Building biologics by chemical synthesis: Practical preparation of Di- and triantennary N-linked glycoconjugates

Article abstract of DOI:10.1021/ja401385v

A unified strategy for the syntheses of bi- and triantennary fully sialylated N-glycans is described. The synthesis capitalizes on a global glycosylation strategy that delivers the desired undeca- and tetradecasaccharide in excellent yields. Finally, conjugation of the glycan to PSMA oligopeptide is described.

Full text of DOI:10.1021/ja401385v

Communication
pubs.acs.org/JACS
Building Biologics by Chemical Synthesis: Practical Preparation of Di-
and Triantennary N‑Linked Glycoconjugates
Maciej A. Walczak,† Joji Hayashida,† and Samuel J. Danishefsky*,†,‡
†Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, New York
10065, United States
‡Department of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027, United States
S
* Supporting Information
Chart 1
ABSTRACT: A unified strategy for the syntheses of bi-
and triantennary fully sialylated N-glycans is described.
The synthesis capitalizes on a global glycosylation strategy
that delivers the desired undeca- and tetradecasaccharide
in excellent yields. Finally, conjugation of the glycan to
PSMA oligopeptide is described.
emodeling of glycan composition in glycoproteins often
results in critical changes in the (inter)cellular environment,
R
and may signal metabolic imbalance.1 These changes can induce
functional modifications of glycoproteins by altering their
structure, stability, and site of localization.2 Given the importance
of glycoproteins as potential therapeutic and diagnostic assets,
their chemical synthesis could well prove to be of paramount
importance in providing viable avenues for the investigations of
covalent posttranslational modifications.3 A great deal of
structural variability comes from N-linked glycans, which are
linked to the peptidic backbone through a strategic asparagine
residue. The parent sialylated glycan is believed to be a
biantennary structure containing terminal N-acetylneuraminic
acid residues connected via an α-2,6- and/or α-2,3-linkage (1,
Chart 1). The nature of these glycosyl bonds depends on the
origin of protein; many glycoproteins contain a mixture of both
linkage regioisomers. The first chemical synthesis of an
undecasaccharide containing an α-2,6-linked sialic acid was
reported by Ogawa.4 However, it was found that this glycan can
be easily obtained in substantial quantities from natural sources
through isolation from egg yolk.5 Another typical regioisomer,
containing α-2,3-linked bonds (1), was prepared via synthetic
means by Ito.6 Not surprisingly, the chemical synthesis of this
structure required lengthy sequences. Synthetic surrogates
strategies against cancer, we investigated the role of glyco-
proteins in the emergence and progression of prostate cancer.11
We have shown that the extent of N-glycan branching in
prostate-specific antigen (PSA) can be differentiated with
synthetic glycoconjugates containing di-, tri-, and tetraantennary
glycans. However, these systems did not contain terminal sialic
acid residues, which are known to be critical components in cell
signaling and promotion of immune response.12 Access to
glycans 1 and 2 would allow for validation of the processing of the
glycans in biological systems and analysis of the impact of various
glycoforms on the structure and function of glycoproteins.
Additionally, given the complexity of these systems, chemical
synthesis would provide ultimate validation of the synthetic
strategies for the preparation of branched oligosaccharides.
At the retrosynthetic level, we envisioned that each antenna
could be installed in a single glycosylation event using a properly
containing unnatural linkages have also been prepared.7
A
higher order structure of N-linked glycan contains additional
trisaccharide branching linked via the 6-OH position of a
mannose residue (2, Chart 1). The structure 2 and its fucosylated
analog have been identified in a panel of glycoproteins, including
erythropoietin (EPO),8 follicle-stimulating hormone (FSH),9
and human chorionic gonadotropin (hCG),10 rendering syn-
thesis of these oligosaccharides a worthwhile pursuit. Here, we
present a practical approach for the synthesis of dibranched
(undecasaccharide) and tribranched (tetradecasaccharide)
structures containing terminal Neu5Ac residues.
As a part of our overall program directed toward the
development of novel therapeutic agents and diagnostic
Received: February 6, 2013
Published: March 5, 2013
© 2013 American Chemical Society
4700
dx.doi.org/10.1021/ja401385v | J. Am. Chem. Soc. 2013, 135, 4700−4703

Journal of the American Chemical Society
Communication
functionalized donor with Neu5Ac residues already in place.13
We were mindful of potential hurdles associated with this
approach since incorporation of three sialylated antennae in a
related system presented a challenging synthetic step requiring
careful orchestration to achieve practical yields.14 However,
because of the convergent nature of our synthetic plan, these
critical steps seemed an attractive possibility to allow for a rapid
assembly of the glycan core in a minimal number of synthetic
operations.
isolated yield. Similarly, glycosylation with anomeric fluoride
proceeded very efficiently and reached completion in 1.5 h at 0
°C to deliver 10 in a similar yield (86% average yield per glycosyl
acceptor).16 It is noteworthy that the reaction did not seem to
produce, at least to the levels of our detection, transient
monoglycosylated products. The stereochemistry of all of the
glycosidic bonds was confirmed by analysis of 1JCH of anomeric
carbon atoms.
With sufficient quantities of undecasaccharide 10 in hand, we
advanced to the deprotection sequence (Scheme 2). First, the
The synthesis began with trisaccharide 3,14 which was
reductively opened to provide diol 4 in an excellent yield
(Scheme 1). This intermediate was then coupled with mannose
a
Scheme 2. Deprotection Sequence for Glycan 1
a
Scheme 1. Synthesis of Protected Undecasaccharide 10
a
Reagents and conditions: (a) NaOMe/NaOH, MeOH/H2O,
CH2Cl2, rt, 24 h; (b) 1,2-ethylenediamine, EtOH, 80 °C, 24 h; (c)
Ac2O, pyridine, rt, 24 h, 35% (over three steps); (d) NaOMe, MeOH,
CH2Cl2 then H2O, rt, 24 h; (e) Na, NH3, THF, −78 °C, 4 h; (f) Ac2O,
NaHCO3 (aq), 0 °C, 77% (over 3 steps); (g) NH4HCO3, H2O, 40 °C,
3 d, 99%.
a
Reagents and conditions: (a) BH3·THF, n-Bu2BOTf, CH2Cl2, 0 °C, 3
h, 83%; (b) 5, NIS, AgOTf, CH2Cl2, −20 °C, 2 h, 89%; (c) NaOMe,
MeOH, THF, rt, 1 h, 99%; (d) 8, NIS, AgOTf, CH2Cl2, −20 °C, 24 h,
75%; (e) 9, Cp2Zr(OTf)2·THF, CH2Cl2, 0 °C, 2 h, 74%.
acetate groups were removed with NaOMe/MeOH and the
methyl ester functionalities were saponified with NaOH. The
resultant diacid was then used in a crude state to remove four
phthalimide groups with 1,2-ethylenediamine in refluxing
ethanol. We found that this set of conditions proved to be a
more robust protocol for the deprotection of nitrogen
functionalities in comparison with the conditions reported
previously,14 which required higher reaction temperatures. The
resultant crude material was then subjected to acetylation
conditions to provide bislactone 11 in 35% yield over a three-step
sequence. The peracetylation step allows for purification of the
oligosaccharide material before the reductive removal of benzyl
groups. To complete the synthesis of 1, saponification of 11 with
NaOMe/NaOH furnished diacid, which was then subjected to
Birch-type reduction to reveal undecasaccharide 1 in an
appreciable yield of 77%. Occasionally, we found that some N-
thioether 514 in 89% yield and the alcohol functionalities were
liberated under basic conditions to afford diol 7. It is well to note
the importance of the dFBz (2,5-difluorobenzoyl) group in this
step. Attempts to remove unsubstituted benzoyl groups in an
analogous pentasaccharide led only to 40% yield of 7, and a
mixture of products arising from a partial cleavage of the
phthalimide group was observed.
At this point we were ready to evaluate the feasibility of
conducting a double glycosylation.15 Accordingly, we inves-
tigated the utility of two trisaccharide donors in this trans-
formation. Thioether 8,15 under NIS/TfO− conditions, did
indeed provide the expected undecasaccharide 10 in 75%
4701
dx.doi.org/10.1021/ja401385v | J. Am. Chem. Soc. 2013, 135, 4700−4703

Journal of the American Chemical Society
Communication
acetamide groups were cleaved under the reductive step, and an
additional acetylation step (Ac2O, sat. NaHCO3(aq), 0 °C) was
necessary to provide homogeneous material. Unlike depro-
tection of fucosylated biantennary glycan, which was shown to
lead to a partial cleavage of the oligosaccharide chain, we
observed only clean removal of the benzyl groups without any
truncated saccharide fragments and/or over-reduction prod-
ucts.15 Compared to the synthesis of 11 reported previously,6 our
approach capitalizes on a more convergent assembly of the core
undecasaccharide and it displays significantly improved yields.
The analytical data for glycan 1 match the previous reports.6,10,17
Having established a synthetic approach for the construction
of the biantennary saccharide, we turned our attention to
tetradecasaccharide 2 (Scheme 3). The synthesis started by
of 17 with anomeric fluoride donor 9 under the conditions
optimized for the synthesis of biantennary glycan 1 provided the
desired triantennary glycan 18 in a surprisingly high yield (89%
per glycosyl acceptor). Notably, we were again unable to detect
biantennary oligosaccharide intermediates upon completion of
the reaction. However, thioether donor 8 afforded 18 in a
significantly diminished yield (42%) and a significant amount of
diglycosylated intermediate was observed. Completion of the
synthesis of 2 is depicted in Scheme 4. A sequence of steps used
a
Scheme 4. Deprotection Sequence for Glycan 2
a
Scheme 3. Synthesis of Protected Tetradecasaccharide 18
a
Reagents and conditions: (a) NaOMe/NaOH, MeOH/H2O, rt, 24 h;
(b) 1,2-ethylenediamine, EtOH, 80 °C, 24 h; (c) Ac2O, pyridine 33%
(over 3 steps); (d) NaOMe, MeOH/CH2Cl2 then H2O, rt, 24 h; (e)
Na/NH3, THF, −78 °C, 71% (over 2 steps); (f) NH4HCO3, H2O, 40
°C, 3 d, 82%.
a
Reagents and conditions: (a) 5, NIS, AgOTf, CH2Cl2, 4 Å MS, −50
to −20 °C, 1.25 h, 91%; (b) BH3·THF, n-Bu2BOTf, CH2Cl2, 0 °C, 4 h,
99%; (c) 15, NIS, AgOTf, CH2Cl2, −10 °C, 2 h, 87%; (d) NaOMe,
MeOH/THF, rt, 1.7 h, 93%; (e) 9, Cp2Zr(OTf)2·THF, CH2Cl2, 0 °C,
1.5 h, 71%.
in the preparation of 1 was used to provide tris-lactone 19. This
compound was taken further to finish the deprotection sequence
under the Birch conditions furnishing free oligosaccharide 2 in
71% yield.
coupling of 3 to a mannose thioether donor 5, which proceed in
an excellent yield. Regioselective benzylidene opening revealed
the desired 6-OH acceptor, as confirmed by 2D NMR analysis.
The alcohol functionality served as the glycosyl acceptor site to
the mannose donor 15 bearing the corresponding protective
groups at positions 2 and 6. Finally, deprotection under basic
conditions allowed for a rapid construction of triol 17 in excellent
yield.
Unlike the case in the synthesis of triantennary system
described previously,14 it could be anticipated that the
glycosylation reaction with the corresponding trisaccharide
donors should be a rather more facile process. Indeed, coupling
In order to evaluate the feasibility of chemical ligation of
saccharides 1 and 2 to a peptide chain, a selected motif of
prostate-specific membrane antigen (PSMA)18 21 was used. This
peptide corresponds to Gly332-Gln340 native sequence of
PSMA, and it was chosen on the basis of the X-ray structure as the
fragment containing one of the most exposed glycosylation sites
in this glycoprotein.19 Thus, Kochetkov amination20 of 1
(Scheme 2) followed by repeated lyophilizations afforded
anomeric amine 12 in an almost quantitative yield. The
subsequent ligation to 21 was carried out under the Lansbury
conditions21 to afford the PSMA glycoconjugate 22 in 54%
4702
dx.doi.org/10.1021/ja401385v | J. Am. Chem. Soc. 2013, 135, 4700−4703

Journal of the American Chemical Society
Communication
Scheme 5. Conjugation of Glycans 1 and 2 to PSMA
Oligopeptide
ACKNOWLEDGMENTS
■
a
Support for this research was provided by the National Institutes
of Health (GM102872 to S.J.D.). M.A.W. acknowledges the
Terrie Brodeur Breast Cancer Foundation for postdoctoral
fellowship. We also thank Dr. George Sukenick, Hui Fang, and
Sylvi Rusli of SKI’s NMR core facility for assistance.
REFERENCES
■
(1) Montreuil, J., Vliegenthart, J. F. G., Schachter, H., Eds. Glycoproteins
and Disease; Elsevier Science: Amsterdam, 1996.
(2) (a) WittmannV., Ed. Glycopeptides and GlycoproteinsSynthesis,
Structure, and Application; Springer: Berlin, 2010. (b) Montreuil, J.
Glycoproteins; Elsevier Science: Amstrdam, 1995.
(3) (a) Davis, B. G. Chem. Rev. 2002, 102, 579. (b) Gamblin, D. P.;
Scanlan, E. M.; Davis, B. G. Chem. Rev. 2008, 109, 131.
(4) Ogawa, T.; Sugimoto, M.; Kitajima, T.; Sadozai, K. K.; Nukada, T.
Tetrahedron Lett. 1986, 27, 5739.
(5) Kajihara, Y.; Suzuki, Y.; Yamamoto, N.; Sasaki, K.; Sakakibara, T.;
Juneja, L. R. Chem.Eur. J. 2004, 10, 971.
(6) Seifert, J.; Lergenmuller, M.; Ito, Y. Angew. Chem., Int. Ed. 2000, 39,
̈
531.
(7) Pratt, M. R.; Bertozzi, C. R. J. Am. Chem. Soc. 2003, 125, 6149.
(8) Jensen, P. H.; Karlsson, N. G.; Kolarich, D.; Packer, N. H. Nat.
Protoc. 2012, 7, 1299.
(9) Gervais, A.; Hammel, Y.-a.; Pelloux, S.; Lepage, P.; Baer, G.; Carte,
N.; Sorokine, O.; Strub, J.-m.; Koerner, R.; Leize, E.; Van Dorsselaer, A.
Glycobiology 2003, 13, 179.
(10) Damm, J.; Kamerling, J.; Dedem, G.; Vliegenthart, J.
Glycoconjugate J. 1987, 4, 129.
(11) (a) Dudkin, V. Y.; Miller, J. S.; Danishefsky, S. J. J. Am. Chem. Soc.
2003, 126, 736. (b) Dudkin, V. Y.; Miller, J. S.; Dudkina, A. S.; Antczak,
C.; Scheinberg, D. A.; Danishefsky, S. J. J. Am. Chem. Soc. 2008, 130,
13598.
(12) Schauer, R. Curr. Opin. Struct. Biol. 2009, 19, 507.
(13) Wu, B.; Hua, Z.; Warren, J. D.; Ranganathan, K.; Wan, Q.; Chen,
G.; Tan, Z.; Chen, J.; Endo, A.; Danishefsky, S. J. Tetrahedron Lett. 2006,
47, 5577.
(14) Walczak, M. A.; Danishefsky, S. J. J. Am. Chem. Soc. 2012, 134,
16430.
a
Reagents and conditions: (a) 12, HATU, DIPEA, DMSO, rt, 1.5 h,
(15) Nagorny, P.; Fasching, B.; Li, X.; Chen, G.; Aussedat, B.;
Danishefsky, S. J. J. Am. Chem. Soc. 2009, 131, 5792.
(16) Allen, J. R.; Allen, J. G.; Zhang, X.-F.; Williams, L. J.; Zatorski, A.;
Ragupathi, G.; Livingston, P. O.; Danishefsky, S. J. Chem.Eur. J. 2000,
6, 1366.
54%; (b) 20, HATU, DIPEA, rt, 1 h, 10%.
(Scheme 5). Similarly, triantennary amine 20 was coupled with
peptide 21 to furnish glycoconjugate 23 in 10% yield.
(17) Ishii, K.; Iwasaki, M.; Inoue, S.; Kenny, P. T.; Komura, H.; Inoue,
Y. J. Biol. Chem. 1989, 264, 1623.
In summary, we have described here a unified strategy for the
synthesis of sialylated di- and tribranched N-linked glycans based
on multiple glycosylations using very efficient anomeric fluoride
donor. This approach allowed for preparation of fully sialylated
glycans in excellent yields in a minimal number of steps. Finally,
conjugation of the saccharides 1 and 2 to a native peptidic
sequence of PSMA was demonstrated, supporting the feasibility
of preparing diagnostic and therapeutic tools based on tumor-
associated carbohydrate antigens related to prostate cancer.
̌
́ ́ ̌ ̌
ha, P.; Sklenar, J.; Man, P.; Bezouska, K.; Slusher,
(18) Barinka, C.; Sac
B. S.; Konvalinka, J. Protein Sci. 2004, 13, 1627.
(19) Davis, M. I.; Bennett, M. J.; Thomas, L. M.; Bjorkman, P. J. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 5981.
(20) Likhosherstov, L. M.; Novikova, O. S.; Derevitskaja, V. A.;
Kochetkov, N. K. Carbohydr. Res. 1986, 146, C1.
(21) Cohen-Anisfeld, S. T.; Lansbury, P. T. J. Am. Chem. Soc. 1993,
115, 10531.
ASSOCIATED CONTENT
* Supporting Information
General experimental procedures, including spectroscopic and
analytical data for new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
s-danishefsky@ski.mskcc.org
■
Notes
The authors declare no competing financial interest.
4703
dx.doi.org/10.1021/ja401385v | J. Am. Chem. Soc. 2013, 135, 4700−4703