Synthesis of a versatile probe for analysis of cytoplasmic peptide-N-glycanase

Article abstract of DOI:10.1002/jccs.201100631

Aclickable alkyne tagged chloroacetamidyl chitobiose derivative was synthesized as a potential inhibitor of cytoplasmic peptide-N-glycanase (PNGase). Construction of a chitobiose structure containing both alkyne and azide functional groups was performed using a thioglycoside donor having a triisopropylsilyl (TIPS) protected alkyne group and a glycosyl azide acceptor. The resultant chitobiosyl azide derivative was reduced to the corresponding glycosyl amine and a chloroacetyl group was introduced. Finally, removal of the TIPS group using tetrabutylammonium fluoride (TBAF) gave the target compound.We conjugated the dansyl derivative, using click conditions and obtained the fluorescent labeled disaccharide derivative, selectively. Our probe can easily lead to a variety of compounds and should help in the understanding of the biological functions of cytoplasmic PNGase.

Full text of DOI:10.1002/jccs.201100631

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Communication
Synthesis of a Versatile Probe for Analysis of Cytoplasmic Peptide-N-Glycanase
Mizuho Masuda,a Shinpei Miyazawa,a Yukishige Itob and Ichiro Matsuoa*
aDepartment of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University,
1-5-1 Tenjin, Kiryu, Gunma 376-8515, Japan
bRIKEN Advanced Science Institute and ERATO Japan Science and Technology Agency, 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan
(Received: Oct. 27, 2011; Accepted: Nov. 28, 2011; Published Online: Dec. 20, 2011; DOI: 10.1002/jccs.201100631)
A clickable alkyne tagged chloroacetamidyl chitobiose derivative was synthesized as a potential inhibitor
of cytoplasmic peptide-N-glycanase (PNGase). Construction of a chitobiose structure containing both
alkyne and azide functional groups was performed using a thioglycoside donor having a triisopropylsilyl
(TIPS) protected alkyne group and a glycosyl azide acceptor. The resultant chitobiosyl azide derivative
was reduced to the corresponding glycosyl amine and a chloroacetyl group was introduced. Finally, re-
moval of the TIPS group using tetrabutylammonium fluoride (TBAF) gave the target compound. We con-
jugated the dansyl derivative, using click conditions and obtained the fluorescent labeled disaccharide de-
rivative, selectively. Our probe can easily lead to a variety of compounds and should help in the under-
standing of the biological functions of cytoplasmic PNGase.
Keywords: PNGase; Synthesis; Inhibitor; Click coupling.
INTRODUCTION
Protein quality control in the endoplasmic reticulum
probes such as specific inhibitors would be powerful tools,
but only few inhibitors are currently available.7 Recently
we developed a specific inhibitor for PNGase based on an
oligosaccharide structure having a thiol-reactive haloace-
toamidyl group at the reducing end. These probes cova-
lently modify the cysteine residue of the catalytic triad.8
We succeeded in inhibiting the intracellular activity of
PNGase.9 Further, the chloroacetamidyl chitobiose deriva-
tive was analyzed by X-ray crystallography of the PNGase
and this revealed the catalytic site of Cys residue at the
atomic level.10
(ER) is a cellular process during which terminally mis-
folded proteins in the lumen of the ER are recognized and
targeted for ER-associated degradation (ERAD).1 Recent
studies have revealed that the N-glycan plays a regulatory
role in determining the fate of newly synthesized glyco-
proteins.2 Misfolded proteins are retrotranslocated into the
cytosol by the action of lectin-like proteins and eventually
degraded by proteasomes.3 During degradation, cytoplas-
mic peptide-N-glycanase (PNGase) plays an important
role, cleaving the N-glycans from the misfolded glycopro-
teins in the cytosol, after which the misfolded proteins are
effectively scavenged by 20S proteasomes.4 Because the
barrel shaped proteasome has its active site in the interior
of the pore, removal of bulky N-glycans from glycopro-
teins is necessary in order to insert the peptide chain into its
degrading chamber.5 Therefore, cytoplasmic PNGase is not
merely a deglycosylating enzyme, but is even more impor-
tant as the key enzyme in the glycoprotein degradation ma-
chinery. Furthermore, deglycosylation independent biolog-
ical functions of PNGase have been described in various
organisms.6 Functional diversification of this protein
should be conducted in order to clarify its interactions at
the molecular level.
To better understand the biological function of
PNGase at the molecular level, a diverse compound library
is needed. We decided to synthesize a chloroacetamidyl
chitobiose derivative bearing an alkyne group on the non-
reducing end. This allows for a variety of functional groups
to be introduced such as detection and purification tags eas-
ily using a Cu(I)-catalyzed[3,2]cycloaddition reaction be-
tween the alkyne group and an azide group, so-called “click
chemistry”.11 In this paper, we report a synthetic study of
the clickable probe as a versatile tool for the analysis of
PNGase and demonstrate attachment of a fluorescent tag.
RESULTS AND DISCUSSION
Synthetic plan for the clickable probe is shown in Fig.
1. The reducing end N-acetylglucosamine was designed as
In order to investigate cytoplasmic PNGase, chemical
Dedicated to the memory of Professor Yung-Son Hon (1955–2011).
* Corresponding author. E-mail: matsuo@gunma-u.ac.jp
J. Chin. Chem. Soc. 2012, 59, 269-272
© 2012 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
269

Communication
Masuda et al.
chitobiose derivative 2 in 83% yield. In order to remove the
benzyl groups, without affecting the azide and alkyne
groups, we employed oxidative debenzylation using 2,3-
dichloro-5,6-dicyano-p-benzoquinone (DDQ).18 Removal
of the benzyl groups of 2 was cleanly achieved using 20
equivalents of DDQ in aq. CH2Cl2 to afford 10 in 95%
yield. In addition, to reduce the amount of DDQ, we exam-
ined regeneration of DDQ from dihydroxybenzoquinone
using MnO2 as a co-oxidizing agent.19 This improved con-
dition allowed us to decrease the amount of DDQ by 1/2 re-
sulting in 10 in quantitative yield. Subsequent dephthaloyl-
ation of 10 was performed under Kanie’s conditions,20
which was followed by the acetylation of the resulting
amine, and deacetylation of hydroxyl groups providing
compound 11. Reduction of 11 was carried out using pro-
panedithiol and the resultant glycosyl amine was chloro-
acetylated to give 12. Removal of the TIPS group from 12
using 2 eq. of TBAF and 4 eq. of acetic acid in THF at 40 oC
gave the target compound 1 in 69% yield (Scheme II).
Fig. 1. Synthetic plan for clickable probe 1.
a glycosyl azide 4 so that late stage coupling of chloro-
acetic acid could be performed via glycosylamine. Cou-
pling of 4 with the glucosamine donor 3 having TIPS pro-
tected alkynyl group should be possible without interfer-
ence of azide and alkyne groups.12 Compound 3, in turn,
was designed to be prepared from thioglycoside 6 and the
propargyl bromide derivative 5.
Scheme II Synthesis of clickable probe 1
OBn
O
OBn
O
NPhth
O
TIPS
BnO
O
BnO
O
N3
SPh
5
4
O
BnO
BnO
6
TIPS
NPhth
NPhth
a)
b)
3
2
OH
O
R
O
TIPS
HO
O
N3
O
HO
Preparation of the propargyl bromide derivative 5
proceeded in three steps starting from commercially avail-
able compound 7 which was first treated with n-butyllith-
ium and triisopropylsilyl chloride to afford 8. Subsequent
deprotection of the tetrahydropyranyl ether using Hon’s
conditions13 and conversion of the resultant alcohol 9 into a
bromide to gave 514 (Scheme I).
HO
R
c) or d)
f)
R
NPhth
NHAc
10
11
e)
Dansyl
N
H
Dansyl
Cl
N
H
N3
N
N
N
OH
O
OH
O
NHAc
NHAc
O
13
h)
R
H
N
H
N
HO
HO
O
HO
O
O
O
HO
O
HO
Cl
HO
NHAc
NHAc
O
O
14
R
TIPS
N
12
1
Dansyl =
g)
H
SO2
Synthesis of propargyl bromide derivative 5
Scheme I
Reagents and conditions: (a) NaH, DMF, 51%; (b) NIS, AgOTf,
CH2Cl2, 83%; (c) DDQ, 10 eq. H2O, CH2Cl2, 95%; (d) DDQ,
MnO2, 8 eq. MeOH, (CH2Cl)2, quant.; (e) 1. 1,2-diaminoethane,
n-BuOH, 2. Ac2O, DMAP, 3. NaOMe, MeOH/THF, 74%; (f) 1.
1,3-propanedithiol, DIEA, MeOH, 2. ClAc2O, MeOH/CH2Cl2,
79%; (g) 2 eq. TBAF, 4 eq. AcOH, THF, 69%; (h) CuI, 2,6-
lutidine, DIPEA, CH3CN/H2O (1/1), 78%.
Reagents and conditions: (a) TIPSCl, BuLi, THF, 84%; (b) Ace-
tonyltriphenylphoshonium Bromide, MeOH, 90%; (c) Ph3P,
CBr4, CH2Cl2, quant.
Target compound in hand, we carried out a Cu(I)-cat-
alyzed[3+2]cycloaddition reaction between 1 and the dan-
syl derivative 13 bearing azide group as shown in Scheme
II. Coupling of 1 and 13 using CuI in CH3CN/H2O (1/1)
gave 14 as a single product in 78% yield.21 The 1H-NMR
spectrum of compound clearly reveals the presence of two
anomeric protons, chloroacetamide methylene protons, the
Introduction of the alkyne group into compound 615
using 5 with NaH in DMF gave thioglycoside donor 3 in
51% yield. Coupling of donor 3 and glycosyl azide accep-
tor 416 using NIS-AgOTf as a promoter,17 resulted in the
270
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© 2012 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Chin. Chem. Soc. 2012, 59, 269-272

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Cytoplasmic Peptide-N-Glycanase
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Fig. 2. 1H-NMR spectra (300 MHz, D2O, 25 ºC, refer-
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ton (Fig. 2).
As described above, we synthesized the clickable
alkyne tagged chloroacetamidyl chitobiose derivative and
demonstrated the introduction of a fluorescent tag by click
chemistry. This versatile compound should be extremely
useful in the preparation of a variety of probes using click
chemistry, enabling the measurement of enzyme activity,
fluorescent labeling, the exploration of a novel PNGase, lo-
calization analysis, and functional analysis of PNGase in
cells. It should also be useful to determining the exact func-
tion of PNGase. Our current research is focused on the in-
vestigation of a novel PNGase from a natural source using
probes with conjugated affinity tags.
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ACKNOWLEDGEMENTS
A part of this work was financially supported by a
Grant-in-Aid for Scientific Research (21580410) from
Ministry of Education, Culture, Sports, Science, and Tech-
nology of Japan and ERATO JST. We gratefully thank Dr.
R. Walton for his helpful discussions in completing this ar-
ticle, and Mr. K. Iino and Ms. K. Kobayashi for technical
assistance.
1
14. Physical data for new compounds given below. H-NMR
spectra were measured on a JEOL AL-300 spectrometer. An-
alytical TLC was performed on aluminium sheets coated
with silica gel 60 F254 (Merck). MALDI-TOF MS spectra
were recorded in the positive ion mode on an AXIMA CFR
Kompact MALDI (Shimazu/KRATOS) equipped with nitro-
1
gen laser with an emission wavelength of 337 nm. 5: H
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© 2012 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Chin. Chem. Soc. 2012, 59, 269-272