4.13 (dd, J ) 10.8, 5.1
Hz, 1H), 3.94 (t, J ) 8.1, 8.1 Hz, 1H), 3.90 (dd, J ) 9.5, 8.9 Hz,
1H), 3.84 (t, J ) 8.8, 8.8 Hz, 1H), 3.75-3.68 (m, 1H), 3.68-3.54
(m, 3H), 3.50 (t, J ) 8.4, 8.4 Hz, 1H), 3.47-3.37 (m, 2H), 3.23
(dt, J ) 9.3, 9.2, 4.8 Hz, 1H), 2.69 (dd, J ) 16.1, 8.9 Hz, 1H),
2.54 (dd, J ) 16.0, 2.4 Hz, 1H), 1.87 (s, 3H), 1.86 (s, 3H); 13C
NMR (100 MHz, CDCl3/MeOD) δ ppm 209.0, 171.9, 171.8, 139.1,
138.6, 138.1, 137.5, 129.2, 128.8, 128.54, 128.46, 128.4, 128.2,
128.0, 127.72, 127.67, 126.2, 101.4, 101.0, 82.4, 81.5, 78.9, 78.1,
76.0, 75.0, 74.3, 74.1, 73.9, 69.1, 68.73, 68.65, 65.9, 55.9, 53.9,
41.9, 23.1, 22.8; FT-IR Vmax (neat)/cm-13278.2, 2870.1, 1717.7,
1658.1, 1651.8, 1557.7, 1538.5, 1497.4, 1455.5, 1373.1, 1321.7,
1210.2, 1173.8, 1072.0, 1027.9, 1011.9; LC/MS tR 8.96 min; linear
gradient 10f90% B in 13.5 min; ESI/MS m/z ) 839.60 (M +
H)+; HRMS (M + H+) calcd for C47H55N2O12 839.37495, found
839.37581; mp 159 °C (start of decomposition).
1
58.2 µmol) as a white solid: H NMR (400 MHz, D2O) δ ppm
6.96 (td, J ) 14.8, 6.8, 6.8 Hz, 1H), 6.66 (d, J ) 15.3 Hz, 1H),
4.57 (d, J ) 8.4 Hz, 1H), 3.91 (dd, J ) 12.4, 1.3 Hz, 1H), 3.82
(dd, J ) 11.9, 1.6 Hz, 1H), 3.77-3.52 (m, 8H), 3.52-3.40 (m,
3H), 3.09 (s, 3H), 2.68-2.59 (m, 1H), 2.51 (td, J ) 15.6, 6.7, 6.7
Hz, 1H), 2.06 (s, 3H), 2.01 (s, 3H); 13C NMR (100 MHz, D2O) δ
ppm 174.6, 174.4, 145.1, 130.0, 101.5, 79.8, 78.1, 76.1, 76.0, 73.8,
73.5, 69.7, 60.6, 60.4, 55.6, 54.5, 41.8, 33.4, 22.2, 22.2; FT-IR
Vmax (neat)/cm-13287.6, 1636.5, 1557.7, 1420.6, 1374.6, 1277.4,
1202.8, 1124.8, 1026.6; LC/MS tR 3.767 min; linear gradient
10f90% B in 13.5 min; ESI/MS m/z ) 527.5 (M + H)+; HRMS
(M + H+) calcd for C20H34N2O12S 527.19052, found 527.19018;
mp 136 °C (start of decomposition).
3-C-(O-(3-O-Benzyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-
ꢀ-D-glucopyranosyl)-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthal-
imido-ꢀ-D-glucopyranosyl)propene (40). Imidate 39 (2.8 g, 3.7
mmol) and acceptor 26 (1.9 g, 3.5 mmol) were coevaporated three
times with toluene before being dissolved in freshly distilled CH2Cl2
(30 mL). Activated molecular sieves 4 Å were added, after which
the solution was cooled to -30 °C. TMSOTf (73 µL) was added
followed by stirring until TLC showed complete conversion. The
reaction was quenched with Et3N, diluted with EtOAc, extracted
with NaHCO3 (satd aq) and brine, dried (Na2SO4), and concentrated.
Silica gel column chromatography (10% EA/PEf30% EA/PE)
furnished disaccharide 40 (97%, 3.35 g, 3.41 mmol) as a thick
3-C-(O-(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-
ꢀ-D-glucopyranosyl)-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
ꢀ-D-glucopyranosyl)-1-(2,6-dimethylbenzoyloxy)propan-2-one (42).
Hydroxy methylketone 41 (20 mg, 24 µmol) was dissolved in
CH2Cl2/DMF (0.5 mL, 3/1 v/v). Methanesulfonic anhydride (9 mg,
50 µmol) and Et3N (14 µL, 0.1 mmol) were added. After 3 h, the
solution was concentrated. DMF (0.4 mL), K2CO3 (20 mg, 0.1
mmol), and 2,6-dimethylbenzoic acid (20 mg, 0.1 mmol) were
added. The reaction was stirred overnight, diluted with CH2Cl2,
washed with NaHCO3 (satd aq), dried, and concentrated. Silica gel
column chromatography (CH2Cl2f3% MeOH/CH2Cl2) gave acy-
loxy methylketone 42 (65%, 15 mg, 15 µmol): 1H NMR (400 MHz,
DMSO) δ ppm 8.10 (d, J ) 8.5 Hz, 1H), 7.99 (d, J ) 8.9 Hz, 1H),
7.44-7.21 (m, 21H), 7.10 (d, J ) 7.6 Hz, 2H), 5.67 (s, 1H), 5.09
(d, J ) 17.2 Hz, 1H), 5.03 (d, J ) 17.2 Hz, 1H), 4.84 (d, J ) 11.1
Hz, 1H), 4.75-4.69 (m, 2H), 4.61-4.51 (m, 4H), 4.04-3.99 (m,
1H), 3.81-3.48 (m, 11H), 3.39 (ddd, J ) 9.9, 4.8, 1.3 Hz, 1H),
3.17-3.10 (m, 1H), 2.66 (dd, J ) 16.4, 7.2 Hz, 1H), 2.61 (dd, J )
16.2, 3.7 Hz, 1H), 2.29 (s, 6H), 1.84 (s, 3H), 1.82 (s, 3H); 13C
NMR (100 MHz, DMSO) δ ppm 201.4, 169.4, 169.3, 168.0, 139.1,
138.7, 138.6, 137.5, 134.6, 132.9, 129.5, 128.7, 128.6, 128.1,
128.01, 127.99, 127.9, 127.4, 127.3, 127.2, 127.1, 126.9, 125.9,
100.7, 99.9, 81.5, 80.9, 78.3, 76.0, 74.2, 73.3, 73.2, 71.8, 68.7, 68.6,
67.7, 65.5, 55.3, 53.7, 53.6, 41.7, 22.9, 22.82, 22.75, 19.2; FT-IR
Vmax (neat)/cm-13266.0, 2865.8, 1728.0, 1651.7, 1538.0, 1452.3,
1370.5, 1264.7, 1086.8, 1060.9; LC/MS tR 10.76 min; linear gradient
10f90% B in 13.5 min; ESI/MS m/z ) 971.27 (M + H)+; HRMS
(M + H+) calcd for C56H63N2O13 971.43247, found 971.43365; mp
211 °C (start of decomposition).
1
syrup: H NMR (400 MHz, CDCl3) δ ppm 7.93-7.83 (m, 1H),
7.77-7.55 (m, 7H), 7.53-7.48 (m, 2H), 7.42-7.23 (m, 8H),
7.07-7.02 (m, 2H), 7.00-6.96 (m, 2H), 6.95-6.85 (m, 6H), 5.64
(tdd, J ) 17.0, 10.0, 6.9, 6.9 Hz, 1H), 5.52 (s, 1H), 5.43 (d, J )
8.3 Hz, 1H), 4.88-4.76 (m, 4H), 4.55-4.40 (m, 5H), 4.29-4.15
(m, 4H), 4.07-4.01 (m, 2H), 3.73 (t, J ) 9.1, 9.1 Hz, 1H), 3.56 (t,
J ) 10.2, 10.2 Hz, 1H), 3.49 (d, J ) 10.9 Hz, 1H), 3.46-3.38 (m,
1H), 3.34 (dd, J ) 11.1, 3.0 Hz, 1H), 3.28 (dd, J ) 9.3, 2.7 Hz,
1H), 2.21-2.05 (m, 2H); 13C NMR (100 MHz, CDCl3) δ ppm
167.8, 167.6, 138.3, 138.1, 137.6, 137.1, 133.83, 133.82, 133.64,
133.56, 133.0, 131.2, 128.8-125.8, 123.1, 123.0, 116.8, 100.9, 97.5,
82.9, 78.2, 77.5, 76.2, 74.24, 74.17, 74.1, 73.8, 72.4, 68.4, 67.9,
65.5, 56.3, 55.5, 36.6; FT-IR Vmax (neat)/cm-13034.0, 2858.4,
1775.6, 1710.9, 1613.0, 1496.0, 1453.1, 1383.6, 1310.6, 1174.1,
1075.6, 1026.5; HRMS (M + Na+) calcd for C59H54N2O12Na
1005.35690, found 1005.35776.
3-C-(O-(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-
ꢀ-D-glucopyranosyl)-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
ꢀ-D-glucopyranosyl)-1-hydroxypropan-2-one (41). Disaccharide
40 (3.33 g, 3.38 mmol) was dissolved in THF/H2O (30 mL, 6/1
v/v). K2OsO4 (25 mg, 68 µmol) and morpholine N-oxide (0.99 g,
8.46 mmol) were added. After being stirred overnight, the reaction
was quenched with 1 M HCl, extracted with EtOAc, washed with
Na2S2O3 (aq), NaHCO3 (satd aq), and brine, dried (Na2SO4), and
concentrated. The resulting diol was redissolved in n-BuOH/
ethylenediamine (50 mL, 9/1 v/v) and activated 4 Å MS were added
after which the reaction was stirred overnight at 90 °C. The reaction
was filtered, concentrated, and coevaporated with toluene to remove
3-C-(O-(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-
ꢀ-D-glucopyranosyl)-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
ꢀ-D-glucopyranosyl)-1-chloropropan-2-one (43). Hydroxymethyl
ketone 41 (183 mg, 218 µmol) was dissolved in DMF (0.5 mL)
and dioxane (5 mL). Methanesulfonic anhydride (180 mg, 1.03
mmol) and Et3N (280 µL, 2.0 mmol) were added. TLC analysis
showed complete conversion to a higher running spot after 3 h.
The solution was concentrated and redissolved in DMF (5 mL),
and LiCl (400 mg) was added followed by stirring overnight. The
J. Org. Chem. Vol. 74, No. 2, 2009 615

Witte et al.
reaction was diluted with CH2Cl2, washed with 10% (aq) citric acid,
dried (Na2SO4), and concentrated. Silica column chromatography
(CH2Cl2 f 4% MeOH/CH2Cl2) afforded fully protected chlorom-
3-C-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-(1f4)-2-
acetamido-2-deoxy-ꢀ-D-glucopyranyl)-1-(diphenylphosphinoy-
loxy)propan-2-one (47). Chloromethyl ketone 45 (6.33 mg, 12.7
µmol) was dissolved in DMF (0.5 mL). Diphenylphosphinic acid
(5.6 mg, 25 µmol), K2CO3 (3.5 mg, 25 µmol), and potassium iodide
(cat.) were added after which the solution was stirred at 50 °C for
16 h. The volatiles were removed, and subsequently, the mixture
was purified. Semipreparative RP-HPLC (linear gradient 11%f26%
B in 14.00 min) afforded title compound 47 (16%, 1.394 mg, 2.05
1
ethyl ketone 43 (59%, 111 mg, 129 µmol) as a white solid: H
NMR (400 MHz, CDCl3/MeOD) δ ppm 7.50-7.25 (m, 20H), 5.51
(s, 1H), 4.87 (d, J ) 11.5 Hz, 1H), 4.86 (d, J ) 11.9 Hz, 1H), 4.65
(d, J ) 11.9 Hz, 1H), 4.65 (d, J ) 11.0 Hz, 1H), 4.62 (d, J ) 12.0
Hz, 1H), 4.59 (d, J ) 8.1 Hz, 1H), 4.52-4.49 (m, 1H), 4.24 (s,
2H), 4.11 (dd, J ) 10.4, 5.0 Hz, 1H), 3.93 (dd, J ) 8.7, 8.3 Hz,
1H), 3.90 (dd, J ) 18.5, 9.2 Hz, 1H), 3.86-3.80 (m, 1H), 3.75-3.69
(m, 1H), 3.68-3.61 (m, 3H), 3.51 (t, J ) 8.7, 8.7 Hz, 1H),
3.47-3.38 (m, 2H), 3.24 (dt, J ) 9.5, 9.3, 4.2 Hz, 1H), 2.83 (dd,
J ) 16.2, 8.9 Hz, 1H), 2.66 (dd, J ) 16.2, 3.1 Hz, 1H), 1.90 (s,
3H), 1.88 (s, 3H); 13C NMR (100 MHz, CDCl3/MeOD) δ ppm
200.4, 171.5, 171.4, 138.5, 137.9, 137.5, 136.8, 128.6, 128.1,
127.84, 127.76, 127.6, 127.4, 127.3, 127.0, 125.6, 100.8, 100.4,
81.7, 81.0, 78.3, 77.6, 75.5, 74.6, 73.7, 73.6, 73.0, 68.1, 68.0, 65.3,
55.4, 53.4, 48.9, 42.5, 22.3, 22.0; FT-IR Vmax (neat)/cm-13287.5,
2865.9, 1727.6, 1645.4, 1557.7, 1454.9, 1368.3, 1319.8, 1175.0,
1071.4; LC/MS tR 9.85 min; linear gradient 10f90% B in 13.5
min; ESI/MS m/z ) 857.07 (M + H)+; HRMS (M + H+) calcd
for C47H54ClN2O11 857.34106, found 857.34206; mp 194 °C (start
of decomposition).
1
µmol) as a white solid: H NMR (400 MHz, D2O) δ ppm 7.84
(tdd, J ) 12.8, 8.4, 1.4, 1.4 Hz, 4H), 7.76-7.71 (m, 2H), 7.62 (dt,
J ) 7.6, 7.4, 3.9 Hz, 4H), 4.82 (d, J ) 8.7 Hz, 2H), 4.56 (d, J )
8.4 Hz, 1H), 3.93 (dd, J ) 12.4, 1.5 Hz, 1H), 3.81-3.70 (m, 3H),
3.68-3.62 (m, 2H), 3.62-3.54 (m, 3H), 3.54-3.45 (m, 3H), 3.33
(ddd, J ) 9.6, 4.6, 2.1 Hz, 1H), 2.75 (dd, J ) 16.2, 7.8 Hz, 1H),
2.70 (dd, J ) 16.1, 3.9 Hz, 1H), 2.08 (s, 3H), 1.99 (s, 3H); 13C
NMR (100 MHz, D2O) δ ppm 205.34, 205.28, 174.5, 133.8, 132.0,
131.6, 131.53, 131.47, 131.4, 129.2, 129.1, 129.0, 127.14, 127.05,
101.4, 79.3, 77.9, 75.9, 73.9, 73.4, 69.7, 69.1, 69.1, 60.5, 60.0, 55.6,
54.4, 41.0, 29.6, 22.1, 22.0; FT-IR Vmax (neat)/cm-13289.9, 1668.0,
1651.7, 1557.7, 1435.9, 1374.0, 1202.2, 1131.0, 1028.1; LC/MS
tR 4.050 min; linear gradient 10f90% B in 13.5 min; ESI/MS m/z
) 681.3 (M + H)+; HRMS (M + H+) calcd for C31H42N2O13P
681.24190, found 681.24198; mp 84 °C (start of decomposition).
3-C-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-(1f4)-2-
acetamido-2-deoxy-ꢀ-D-glucopyranyl)-1-thiophenylpropan-2-
one (48). Chloromethyl ketone 45 (31.5 mg, 66 µmol) was dissolved
in MeOH (2 mL) before thiophenol (20 µL, 150 µmol) and K2CO3
(18 mg, 132 µmol) were added. After 16 h of stirring, the volatiles
were removed; the reaction was diluted in H2O/Et2O, and the layers
were separated. Concentration in vacuo followed by semipreparative
RP-HPLC (linear gradient 17.5 f 26.5% B in 14.00 min) gave 48
(64%, 24.23 mg, 42.3 µmol) as a white solid: 1H NMR (400 MHz,
D2O/MeOD) δ ppm 7.34 (d, J ) 3.9 Hz, 4H), 7.32-7.24 (m, 1H),
4.53 (d, J ) 8.4 Hz, 1H), 3.91 (s, 1H), 3.88 (dd, J ) 12.4, 1.0 Hz,
1H), 3.80-3.37 (m, 12H), 3.34-3.23 (m, 1H), 2.86 (dd, J ) 16.8,
8.8 Hz, 1H), 2.73 (dd, J ) 16.8, 2.7 Hz, 1H), 2.03 (s, 3H), 1.94 (s,
3H); 13C NMR (101 MHz, D2O/MeOD) δ ppm 207.9, 175.4, 175.3,
134.8, 130.6, 130.4, 128.3, 102.4, 80.4, 78.9, 77.0, 75.1, 74.6, 74.5,
70.8, 61.6, 61.0, 56.6, 55.4, 44.7, 23.10, 23.06; FT-IR Vmax (neat)/
cm-13275.0, 1713.4, 1636.0, 1555.6, 1435.9, 1373.3, 1319.6,
1203.1, 1164.4, 1028.6; LC/MS tR 4.52 min; linear gradient
10f90% B in 13.5 min; ESI/MS m/z ) 573.00 (M + H)+; HRMS
(M + H+) calcd for C25H36N2O11S 573.21126, found 573.21112;
mp 229 °C (start of decomposition).
3-C-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-(1f4)-2-
acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-1-(2,6-dimethylbenzoy-
loxy)propan-2-one (44). Reductive removal of the protective
groups of disaccharide 42 (70 mg, 72 µmol) was performed as
described in general procedure 1. Semipreparative RP-HPLC (linear
gradient 25%f34.375% B in 12.5 min) afforded title compound
44 (83%, 36.7 mg, 60 µmol) as a white solid: 1H NMR (400 MHz,
D2O) δ ppm 7.38 (t, J ) 7.6, 7.6 Hz, 1H), 7.22 (d, J ) 7.6 Hz,
2H), 5.23 (s, 2H), 4.72 (d, J ) 8.4 Hz, 1H), 4.04 (d, J ) 12.2 Hz,
1H), 3.99 (dd, J ) 17.5, 8.6 Hz, 1H), 3.93-3.81 (m, 5H), 3.80-3.66
(m, 3H), 3.65-3.49 (m, 3H), 2.95 (dd, J ) 16.1, 9.1 Hz, 1H), 2.85
(dd, J ) 16.2, 2.1 Hz, 1H), 2.43 (s, 6H), 2.16 (s, 3H), 2.13 (s, 3H);
13C NMR (100 MHz, D2O) δ ppm 204.9, 175.2, 175.1, 171.4, 136.7,
133.7, 131.5, 129.1, 102.9, 81.1, 79.5, 77.5, 75.6, 75.0, 74.8, 71.3,
70.5, 62.1, 61.5, 57.0, 56.0, 42.8, 31.3, 23.6, 23.5, 20.3; FT-IR
Vmax (neat)/cm-13271.6, 1728.5, 1651.8, 1562.9, 1557.7, 1423.9,
1373.9, 1269.0, 1203.0, 1165.7, 1106.4, 1077.5, 1049.0; LC/MS
tR 8.46 min; linear gradient 0f50% B in 13.5 min; ESI/MS m/z )
613.07 (M + H)+; HRMS (M + H+) calcd for C28H41N2O13
613.26032, found 613.26018; mp 190 °C (start of decomposition).
3-C-(O-(2-Acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-(1f4)-2-
acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-1-chloropropan-2-one
(45). Chloromethyl ketone 43 (80 mg, 95 µmol) was coevaporated
three times with toluene before being suspended in freshly distilled
CH2Cl2. The suspension was cooled to 0 °C after which FeCl3 (250
mg, 0.152 mmol) was added. TLC analysis showed conversion to
a very polar product after 5 h. The reaction was quenched by the
addition of H2O and washed with CH2Cl2. The aqueous layer was
concentrated to give the crude title compound. Semipreprative RP-
HPLC purification (linear gradient 0f12% B in 15.7 min) afforded
pure title compound 45 (66%, 31.46 mg, 63 µmol): 1H NMR (400
MHz, D2O) δ ppm 4.59 (d, J ) 8.4 Hz, 1H), 4.47 (s, 2H), 3.93
(dd, J ) 12.3, 1.7 Hz, 1H), 3.89 (ddd, J ) 7.4, 4.8, 2.1 Hz, 1H),
3.80 (dd, J ) 12.4, 2.2 Hz, 1H), 3.79-3.66 (m, 5H), 3.65-3.55
(m, 3H), 3.54-3.43 (m, 3H), 2.88 (dd, J ) 15.3, 5.7 Hz, 1H), 2.83
(dd, J ) 12.5, 1.0 Hz, 1H), 2.08 (s, 3H), 2.02 (s, 3H); 13C NMR
(100 MHz, D2O) δ ppm 203.9, 174.6, 101.5, 79.5, 78.1, 75.9, 74.0,
73.6, 73.5, 69.7, 60.5, 60.2, 55.6, 54.5, 49.5, 42.3, 22.1; FT-IR
Vmax (neat)/cm-13289.0, 1651.7, 1634.2, 1557.8, 1373.8, 1033.8;
LC/MS tR 3.16 min; linear gradient 0f50% B in 13.5 min; ESI/
MS m/z ) 498.93 (M + H)+; HRMS (M + H+) calcd for
C19H32ClN2O11 499.16891, found 499.16869; mp 140 °C (start of
decomposition).
Acknowledgment. PNGase was provided by S. Misaghi and
H. L. Ploegh, Whitehead Institute for Biomedical Research. This
work was supported by The Netherlands Organization for
Scientific Research (NWO) and The Netherlands Proteomic
Centre (NPC). This work was supported by the Dutch Cancer
Society (Grant No. UL 2005-3259 to D.H.).
Note Added after ASAP Publication. A current address was
added to the version published ASAP on January 2, 2009.
Supporting Information Available: General experimental
procedures. Synthesis and characterization of compounds 5, 7,
26, 39, and 49. Procedures for in vitro and cell culture inhibitory
studies. 1H NMR and 13C NMR spectra of all new compounds.
LC/MS spectra of 10-14, 17-21, 24, 25, 30-33, 38, 41-45,
47, and 48. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO801906S
616 J. Org. Chem. Vol. 74, No. 2, 2009