Synthesis of the fucosylated biantennary N-glycan of erythropoietin

Article abstract of DOI:10.1016/j.tetlet.2006.05.086

A synthesis of the protected biantennary N-glycan of the naturally occurring glycoprotein, erythropoietin, is described.

Full text of DOI:10.1016/j.tetlet.2006.05.086

Tetrahedron Letters 47 (2006) 5577–5579
Synthesis of the fucosylated biantennary N-glycan of erythropoietin
Bin Wu,a Zihao Hua,a J. David Warren,a Krishnakumar Ranganathan,a
Qian Wan,a Gong Chen,a Zhongping Tan,a Jiehao Chen,a
Atsushi Endoa and Samuel J. Danishefskya,b,
*
aLaboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY 10021, USA
bDepartment of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA
Received 21 April 2006; accepted 16 May 2006
Available online 19 June 2006
Abstract—A synthesis of the protected biantennary N-glycan of the naturally occurring glycoprotein, erythropoietin, is described.
Ó 2006 Elsevier Ltd. All rights reserved.
Our laboratory is currently committed to a major initia-
tive seeking to accomplish the de novo synthesis of the
naturally occurring glycoprotein, erythropoietin (EPO).
Aside from the obvious synthetic allure of this highly
complex and challenging target, we were particularly
drawn to EPO as a function of its remarkable therapeu-
tic profile. EPO, a 166-residue, multiply glycosylated
protein, is widely used in the treatment of anemia.1
EPO possesses four carbohydrate domains, three of
which are N-linked to asparagines and one of which is
O-linked to a serine. Various studies have established
the importance of the carbohydrate motifs in mediating
the stability and in vivo activity of EPO. However, EPO
is currently available only as a mixture of glycoforms.
Without access to homogeneous EPO, it is difficult to
perform rigorous comparative studies on the properties
of the different glycoforms. Through recourse to total
synthesis, we will be in the unique position of having
potential access to a number of different EPO glyco-
forms for biological studies.
backbone of EPO, we have developed a novel cysteine-
free glycopeptide coupling protocol.4 With these
enabling methodologies in hand, we next approached
the syntheses of the carbohydrate domains of EPO. We
report herein the synthesis of the biantennary N-linked
type glycan (cf. 1), incorporating the complicating
fucose and sialic acid motifs, which are believed to be
important for the in vivo activity of EPO (Fig. 1).
Our laboratory has had a longstanding interest in devel-
oping improved methods for the synthesis of complex
carbohydrates.5 In 2003, we disclosed a synthesis
of a pentasaccharide N-glycan featuring application of
Crich’s direct coupling method for the formation of
the key mannose–chitobiose linkage.6 More recently,
we completed the synthesis of multibranched N-acetyl-
lactosamine type PSA glycans.7 In the course of this
endeavor, we developed a useful and direct protocol
for the appendage of complex glycans to peptides,
HO
O
OH
O
HO
HO2C
O
OH
The total synthesis of a glycoprotein such as EPO would
be a complicated matter. Success would be predicted on
the ability to devise solutions to a range of synthetic
challenges that will arise. In order to address some anti-
cipated issues, we first applied a cysteine-based glyco-
peptide ligation strategy.2 This was recently extended to
accommodate reiterative couplings, where glycopeptides
are sequentially coupled to form a tri-glycosylated pep-
tide.3 In light of the paucity of cysteine residues on the
HO
O
HO
O
AcN
H
O
HO
OH
HO
AcN
H
b
HO
HO
HO
OH
O
HO
OH
a
a
Me
O
OH
O
O
O
HO
O
HO
O
O
O
O
HO
OH
HO
HO
HO
AcN
H
AcN
H
HO
O
b
AcHN
O
HO
OH
O
HO
O
O
AcHN
HO
O
O
(1) Biantennary N-Linked Glycan of EPO
HO
HO
OH
HO2C
OH
HO
*
Corresponding author. Tel.: +1 212 639 5501; fax: +1 212 772
8691; e-mail: danishes@mskcc.org
Figure 1. Biantennary N-linked glycan (1).
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.05.086

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B. Wu et al. / Tetrahedron Letters 47 (2006) 5577–5579
commencing with global deprotection of the glycan
domain (Na/NH3) followed by a Kochetkov amination–
Lansbury aspartylation sequence.8 We expect to apply
these advances to the synthesis of 1 and its appendage
to the peptide domain. We envisioned a synthetic path-
way to 1 that would build from the tetrasaccharide core
and would involve sequential diglycosylation steps (a
and b) for the installation of the antennary portions of
the molecule.
acceptor, 8. Subsequent triflate-mediated mannosylation
of 8 with a slight excess of donor 9 proceeded smoothly
at À78 °C to give an anomeric mixture of 10 with an a/b
ratio of 1:5 (75–84% yield). The undesired a–isomer
could be removed through deprotection of the C4
PMB group with cerium ammonium nitrate (CAN), to
yield tetrasaccharide 11 as a single isomer. Regioselec-
tive cleavage of the benzylidene acetal with borane in
the presence of dibutyl boron triflate afforded the
3,6-diol, 12. Twofold glycosylation of the latter was
Our synthesis commenced with known monosaccharides
2 and 3,9 which are available from D-glucal (Scheme 1).
Methyl triflate-mediated glycosylation between 2 and 3,
followed by removal of the C6 –TIPS group, provided
disaccharide 4. The fucose motif was introduced
through glycosyl fluoride 5. To simplify the final steps
of the synthesis, we elected to functionalize the reducing
end double bond at this early trisaccharide stage. To
that end, 6 was subjected to iodosulfonamidation,
hydrolysis, and TBS protection to provide protected tri-
saccharide 7. Saponification of 7 furnished the requisite
accomplished through Sinay radical activation, to afford
¨
hexasaccharide 14.10 Subsequent cleavage of the two
benzoates provided 15.
With the hexasaccharide in hand, we turned our atten-
tion to the preparation of trisaccharide thiol donor
(Scheme 2). The key disaccharide intermediate in this
sequence (21) could be prepared through two alternate
pathways, as shown. According to Path A, the route
to 21 commenced with the known disaccharide 16, avail-
able through DMDO mediated glycal epoxidation fol-
BnO
BnO
OBn
BnO
OBn
OBn
OBn
OBn
O
BnO
AcO
BnO
HO
O
BnO
BnO
AcO
BnO
OBn
F
O
O
O
5
TIPSO
a, b
O
O
SEt
O
d-f
HO
BnO
RO
BnO
O
O
BnO
O
O
O
+
O
PhSO2N
H
AcO
PhSO2N
H
O
O
c
BnO
BnO
BnO
BnO
PhSO2N
H
PhSO2N
PhSO2N
H
OTBS
2
H
7
3
4
6
R = Ac
g
8
R = H
OR
O
BnO
BnO
BnO
OBn
BnO
BnO
OBn
O
OBn
OBn
BnO
j
O
h
OBn
OBn
k
OBn
O
O
O
OBn
O
BnO
OBn
O
O
O
OBn
O
Ph
O
BnO
O
O
HO
BnO
HO
OBz
O
BnO
BnO
BnO
BnO
BnO
O
Ph
O
O
O
O
O
O
PhSO2N
O
O
O
O
BnO
O
O
BnO
RO
O
O
BnO
PMBO
BnO
BnO
PhSO2N
H
PhSO2N
H
BnO
BnO
BnO
BnO
OTBS
PhSO2N
H
PhSO2N
H
PhSO2N
H
OTBS
O
OR
S
OTBS
13
H
SEt
O
Ph
10 R = PMB
11 R = H
9
14 R = Bz
15 R = H
12
i
l
Scheme 1. Reagents and conditions: (a) MeOTf, DTBP, DCM, 64%; (b) TBAF, AcOH, THF, 96%; (c) Sn(OTf)2, DTBP, THF, 65–72%; (d) IDCP,
PhSO2NH2, THF; (e) H2O, Et3N, THF; (f) TBSOTf, 2,6-lutidine, DCM; (g) NaOMe, MeOH, 44–55% (four steps); (h) Tf2O, TBP, DCM, 75–84%; (i)
CAN, MeCN, H2O, 65–70%; (j) BH3ÆTHF, Bu2BOTf, 70–74%; (k) (BrC6H4)3NSbCl6, MeCN, 90%; (l) NaOMe, MeOH, 83–85%.
Path A:
AcO
AcO
O
O
OBn
O
OBn
O
AcO
AcO
OBn
O
OBn
O
O
O
OBn
O
OBn
O
OBn
O
OBn
O
b, c
O
O
f, g
O
d,e
O
O
O
SEt
SEt
BnO
BnO
SEt
BnO
BnO
BnO
OR
BnO
BnO
NPhth
NH
NH
h
16 R = H
TMSCH2CH2SO2
20
TMSCH2CH2SO2
19
18
a
17 R = Bn
HO
HO
OBn
O
OBn
O
SEt
NPhth
O
Path B:
BnO
BnO
21
OBn
O
AcO
AcO
O
OAc
O
AcO
AcO
23
l, m
OH
O
OAc
O
OBn
O
O
OBn
O
HO
BnO
j, k
i
+
SEt
O
O
SEt
SEt
BnO
BnO
NPhth
PivO
OH
PivO
NPhth
NPhth
O
CCl3
22
24
25
NH
OAc
MeO2C
RO
O
HO
HO
OBn
O
AcO
OAc
OAc
OBn
O
SEt
NPhth
OP(OBn)2
CO2Me
OBn
O
OBn
O
SEt
NPhth
AcO
OAc
n
O
O
AcHN
O
O
+
BnO
AcHN
BnO
AcO
BnO
BnO
AcO
27 R = H
21
26
o
28 R = Ac
Scheme 2. Reagents and conditions: (a) BnBr, NaH, DMF, 70–80%; (b) IDCP, TMSCH2CH2NH2, THF, (c) LHMDS, EtSH, DMF, 92% (two
steps) (d) NaOMe, MeOH, 90%; (e) Ac2O, pyr, 92%; (f) CsF, DMF, 64–77%; (g) phthalic anhydride, pyr, Piv2O, 74–85%; (h) NaOMe, MeOH, 86–
90%; (i) TMSOTf, DCM, À78 °C, 79%; (j) NaOMe, MeOH, 97%; (k) DMP, CSA, 68%; (l) NaH, BnBr, TBAI, THF, 85%; (m) aq HOAc, 60 °C, 92%;
(n) TMSOTf, MeCN, 55–62%; (o) Ac2O, pyr, 84–87%.

B. Wu et al. / Tetrahedron Letters 47 (2006) 5577–5579
5579
OH
O
OAc
BnO
AcO
OBn
O
MeO2C
OAc
BnO
BnO
OBn
O
OBn
O
AcO
OBn
BnO
O
O
O
AcHN
SEt
BnO
OBn
O
O
BnO
a
AcO
BnO
O
PhthN
28
BnO
O
O
O
O
O
BnO
BnO
PhSO2N
H
PhSO2N
H
BnO
OTBS
BnO
BnO
O
OH
15
OAc
AcO
O
MeO2C
OBn
O
AcO
OAc
BnO
O
O
O
AcHN
O
BnO
AcO
BnO
PhthN
BnO
BnO
OBn
O
O
BnO
OBn
BnO
OBn
O
O
BnO
O
BnO
O
O
O
PhSO2N
O
O
BnO
BnO
PhSO2N
BnO
BnO
BnO
OTBS
H
H
O
OAc
AcO
O
MeO2C
OBn
O
AcO
OAc
BnO
O
O
O
29
O
AcHN
BnO
BnO
AcO
PhthN
Scheme 3. Reagents: (a) (BrC6H4)3NSbCl6, MeCN, 50%.
lowed by direct glycosylation.11 Following protection at
C2, the reducing end olefin was functionalized through
iodosulfonamidation followed by roll-over, as shown.
Intermediate 18 was advanced to disaccharide 20
through a series of protecting group manipulations. Fol-
lowing saponification of 20, the requisite diol 21 was in
hand. An alternate route to 21 (Path B) commenced
with monosaccharide 22.12 Coupling with trichloroimi-
date donor 23, equipped with a pivaloate at C2, pro-
vided disaccharide 24. Hydrolysis followed by selective
acetonide formation yielded 25, which was then con-
verted to disaccharide 21 in two steps, as shown.13
Under previously reported conditions, the regioselective
coupling between 21 and glycosyl phosphite 26 pro-
ceeded smoothly to afford trisaccharide 27 in 55–62%
yield.14 Acetylation at C4 furnished the requisite trisacc-
charide donor 28.
References and notes
1. (a) Szymkowski, D. E. Curr. Opin. Drug Discovery
Develop. 2005, 8, 590–600; (b) Pavlou, A. K.; Reichert,
J. M. Nature Biotech. 2004, 22, 1513–1519; (c) Jelkmann,
W.; Wagner, K. Ann. Hematol. 2004, 83, 673–686; (d)
Ridley, D. M.; Dawkins, F.; Perlin, E. J. Natl. Med.
Assoc. 1994, 86, 129–135.
2. Warren, J. D.; Miller, J. S.; Keding, S. J.; Danishefsky, S.
J. J. Am. Chem. Soc. 2004, 126, 6576–6578.
3. Wu, B.; Warren, J. D.; Chen, J.; Chen, G.; Hua, Z.;
Danishefsky, S. J. Tetrahedron Lett., in press, doi:10.1016/
j.tetlet.2006.04.132.
4. Wu, B.; Chen, J.; Warren, J. D.; Chen, G.; Hua, Z.;
Danishefsky, S. J. Angew. Chem., Int. Ed. 2006, 45, 4116–
4125.
5. (a) Danishefsky, S. J.; Bilodeau, M. T. Angew. Chem., Int.
Ed. 1996, 35, 1380–1419; (b) Danishefsky, S. J.; Allen, J.
R. Angew. Chem., Int. Ed. 2000, 39, 836–863.
6. Dudkin, V. Y.; Miller, J. S.; Danishefsky, S. J. Tetra-
hedron Lett. 2003, 44, 1791–1793.
In the event, we were pleased to find that glycosylation
of 15 with excess amounts of thioglycoside 28 under
Sinay radical cation activation provided the dodecasac-
¨
7. Dudkin, V. Y.; Miller, J. S.; Danishefsky, S. J. J. Am.
Chem. Soc. 2004, 126, 736–738.
charide 29 in 50% yield, along with a small amount of
the monocoupled product (Scheme 3).
8. (a) Iserloh, U.; Dudkin, V.; Wang, Z. G.; Danishefsky, S.
J. Tetrahedron Lett. 2002, 43, 7027–7030; (b) Likhoshers-
tov, L. M.; Novikova, O. S.; Derevitskaja, V. A.;
Kochetkov, N. K. Carbohydr. Res. 1986, 146, C1–C5; (c)
Cohen-Anisfeld, S. T.; Lansbury, P. T. J. Am. Chem. Soc.
1993, 115, 10531–10537.
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Bilodeau, M. T.; Danishefsky, S. J. Enantiomer 1996, 1,
311–323; (b) Schell, P.; Orgueira, H. A.; Roehrig, S.;
Seeberger, P. H. Tetrahedron Lett. 2001, 42, 3811–3814; (c)
Lohman, G. J. S.; Seeberger, P. H. J. Org. Chem. 2003, 68,
7541–7543.
In summary, we have developed a convergent strategy
toward the synthesis of complex biantennary N-linked
glycan of EPO. Studies which interface the total synthesis
of the fucose and sialic acid dodecameric oligosaccha-
rides described above with various ligation strategies
are well underway and the results will be reported in
due course.
10. (a) Zhang, Y. M.; Mallet, J. M.; Sinay, P. Carbohydr. Res.
¨
Acknowledgements
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This work was supported by the NIH (CA28824 to
S.J.D.). We thank Dr. George Sukenick, Ms. Sylvi Rusli
and Ms. Hui Fang of the Sloan-Kettering Institute’s
NMR core facility for mass spectral and NMR spectro-
scopic analysis (SKI core Grant no.: CA02848). Post-
doctoral fellowship support is gratefully acknowledged
by BW (New York State Department of Health, New
York State Breast Cancer Research and Education
Fund) and JDW (NIH, CA62948).
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