Total synthesis of the 2,6-sialylated immunoglobulin G glycopeptide fragment in homogeneous form

Article abstract of DOI:10.1021/ja907136d

(Figure Presented) The 2,6-sialylated tridecasaccharide 1 associated with the Fc fragment of intravenous immunoglobulin has been synthesized.

Full text of DOI:10.1021/ja907136d

Published on Web 11/03/2009
Total Synthesis of the 2,6-Sialylated Immunoglobulin G Glycopeptide
Fragment in Homogeneous Form
Ping Wang,† Jianglong Zhu,† Yu Yuan,† and Samuel J. Danishefsky*,†,‡
Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York AVenue, New York,
New York 10065, and Department of Chemistry, Columbia UniVersity, HaVemeyer Hall, 3000 Broadway,
New York, New York 10027
Received August 24, 2009; E-mail: s-danishefsky@ski.mskcc.org
Carbohydrate domains mediate the stability, folding, and biologi-
cal activity of glycoproteins.1 Quality investigations into the impact
of specific glycosylation patterns are complicated by formation of
horrific mixtures of difficult-to-separate glycoforms. A major
program underway in our laboratory seeks to address the challenge
of obtaining single glycoforms of biologics through the de novo
chemical synthesis of homogeneous glycopeptides and glycoproteins
of established therapeutic value.2
chitobiose (rings 1 and 2). It also presents the complex ꢀ-mannose
linkage joining a ꢀ-mannose (ring 3) to the chitobiose core system.
The ꢀ-mannoside (ring 3) in turn is linked in a biantennary fashion
from its C3 and C6 hydroxyls to two R-linked mannosides (see rings
6 and 7). The C2 hydroxyls of these mannosides are linked in a
ꢀ-fashion to two lactosamines, joined in 2,6-linkages to sialic acids
(see rings 8-13). The ꢀ-linked mannose (ring 3) is also joined at
its equatorial C4 oxygen in a ꢀ-linkage to a GlcNAc residue (see
ring 4).
Accordingly, we became interested in monomeric immuno-
globulin G (IgG) purified from intravenous immunoglobulin
(IVIG). This therapeutically valuable anti-inflammatory agent
is commonly employed in the treatment of autoimmune disorders,
such as rheumatoid arthritis and immune thrombocytopenia. The
biological activity of IVIG derives from its Fc fragment, which
presents a biantennary tridecasaccharide attached through an
N-linkage to the Asn297 residue. Recently, Ravetch and co-
workers discovered the critical role of the terminal 2,6-linked
sialic acids of the glycan in mediating the anti-inflammatory
activity of IVIG.3 Although the IVIG glycan is biosynthesized
as a heterogeneous mixture containing both 2,3- and 2,6-
sialylated carbohydrate domains, only the Fc fragment possessing
the 2,6-sialylated glycoform demonstrates appreciable anti-
inflammatory activity in arthritic mice. Moreover, an IVIG Fc
fragment possessing exclusively 2,6-sialic acid linkages was
shown to be 10-fold more active in suppressing inflammation
in mice than was an Fc fragment isolated from IVIG (containing
both 2,6- and 2,3-linkages). These results suggest that an Fc
peptide fragment presenting a homogeneous 2,6-sialylated glycan
domain (e.g., 1) could be more potent and effective than the
heterogeneous mixture currently employed in clinical settings.
We describe herein the chemical synthesis of the homogeneous
tridecasaccharide 1 possessing terminal 2,6-sialic acid linkages. We
further describe the aspartylation of 1 with a peptide domain,
thereby demonstrating the ability to convert it into a glycopolypep-
tide (see 28). Accordingly, glycopeptidic constructs corresponding
to the carbohydrate domain of IgG can now be synthesized and
screened.
On the basis of earlier literature in this general area,6 it seemed
likely that the most demanding phase of the synthesis would involve
the attachment of the two trisaccharide ensembles, rings 8-10 and
rings 11-13, to their respective axial hydroxyl acceptor sites in
ring 6 and ring 7. Looming particularly difficult was the prospect
of introducing additional appendages at the interior C2 axial
hydroxyl of ring 7. Indeed, it was our hope for conciseness to
conduct simultaneous azaglycosylations at both of these two
acceptor sites to introduce ꢀ-glucosamine donor residues (see rings
8 and 11). These rings would in turn be joined at their C4 equatorial
hydroxyl groups via ꢀ-linkages to C6-sialylated galactosyl donors.
Ring 7 emanates from the particularly hindered C3 hydroxyl site
of ring 3 nestled between the glucosamine moiety at C4, the axial
hydroxyl at C2, and the complex core substructure (rings 1 and 2).
In fact, previous attempts to use the more exposed C6 hydroxyl of
ring 3 as a viable acceptor site met with only the slightest of success.
No glycosylation at all had been achieved at the much more
hindered secondary hydroxyl acceptor site at C3 of ring 3. Indeed,
the key breakthrough involved the use of a phenylsulfamido
function on the future ring 4 (see the * in compound 4) to allow
for introduction of rings 5, 6, and 7 (see below).
In Scheme 1, by focusing on key building blocks, we recapitulate
the strategic analysis that eventually achieved success. Thus, as
shown, we envisioned reaching tetrasaccharide 4 through coupling
of trisaccharide 5 with sulfonamide donor 6.7 As noted, the presence
of the sulfonamide functionality on diol 4 would prove to be key
to the efficient installation of the “wing” mannose and fucose units,
furnishing core heptasaccharide 3.8 Elongation of the mannose units
with a ꢀ-GlcNAc donor would yield the nonasaccharide, 2, which
would finally be subjected to diglycosylation with disaccharide 10,
affording the target compound, 1. Happily, in this synthetic analysis,
we were able to revisit the chemistry developed in connection with
our longstanding glycal assembly program.9
The power of prioritized strategic bond disconnection as a means
of guiding synthetic analysis, formalized by the Corey school,4 is
well-appreciated by students of chemical synthesis of complex target
systems. Less well appreciated is a type of pattern recognition
analysis that can be very helpful in devising total synthesis programs
directed toward biologic-level oligosaccharides.5 System 1 consti-
tutes a significant challenge to chemical synthesis. In particular,
the 13-mer contains an often troublesome L-R-fucosyl ring (ring
5) branching from the “reducing-end” GlcNAc of the terminal
Our synthesis of key intermediate 2 commenced with the known
monosaccharides 1110 and 12 (Scheme 2). Glycosylation of 12 with
donor 11 through exposure to NIS/TMSOTf11 under sonication
conditions proceeded rapidly to afford the disaccharide in 82%
yield.12 Subsequent saponification furnished acceptor 13. The latter
was then glycosylated with donor 14,13 which gave trisaccharide
15 with good selectivity (ꢀ/R ) 7:1). The ꢀ-isomer was isolated
† Sloan-Kettering Institute for Cancer Research.
‡ Columbia University.
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10.1021/ja907136d CCC: $40.75  2009 American Chemical Society
J. AM. CHEM. SOC. 2009, 131, 16669–16671 16669

C O M M U N I C A T I O N S
Scheme 1. Synthetic Strategy Toward Tridecasaccharide 1
Scheme 2. Synthesis of Intermediate 2a
a Key: (a) 1. NIS/TMSOTf, CH2Cl2, sonication, 82%; 2. NaOMe/MeOH, 92%. (b) 14, Tf2O, DTBP, CH2Cl2, 65%. (c) 1. Zn(OTf)2/EtSH, CH2Cl2, 75%;
2. BzCl, Py, CH2Cl2, 93%. (d) 6, MeOTf, DTBP, CH2Cl2, 92% (ꢀ/R ) 2.7:1). (e) 1. NaOMe/MeOH, 83%; 2. TMSOTf, CH2Cl2, 93% (CAN, 66%). (f) 7,
NIS/TMSOTf, CH2Cl2, 90%. (g) HF/Py, THF, sonication, 88%. (h) 8, Cp2Zr(OTf)2, DTBP, toluene/THF, 93% (R/ꢀ ) 12:1). (i) N2H4 ·H2O, Py/AcOH (1:1),
CH2Cl2, 93%. (j) 9, toluene, BF3 ·OEt2. (k) K2CO3, MeOH, 52%.
Scheme 3. Synthesis of Disaccharide Donor 10a
in 65% yield. Following removal of the benzylidene acetal14 and
selective benzoylation of the resulting 4,6-diol, compound 5 was
in hand. MeOTf-promoted15 glycosylation with glucosamine build-
ing block 6 afforded a 92% yield of 16 (ꢀ/R ) 2.7:1).
Sequential removal of the benzoate and PMB protecting groups
of tetrasaccharide 16 afforded 3,6-diol 4 in 77% yield over two
steps. Twofold glycosylation of 4 was accomplished with an excess
of donor 7 and NIS/TMSOTf, providing hexasaccharide 17 in 90%
a Key: (a) TMSOTf, toluene/THF, 81%. (b) 1. PhI(OAc)2/BF3 · OEt2,
yield. The TBDPS group was cleaved upon exposure to HF/Py (88%
CH2Cl2; 2. Py, Ac2O, 89%. (c) 1. N2H4 ·AcOH, DMF; 2. DAST, CH2Cl2,
yield). To the resultant free hydroxyl group was appended fluoride
82%.
donor 816 under Cp2Zr(OTf)2 mediation17 to afford compound 19
in 93% yield and excellent selectivity (R/ꢀ ) 12:1). Following
removal of the levulinoyl protecting group, intermediate 3 was in
hand. BF3 ·OEt2-promoted diglycosylation of 3 was achieved
through the use of excess donor 9.18 Finally, removal of the
chloroacetate groups on the glucosamine units provided the target
nonasaccharide 2 in 52% yield over two steps.
Having completed the synthesis of compound 2, we then turned
to the assembly of disaccharide donor 10 (Scheme 3). In the event,
coupling of phosphite 2119 and galactal 2220 proceeded smoothly
to afford disaccharide 23 with good selectivity (R/ꢀ ) 11:1). The
predominant R isomer was isolated in 81% yield. Oxidation of 23
21
with PhI(OAc)2 followed by acetylation furnished disaccharide
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C O M M U N I C A T I O N S
Scheme 4. Synthesis of Tridecasaccharide 1 and Glycopeptide 28a
In summary, we have synthesized the homogeneous 2,6-sialylated
tridecasaccharide of the Fc sector of IVIG and successfully coupled
the carbohydrate with a peptide domain to provide a glycopeptide
adduct (28). The merger of 1 with appropriate Fc-derived peptide
fragments and the results of biological evaluations of the glyco-
peptides will be reported in due course.
Acknowledgment. Support was provided by the NIH (CA28824
to S.J.D.). We thank Dr. George Sukenick, Hui Fang, and Sylvi
Rusli of SKI’s NMR core facility for mass spectral and NMR
analysis; Rebecca Wilson for editorial assistance; and Dana Ryan
for assistance with the preparation of the manuscript.
Supporting Information Available: Experimental procedures,
characterization, and copies of spectral data. This material is available
free of charge via the Internet at http://pubs.acs.org.
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