Chemical Synthesis of a Bisphosphorylated Mannose-6-Phosphate N-Glycan and its Facile Monoconjugation with Human Carbonic Anhydrase II for in vivo Fluorescence Imaging

Article abstract of DOI:10.1002/cbic.201000785

Red-ily made: The first chemical synthesis of a fully elaborated bisphosphorylated triantennary M6P N-glycan was achieved through a highly efficient late-stage phosphorylation strategy. A human carbonic anhydrase II-based fluorescently tagged neoglycoprotein platform was successfully constructed in order to evaluate the M6P N-glycan-directed protein internalization process in cell-based assays by confocal fluorescence imaging.

Full text of DOI:10.1002/cbic.201000785

DOI: 10.1002/cbic.201000785
Chemical Synthesis of a Bisphosphorylated Mannose-6-Phosphate N-Glycan
and its Facile Monoconjugation with Human Carbonic Anhydrase II for in
vivo Fluorescence Imaging
Yunpeng Liu,[a] Yan Mei Chan,[a] Jianhui Wu,[a, c] Chen Chen,[a] Alan Benesi,[a] Jing Hu,[b] Yanming Wang,[b] and
Gong Chen*[a]
Dedicated to Professor Raymond Funk on the occasion of his 60th birthday.
Mannose-6-phosphate (M6P) containing N-linked glycans
(N-glycans) belongs to a class of high-mannose-type oligo-
saccharides with phosphorylation on the C6-OH groups of
peripheral mannoses.[1] These M6P N-glcans function as a
universal organelle-targeting signal that directs the trans-
port of some 60 newly synthesized hydrolases and some
non-hydrolase proteins from Golgi to endosome/lysosome
compartments through specific interaction with two p-type
lectins, cation-dependent and cation-independent M6P re-
ceptors (CD- and CI-MPRs) in eukaryotic cells.[2] On the cell
surface, CI-MPR controls the level of a number of extracellu-
lar hydrolases and insulin-like growth factor II through re-
ceptor-mediated endocytosis. Defects in this carbohydrate–
lectin recognition system are known to cause severe physio-
logical conditions such as lysosomal storage diseases, and
are closely related to the progress of cancers and liver fibro-
sis.[3] The current understanding of this complex protein-
transport system is incomplete, particularly with regard to
the detailed structure–activity relationship of the M6P
sugar-coding sytem at the molecular level. Only limited
knowledge is available on how the sugar structure, the loca-
tion of the phosphate group(s), and the valency of M6P in
N-glycans affect their binding to MPRs (Scheme 1A).[4] Ef-
forts to carry out detailed glycan receptor binding studies
have been hampered by the inherent heterogeneity of M6P
N-glycans and the formidable difficulties in obtaining ade-
quate amounts of different, structurally well defined M6P N-
glycans in good purity from natural resources. Powered by
the recent advance of chemical synthesis of complex carbo-
hydrates, total synthesis of M6P N-glycans with defined gly-
coform and phosphoform would provide an attractive solu-
tion to this long-standing problem.[5,6]
[a] Dr. Y. Liu, Y. M. Chan, Dr. J. Wu, Dr. C. Chen, Prof. Dr. A. Benesi,
Prof. Dr. G. Chen
Department of Chemistry, The Pennsylvania State University
104 Chemistry Building, University Park, PA 16802 (USA)
Fax: (+1)814-863-5319
E-mail: guc11@psu.edu
[b] J. Hu, Prof. Dr. Y. Wang
Department of Biochemistry and Molecular Biology
The Pennsylvania State University
454A North Frear Laboratory, University Park, PA 16802 (USA)
[c] Dr. J. Wu
Scheme 1. Structure and synthetic strategies for M6P N-glycans.
College of Pharmaceutical Sciences, Capital Medical University
Beijing, 100069 (China)
Herein, we report the first chemical synthesis of a fully ela-
borated bis-phosphorylated triantennary M6P N-glycan
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000785.
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685

through a a highly efficient late-stage phosphorylation strat-
egy. In addition, we have constructed a monosubstituted neo-
glycoprotein platform based on human carbonic anhydrase II
(HCAII) in a site-specific manner. Finally, using a non-covalent
fluorescent tag that binds HCAII, we have successfully demon-
strated that a single bivalent M6P N-glycan can facilitate MPR-
mediated cellular uptake of the neoglycoprotein by Hep G2
cells.
to protect the amino groups of glucosamines. However, it was
soon found that the benzyl-protected phosphate groups failed
to survive all attempts to remove the Phth group of glucosa-
mine with any nucleophilic treatment tested.[9] The chemical la-
bility of the Bn-protected phosphate group in this N-glycan
system forced us to pursue the seemingly riskier late-stage
phosphorylation strategy.
Accordingly, chitobiose 3[10] was prepared from the easily ac-
cessible benzylidene-protected glucosamine thioether donor 1
and glucosamine acceptor 2 under promotion by MeOTf (Tf=
triflate, Scheme 2). Glycosylation of mannose sulfoxide donor 4
and the chitobiose acceptor under Crich–Kahne conditions
gave the desired trisaccharide intermediate in satisfying yield
and b-selectivity.[11] Upon oxidative deprotection of the p-
methoxybenzyl group, the core trisaccharide acceptor 5 was
readily prepared in large quantity. The triisopropylsilyl (TIPS)
protecting group, which tolerates the acidic treatment re-
quired for the glycosylation and the benzylidene opening
steps, was chosen to mask the latent C6-OH phosphorylation
site on the mannose residues (Scheme 2).[12] Man2 thioether
donor 9 was prepared and then stereoselectively installed
onto trisaccharide core 5 with N-iodosuccinimide (NIS)/TfOH.
Upon benzylidene opening with Et3SiH/PhBCl2, pentasacchar-
ide acceptor 10 was obtained in excellent yield.
The majority of naturally occurring M6P N-glycans carry one
or two phosphate groups at different mannoses, and multiva-
lent M6P N-glycans generally bind more tightly to the MPRs.[1]
As a result of this, a bis-phosphorylated octasaccharide N-
glycan was chosen as our initial synthetic target (Scheme 1B).
Multiphosphorylated M6P N-glycan poses a greater synthetic
challenge than ordinary N-glycans. New synthetic strategies
had to be delicately orchestrated and executed in order to
incorporate labile phosphate groups.[7,8] In addition, due to the
difficulties associated with the purificationof intermediates and
the final product, high-yielding and stereoselective transforma-
tions are necessary for all the key steps, especially those in the
late stages. In principle, two general synthetic strategies can
be employed to prepare the target multi-phosphorylated N-
glycans (Scheme 1B). A global phosphorylation strategy can be
applied to install the phosphate moieties at a late stage of the
synthesis. Though this approach
offers a convenient solution to
preparing a variety of M6P N-
glycans, we were initially con-
cerned that it would suffer from
potential complications due to
incomplete multi-phosphoryla-
tion steps near the end of the
synthesisand difficulties in puri-
fying partially phosphorylated
products from our target.
Alternatively, a properly pro-
tected phosphate moiety could
be first incorporated into simple
mannose building blocks, which
could be assembled in a modu-
lar fashion to provide the final
multi-phosphorylated product.
These protected phosphate moi-
eties should withstand glycosyla-
tion and other procedures, and
can be quantitatively removed
at the end. Due to its conver-
gence, the pre-phosphorylation
strategy was tested in our initial
synthesis of the octasaccharide
M6P N-glycan. O-Benzyl groups
Scheme 2. Synthesis of compound 10. Reagents and conditions: a) TMSN3, MeOTf, MS 4 ꢁ, CH2Cl2, RT, 92%;
b) MeOTf, MS 4 ꢁ, CH2Cl2, RT, 82%; c) NaBH3CN, HCl (1m in Et2O), MS 3 ꢁ, THF, RT, 93%; d) 4, Tf2O, TTBP, MS 3 ꢁ,
were chosen for the protection
of all the hydroxyl and phos- CH2Cl2, ꢀ408C; e) CAN, CH3CN, phosphate buffer (pH 7.0), RT, 50% over two steps; f) i: HgBr2, EtSH, CH3CN, RT,
90%; ii: NBS, acetone/H2O, ꢀ208C, 92%; iii: DBU, CCl3CN, CH2Cl2, ꢀ208C, 92%; g) i: 8, TMSOTf, MS 4 ꢁ, CH2Cl2,
phate groups; an azido group
ꢀ208C, 80%; ii: NaOMe, MeOH, RT, 94%; iii: BnBr, NaH, TBAI, DMF, RT, 89%; h) i: 5, NIS, TfOH, Et2O, MS 4 ꢁ,
was used to mask the nitrogen
ꢀ208C!RT, 92%; ii: PhBCl2, Et3SiH, CH2Cl2, ꢀ788C, 92%. TMSN3 =trimethylsilyl azide, MS=molecular sieves,
on the reducing end, and phtha-
TTBP=tri-tert-butylpyridine, CAN=cerium ammonium nitrate, NBS=N-bromosuccinimide, DBU=1,5-diazabicyclo-
limide (Phth) groups were used (5.4.0)undec-5-ene, TMSOTf=trimethylsilyl triflate, TBAI=tetrabutylammonium iodide.
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TIPS-protected Man3 branch 14 was prepared in the thioeth-
er form by using standard protocols (Scheme 3). After an ex-
tensive screening, the NIS/AgOTf conditions were found to be
superior in facilitating a highly efficient and stereoselective in-
stallation of the upper-branch 14 to acceptor 10 (a/b>15:1,
minimize the number of post-phosphorylation synthetic steps,
the N-Phth groups of the resulting octasaccharide were trans-
formed to NH-Ac groups, and the azido group was reduced by
propanedithiol and coupled with Cbz-b-alanine by HATU.[14]
TIPS groups were then removed with tetrabutylammonium
fluoride (TBAF) to give compound 16. Gratifyingly, final bi-
sphosphorylation of 16 through dibenzyl N,N-diiso-
propyl phosphoramidite/mCPBA oxidation gave the
desired bisphosphorylated product 17 in near quanti-
tative yield.[15] The final global deprotection of Bn
groups and the Cbz group proceeded cleanly by first
catalytic hydrogenolysis with Pd(OH)2 in MeOH/
EtOAc/H2O (15:8:3), followed by Pd/C in MeOH/H2O
under H2 (1 atm; Figure 1A).[16] Analytically pure N-
glycan product 18 bearing an amino group handle
was obtained in near quantitative yield without chro-
matographic purification (Figure 1B, see the Support-
ing Information for 13C, 31P NMR and MS characteriza-
tion).
1
stereochemistry was confirmed by J1CH of Man3[13]). In order to
With this M6P N-glycan in hand, we constructed a
neoglycoprotein in order to evaluate the contribution
of the N-glycan in ligand/CI-MPR interactions and cel-
lular uptake. As stated before, preparing homoge-
nous, naturally occurring glycoprotein by using cur-
rent methodologies remains a formidable technical
challenge.[17] We feel that a chemically well defined
neoglycoprotein that carries a monosubstitution of
M6P N-glycan linked to a protein molecule at a pre-
cise location would provide an excellent model with
which to study the function of an individual N-
glycan.[18]
The resulting neoglycoprotein could be further flu-
orescently tagged and utilized to track the M6P N-
glycan-directed protein-transport process inside cells
by using fluorescence imaging. Human carbonic an-
hydrase II (HCAII), a 29 kD globular protein that car-
ries an unpaired Cys206, is particularly suited for this
purpose for two reasons.[19] Firstly, the SH group of
Cys206 is remote from the active site and so can be
used to conjugate agents carrying an iodoacetamide
group; the resulting HCAII conjugate retains its cata-
lytic and ligand-binding ability.[20] Secondly, HCAII
forms a tight 1:1 noncovalent complex with simple
arylsulfonamide-derived ligands. A fluorescent tag
tethered to an arylsulfonamide ligand can be used to
complex the HCAII conjugate to provide a fluores-
cently tagged neoglycoprotein probe.[21]
Accordingly, we prepared the monosubstituted
neoglycoprotein probe with the synthetic M6P N-
Scheme 3. Synthesis of compound 17. Reagents and conditions: a) i: TMSOTf, CH2Cl2, MS
4 ꢁ, ꢀ208C, 79%; ii: thiourea, 2,6-lutidine, MeOH, 708C, 98%; b) i: TMSOTf, MS 4 ꢁ,
CH2Cl2, ꢀ208C, 70%; ii: NaOMe, MeOH, RT, 80%; iii: BnBr, NaH, TBAI, DMF, RT, 94%; c) 10,
NIS, AgOTf, Et2O, MS 4 ꢁ, ꢀ208C-RT, 72%, (a/b >15:1); d) i: ethylenediamine, n-butanol,
908C; ii: Ac2O, pyridine, RT, 63% over two steps; iii: propanedithiol, DIEA, MeOH, RT; iv:
Cbz-b-alanine, HATU, DIPEA, N-methyl-2-pyrrolidone, RT, 50% over two steps; v: TBAF,
THF, RT, 90%; e) i: (BnO)2PN(iPr)2, tetrazole, MS 4 ꢁ, CH2Cl2, RT; ii: mCPBA, RT, 91% over
two steps. HATU=2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluoro-
phosphate methanaminium, DIPEA=N, N-diisopropylethylamine, (BnO)2PN(iPr)2 =dibenz-
yl N, N-diisopropylphosphoramidite, mCPBA=m-chloroperoxybenzoic acid.
glycan. M6P N-glycan 19 bearing an a-iodoacetamide
handle was obtained in high yield by treating glycan
18 with iodoacetic anhydride (Figure 1A). The conju-
gation reaction between 19 (10 equiv) and Cys206 of
HCAII was effected by using a well-known strategy,
firstly under denaturing conditions (5m guanidine
HCl, pH 7.6) for 12 h at 258C.[22] Upon dilution (to
0.3m guanidine) and standing, the modified HCAII
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readily refolded, and its enzymatic activity was re-
stored.[23] After the removal of excess 19 and buffer
exchange, the desired monolabeled product (M-
HCAII) was obtained in ~70% purity based on pro-
tein gel staining (Figure 1C, D). ESR-TOF MS analysis
confirmed that a single N-glycan molecule was conju-
gated to HCAII.
The arylsulfonamide-derived fluorescent affinity
ligand (LF) was easily prepared by standard amide
couplings (Figure 2A). p-Sulfonamidobenzoic acid
(21) was first extended with the linker hexane-1,6-di-
amine (22) and then capped with Texas Red succini-
midyl ester. Upon mixing LF and M-HCAII in Tris
buffer (100 mm, pH 7.6) for 2 h at room temperature,
a tight protein complex (M-HCAII-LF) formed that
was readily purified on a short Sephadex G25 column
(Figure 2B); the stoichiometry of the ligand/protein
complex was estimated to be 0.7:1 by UV spectrome-
try (Supporting Information). A control probe (with-
out the N-glycan-modification, HCAII-LF) was also
prepared by using the same procedure. The dissocia-
tion constants, Kd, of LF/M-HCAII (90 nm) and LF/
HCAII (65 nm) complexes were determined from fluo-
rescence-polarization assays (Supporting Informa-
tion).
The fluorescently labeled protein probes were then
subjected to cell-based internalization assay. CI-MPR
at the cell membrane is responsible for the receptor-
mediated endocytosis process for extracellular pro-
teins bearing M6P N-glycans.[24] Hep G2 hepatocarci-
noma cells were used as a model in our initial assays
because high levels of CI-MPR were detected on the
membrane of these cells.[25] Briefly, Hep G2 cells were
incubated with the M-HCAII-LF and HCAII-LF probes
respectively for 4 h, washed extensively with blank
buffer, and analyzed by confocal fluorescence micros-
copy. The fluorescence imaging study indicated that
M-HCAII-LF was readily internalized and accumulated
in endocytotic compartments, while HCAII-LF showed
only background levels of internalization (Figure 2C).
These experiments clearly demonstrated that the
HCAII-based neoglycoprotein platform will allow us
to carry out well-controlled, cell-based assays in
order to investigate the cellular function of the indi-
vidual M6P N-glycan and to analyze the dynamics of
this M6P-directed protein-transport process.
In summary, we have accomplished the first chemi-
cal synthesis of a fully elaborated bis-M6P triantenna-
ry N-glycan through a highly efficient, late-stage
phosphorylation strategy. The synthetic N-glycan was
then monoconjugated with the model protein HCAII
to provide a functional neoglycoprotein that was fur-
ther fluorescently tagged by facile, noncovalent com-
plexation with a fluorescent affinity ligand. Confocal
fluorescence imaging analysis of the cell-based inter-
nalization assays was successfully carried out to eval-
uate the M6P-facilitated cellular uptake of the neo-
Figure 1. Global deprotection and monoconjugation of the M6P N-glycan with HCAII.
A) Reagents and conditions: a) i: Pd(OH)2, H2 (1 atm), MeOH/EtOAc/H2O (15:8:3), RT, 8 h;
ii: 10% Pd/C, H2 (1 atm), MeOH/H2O, RT, 12 h, 92% over two steps; b) iodoacetic anhy-
dride, NaHCO3, H2O, RT, 12 h, 91%; c) HCAII labeling: HCAII, 19 (10 equiv), guanidine HCl
(5m), Tris buffer pH 7.6, 258C, 12 h; then quenched with 2-mercaptoethanol and diluted
into guanidine (0.3m) for HCAII refolding; B) 1H NMR spectrum of 18 (D2O, 850 MHz);
C) ESR-TOF MS of M-HCAII conjugate; D) SDS-PAGE of HCAII and M-HCAII (stained by
coomassie blue).
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Acknowledgements
We thank the Pennsylvania State University for its sup-
port of this work.
Keywords: fluorescence imaging · HCAII · mannose-
6-phosphate · MPR · neoglycoproteins · N-glycans
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Received: December 23, 2010
Published online on February 18, 2011
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