Chemoenzymatic synthesis and Fcγ receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcγIIIa receptor

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pubs.acs.org/JACS

Chemoenzymatic Synthesis and Fcγ Receptor Binding of

Homogeneous Glycoforms of Antibody Fc Domain. Presence of a

Bisecting Sugar Moiety Enhances the Affinity of Fc to FcγIIIa Receptor

Guozhang Zou, Hirofumi Ochiai, Wei Huang, Qiang Yang, Cishan Li, and Lai-Xi Wang*

Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine,

Baltimore, Maryland 21201, United States

S Supporting Information

b

ABSTRACT: Structurally well-deﬁned IgG-Fc glycoforms are

highly demanded for understanding the eﬀects of glycosylation

on an antibody’s eﬀector functions. We report in this paper

chemoenzymatic synthesis and Fcγ receptor binding of an array

of homogeneous IgG-Fc glycoforms. The chemoenzymatic

approach consists of the chemical synthesis of deﬁned N-glycan

oxazolines as donor substrates, the expression of the Fc domain

in a CHO cell line in the presence of an α-mannosidase inhibitor kifunensine, and an endoglycosidase-catalyzed glycosylation of the

deglycosylated Fc domain (GlcNAc-Fc homodimer) with the synthetic glycan oxazolines. The enzyme from Arthrobacter

protophormiae (Endo-A) was found to be remarkably eﬃcient to take various modiﬁed N-glycan core oxazolines, including the

bisecting sugar-containing derivatives, for Fc glycosylation remodeling, resulting in the formation of the corresponding

homogeneous Fc glycoforms. Nevertheless, neither Endo-A nor the Mucor hiemalis endoglycosidase mutants (EndoM-N175A

and EndoM-N175Q) were able to transfer full-length complex-type N-glycan to the Fc domain, implicating the limitations of these

two enzymes in Fc glycosylation remodeling. Surface plasmon resonance (SPR) binding studies with the synthetic IgG-Fc

glycoforms unambiguously proved that the presence of a bisecting GlcNAc moiety could signiﬁcantly enhance the binding of Fc to

FcγRIIIa, the activating Fcγ receptor, independent of Fc core-fucosylation. Interestingly, the Fc glycoforms carrying an unusual

bisecting sugar moiety such as a mannose or a LacNAc moiety also demonstrated enhanced aﬃnity to FcγRIIIa. On the orther hand,

the presence of a bisecting GlcNAc or core-fucosylation had little eﬀect on the aﬃnity of Fc to the inhibitory Fcγ receptor, FcγRIIb.

Our experimental data also showed that the α-linked mannose residues in the pentasaccharide Man3GlcNAc2 core was essential to

maintain a high aﬃnity of Fc to both FcγRIIIa and FcγRIIb. The synthetic homogeneous Fc glycoforms thus provide a useful tool

for elucidating how a ﬁne Fc N-glycan structure precisely aﬀects the function of the Fc domain.

’ INTRODUCTION

IgG-Fc N-glycans are typical biantennary complex-type N-gly-

cans with considerable structural heterogeneity.^5À7More than

30 diﬀerent Fc oligosaccharides were characterized, in which the

core Asn-linked heptasaccharide GlcNAc2Man3GlcNAc2-Asn

can be diﬀerentially decorated with core-fucosylation, bisecting

GlcNAc attachment, and varied terminal galactosylation and

sialylation (Figure 1).

Recent advances in glycobiology and immunology have

suggested that antibody Fc glycosylation can signiﬁcantly impact

the structure and biological function of antibodies.^2,8It has been

demonstrated that aglycosylated or deglycosylated IgG antibo-

dies are almost completely devoid of Fc-mediated eﬀector

functions as a result of reduced or ablated binding to Fcγ

receptors or those proteins in the complement system.^7,9Crystal-

lographic studies and NMR analysis have shown that the Fc N-

glycans have multiple noncovalent interactions with the Fc

protein domain, implicating an important role of the N-glycan

Monoclonal antibodies (MAbs) are a class of therapeutic

glycoproteins used for the treatment of various human diseases

including cancer and inﬂammatory disorders.^1À3Almost all the

therapeutic monoclonal antibodies currently used for disease

treatment are of the immunoglobulin G (IgG) type, which

are composed of two light chains and two heavy chains that

are associated to form three distinct protein domains linked by a

ﬂexible hinge region. The two identical Fab domains are speciﬁc

for antigen binding, while the Fc domain, a homodimer con-

sisting of the C_H2 and C_H3 subdomains, is engaged in the

“downstream” eﬀector functions of antibodies including anti-

body-dependent cellular cytotoxicity (ADCC) and complement-

dependent cytotoxicity (CDC).^2,4ADCC and CDC eﬀector

functions are mediated by the interactions of the Fc domain

with respective Fcγ receptors (such as FcγRIIIa and FcγRIIb)

on eﬀector cells and the C1q component of the complement

cascade, respectively.⁴The Fc homodimer carries two N-glycans

at each of the conserved N-glycosylation sites (Asn-297) of the

two C_H2 domains. Structural analysis has indicated that human

Received: September 6, 2011

Published: October 17, 2011

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2011 American Chemical Society

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Figure 1. Schematic presentations of the natural and synthetic human IgG1-Fc glycoforms. (A) Natural heterogeneous Fc glycforms; (B) synthetic

homogeneous Fc glycoforms. The IgG1-Fc structure was modeled on the basis of the crystal structure of an anti-HIV antibody, b12 (PDB code, 1hzh)

(Saphire et al. Science, 2001, 293, 1155). GlcNAc, N-acetylglucosamine; Man, mannose; Gal, galactose; Glc, glucose; Fuc, ^L-fucose; Sia, sialic acid. The

dash lines represent variable decorations.

in maintaining an appropriate Fc domain conformations required

for the speciﬁc interactions between Fc and Fc receptors for

antibody’s eﬀector functions.^6,9À16It has been further demon-

strated that the distinct ﬁne structures of Fc N-glycans can deﬁne

whether the antibody goes for activation (such as ADCC) or

inhibitory (such as anti-inﬂammatory) eﬀector functions.^2,17For

example, core-fucosylation has been shown to signiﬁcantly

decrease the aﬃnity of Fc to FcγIIIa receptor that correlates

with the antibody’s ADCC function and anticancer eﬃcacy

in vivo.^18À22On the other hand, speciﬁc terminal α-2,6-sialyla-

tion of IgG-Fc N-glycan, a minor population (about 5%) of the

intravenous immunoglobulin (IVIG), has recently been demon-

strated to be responsible for the anti-inﬂammatory activity of

IVIG in the treatment of autoimmune diseases.^4,23À25As to the

role of the bisecting GlcNAc residue in the Fc N-glycans, studies

have shown that increasing the population of the bisecting

GlcNAc contents leads to enhanced aﬃnity to FcγIIIa receptor

and thus enhanced ADCC function of the antibodies.^21,26À28

However, another study has suggested that the lack of the core

fucose, rather than the presence of a bisecting GlcNAc, plays the

key role in enhancing the ADCC, as preaddition of the bisecting

GlcNAc residue to the core-β-mannose inhibits core-fucosylation

in the biosynthetic pathway, resulting in an increased population

of nonfucosylated glycoforms that demonstrate enhanced

ADCC.¹⁹It should be pointed out that many of the structureÀ

activity relationship studies so far reported have used mixtures of

Fc glycoforms, although some of the speciﬁc glycoforms were

enriched by lectin aﬃnity puriﬁcation and/or enzymatic trim-

ming. This situation sometimes makes a conclusive interpreta-

tion of the experimental results diﬃcult.

The urgent need of various glycosylation-deﬁned pure IgG-Fc

glycoforms, which are diﬃcult to isolate from natural sources, for

structureÀactivity relationship studies and for biomedical appli-

cations has stimulated an intense interest in developing methods

to control Fc glycosylation. These include glycan biosynthetic

pathway engineering in plant, mammalian, and yeast expression

systems to produce nonfucosylated IgG-Fc glycoforms with

improved ADCC activity;^26,29À33sequential enzymatic trimming

of Fc N-glycans to produce truncated IgG-Fc glycoforms for

structural analysis and Fcγ receptor binding studies;^13,28,34and

chemoselective Fc glycosylation by site-directed mutagenesis at

the glycosylation site (N297C) followed by chemoselective liga-

tion with a glycan.³⁵We have previously described a convergent

chemoenzymatic method for IgG-Fc glycosylation remodeling

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Figure 2. Structures of synthetic sugar oxazolines.

that takes advantage of the transglycosylation activity of the

Arthrobacter endoglycosidase (Endo-A) and the highly active

N-glycan oxazoline as the donor substrates.³⁶In this approach, an

IgG-Fc was ﬁrst expressed in yeast to give the IgG-Fc carrying

yeast N-glycans at the Fc domain. Then the heterogeneous N-

glycans were cleaved to leave only the innermost GlcNAc

attached at the glycosylation site (Asn-297). Finally a deﬁned

core N-glycan was transferred to the GlcNAc moiety by Endo-A

to provide a homogeneous IgG-Fc glycoform. This enzymatic

transglycosylation approach preserves the natural N-glycan core

structure in the Fc domain, which appears essential for the

eﬀector functions of antibodies.²Our initial studies have sug-

gested that the enzymatic transglycosylation was feasible for

glycosylation remodeling of recombinant IgG-Fc dimers under

mild conditions without the need of denaturing the Fc protein

domain.³⁶Despite this initial success, it is still to be demonstrated

whether diﬀerent types of N-glycans can be introduced at the Fc

domain by this method. Glycosylation remodeling of the IgG-Fc

homodimer could be particularly challenging as the two Fc N-

glycans at the glycosylation sites (Asn-297) are sandwitched

between the two Fc domains, which might be less accessible for

enzymatic reactions.^9À15Our initial success in the chemoenzy-

matic glycosylation remodeling of IgG-Fc,³⁶together with recent

advances in the method development,^37À48prompted us to

expand the chemoenzymatic approach to the synthesis of various

homogeneous IgG-Fc glycoforms while examining the scope and

limitations of the endoglycosidase-catalyzed transglycosylation

for IgG-Fc glycosylation remodeling. We report in this paper the

chemoenzymatic synthesis of an array of speciﬁc, homogeneous

IgG-Fc glycoforms (Figure 1, glycoforms Fc-1 to Fc-6), with a

focus on elucidating the roles of individual sugar residues within

and/or ﬂanking the Fc N-glycan core in the binding to Fcγ

receptors FcγRIIIa and FcγRIIb. In particular, several bisecting

sugar-containing Fc glycoforms, including the unusual bisecting

LacNAc Fc glycoform that was recently discovered as a minor

IgG-Fc glycoform with unknown function,⁴⁹were synthesized.

Our experimental data indicate that Endo-A is remarkably

eﬃcient to take various modiﬁed N-glycan core oxazolines,

including the bisecting sugar-containing derivatives, for Fc glyco-

sylation remodeling. Nevertheless, neither Endo-A nor the

Mucor hiemalis endoglycosidase mutant (EndoM-N175A and

EndoM-N175Q) was able to transfer full-length complex-type

N-glycan oxazoline to the Fc domain, implicating the limitations

of these two enzymes in Fc glycosylation remodeling. Our

surface plasmon resonance (SPR) binding studies with well-

deﬁned synthetic IgG-Fc glycoforms provide unambiguous evi-

dence indicating that the presence of a bisecting GlcNAc moiety

could directly enhance the interaction between Fc to FcγRIIIa,

independent of core-fucosylation, whereas the presence of a

bisecting GlcNAc has little eﬀect on the aﬃnity of Fc to FcγRIIb.

Our experimental data also showed that the two α-linked

mannose residues in the pentasaccharide Man3GlcNAc2 core

is essential to maintain a high aﬃnity of Fc to the FcγRIIIa, while

the core-β-mannose residue could be changed to a β-glucose

moiety without signiﬁcantly aﬀecting the binding to FcγRIIIa.

’ RESULTS AND DISCUSSION

Synthesis of Bisecting GlcNAc-Containing N-Glycan Ox-

azoline (1) and Bisecting LacNAc-Containing N-Glycan Ox-

azoline (2). Chemoenzymatic synthesis of the homogeneous Fc

glycoforms by the endoglycosidase-catalyzed transglycosylation

requires the preparation of the corresponding N-glycan oxazo-

lines as donor substrates. The structures of sugar oxazolines

synthesized and used for the present study are listed in Figure 2.

These glycan oxazolines were designed for the preparation of the

well-defined homogeneous Fc glycoforms that will allow a

detailed probing of the effects of varied glycan structures on

the binding of Fc to respective Fcγ receptors that are essential for

antibody effector functions. Chemical synthesis of bisecting

GlcNAc-containing N-glycans of varied size has been reported

by several research groups.^50À55These studies suggested that the

4-hydroxyl group of the core-β-mannose was sterically hindered

and made difficult to glycosylate when the 3- and 6-positions of

the core-β-mannose were preoccupied with bulky sugar residues.

Thus, for the synthesis of the bisecting GlcNAc-containing sugar

oxazoline (1), we adopted a synthetic strategy of introducing the

bisecting GlcNAc residue prior to the glycosylation at the 3- and

6-positions of the β-mannose moiety (Scheme 1).

Glycosylation of monosaccharide 7⁵⁶with thioglycoside do-

nor 8⁵⁷under the promotion of 1-benzenesulﬁnyl piperidine

(BSP)/2,4,6-tri-tert-butylpyrimidine (TTBP)/triﬂuoromethane-

sulfonic anhydride (Tf₂O)⁵⁶gave the corresponding disacchar-

ide product (β/α = 6:1), from which the desired β-isomer 9 was

isolated in 56% yield. Removal of the benzylidene group by mild

acidic hydrolysis provided diol 10 in 80% yield. The primary

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Scheme 1. Synthesis of Bisecting GlcNAc- and LacNAc-Containing N-Glycan Oxazolines^a

^aReagents and conditions: (a) BSP, TTBP, Tf₂O, CH₂Cl₂, 56%. (b) 80% aq. AcOH, 80%. (c) TBDMSCl, pyridine, 95%. (d) BF₃OEt₂, CH₂Cl₂, 76%.

3

(e) TFA, CH₂Cl₂, 88%. (f) TMSOTf, CH₂Cl₂, 54%. (g) (1) NH₂NH₂monohydrate, EtOH, H₂O; (2) Ac₂O, pyridine, 77% (2 steps). (h) (1) Pd(OH)₂-

C, H₂, CH₂Cl₂, MeOH; (2) Ac₂O, pyridine, 89% (2 steps). (i) TMSBr, BF₃OEt₂, 2,4,6-collidine, CH₂Cl₂, 48%. (j) MeONa, MeOH, quantitative.

3

(k) MeONa, MeOH, quantitative. (l) DMC, Et₃N, H₂O, quantitative. (m) UDP-Gal, β-1,4-galactosyltransferase, buﬀer, quantitative. (n) DMC, Et₃N,

H₂O, quantitative.

hydroxyl group was then selectively protected with TBDMSCl in

yield (48%) of 19, mainly due to decomposition of the oligo-

saccharide under the acidic reaction conditions. De-O-acetyla-

tion of 19 with a catalytic amount of MeONa in MeOH provided

pentasaccharide oxazoline 1. Recently, a method for one-step

conversion of unprotected N-acetyl-2-amino sugars to sugar

oxazolines using a chloroformamidinium reagent in water was

reported,⁶¹and we have also shown that this method was equally

eﬃcient for making even very large complex N-glycan oxazolines

from free N-glycans.⁴¹Thus, we tested this method for preparing

oxazoline 1 from free pentasaccharide 20, which was obtained by

de-O-acetylation of 18. Treatment of 20 with an excess of

2-chloro-1,3-dimethylimidazolinium chloride (DMC) and Et₃N

in water aﬀorded the desired sugar oxazoline 1 in quantitative yield,

which was readily puriﬁed by gel ﬁltration on a Sephadex G-10

column. These results clearly indicate that the one-step conver-

sion of free glycan to the sugar oxazoline in an aqueous solution is

much more eﬃcient than the Lewis acid-catalyzed oxazoline ring

pyridine to give disaccharide 11 in excellent yield. BF₃Et₂O-

3

catalyzed glycosylation of the 4-OH with glucosamine build

ing block 12⁵⁸gave the trisaccharide 13 in 76% yield, in which the

bisecting glucosamine moiety was introduced with a complete

β-selectivity. The TBDMS and PMB groups in trisaccharide 13

were simultaneously removed by treatment with TFA in CH₂Cl₂

to give diol 14 in 88% yield. Diglycosylation of 14 was achieved

with an excess of glycosyl donor 15 under the catalysis of

TMSOTf, giving pentasaccharide 16 in 54% yield. The phthal-

imido groups were then changed to the acetamido groups in two

steps by treatment with hydrazine hydrate followed by acetyla-

tion with Ac₂O/pyridine to aﬀord 17, which was subject to

catalytic hydrogenation and subsequent O-acetylation to give the

peracetate (18) in 89% yield. Initial attempts to form the sugar

oxazoline by the Lewis acid-catalyzed reaction using TMSBr/BF₃

3

Et₂O/2,4,6-collidine as the promoter^59,60gave only a moderate

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Scheme 2. Synthesis of a Glucose-Containing N-Glycan Oxazoline ^a

^aReagents and conditions: (a) NIS, TfOH, dichloroethane, 63%. (b) DDQ, CH₂Cl₂, H₂O, 87%. (c) Et₃SiH, PhBCl₂, CH₂Cl₂, 94%. (d) TMSOTf,

CH₂Cl₂, 95%. (e) AcSH, pyridine, CHCl₃, 78%. (f) 1) MeONa, MeOH, then Dowex (H⁺); 2) Pd(OH)₂/C, H2, MeOH; 3) Ac₂O, pyridine, 92% (three

steps). (g) 1) MeONa, MeOH, then Dowex (H⁺); 2) DMC, Et₃N, H₂O, 95% (two steps).

formation from the O-acetylated derivative. The synthesis of the

bisecting LacNAc-containing sugar oxazoline (2) started with

enzymatic extension of the sugar chain in tetrasaccharide 20. We

found that addition of a β-1,4-linked galactose to the bisecting

GlcNAc could be readily achieved by incubation of 20 with UDP-

Gal and a β-1,4-galactosyltransferase from bovine milk in a

phosphate buﬀer, giving hexasaccharide 21 in quantitative yield.

Compound 21 was then converted into hexasaccharide oxazoline

2 in a single step upon treatment with an excess of DMC and

Et₃N (Scheme 1). The bisecting mannose-containing oxazoline,

Man4GlcNAc-oxazoline (3), was synthesized following the pre-

viously reported procedure.⁴⁵

Synthesis of Core-Glucose-Containing Glycan Oxazoline

(4). Another sugar oxazoline that would be interesting for structureÀ

activity relationship studies related to Fc glycosylation is the tetra-

saccharide oxazoline (4), in which the core-β-mannose moiety

was replaced with a glucose moiety. Oxazoline 4 was previously

synthesized by Fairbanks and co-workers and was shown to be a

substrate for Endo-A.⁴⁶We describe here an alternative synthesis

of 4 (Scheme 2). Briefly, coupling of thioglycoside 22⁶²and

acceptor 23⁵⁶gave the β-linked disaccharide 24. the PMB group

was selectively removed by DDQ to provide 25, which was then

converted to diol 26 in 94% yield by regioselective reductive ring-

opening of the benzylidene group using Et₃SiH/PhBCl₂in

CH₂Cl₂. Glycosylation of 26 with glycosyl donor 27 under the

catalysis of TMSOTf, with simultaneous introduction of two

mannose residues, afforded tetrasaccharide 28 in 95% yield. A

one-step transformation of the azido group to an acetamido

group was carried out by treatment of 28 with AcSH in pyridine

to give 29 (78% yield), which was then converted into the per-

acetate derivative (30) by sequential de-O-acylation, catalytic

hydrogenation, and O-acetylation. Finally, compound 30 was

de-O-acetylated and the resulting free oligosaccharide was con-

verted to sugar oxazoline 4 in excellent yield through the single-

step, DMC-promoted oxazoline-forming reaction (Scheme 2).

The synthesis of Man3GlcNAc-oxazoline (5) and ManGlcNAc-

oxazoline (6) was previously reported.⁶⁰

Expression of Human IgG1-Fc Homodimer Carrying a

Homogeneous Man9GlcNAc2 Glycan at Each of the Fc

Glycosylation Sites (Asn-297). We have previously expressed

human IgG1-Fc in yeast Pichia pastoris to obtain a recombinant

IgG1-Fc carrying yeast N-glycans.³⁶The N-glycans were a

mixture of yeast high-mannose type glycans ranging from

Man9GlcNAc2 to Man13GlcNAc2. To obtain a homogeneous,

human-like Man9GlcNAc2 glycoform of the Fc domain, we

expressed human IgG1-Fc in Chinese hamster ovary (CHO)

cell lines in the presence of kifunensine, a potent class I

α-mannosidase inhibitor that blocks further processing of the

Man9GlcNAc2 N-glycan during glycoprotein biosynthesis.^63À65

It has been previously reported that expression of a monoclonal

antibody in CHO or human HEK293 cell lines in the presence of

kifunensine could efficiently control the N-glycosylation at the

high-mannose type stage.^11,66Recently we have successfully

overproduced Man9GlcNAc2-glycoform of Fc-fused HIV-1 V3

domain using kifunensine as the α-mannosidase inhibitor.⁶⁷

Following this protocol, we overproduced human IgG1-Fc in

CHO cell lines in the presence of kifunensine (2 μg/mL). The

recombinant Fc was purified by protein A affinity chromatogra-

phy. SDS-PAGE analysis indicated that the recombinant Fc

expressed in the presence of kifunensine appeared as a single

band of a size of ∼65 kDa and ∼33 kDa under nonreducing and

reducing conditions, respectively (Figure 3A, lanes 1 and 2). This

result suggests that the recombinant IgG1-Fc was produced as a

homodimer. Treatment of the IgG1-Fc with peptide:N-glycosi-

dase F (PNGase F), which would completely deglycosylate the

glycoprotein by hydrolyzing the glycosylamide linkage between

N-glycan and the Asn residue, gave a new single band that was

about 2 kDa less than the glycosylated monomeric IgG1-Fc

(Figure 3A, lane 3, shown under reducing SDS-PAGE con-

ditions). These results implicate the attachment of two N-glycans

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Figure 3. SDS-PAGE and MALDI-TOF MS analysis of recombinant IgG1-Fc. (A) SDS-PAGE: Lane M, protein marker; lane 1, HM-Fc (nonreducing

conditions); lane 2, HM-Fc, reducing conditions; lane 3, PNGase F-treatment of HM-Fc; (B) MALDI-TOF MS of HM-Fc; (C) MALDI-TOF MS of

N-glycans released from HM-Fc; (D) MALDI-TOF MS of N-glycans released from CHO-expressed Fc (CT-Fc).

Scheme 3. Chemoenzymatic Synthesis of Homogeneous Glycoforms of Human IgG1-Fc ^a

^aReagents and conditions. The high-mannose IgG1-Fc glycoform was produced in CHO cells in the presence of kifunensine. Then the high-mannose

N-glycan was removed by Endo-H to give the GlcNAc-containing IgG1-Fc, which served as an acceptor for the Endo-A-catalyzed transglycosylation with

respective synthetic sugar oxazolines (1 to 6) to aﬀord the corresponding homogeneous IgG1-Fc glycoforms.

in the HM-Fc homodimer. The recombinant HM-Fc was also

characterized by direct MALDI-TOF MS analysis of the Fc

homodimer, which gave a species with an average m/z of

65447 (Figure 3B). The experimental data was in good agree-

ment with the theoretical molecular weight (M = 65444 Da) of

Fc homodimer carrying two high-mannose type (Man9Glc-

NAc2) N-glycans, which was calculated on the basis of the amino

acid sequence of the recombinant Fc plus two Man9GlcNAc2

glycans (Figure S1, Supporting Information). The N-glycans in

the recombinant IgG1-Fc was further verified by MALDI-TOF

MS analysis. The MS spectrum of the PNGase F-released N-

glycans indicated that more than 90% of the N-glycans attached

were Man9GlcNAc2, with Man8GlcNAc2 being a minor fraction

(Figure 3C). In contrast, the N-glycans released from the wild-

type recombinant IgG1-Fc produced in CHO cell lines in the

absence of kifunensine, designated as CT-Fc, were a mixture of

biantennary complex-type N-glycans that were fucosylated and

contained 0, 1, and 2 terminal galactose residues, respectively

(designated as G0F, G1F, and G2F glycoforms) (Figure 3D).

Synthesis of IgG-Fc Homogeneous Glycoforms through

Chemoenzymatic Glycosylation Remodeling. Our prelimin-

ary studies have shown that the Endo-A-catalyzed transglycosy-

lation with Man3GlcNAc oxazoline (5) could be efficiently

applied to the seemingly hindered GlcNAc residues of the Fc

homodimer (GlcNAc-Fc) to make a homogeneous glycoform of

IgG-Fc under very mild conditions, without the need of denatur-

ing the Fc domain. Thus, the native structure of IgG1-Fc

homodimer was kept intact during the enzymatic deglycosylation

and subsequent enzymatic transglycosylation.³⁶In the present

work, we used the high-mannose type IgG-Fc homodimer (HM-

Fc) that was expressed in CHO cell line in the presence of

kifunensine as the starting material for glycosylation remodeling

to prepare a panel of new homogeneous IgG-Fc glycoforms, as

shown in Scheme 3. Treatment of HM-Fc with Endo-H under

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Figure 4. SDS-PAGE and MALDI-TOF MS analysis of GlcNAc-Fc and transglycosylation product Fc-1. A, SDS-PAGE: Lane 1, HM-Fc; lane 2,

GlcNAc-Fc; lane 3, transglycosylation product Fc-1; lane 4, PNGase F-treatment of Fc-1; B, MALDI-TOF MS of GlcNAc-Fc; C, MALDI-TOF MS of

product Fc-1; D, MALDI-TOF MS of N-glycans released from Fc-1.

mild conditions (phosphate buffer, pH 6.5, 23 °C) led to an

efficient conversion of HM-Fc (Figure 4A lane 1) to the GlcNAc-

Fc (Figure 4A, lane 2). The identity of the GlcNAc-Fc homo-

dimer was confirmed by its MALDI-TOF MS (Calculated, M =

62119 Da; found (m/z), 62122) (Figure 4B). We first examined

the Endo-A-catalyzed reaction of bisecting GlcNAc oxazoline 1

and the GlcNAc-Fc homodimer (donor/acceptor, 20:1, molar

ratio; 10 mol equiv to each GlcNAc moiety of the homodimer) in

a phosphate buffer (pH 7.0), and monitored the reaction by SDS-

PAGE. We found that the bisecting GlcNAc-containing oxazo-

line (1) was an excellent substrate for Endo-A and the transgly-

cosylation reaction went smoothly to give a product, which

appeared as a new band that was about 1 kDa larger than the

GlcNAc-Fc under reducing SDS-PAGE conditions (Figure 4A,

lane 3 vs lane 2). After 3 h, all the GlcNAc-Fc was converted to

the new product. The single band for the glycosylated IgG-Fc

under the reducing conditions suggests that both the glycosyla-

tion sites in the Fc homodimer were glycosylated. (The Fc

homodimer would be reduced to monomeric Fc on SDS-PAGE

in the presence of 2-mercaptoethanol. If any one of the two

glycosylation sites were not transglycosylated, it would show up

as the monomeric GlcNAc-Fc in SDS-PAGE.) The product, Fc-1,

was readily purified by affinity chromatography on a protein A

column. Treatment of glycoform Fc-1 with PNGase F led to

the removal of the N-glycans, giving a single band that was

about 1 kDa smaller than the Fc-1 under reducing conditions

(Figure 4A, lane 4 vs lane 3). Since PNGase F specifically

hydrolyzes the amide linkage between the N-glycan and the

Asn residue in the protein, the result suggests that the transferred

glycans were specifically attached to the GlcNAc moieties in the

GlcNAc-Fc homodimer to form an intact N-glycan in the

product, rather than on any other amino acid residues. MAL-

DI-TOF MS spectrum (Figure 4C) of the Fc-1 showed a single

species (m/z) at 63909, which matched well with the expected

molecular mass (63905 Da) of the Fc homodimer carrying two

bisecting-GlcNAc-Man3GlcNAc2 glycans. Further characteriza-

tion of the product was performed by analysis of the attached

N-glycans. The N-glycans were first released by PNGase F and

were then analyzed by MALDI-TOF MS: calculated for GlcNAc-

Man3GlcNAc2, M = 1113.41 Da; Found (m/z), 1136.80 [M +

Na]⁺(Figure 4D). It should be noted that we have previously

demonstrated that terminal GlcNAc in an N-glycan was itself

eligible to serve as an acceptor for enzymatic transglycosyla-

tion.^42,68In the present case, we did not observe cross-trans-

glycosylation to the bisecting GlcNAc moiety under the trans-

glycosylation conditions used (5 mM sugar oxazoline, 0.5 mM

GlcNAc-Fc). This result suggests that the bisecting GlcNAc

moiety might be relatively hindered in comparison with the

terminal GlcNAc on the α1,3- and α1,6- arms of the N-glycan

core.⁴²Nevertheless, we did observe self-condensation of the

bisecting GlcNAc-containing Man3GlcNAc-oxazoline (1) when

a much higher concentration (50 mM) of the sugar oxazoline (1)

alone was incubated with a larger amount of Endo-A, leading to

the formation of a series of novel oligomers (data not shown). A

more detailed study on this self-condensation reaction of the

sugar oxazoline will be described elsewhere.

Transglycosylation of GlcNAc-Fc with the bisecting LacNAc-

containing glycan oxazoline (2) was performed in the similar way

as for the preparation of the bisecting GlcNAc glycoform (Fc-1).

It was found that the bisecting LacNAc-containing glycan oxazo-

line (2) was also an excellent substrate for Endo-A, leading to the

formation of another Fc glycoform, Fc-2. Again, when an excess

of sugar oxazoline was used (10-fold molar excess for each

GlcNAc acceptor in the GlcNAc-Fc homodimer), the transgly-

cosylation could go to completion and the product (Fc-2) was

readily puriﬁed by Protein A aﬃnity chromatography. The

identity of glycoform Fc-2 was characterized by MALDI-TOF

MS analysis of both the intact glycoprotein and the N-glycan

released by PNGase F treatment: MALDI-TOF MS of Fc-2,

Calculated for Fc-2, M = 64229 Da; found (m/z), 64222.

MALDI-TOF MS of the attached N-glycan, calculated for

LacNAc-Man3GlcNAc2, M = 1275.46 Da; found (m/z),

1298.40 [M + Na]⁺. The Endo-A-catalyzed transglycosylation

of GlcNAc-Fc with the other sugar oxazolines, including Man4-

GlcNAc-oxazoline (3), Man2GlcGlcNAc-oxazoline (4), Man3-

GlcNAc-oxazoline (5),⁵⁸and ManGlcNAc-oxazoline (6),⁶⁰was

performed in a similar way to give the corresponding glycoforms,

Fc-3 to Fc-6, respectively (Scheme 3). Except for the enzymatic

reaction with the disaccharide oxazoline (6), which was much

slower than the reaction with sugar oxazoline 1, all the enzymatic

transglycosylations gave the corresponding products eﬃciently.

In the case of disaccharide oxazoline 6, more sugar oxazoline (15-

fold molar excess for each GlcNAc residue in the GlcNAc-Fc

homodimer) was added to push the transglycosylation to com-

pletion. Again, the Fc glycosylation products were puriﬁed by

protein A aﬃnity chromatography and characterized by MALDI-

TOF MS of the intact glycoprotein as well as the N-glycans

released (see Experimental Section).

We also attempted to make a homogeneous Fc glycoform

carrying a full-length biantennary complex-type N-glycan. Endo-

A is an endoglycosidase speciﬁc for the hydrolysis and transgly-

cosylation of high-mannose type N-glycans, but we have recently

found that Endo-A also possesses a low activity on complex-

type N-glycan oxazoline for transglycosylation without product

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Figure 5. SPR sensorgrams of the binding of various Fc glycoforms to FcγIIIa receptor from a representative experiment. Fc glycoforms were

immobilized by Protein A capture, and the binding was analyzed by injecting the respective Fcγ receptors at varied concentrations.

hydrolysis when a large amount of Endo-A and a high concen-

tration of substrates were used.⁴¹Thus, we tested the ability of

Endo-A to glycosylate GlcNAc-Fc using a complex type of glycan

oxazoline as the donor substrate (see Figure S2, Supporting

Information [SI]). SDS-PAGE analysis of the reaction mixture

after 3 h incubation did not indicate the formation of a

transglycosylation product (SI, Figure S2, lane 3). This result

suggests that, although Endo-A is eﬃcient to make various Fc

glycoforms carrying the core N-glycans, its activity is not

suﬃcient to transfer the full-length complex-type N-glycan to

GlcNAc-Fc domain. We then tested the mutants of the fungus

enzyme, EndoM-N175A and EndoM-175Q. We have previously

reported that the two glycosynthase mutants were able to transfer

complex-type glycan oxazolines to GlcNAc-ribonuclease and

GlcNAc-peptides, using the corresponding sugar oxazoline

(CT-oxazoline) as the donor substrate.^38,43,44,69When the CT-

oxazoline and GlcNAc-Fc were incubated with EndoM-N175A,

SDS-PAGE analysis did not reveal the formation of any trans-

glycosylation product (SI, Figure S2, lane 4). Under the same

reaction conditions, the EndoM-N175Q mutant did not glyco-

sylate the GlcNAc-Fc either (data not shown). Surprisingly,

Endo-M (either the wild-type or the mutants, N175A or

N175Q) also failed to transfer the simple Man3GlcNAc-oxazo-

line to GlcNAc-Fc. Given the fact that EndoM-N175A and

EndoM-N175Q could eﬃciently transfer the complex-type

sugar oxazoline and simple Man3GlcNAc-oxazoline to GlcNAc-

peptides and even GlcNAc-containing ribonuclease B,^38,43,44,69

the present results suggest that the active site of Endo-M enzyme

might not be able to access to the GlcNAc moiety in acceptor

GlcNAc-Fc, which was sandwiched by the two Fc domains and

could be sterically hindered. As Endo-A could eﬃcient transfer

various core-modiﬁed N-glycan oxazolines to GlcNAc-Fc, the

failure of Endo-A to transfer complex-type glycan oxazoline

might simply reﬂect the intrinsic low activity of Endo-A on

complex-type N-glycans, rather than its recognition to acceptor

GlcNAc-Fc. These results indicate the diﬀerence in acceptor

substrate ﬂexibility for the two endoglycosidases (Endo-A and

Endo-M). We are currently working on site-directed mutagenesis

of Endo-A and are also testing other endoglycosidases in order to

ﬁnd an enzyme or mutants capable of eﬀectively attaching full-

length complex-type N-glycans to IgG-Fc to make homogeneous

complex-type IgG-Fc glycoforms.

Binding of IgG-Fc Glycoforms with the Stimulatory

Fcγ Receptor (FcγRIIIa) and the Inhibitory Fcγ Receptor

(FcγRIIb). With the recombinant and synthetic Fc glycoforms

in hands, we evaluated their affinity to Fcγ receptors FcγRIIIa

and FcγRIIb using SPR technology. In our previous study,³⁶we

directly immobilized FcγRIIIa on the chips and flowed different

IgG-Fc glycoforms at varied concentrations. While the direct

“random” immobilization provided a quick comparison of the

binding of different Fc glycoforms when the same chip was used,

the affinity as measured by the apparent K_Dvalues (found at μM

concentrations for the interaction between wild-type IgG-Fc and

FcγRIIIa) seemed to be significantly underestimated, probably

due to partial denaturing of the receptor structure during random

immobilization. In the present study, we measured the affinity by

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Table 1. Binding Aﬃnities of IgG-Fc Glycoforms to Fcγ Receptors FcγRIIIa and FcγRIIb^a

^aThe aﬃnity was measured on a BIAcore T100, and the K_Dvalues were calculated using an equilibrium binding model by ﬁtting the SPR data. ^b)The K_D

data (mean ( S.D.) were obtained from a set of two independent expreiments. ^c)Relative aﬃnity was calculated as K_D(natural complex-type Fc)/K_D

(variant Fc glycoforms). ^d)Not determined.

site-specific immobilization of individual Fc glycoforms through

their affinity capture with preimmobilized protein A on the chips,

as demonstrated in previous reports for site-specific immobiliza-

tion of monoclonal antibodies.^34,70The Fcγ receptors at various

concentrations were injected as analytes. A typical SPR profile for

the interactions between Fc glycoforms and the FcγRIIIa is

shown in Figure 5. The K_Dvalues for the binding of the various

Fc glyocoforms to Fcγ receptors FcγRIIIa and FcγRIIb, which

were obtained by fitting the binding data with a 1:1 steady-state

binding model using BIAcore T100 evaluation software, are

listed in Table 1. We found that the site-directed immobilization

of the Fc glycoforms by affinity capture provided an accurate and

highly reproducible measurement of the affinity of Fc glycoforms

to Fcγ receptors, as it provides a consistent surface orientation of

the immobilized proteins from experiment to experiment. The

protein A chips could be easily regenerated and reused for

capturing and measuring different Fc glycoforms. It should be

pointed out that protein A binding (capture) would not interfere

with the interaction between Fc domain and Fcγ receptors, as

the protein A binds the C_H2/C_H3 interface of the Fc domain,

while the Fcγ receptors’ binding sites locate presumably at the

interface of the low hinge region and the up C_H2 domain.⁷¹

Using the K_Das an estimate of the relative aﬃnity, we found

that the simple GlcNAcMan3GlcNAc2-Fc glycoform (Fc-1)

carrying a bisecting GlcNAc moiety had the highest aﬃnity for

FcγRIIIa (K_D= 22 nM). This is about 6-fold as high as that of

the wild-type IgG-Fc (CT-Fc) carrying the fucosylated complex-

type N-glycans (K_D= 136 nM), 2-fold as high as that of the

Man9GlcNAc2 glycoform (HM-Fc) (K_D= 45 nM), and

about 3-fold as high as that of the Man3GlcNAc2-glycoform

(Fc-5, K_D= 58 nM) (Table 1). Interestingly, the Fc glycoforms with

an unnatural bisecting LacNAc moiety (Fc-2) (K_D= 41 nM) or a

mannose moiety (Fc-3) (K_D= 47 nM) also showed enhanced

aﬃnity in comparison with the Man3GlcNAc2 Fc-glycoform

(Fc-5, K_D= 58 nM), although the enhancement is less signiﬁcant

than the addition of the natural bisecting GlcNAc moiety. These

results indicate that adding a bisecting sugar moiety to the core

N-glycan could enhance the aﬃnity of Fc domain to FcγIIIa

receptor, but the enhancement depends on the nature of the

sugar moiety inserted. Trimming the high-mannose structure

from Man9GlcNAc2 down to the pentasaccharide core-Man3-

GlcNAc2 only slightly decreased the aﬃnity (Man9-Fc, K_D

=

45 nM; Fc-5, K_D= 58 nM). However, removal of the two outer

mannose residues from the Man3GlcNAc2 core to the Man-

GlcNAc2 glycoform (Fc-6, K_D= 354 nM) resulted in about a

6-fold drop of the Fc aﬃnity to FcγRIIIa. Further trimming of the

oligosaccharide down to the glycoform (GlcNAc-Fc, K_D> 3000

nM), which carries only the innermost GlcNAc moiety, resulted

in dramatic loss of the Fc aﬃnity. These results clearly indicate

the essential role of the pentasaccharide core at the Fc domain for

its high aﬃnity binding to FcγRIIIa. On the other hand, changing

the core-β-mannose moiety in the Man3GlcNAc2- glycoform

(Fc-5, K_D= 58 nM) to a glucose moiety such as the Man2-

GlcGlcNAc2-glycoform (Fc-4, K_D= 79 nM) did not have

signiﬁcant eﬀect on the Fc aﬃnity, suggesting that the C-2

conﬁguration of the core-β-mannose moiety in the Fc N-glycan

is not critical for Fc recognition of FcγRIIIa. Taken together, our

experimental data clearly indicate that the Fc domain aﬃnity to

FcγRIIIa could be precisely tuned by the ﬁne structures of the Fc

N-glycan. Our experimental data with the pure, homogeneous

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synthetic Fc glycoforms provided unambiguous evidence show-

ing that the presence of a bisecting GlcNAc moiety on the Fc

glycan core could directly enhance the binding of Fc to FcγRIIIa.

This result clariﬁes a long-standing controversial problem in the

ﬁeld as to the role of bisecting GlcNAc.^19,21,26À28The bisecting

GlcNAc moiety plays both a direct and an indirect role in en-

hancing antibody Fc aﬃnity to FcγRIIIa. On one hand, the

presence of a bisecting GlcNAc moiety can directly impact the Fc

aﬃnity to FcγRIIIa, independent of core-fucosylation. On the

other hand, preaddition of a bisecting moiety to the N-glycan

core can inhibit the enzymatic attachment of a core-fucose

moiety during biosynthesis, playing an indirect role in enhancing

the aﬃnity of Fc to FcγRIIIa resulting from reduced population

of the nonfucosylated Fc glycoforms.

We also measured the aﬃnity of selected Fc glycoforms to the

inhibitory Fcγ receptor FcγRIIb. FcγRIIb is an important

regulator of both adaptive and innate immunity.⁴We found that

the Fc glycoforms with or without the bisecting sugar moiety

(comparing Fc-1, Fc-2, and Fc-3 to Fc-5) showed similar aﬃnity

to FcγRIIb (K_D= 2À3 μM) (Table 1). Thus, in contrast to the

observation that addition of a bisecting GlcNAc moiety signiﬁ-

cantly enhanced Fc aﬃnity to the activating receptor (FcγRIIIa),

the presence of a bisecting sugar moiety had little eﬀect on the

aﬃnity to the inhibitory Fcγ receptor. The similar binding

aﬃnity of the Fc glycoform carrying the core-fucosylated

N-glycan (CT-Fc) to those without a core fucose moiety (HM-Fc,

Fc-1, Fc-2, Fc-3, and Fc-5) also indicated that the presence of a

core fucose moiety in the N-glycan did not have much inﬂuence

on the binding of Fc to FcγRIIb. However, when the Fc N-glycan

was trimmed oﬀ to leave only the trisaccharide ManGlcNAc2

core or the innermost GlcNAc moiety (Fc-6 and GlcNAc-Fc),

the binding of the Fc domain to FcγRIIb became very weak, for

which we did not obtain an accurate K_Dvalue. This observation is

consistent with the previously reported observation on FcγRIIb-

binding of core-fucosylated and truncated IgG-Fc glycoforms.³⁴

Thus, it seems that the pentasaccharide (Man3GlcNAc2) core of

the Fc N-glycan is essential and suﬃcient for the binding of Fc

domain to FcγRIIb, but further decoration such as addition of a

bisecting GlcNAc moiety to the core has little eﬀect on the

aﬃnity.

Identiﬁcation of high-aﬃnity FcγRIIIa-binding glycoforms

with enhanced ADCC function is clinically signiﬁcant to address

the issue of Fcγ receptor polymorphism found in cancer patients

who are less or not responsive to the treatment with common

MAbs. In these patients, the FcγRIIIa-F158 allele has low aﬃnity

to therapeutic antibody such as rituximab in comparison with

the high-aﬃnity receptor FcγRIIIa-V158 allele.^72À74ADCC func-

tion was also reported to be an important mechanism for

achieving protective immunity for HIV-neutralizing antibodies.⁷⁵

Our synthetic and Fcγ receptor-binding studies of various pure

Fc glycoforms demonstrate that addition of an appropriate

bisecting sugar moiety at the N-glycan core could signiﬁcantly

enhance the aﬃnity of Fc to the activating receptor (FcγRIIIa)

while having little eﬀects on aﬃnity to the inhibitory receptor

(FcγRIIb), pointing to a way to enhance the ADCC function of

antibodies. It should be pointed out that previous structural and

functional studies have suggested that the impact of Fc glycan

structures on the aﬃnity of Fc domain to Fcγ receptors are

mainly due to their eﬀects on the functional conformations of the

Fc domain that are critical for the interactions between Fc

domain and respective Fcγ receptors.^9À15Indeed, the Fc oligo-

saccharide can form multiple noncovalent interactions with the

protein surface of the C_H2 domain and, as shown in the crystal

structures, a large portion of the glycans appears sandwiched

between the two C_H2 domains. Nevertheless, it is still not clear

how the addition of the bisecting GlcNAc or other (unnatural)

bisecting sugar moiety precisely aﬀects the local and/or global

conformations of the Fc domain to favor its binding to FcγRIIIa.

The synthetic, homogeneous Fc glycoforms obtained in the

present work should be valuable for NMR and X-ray crystal-

lographic structural studies for deciphering the precise molecular

mechanism by which ﬁne Fc N-glycans modulate the interaction

between Fc domain and Fcγ receptors.

’ CONCLUSION

A convergent chemoenzymatic synthesis of an array of homo-

geneous IgG-Fc glycoforms was achieved through the Endo-A-

catalyzed glycosylation remodeling approach. The results indi-

cate that Endo-A is remarkably eﬃcient to introduce various

modiﬁed N-glycan core to Fc domain by transglycosylation with

the N-glycan core oxazolines. However, either Endo-A or the

Endo-M mutant (EndoM-N175A and EndoM-N175Q) was

unable to eﬃciently transfer a full-length complex-type N-glycan

to the Fc domain, implicating the limitations of Endo-A and

Endo-M for IgG-Fc glycosylation remodeling. The availability of

the synthetic homogeneous Fc glycoforms allowed a clear

assessment on how the individual sugar residues within or

ﬂanking the pentasaccharide Man3GlcNAc2 core aﬀect the

binding of Fc to Fcγ receptors, FcγRIIIa and FcγRIIb. Speciﬁ-

cally, our SPR binding studies provide unambiguous evidence

that the presence of a bisecting sugar moiety at the N-glycan core,

such as a GlcNAc or a mannose moiety, could directly enhance

the aﬃnity of Fc to FcγIIIa receptor, independent of Fc

fucosylation. In contrast, the presence of a bisecting sugar moiety

shows no eﬀect on the intercation between Fc and the inhibitory

Fcγ receptor FcγRIIb. These results implicate a new way to

enhance antibody’s ADCC function by introducing an appro-

priate bisecting sugar moiety at the Fc glycan. To further expand

the scope of this chemoenzymatic method for IgG-Fc glycosyla-

tion remodeling, we are currently performing site-directed

mutagenesis of Endo-A and are also testing other endoglycosi-

dases in order to ﬁnd an enzyme or mutants capable of eﬀectively

transferring full-length complex-type N-glycans to IgG-Fc do-

main. We look forward to reporting new ﬁndings from these

studies.

’ EXPERIMENTAL SECTION

Materials and Methods. Endo-β-N-acetylglucosaminidase

from Arthrobacter protophormiae (Endo-A) was overproduced

in E. coli following the reported procedure.⁷⁶Bovine milk β-1,

4-galactosyltransferase was purchased from Sigma-Aldrich (St.

Louis, MO). PNGase F was purchased from New England

Biolabs (Ipswich, MA). UDP-Gal was purchased from EMD

Chemicals Inc. (Gibbstown, NJ). All other reagents were pur-

chased from Sigma-Aldrich (St. Louis, MO) without further

purification. TLC was performed using Silica-gel on aluminum

plates (Sigma-Aldrich). Flash column chromatography was per-

formed on silica gel 60 (230À400 mesh). NMR spectra were

recorded on JEOL ECX 400 MHz spectrometer. The chemical

shifts were assigned in ppm. Analytical RP-HPLC was performed

on a Waters 626 HPLC instrument with a Symmetry300^TMC18

column (3.5 μm, 4.6 mm Â 250 mm) at 40 °C. The column was

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eluted with a linear gradient 0À90% MeCN containing 0.1%

TFA for 20 min at the flow rate of 1.0 mL/min. MALDI-TOF/

MS spectra were recorded on an Autoflex MALDI-TOF mass

spectrometer (Bruker Daltonics, Billerica, MA) by using 2,5-

dihydroxybenzoic acid as a matrix under positive ion conditions.

High-resolution mass spectra (HRMS) were measured on a

MALDI-TOF/TOF 4800 spectrometer (Applied Biosystems)

with 2,5-dihydroxybenzoic acid as matrix and Cal 4700 standard

peptide mixture (Applied Biosystems) was used as the internal

standard.

Synthesis of Benzyl 2-O-Benzyl-6-O-tert-butyldimethylsi-

lyl-3-O-p-methoxybenzyl-β-^D-mannopyranosyl-(1f4)-3,6-

di-O-benzyl-2-deoxy-2-phthalimido-β-^D-glucopyranoside

(11). To a solution of 10 (455 mg, 0.48 mmol) in pyridine (5 mL)

was added tert-butyldimethylchlorosilane (144 mg, 0.96 mmol)

at room temperature. After 2 h, the reaction solution was diluted

with CH₂Cl₂, washed with saturated NaHCO₃and brine, and

dried over MgSO₄. The solvent was evaporated and the residue

was purified by column chromatography on silica gel (hexanes/

EtOAc, 3:1) to provide 11 (483 mg, 95%) as a white amorphous

powder. ESI-MS: calcd for C₆₂H₇₁NO₁₃Si, M = 1066.31; Found

(m/z), 1089.58 [M + Na]⁺; ¹H NMR (400 MHz, CDCl₃, TMS)

δ 7.87À6.74 (m, 28H), 5.11 (m, 1H), 4.87À4.75 (m, 4H), 4.67

(d, 1H, J = 11.9 Hz), 4.53À4.38 (m, 6H), 4.22À4.18 (m, 2H),

4.04À3.94 (m, 2H), 3.81 (dd, 1H, J = 10.1, 4.6 Hz), 3.78 (s, 3H),

3.74À3.67 (m, 2H), 3.65À3.59 (m, 2H), 3.51 (m, 1H), 3.36 (s,

1H), 3.22À3.13 (m, 2H), 0.84 (s, 9H), 0.02, À0.01 (2s, 6H); ¹³C

NMR (100 MHz, CDCl₃) δ 167.67, 159.11, 138.89, 138.83,

137.86, 137.16, 133.48, 131.59, 130.45, 129.18, 128.46, 128.07,

128.05, 127.80, 127.75, 127.68, 127.60, 127.55, 127.49, 127.24,

126.67, 123.07, 113.73, 101.29, 97.32, 81.23, 78.98, 76.79, 75.07,

74.74, 74.32, 74.20, 74.11, 73.48, 71.53, 70.65, 68.57, 65.38,

55.65, 55.20, 25.74, 18.02, À5.65, À5.70.

Synthesis of Benzyl 2-O-Benzyl-4,6-O-benzylidene-3-O-

p-methoxybenzyl-β-^D-mannopyranosyl-(1f4)-3,6-di-O-ben-

zyl-2-deoxy-2-phthalimido-β-^D-glucopyranoside (9). A mixture

of phenyl 2-O-benzyl-4,6-O-benzylidene-3-O-p-methoxybenzyl-

1-thio-α-^D-mannopyranoside 8⁵⁷(140 mg, 0.25 mmol), BSP

(56.7 mg, 0.27 mmol), TTBP (122 mg, 0.49 mmol), and

activated 3 Å molecular sieves (588 mg) in CH₂Cl₂(4.9 mL) was

stirred for 20 min at À60 °C under an argon atmosphere. Then

Tf₂O (50 μL, 0.30 mmol) was added, followed by the addition of

a solution of benzyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-^D-

glucopyranoside 7 (111 mg 0.19 mmol) in CH₂Cl₂(4.8 mL).

The mixture was stirred at À60 °C for 1 h and then warmed to

room temperature over a period of 1 h. The mixture was filtered

through a Celite pad. The filtrate was diluted with CH₂Cl₂,

washed sequentially with saturated NaHCO₃and brine. The

organic layer was dried over MgSO₄, filtered, and concentrated.

The residue was subjected to silica gel column chromatography

(hexanes/EtOAc, 7:2) to afford 9 (112 mg, 56%) as a white

amorphous powder. ESI-MS: calcd for C₆₃H₆₁NO₁₃, M =

Synthesis of Benzyl 3,4,6-Tri-O-acetyl-2-deoxy-2-phthali-

mido-β-^D-glucopyranosyl-(1f4)-2-O-benzyl-6-O-tert-butyl-

dimethylsilyl-3-O-p-methoxybenzyl-β-^D-mannopyranosyl-

(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-^D-glucopyra-

noside (13). A solution of 11 (23 mg, 22 μmol) and 2,3,4-tri-O-

acetyl-2-deoxy-2-phthalimido-β-^D-glucopyranosyl tricholoroa-

cetimidate 12⁵⁸(25 mg, 44 μmol) in CH₂Cl₂(1 mL) containing

activated 4 Å molecular sieves (120 mg) was stirred under an

atmosphere of argon at room temperature for 30 min. After

1

1039.41; Found (m/z), 1062.43 [M + Na]⁺; H NMR (400

MHz, CDCl₃, TMS) δ 7.77À6.79 (m, 33H), 5.51 (s, 1H), 5.11

(d, 1H, J = 8.3 Hz), 4.89À4.78 (m, 2H), 4.68À4.64 (m, 2H),

4.55À4.47 (m, 3H), 4.43À4.39 (m, 2H), 4.26À4.15 (m, 3H),

4.08À4.00 (m, 2H), 3.79 (s, 3H), 3.72 (m, 1H), 3.67 (dd, 1H, J =

11.3, 1.6 Hz), 3.59À3.53 (m, 2H), 3.47 (m, 1H), 3.41 (dd, 1H,

J = 9.9, 3.0 Hz), 3.14 (dt, 1H, J = 9.6, 4.6 Hz); ¹³C NMR (100

MHz, CDCl₃) δ 167.65, 159.08, 138.70, 138.59, 137.74, 137.61,

137.13, 133.53, 131.57, 130.54, 129.00, 128.75, 128.48, 128.23,

128.10, 127.90, 127.76, 127.70, 127.55, 127.51, 127.47, 126.82,

126.04, 123.11, 113.66, 101.92, 101.25, 97.32, 79.44, 78.60,

77.93, 76.95, 76.90, 74.90, 74.64, 74.55, 73.51, 72.21, 70.66,

68.50, 68.46, 67.27, 55.64, 55.20.

cooling to À20 °C, a solution of BF₃OEt₂in CH₂Cl₂(0.1M, 87μL,

3

8.7 μmol) was added, and the resulting mixture was stirred at

room temperature overnight. Triethylamine (10 μL) was then

added, and the mixture was filtered through a Celite pad. The

filtrate was sequentially washed with saturated NaHCO₃and

brine, dried over MgSO₄, filtered, and concentrated. The residue

was subjected to flash silica gel column chromatography

(hexanes/EtOAc, 5:2) to provide 13 (25 mg, 76%) as a white

amorphous powder. ESI-MS: calcd for C₈₂H₉₀₁N₂O₂₂Si, M =

1482.57; Found (m/z), 1505.81 [M + Na]⁺; H NMR (400

MHz, CDCl₃, TMS) δ 7.86À6.59 (m, 32H), 5.76 (dd, 1H, J =

10.8, 9.0 Hz), 5.56 (d, 1H, J = 8.7 Hz), 5.11 (t, 1H, J = 9.6 Hz),

5.03 (m, 1H), 4.79À4.69 (m, 4H), 4.63À4.58 (m, 2H),

4.47À4.36 (m, 5H), 4.26 (dd, 1H, J = 10.8, 8.5 Hz),

4.15À4.06 (m, 4H), 3.95À3.89 (m, 2H), 3.82 (s, 3H),

3.69À3.55 (m, 5H), 3.46 (m, 1H), 3.27À3.21 (m, 2H), 2.97

(m, 1H), 1.99, 1.93, 1.84 (3s, 9H), 0.77 (s, 9H), À0.04, À0.14

(2s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 170.69, 170.04,

169.48, 167.52, 158.80, 138.96, 138.90, 137.84, 137.15, 134.35,

133.26, 131.60, 131.20, 130.97, 128.40, 128.15, 128.02, 127.92,

127.82, 127.72, 127.51, 127.47, 127.43, 127.04, 126.47, 123.62,

122.97, 113.66, 100.95, 97.23, 97.07, 79.86, 78.73, 76.86, 76.23,

75.68, 74.65, 74.26, 73.99, 73.49, 73.41, 71.32, 71.13, 70.69, 70.57,

68.92, 68.60, 61.84, 61.78, 55.58, 55.31, 55.22, 25.68, 20.60, 20.56,

20.39, 17.98, À5.41, À5.56.

Synthesis of Benzyl 2-O-Benzyl-3-O-p-methoxybenzyl-β-

D-mannopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-ph-

thalimido-β-^D-glucopyranoside (10). A solution of 9 (50 mg,

48 μmol) in 80% aqueous solution of AcOH (5 mL) was stirred

at 40 °C. After stirring for 6 h, the mixture was concentrated and

purified by silica gel column chromatography (hexanes/EtOAc,

1:2) to afford 10 (36 mg, 80%) as a white amorphous powder.

ESI-MS: calcd for C₅₆H₅₇NO₁₃, M = 951.38; Found (m/z),

1

973.52 [M + Na]⁺; H NMR (400 MHz, CDCl₃, TMS) δ

7.87À6.82 (m, 28H), 5.14 (d, 1H, J = 7.8 Hz), 4.89À4.71 (m,

5H), 4.55À4.49 (m, 3H), 4.43À4.37 (m, 2H), 4.29À4.20 (m,

3H), 4.02 (dd, 1H, J = 9.6, 8.2 Hz), 3.81À3.69 (m, 8H), 3.60 (m,

1H), 3.45 (m, 1H), 3.17 (m, 1H), 3.11 (dd, 1H, J = 9.6, 2.8 Hz),

2.18 (d, 1H, J = 1.8 Hz), 1.98 (t, 1H, J = 6.6 Hz); ¹³C NMR (100

MHz, CDCl₃) δ 167.66, 159.37, 138.57, 138.42, 137.82, 137.14,

133.61, 131.56, 129.72, 129.25, 128.53, 128.20, 128.11, 127.84,

127.58, 127.53, 127.50, 127.41, 127.00, 123.17, 113.92, 101.10,

97.33, 81.63, 78.91, 76.80, 75.67, 74.71, 74.44, 74.29, 74.18,

73.62, 70.93, 70.67, 68.69, 67.15, 62.75, 55.63, 55.24.

Synthesis of Benzyl 3,4,6-Tri-O-acetyl-2-deoxy-2-phthali-

mido-β-^D-glucopyranosyl-(1f4)-2-O-benzyl-β-D-mannopy-

ranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-^D-

glucopyranoside (14). To a stirred solution of compound 13

(302 mg, 0.20 mmol) in CH₂Cl₂(20 mL) was added TFA

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(3.75 mL) at 0 °C. The solution was stirred at 0 °C for 1 h, and

MeOH (2 mL) was added. The mixture was sequentially washed

with saturated NaHCO₃and brine. The organic layer was then dried

over MgSO₄and filtered. The filtrate was concentrated by evapora-

tion, and the residue was purified by column chromatography on

silica gel (hexanes/EtOAc, 1:1) to yield 14 (224 mg, 88%) as a white

solid. ESI-MS: calcd for C₆₈H₆₈N₂O₂₁, M = 1248.43; Found (m/z),

91 μmol) was dissolved in EtOH/H₂O/NH₂NH₂monohydrate

(10:1:1, 12 mL), and the mixture was stirred at 80 °C overnight.

The mixture was concentrated in vacuo, and the residue was treated

with Ac₂O/pyridine (1:1, 10 mL) at room temperature overnight.

The mixture was concentrated, diluted with CH₂Cl₂, and washed

sequentially with 1 M HCl, saturated NaHCO₃, and brine. The

organic layer was dried over MgSO₄and filtered. The filtrate was

concentrated. Silica gel column chromatography (CH₂Cl₂/MeOH,

30:1) of the residue afforded compound 17 (122 mg, 77%) as a

white powder. Without further purification, compound 17 thus

obtained was dissolved in CH₂Cl₂ÀMeOHÀAcOH (20:80:1, v/v,

12 mL), and 20% palladium(II) hydroxide on activated carbon

(120 mg) was added. The mixture was vigorously stirred at room

temperature under hydrogen atmosphere overnight and then filtered

through a Celite pad. The filtrate was concentrated in vacuo. Pyridine

(10 mL) andAc₂O (10 mL) were added, and the mixture was stirred

at room temperature overnight. The mixture was concentrated,

diluted with CH₂Cl₂, washed sequentially with 1 M HCl, saturated

NaHCO₃, and brine. The organic layer was dried over MgSO₄and

filtered. The filtrate was concentrated. Silica gel column chromato-

graphy (CH₂Cl₂/MeOH, 45:1) of the residue afforded 18 (97 mg,

89% from 17) as a white amorphous powder. ESI-MS: calcd for

C₆₄H₈₈N₂O₄₁, M = 1541.37; Found (m/z): 1563.75 [M + Na]⁺;¹H

NMR (400 MHz, CDCl₃, TMS, selected signals) δ 6.07 (d, 1H, J =

3.7 Hz), 5.96À5.88 (m, 2H), 4.69 (d, 1H, J = 8.7 Hz), 2.21À1.94

(m, 51H); ¹³C NMR (100 MHz, CDCl₃) δ 171.02, 170.88, 170.55,

170.40, 170.35, 170.27, 170.23, 170.00, 169.83, 169.68, 169.60,

169.49, 169.06, 168.98, 100.07, 97.39, 96.80, 90.46, 75.13, 73.60,

73.14, 72.39, 72.05, 71.86, 71.20, 69.78, 68.87, 68.74, 68.48, 68.40,

66.37, 65.72, 65.23, 62.56, 62.06, 61.75, 61.67, 54.06, 50.59, 22.85,

22.61, 20.73, 20.57, 20.45, 20.36, 20.29, 20.20.

1

1271.58 [M + Na]⁺; H NMR (400 MHz, CDCl₃, TMS) δ

7.88À6.73 (m, 28H), 5.78 (dd, 1H, J = 10.6, 9.2 Hz), 5.42 (d,

1H, J = 8.7 Hz), 5.12 (t, 1H, J = 9.6 Hz), 5.05 (m, 1H), 4.86À4.65

(m, 5H), 4.47À4.41 (m, 3H), 4.36À4.30 (m, 2H), 4.27 (dd, 1H, J=

12.4, 2.3 Hz), 4.27 (dd, 1H, J = 12.4, 6.5 Hz), 4.16À4.12 (m, 2H),

3.98 (m, 1H), 3.89 (m, 1H), 3.85 (d, 1H, J= 2.8 Hz), 3.73 (t, 1H, J=

9.2 Hz), 3.67À3.63 (m, 2H), 3.56 (dd, 1H, J = 11.0, 3.3 Hz),

3.44À3.38 (m, 2H), 3.14 (m, 1H), 2.08, 2.04, 1.85 (3s, 9H); ¹³C

NMR (100 MHz, CDCl₃) δ 170.58, 169.91, 169.44, 167.68, 138.70,

138.43, 137.56, 137.09, 134.47, 133.49, 131.50, 128.48, 128.05,

127.92, 127.71, 127.50, 127.46, 127.34, 127.28, 126.80, 123.64,

123.08, 100.66, 98.53, 97.23, 79.13, 78.62, 77.43, 76.69, 74.85, 74.54,

74.26, 74.06, 73.51, 72.96, 71.84, 70.59, 70.34, 68.81, 68.02, 61.94,

61.15, 55.54, 54.53, 20.55, 20.52, 20.32.

Synthesis of Benzyl 2,3,4,6-Tetra-O-acetyl-α-D-mannopy-

ranosyl-(1f3)-[3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-

β-^D-glucopyranosyl-(1f4)]-[2,3,4,6-tetra-O-acetyl-α-D-manno-

pyranosyl-(1f6)]-2-O-benzyl-β-^D-mannopyranosyl-(1f4)-3,6-

di-O-benzyl-2-deoxy-2-phthalimido-β-^D-glucopyranoside (16).

A solution of 14 (50 mg, 40 μmol) and 2,3,4,6-tetra-O-acetyl-α-D-

mannopyranosyl tricholoroacetimidate 15⁷⁷(197 mg, 0.40 mmol) in

CH₂Cl₂(2.5 mL) containing activated 4 Å molecular sieves (296

mg) was stirred under an atmosphere of argon at room temperature

for 30 min. After cooling to À20 °C, a solution of TMSOTf in

CH₂Cl₂(1 M, 80 μL, 80 μmol) was added and the resulting mixture

was stirred at room temperature overnight. Triethylamine (10 μL)

was then added, and the mixture was filtered through a Celite pad.

The filtrate was sequentially washed with saturated NaHCO₃and

brine, dried over MgSO₄, and filtered. The filtrate was concentrated,

and the residue was purified by silica gel column chromatography

(hexanes/EtOAc, 2:3) to provide 16 (41 mg, 54%) as a white

amorphous powder. ESI-MS: calcd for C₉₆H₁₀₄N₂O₃₉, M = 1909.84;

Synthesis of 2-Methyl-[2,3,4,6-tetra-O-acetyl-α-D-manno-

pyranosyl-(1f3)-[2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-

β-^D-glucopyranosyl-(1f4)]-[2,3,4,6-tetra-O-acetyl-α-D-

mannopyranosyl-(1f6)]-2-O-acetyl-β-^D-mannopyranosyl-

(1f4)-3,6-di-O-acetyl-1,2-dideoxy-α-^D-glucopyrano]-[2,1-d]-

2-oxazoline (19). To a solution of 18 (55 mg, 36 μmol) in

dichloroethane (3.5 mL) containing activated 4 Å molecular sieves

(420 mg) were added 2,4,6-collidine (71 μL, 0.53 mmol), TMSBr

1

Found (m/z): 1910.68 [M + H]⁺; H NMR (400 MHz, CDCl₃,

(69 μL, 0.53 mmol), and BF₃OEt₂(67 μL, 0.53 mmol). The

3

TMS) δ 7.92À6.61 (m, 28H), 5.77 (dd, 1H, J = 10.5, 8.7 Hz), 5.64

(m, 1H), 5.45À5.39 (m, 2H), 5.34À5.20 (m, 4H), 5.11À5.04 (m,

3H), 4.93À4.81 (m, 3H), 4.77À4.73 (m, 2H), 4.64 (d, 1H, J = 12.8

Hz), 4.50À4.39 (m, 3H), 4.35À4.25 (m, 3H), 4.23À4.10 (m, 6H),

4.09À3.92 (m, 5H), 3.73 (m, 1H), 3.68À3.62 (m, 2H), 3.60À3.55

(m, 3H), 3.47(m,1H),3.22(dd, 1H,J= 9.6, 2.7 Hz), 2.32, 2.13, 2.10,

2.08, 2.06, 2.02, 2.01, 1.96, 1.92, 1.90, 1.86 (11s, 33H); ¹³C NMR

(100 MHz, CDCl₃) δ170.59, 170.56, 170.39, 170.30, 170.08, 169.60,

169.56, 169.39, 168.09, 167.50, 167.11, 138.11, 138.04, 137.67,

136.96, 134.96, 134.49, 133.51, 133.15, 131.53, 131.39, 131.08,

130.94, 128.37, 128.35, 128.23, 127.93, 127.73, 127.65, 127.52,

127.38, 127.31, 126.92, 123.61, 123.47, 123.18, 122.84, 99.58,

99.41, 97.20, 97.11, 96.88, 78.08, 77.12, 77.07, 75.85, 74.85,

74.59, 74.20, 74.05, 73.29, 72.31, 70.66, 70.46, 69.53, 69.48,

69.22, 68.98, 68.65, 68.40, 68.16, 67.86, 66.67, 65.62, 65.32,

62.67, 62.51, 62.44, 55.52, 54.16, 20.98, 20.93, 20.77, 20.66,

20.63, 20.56, 20.53, 20.50, 20.39, 20.32.

reaction mixture was stirred at room temperature overnight. The

mixture was diluted with CH₂Cl₂, filtered through a Celite pad, and

washed sequentially with saturated NaHCO₃and brine. The organic

layer was dried over MgSO₄and filtered. The filtrate was concen-

trated, and the residue was subjected to silica gel chromatography

(CH₂Cl₂/MeOH, 50:1) on silica gel to give 19 (25 mg, 48%) as a

white amorphous powder. ESI-MS: calcd for C₆₂H₈₄N₂O₃₉, M =

1480.47; Found (m/z): 1481.69 [M + H]⁺; ¹H NMR (400 MHz,

CDCl₃, TMS) δ 6.00 (d, 1H, J = 9.1 Hz), 5.91 (d, 1H, J = 7.4 Hz),

5.54 (m, 1H), 5.36À5.04 (m, 10H), 5.01 (d, 1H, J = 1.4 Hz), 4.75

(s, 1H), 4.66 (d, 1H, J = 8.7 Hz), 4.39À3.76 (m, 17H), 3.59 (m,

1H), 3.50 (m, 1H), 3.43 (m, 1H), 2.21À1.94 (m, 48H); ¹³C NMR

(100 MHz, CDCl₃) δ 171.14, 170.84, 170.64, 170.60, 170.55,

169.98, 169.86, 169.73, 169.60, 169.57, 169.55, 169.49, 169.25, 165.93,

100.36, 99.08, 99.03, 98.41, 97.71, 76.07, 74.48, 74.38, 72.65, 72.40,

72.11, 69.77, 69.60, 69.39, 68.97, 68.93, 68.84, 68.81, 68.59, 68.48, 67.78,

66.76, 65.98, 65.79, 64.60, 63.46, 63.18, 62.35, 62.22, 54.36, 23.03, 20.92,

20.82, 20.78, 20.71, 20.68, 20.64, 20.61, 20.56, 20.54, 20.47, 13.60.

Synthesis of 2-Methyl-[α-D-mannopyranosyl-(1f3)]-[2-ace-

tamido-2-deoxy-β-^D-glucopyranosyl-(1f4)]-[α-D-mannopy-

ranosyl-(1f6)]-β-^D-mannopyranosyl-(1f4)-1,2-dideoxy-α-

D-glucopyrano-[2,1-d]-2-oxazoline (1). To a solution of 19

Synthesis of 2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl-

(1f3)-[2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-^D-glucopyrano-

syl-(1f4)]-[2,3,4,6-tetra-O-acetyl-α-^D-mannopyranosyl-(1f6)]-2-

O-acetyl-β-^D-mannopyranosyl-(1f4)-2-acetamido-1,3,6-tri-O-

acetyl-2-deoxy-^D-glucopyranose (18). Compound 16 (173 mg,

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(23 mg, 16 μmol) in MeOH (2 mL) was added MeONa in

MeOH (0.5 M, 3.2 μL, 1.6 μmol). After stirring at room

temperature overnight, the reaction mixture was concentrated

to dryness. The residue was dissolved in water and lyophilized to

give sugar oxazoline 1 (14 mg, quantitative). ESI-MS: calcd for

C₃₄H₅₆N₂O₂₅, M = 892.32; Found (m/z), 893.78 [M + H]⁺;

MALDI-TOF HRMS: calcd for [M + Na]⁺, 915.3070; found (m/

z), 915.3048; ¹H NMR (400 MHz, D₂O) δ 5.96 (d, 1H, J = 7.3

Hz), 5.10 (s, 1H), 4.89 (s, 1H), 4.59 (m, 1H), 4.40 (d, 1H, J = 8.2

Hz), 4.24 (m, 1H), 4.09À3.23 (m, 29H), 1.94À1.92 (m, 6H);

¹³C NMR (100 MHz, D₂O) δ 174.61, 168.73, 101.83, 101.18,

101.06, 100.31, 99.93, 77.82, 77.24, 76.43, 74.26, 73.44, 73.15,

70.85, 70.48, 70.40, 70.23, 69.88, 69.84, 69.14, 66.91, 66.87,

65.70, 65.10, 61.70, 61.47, 61.13, 56.16, 22.19, 13.00.

Preparation of Free Bisecting GlcNAc-Containing Oligo-

saccharide 20. A solution of peracetylated pentasaccharide 18

(50 mg, 32.4 μmol) in MeOH (2 mL) containing a catalytic

amount of MeONa was stirred at room temperature overnight.

The reaction mixture was neutralized with Dowex 50W-X8 (H⁺

form) and filtered. The filtrate was concentrated to dryness. The

residue was dissolved in water and lyophilized to give free

pentasaccharide 20 (30 mg, quantitative). ESI-MS: calcd for

C₃₄H₅₈N₂O₂₆, M = 910.33, Found (m/z): 933.37 [M + Na]⁺; ¹H

NMR (400 MHz, D₂O) δ 5.10 (s, 1H), 5.07 (m, 0.7H, αH-1 of

GlcNAc-1), 4.82 (s, 1H), 4.63 (m, 1H), 4.40 (m, 1H), 4.16À3.23

(m, 30H), 1.93 (m, 6H); ¹³C NMR (100 MHz, D₂O) δ174.61,

101.93, 101.18, 100.86, 100.11, 100.03, 94.92, 79.92, 77.34,

76.43, 74.56, 73.44, 73.15, 70.65, 70.48, 70.24, 70.03, 69.98,

69.84, 69.14, 66.91, 66.67, 65.80, 65.10, 61.40, 61.17, 60.03,

56.16, 22.19, 21.90.

Synthesis of Sugar Oxazoline 1 by the DMC-Based One-

Pot Method. A solution of pentasaccharide 20 (15 mg, 16.5

μmol), 2-chloro-1,3-dimethylimidazolinium chloride (DMC)

(50 mg, 295 μmol) and Et₃N (90 μL, 648 μmol) in water

(400 μL) was stirred at 4 °C for 1 h. The reaction mixture was

subjected to gel filtration chromatography on a Sephadex G-10

column eluted by 0.05% aqueous Et₃N. The fractions containing

the product were combined and lyophilized to give oxazoline 1 as

a white powder (15 mg, quantitative yield). The sugar oxazoline

obtained by this method was identical to the above synthesized

compound as confirmed by ¹H NMR, ¹³C NMR, and ESI-MS.

Synthesis of Bisecting LacNAc-Containing Hexasacchar-

ide 21. A solution of pentasaccharide 20 (15 mg, 16.5 μmol) and

UDP-Gal (15 mg, 24.9 μmol) in a HEPES buffer (50 mM, pH

7.5, 1.5 mL) containing α-lactalbumin (0.2 mg/mL) and Mn²⁺

(20 mM) was incubated with β-1,4-galactosyltransferase (1 U) at

37 °C for 6 h. The reaction mixture was subject to gel filtration

chromatography on a Sephadex G-10 column eluted by 0.05%

aqueous Et₃N. The fractions containing the product were

combined and lyophilized to give hexasaccharide 21 as a white

solid (18 mg, quantitative yield). ESI-MS: calcd for

C₄₀H₆₈₁N₂O₃₁, M = 1072.38, Found (m/z): 1095.03 [M +

Na]⁺. H NMR (400 MHz, D₂O) δ 5.10 (s, 1H), 5.08 (m,

0.65H, αH-1 of GlcNAc-1), 4.81 (s, 1H), 4.61 (m, 1H), 4.42 (d,

1H, J = 6.8 Hz), 4.32 (d, 1H, J = 7.6 Hz), 4.24À3.36 (m, 35H),

3.08 (m, 1H), 1.93 (m, 6H). ¹³C NMR (100 MHz, D₂O) δ

174.62, 101.95, 101.02, 100.89, 100.28, 100.14, 100.07, 94.92,

79.96, 77.38, 76.40, 74.59, 74.19, 73.86, 73.56, 73.42, 73.33,

73.10, 72.24, 70.50, 70.41, 70.27, 70.21, 70.02, 69.92, 69.88,

69.68, 69.12, 67.65, 66.90, 66.83, 65.78, 65.14, 61.47, 61.18,

60.06, 56.12, 53.56, 22.16, 21.95.

Synthesis of Bisecting LacNAc-Containing Sugar Oxazo-

line 2. A solution of hexasaccharide 21 (15 mg, 14.0 μmol),

2-chloro-1,3-dimethylimidazolinium chloride (DMC) (50 mg,

295 μmol) and Et₃N (90 μL, 648 μmol) in water (400 μL) was

stirred at 4 °C for 1 h. The reaction mixture was subjected to gel

filtration chromatography on a Sephadex G-10 column eluted by

0.05% aqueous Et₃N. The fractions containing the product were

combined and lyophilized to give oxazoline 2 (15 mg, quantita-

tive yield) as a white powder. ESI-MS: calcd for C₄₀H₆₆N₂O₃₀,

M = 1054.37, Found (m/z): 1055.42 [M + H]⁺. MALDI-TOF

HRMS: calcd for [M + Na]⁺, 1077.3598; found (m/z),

1077.3638; ¹H NMR (400 MHz, D₂O) δ 5.97 (d, 1H, J = 6.8

Hz), 5.10 (s, 1H), 4.88 (d, 1H, J = 1.6 Hz), 4.59 (s, 1H), 4.43 (d,

1H, J = 7.6 Hz), 4.32 (d, 1H, J = 8.0 Hz), 4.24 (m, 1H),

1.94À1.92 (m, 6H). ¹³C NMR (100 MHz, D₂O, selected from

13

¹HÀ C HMQC) δ 102.35, 101.85, 101.33, 100.46, 99.67, 99.56,

82.58, 79.23, 77.87, 76.02, 74.33, 74.18, 72.68, 72.17, 71.25,

70.97, 70.57, 70.12, 69.27, 68.87, 68.53, 67.32, 66.56, 65.65,

61.32, 56.02, 53.88, 22.56, 13.27.

Synthesis of Benzyl 2-O-Benzoyl-4,6-O-benzylidene-3-O-

p-methoxybenzyl-β-^D-glucopyranosyl-(1f4)-2-azido-3,6-di-

O-benzyl-2-deoxy-β-^D-glucopyranoside (24). Ethyl 2-O-ben-

zyl-4,6-O-benzylidene-3-O-p-methoxybenzyl-1-thio-β-^D-gluco-

pyranoside 22⁶²(677 mg, 1.26 mmol), benzyl 2-azido-3,6-di-O-

benzyl-2-deoxy-β-^D-glucopyranoside 23 (500 mg, 1.05 mmol)

and 4 Å molecular sieves (1.44 g) were stirred in dichloroethane

(12 mL) at room temperature for 30 min and the mixture was

then cooled to 0 °C. A solution of NIS (290 mg, 1.29 mmol) and

TfOH (12.6 μL, 142 μmol) in CH₂Cl₂ÀEt₂O (1:1, 12.6 mL)

was added to the mixture, and the resulting mixture was stirred

for 30 min at 0 °C. Et₃N (60 μL) was added, and the reaction

mixture was filtered through a Celite pad. The filtrate was diluted

with CH₂Cl₂, and washed sequentially with 10% Na₂S₂O₃,

saturated NaHCO₃, and brine. The organic layer was dried over

MgSO₄, filtered, and concentrated. The residue was subjected to

silica gel column chromatography (hexanes/EtOAc, 6:1) to give

24 (632 mg, 63%) as a white amorphous powder. ESI-MS: calcd

for C₅₅H₅₅N₃O₁₂, M = 949.3786; Found (m/z): 950.51 [M +

H]⁺. ¹H NMR (400 MHz, CDCl₃, TMS) δ 7.86À6.58 (m, 29H),

5.52 (s, 1H), 5.19 (t, 1H, J = 8.5 Hz), 4.93 (d, 1H, J = 10.6 Hz),

4.84 (d, 1H, J = 11.9 Hz), 4.73 (d, 1H, J = 10.5 Hz), 4.72 (d, 1H,

J = 11.5 Hz), 4.68 (d, 1H, J = 7.8 Hz), 4.66 (d, 1H, J = 12.4 Hz),

4.58 (d, 1H, J = 11.9 Hz), 4.57 (d, 1H, J = 11.9 Hz), 4.30 (d, 1H,

J = 12.4 Hz), 4.20À4.16 (m, 2H), 3.99 (t, 1H, J = 9.4 Hz),

3.75À3.63 (m, 5H), 3.59 (dd, 1H, J = 11.3, 3.0 Hz), 3.49À3.41

(m, 3H), 3.33À3.22 (m, 2H), 3.08 (m, 1H); ¹³C NMR (100

MHz, CDCl₃) δ 164.70, 159.06, 138.34, 137.91, 137.23, 136.72,

133.24, 129.89, 129.79, 129.63, 129.43, 129.03, 128.52, 128.37,

128.26, 128.21, 128.03, 127.97, 127.85, 127.81, 127.60, 126.01,

113.51, 101.21, 100.66, 100.31, 81.85, 81.11, 77.21, 76.13, 75.30,

74.50, 73.78, 73.54, 73.46, 70.80, 68.50, 67.29, 66.04, 65.71,

55.07.

Synthesis of Benzyl 2-O-Benzoyl-4,6-O-benzylidene-β-D-

glucopyranosyl-(1f4)-2-azido-3,6-di-O-benzyl-2-deoxy-β-

D-glucopyranoside (25). To a solution of 24 (47 mg, 50 μmol)

in CH₂Cl₂ÀH₂O (17.5/1, 2.1 mL) was added DDQ (26 mg, 115

μmol) at 0 °C. After 30 min, the reaction mixture was warmed to

room temperature and further stirred for 2 h. The reaction

mixture was diluted with CH₂Cl₂, washed with saturated NaH-

CO₃and brine, and dried over MgSO₄. The mixture was filtered,

and the filtrate was concentrated. The residue was subjected

to silica gel column chromatography (hexanes/EtOAc, 4:1) to

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provide 25 (36 mg, 87%) as a white amorphous powder. ESI-MS:

calcd for C₄₇H₄₇N₃O₁₁, M = 829.3211; Found (m/z): 830.58

[M + H]⁺. ¹H NMR (400 MHz, CDCl₃, TMS) δ 7.99À7.26 (m,

25H), 5.48 (s, 1H), 5.16 (t, 1H, J = 8.7 Hz), 4.93 (d, 1H, J = 10.5

Hz), 4.86 (d, 1H, J = 12.4 Hz), 4.79 (d, 1H, J = 8.3 Hz), 4.75 (d,

1H, J = 11.5 Hz), 4.71 (d, 1H, J = 12.4 Hz), 4.59 (d, 1H, J = 11.9

Hz), 4.21À4.17 (m, 2H), 4.03 (t, 1H, J = 9.2 Hz), 3.89À3.83 (m,

1H), 3.69 (dd, 1H, J = 11.0, 3.2 Hz), 3.58À3.43 (m, 4H),

3.35À3.25 (m, 2H), 3.14 (m, 1H), 2.51 (d, 1H, J = 3.2 Hz); ¹³C

NMR (100 MHz, CDCl₃) δ 165.35, 138.33, 137.93, 136.81,

136.70, 133.44, 129.80, 129.34, 129.24, 128.53, 128.50, 128.37,

128.34, 128.21, 128.03, 127.91, 127.87, 127.81, 127.78, 127.62,

126.25, 101.86, 100.45, 100.32, 81.12, 80.87, 76.24, 75.26, 74.78,

74.52, 73.53, 72.29, 70.81, 68.40, 67.41, 66.01, 65.73.

Synthesis of Benzyl 2-O-Benzoyl-4-O-benzyl-β-D-glucopy-

ranosyl-(1f4)-2-azido-3,6-di-O-benzyl-2-deoxy-β-^D-glucopy-

ranoside (26). To a suspension of 25 (50 mg, 60 μmol) and 4 Å

molecular sieves (192 mg) in CH₂Cl₂(1.6 mL) were added

successively Et₃SiH (29 μL, 181 μmol) and PhBCl₂(27 μL, 205

μmol) at À78 °C. After stirring at À78 °C for 1 h, Et₃N (116 μL)

and MeOH (116 μL) were added. The reaction mixture was

diluted with CH₂Cl₂, filtered through a Celite pad, and washed

sequentially with saturated NaHCO₃and brine. The organic

layer was dried over MgSO₄, filtered, and concentrated. The

resulting residue was subjected to silica gel column chromato-

graphy (hexanes/EtOAc, 3:1) to afford 26 (47 mg, 94%) as a

white amorphous powder. ESI-MS: calcd for C₄₇H₄₉N₃O₁₁, M =

831.3367; Found (m/z), 832.50 [M + H]⁺. ¹H NMR (400 MHz,

CDCl₃, TMS) δ 7.95À7.26 (m, 25H), 5.02 (dd, 1H, J = 9.2, 8.3

Hz), 4.95 (d, 1H, J = 11.0 Hz), 4.86 (d, 1H, J = 12.4 Hz),

4.79À4.67 (m, 5H), 4.60 (d, 1H, J = 11.9 Hz), 4.41 (d, 1H, J =

11.9 Hz), 4.20 (d, 1H, J = 7.8 Hz), 3.97 (t, 1H, J = 9.3 Hz), 3.75

(dt, 1H, J = 9.2, 3.7 Hz), 3.70À3.65 (m, 2H), 3.53 (dd, 1H, J =

11.0, 1.4 Hz), 3.49À3.43 (m, 2H), 3.41À3.29 (m, 2H), 3.21

(ddd, 1H, J = 9.5, 4.5, 2.6 Hz), 3.15 (m, 1H), 2.31 (d, 1H, J = 4.1

Hz), 1.40 (t, 1H, J = 7.1 Hz); ¹³C NMR (100 MHz, CDCl₃) δ

165.65, 138.36, 137.88, 137.79, 136.69, 133.38, 129.71, 129.25,

128.53, 128.51, 128.45, 128.39, 128.34, 127.99, 127.96, 127.85,

127.78, 127.69, 127.10, 100.26, 99.88, 81.06, 77.97, 76.13, 75.51,

75.13, 75.02, 74.70, 74.65, 74.48, 73.54, 70.75, 67.33, 65.79,

61.54.

Synthesis of Benzyl 6-O-Acetyl-2,3,4-tri-O-benzoyl-α-D-

mannopyranosyl-(1f3)-[6-O-acetyl-2,3,4-tri-O-benzoyl-α-^D-

mannopyranosyl-(1f6)]-2-O-benzoyl-4-O-benzyl-β-^D-gluco-

pyranosyl-(1f4)-2-azido-3,6-di-O-benzyl-2-deoxy-β-^D-gluco-

pyranoside (28). A solution of compound 26 (75 mg, 90 μmol)

and 2,3,4-tri-O-acetyl-6-O-benzoyl-α-^D-mannopyranosyl tricho-

loroacetimidate 27⁷⁸(305 mg, 449 μmol) in CH₂Cl₂(3.4 mL)

containing activated 4 Å molecular sieves (456 mg) was stirred

under an atmosphere of argon at room temperature for 30 min.

After this mixture cooled to À40 °C, a solution of TMSOTf in

CH₂Cl₂(0.1 M, 450 μL, 45 μmol) was added and the resulting

mixture was stirred at room temperature overnight. The mixture

was filtered through a Celite pad. The filtrate was poured into

saturated NaHCO₃, and extracted with CH₂Cl₂. The organic

layer was washed with brine, dried over MgSO₄, and filtered. The

filtrate was concentrated, and the residue was subjected to silica

gel column chromatography (hexanes/EtOAc, 5:2) to provide

28 (159 mg, 95%) as a white amorphous. ESI-MS: calcd for

C₁₀₅H₉₇N₃O₂₉; M = 1864.90; Found (m/z): 1865.83 [M + H]⁺.

¹H NMR (400 MHz, CDCl₃, TMS) δ 8.10À7.16 (m, 55H),

5.95À5.83 (m, 3H), 5.77À5.74 (m, 2H), 5.61 (t, 1H, J = 2.0 Hz),

5.51 (s, 1H), 5.37 (t, 1H, J = 8.7 Hz), 5.13 (d, 1H, J = 0.9 Hz),

5.07 (d, 1H, J = 11.0 Hz), 4.90 (d, 1H, J = 11.5 Hz), 4.84À4.80

(m, 2H), 4.76À4.69 (m, 3H), 4.54 (d, 1H, J = 11.9 Hz), 4.36 (d,

1H, J = 11.9 Hz), 4.28À4.02 (m, 7H), 3.89 (dd, 1H, J = 12.1, 2.6

Hz), 3.81 (d, 1H, J = 11.0 Hz), 3.73À3.61 (m, 3H), 3.54À3.43

(m, 4H), 3.28 (t, 1H, J = 9.4 Hz), 3.11 (d, 1H, J = 9.6 Hz), 2.08 (s,

3H), 1.99 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 170.61,

170.32, 165.44, 165.32, 165.26, 165.12, 165.04, 165.01, 164.65,

138.33, 137.90, 137.06, 136.82, 133.56, 133.49, 133.44, 133.43,

133.35, 133.27, 133.23, 133.07, 132.94, 130.05, 129.80, 129.76,

129.67, 129.59, 129.33, 129.23, 129.07, 128.98, 128.94, 128.80,

128.61, 128.56, 128.42, 128.34, 128.28, 128.23, 128.20, 128.15,

128.11, 128.07, 127.93, 127.88, 127.76, 127.71, 127.51, 100.60,

99.71, 98.60, 98.03, 80.77, 80.17, 79.28, 75.64, 75.26, 75.01,

74.70, 74.39, 73.63, 73.02, 70.76, 70.26, 69.95, 69.85, 69.70,

69.12, 68.91, 67.63, 67.49, 66.40, 65.87, 65.40, 62.30, 61.70,

20.66, 20.53.

Synthesis of Benzyl 6-O-Acetyl-2,3,4-tri-O-benzoyl-α-D-

mannopyranosyl-(1f3)-[6-O-acetyl-2,3,4-tri-O-benzoyl-α-^D-

mannopyranosyl-(1f6)]-2-O-benzoyl-4-O-benzyl-β-^D-gluco-

pyranosyl-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-^D-

glucopyranoside (29). Toa solution of 28 (604 mg, 0.324 mmol)

in CHCl₃(6 mL) was added pyridine (6 mL) and thioacetic acid

(AcSH) (6 mL) at room temperature. After stirring for 48 h, the

mixture was concentrated and the residue was subjected to silica

gel column chromatography (hexanes/EtOAc, 1:1) to afford 29

(474 mg, 78%). ESI-MS: calcd for C₁₀₇H₁₀₁NO₃₀; M = 1880.94;

Found (m/z), 1881.68 [M + H]⁺. ¹H NMR (400 MHz, CDCl₃,

TMS) δ 8.12À7.12 (m, 55H), 6.01À5.86 (m, 3H), 5.79À5.74

(m, 3H), 5.63 (d, 1H, J = 8.7 Hz), 5.54 (s, 1H), 5.43 (t, 1H, J = 8.9

Hz), 5.26 (s, 1H), 5.15 (d, 1H, J = 11.5 Hz), 4.83 (d, 1H, J = 11.5

Hz), 4.70 (d, 1H, J = 8.2 Hz), 4.65À4.53 (m, 5H), 4.35 (d, 1H, J =

12.4 Hz), 4.29 (dd, 1H, J = 12.2, 4.4 Hz), 4.24À4.07 (m, 6H),

3.96À3.83 (m, 4H), 3.77À3.69 (m, 3H), 3.64À3.62 (m, 2H),

3.51À3.46 (m, 2H), 2.09 (s, 3H), 1.99 (s, 3H), 1.81 (s, 3H); ¹³C

NMR (100 MHz, CDCl₃) δ 170.65, 170.33, 170.04, 165.49,

165.46, 165.31, 165.24, 165.14, 165.05, 164.89, 138.44, 138.10,

137.66, 137.32, 133.54, 133.44, 133.33, 133.25, 133.10, 133.06,

129.82, 129.77, 129.65, 129.43, 129.30, 129.25, 129.00, 128.91,

128.76, 128.68, 128.61, 128.42, 128.25, 128.22, 128.06, 128.03,

127.95, 127.89, 127.73, 127.37, 127.31, 99.84, 99.05, 98.52, 98.10,

80.44, 78.77, 77.17, 75.35, 74.75, 74.36, 73.45, 72.80, 70.55, 70.25,

70.04, 69.87, 69.73, 69.01, 68.91, 66.80, 66.63, 65.86, 62.50, 61.77,

53.33, 23.25, 20.66, 20.53.

Synthesis of 2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl-

(1f3)-[2,3,4,6-tetra-O-acetyl-α-^D-mannopyranosyl-(1f6)]-

2,4-di-O-acetyl-β-^D-glucopyranosyl-(1f4)-2-acetamido-1,3,6-

tri-O-acetyl-2-deoxy-^D-glucopyranose (30). To a solution of

29 (95 mg, 54 μmol) in MeOH/CH₂Cl₂(5:1, 10 mL) was added

a solution of MeONa in MeOH (0.5 M, 400 μL). The resulting

mixture was stirred at room temperature overnight and then

neutralized with Dowex 50W-X8 (H⁺form). The mixture was

filtered, and the filtrate was concentrated. The residue was

dissolved in MeOH (5 mL), and palladium(II) hydroxide

(20%) on activated carbon (40 mg) was added. The resulting

mixture was vigorously stirred at room temperature under

hydrogen atmosphere for 24 h. The reaction mixture was filtered

through a Celite pad, and the filtrate was concentrated in vacuo

to dryness. The residue was dissolved in pyridine (3 mL) and

Ac₂O (3 mL) for acetylation. The mixture was stirred at room

temperature for 18 h and then concentrated in vacuo to dryness.

The residue was subjected to silica gel column chromatography

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(CH₂Cl₂/MeOH, 60:1 to 40:1) to give 30 (62 mg, 92% in three

steps) as a white solid. ESI-MS: calcd for C₅₂H₇₁NO₃₄; M =

Scientific, Rockford, IL). Each elution fraction was immediately

neutralized with 1 M Tris, pH 8.8. Subsequently, the elution

fractions were combined and concentrated by centrifugal filtra-

tion (Amicon Ultra centrifugal filter, Millipore, Billerica, MA).

The integrity and purity of the recombinant IgG-Fc that carries

high-mannose type N-glycans (HM-Fc) was checked by SDS-

PAGE. MALDI-TOF MS of HM-Fc, calcd M = 65444 Da; found

(m/z), 65447. MALDI-TOF MS of the PNGase F released N-

glycan, calcd for Man9GlcNAc2, M = 1883.67 Da; found (m/z)

1906.40 [M + Na⁺]. For the production of the natural complex-

type glycoform of IgG-Fc, the transfected CHO cells were grown

in the absence of the α-mannosidase inhibitor kifunensine, and

the recombinant IgG-Fc was purified following the same pro-

cedure.

Preparation of GlcNAc-Fc Homodimer. The recombinant

IgG-Fc (HM-Fc) (15 mg) was treated with Endo-H (0.8 unit) in

a phosphate buffer (3 mL, 50 mM, pH 6.5) at 30 °C. After 10 h,

SDS-PAGE indicated the completion of the deglycosylation. The

GlcNAc-Fc homodimer was then purified by protein A affinity

chromatography. MALDI-TOF MS of GlcNAc-Fc: calcd M =

62119 Da; found (m/z), 62112.

Synthesis of the Bisecting-GlcNAc Fc Glycoform (Fc-1) by

the Endo-A-Catalyzed Transglycosylation of GlcNAc-Fc. A

solution of GlcNAc-Fc homodimer (1.20 mg, 19.3 nmol) and bi-

GlcNAc-Man₃GlcNAc oxazoline 1 (0.345 mg, 386 nmol, about

10 equivalent for each of the GlcNAc moiety in the GlcNAc-Fc

homodimer) in a phosphate buffer (50 mM, pH 7.0, 200 μL) was

incubated with Endo-A (8 μg) at 23 °C. The enzymatic reactions

were monitored by SDS-PAGE analysis and MALDI-TOF MS

analysis of an aliquot of the reaction mixture. When all the

GlcNAc-Fc was converted (after 3 h), the reaction mixture was

subject to affinity chromatography on a protein A-agarose

column, following the procedure described above for purification

of the recombinant IgG1-Fc. The fractions containing the IgG-Fc

glycoform were combined, desalted by ultracentrifugation, and

lyophilized to give Fc-1 (1.1 mg) (quantified using the antihu-

man IgG1 Fc ELISA quantification kit). The purified IgG-Fc

glycoform (Fc-1) was analyzed by MALDI-TOF MS. The

homogeneity of the product was confirmed by SDS-PAGE

analysis under reducing conditions. The attached N-glycan was

released by PNGase F treatment and analyzed by MALDI-TOF

MS following the previously reported procedure.³⁶MALDI-

TOF MS of Fc-1, calcd M = 63905 Da; found (m/z), 63909.

PNGase F released N-glycan (bisecting-GlcNAc-Man3GlcNAc2):

calcd for C₄₂H₇₁N₃O₃₁, M = 1113.41 Da; found (m/z), 1136.80

[M + Na]⁺.

Synthesis of Other Fc Glycoforms (Fc-2 to Fc-6) by the

Endo-A-Catalyzed Transglycosylation of GlcNAc-Fc. The

synthesis of glycoforms Fc-2, Fc-3, Fc-4, Fc-5, and Fc-6 was

performed in the same way as described for the preparation of

Fc-1, using excess of the corresponding sugar oxazoline (10À15 mol

equiv of each of the GlcNAc moiety in the GlcNAc-Fc homo-

dimer). After SDS-PAGE indicated the completion of the Glc-

NAc-Fc transformation, the product was purified on a protein A

affinity column. The purity of the product was confirmed by

SDS-PADE analysis, and the identity was characterized by

MALDI-TOF MS analysis of the intact Fc glycoforms and the

released N-glycans.

1

1253.3858; found (m/z): 1254.68 [M + H⁺]. H NMR (400

MHz, CDCl₃, TMS) δ 5.92 (d, 1H, J = 7.3 Hz), 5.78 (d, 1H, J =

2.7 Hz), 5.36À5.21 (m, 4H), 5.14À5.08 (m, 2H), 5.03À4.93 (m,

2H), 4.89 (d, 1H, J = 1.8 Hz), 4.79 (s, 1H), 4.70 (d, 1H, J = 7.8

Hz), 4.39 (dd, 1H, J = 12.3, 4.5 Hz), 4.24À4.18 (m, 2H),

4.16À3.99 (m, 5H), 3.95 (dt, 1H, J = 10.1, 2.8 Hz), 3.89À3.74

(m, 3H), 3.57 (m, 1H), 3.49À3.42 (m, 2H), 2.16À2.13 (m, 9H),

2.11À2.07 (m, 18H), 2.01 (s, 3H), 1.95À1.94 (m, 9H); ¹³C

NMR (100 MHz, CDCl₃) δ 170.68, 170.52, 170.49, 170.00,

169.96, 169.87, 169.54, 169.48, 169.33, 169.23, 166.21, 101.56,

99.67, 98.83, 97.46, 81.70, 78.24, 72.53, 71.98, 70.02, 69.52,

69.31, 69.19, 68.92, 68.82, 68.73, 68.40, 67.29, 65.68, 64.99,

64.36, 63.65, 61.97, 61.49, 20.89, 20.85, 20.78, 20.77, 20.68,

20.62, 20.56, 20.47, 20.44, 13.78.

Synthesis of 2-Methyl-[α-D-mannopyranosyl-(1f3)]-[α-D-

mannopyranosyl-(1f6)]-[β-^D-glucopyranosyl-(1f4)]-1,2-

dideoxy-α-^D-glucopyrano]-[2,1-d]-2-oxazoline (4). To a solu-

tion of 30 (58 mg, 46 μmol) in MeOH (2 mL) was added a

catalytic amount of MeONa and the solution was stirred at room

temperature overnight. The reaction mixture was neutralized

with Dowex 50W-X8 (H⁺form) and filtered. The filtrate was

concentrated to dryness, and the residue was dissolved in water

and lyophilized to give the free tetrasaccharide (33 mg,

quantitative) (ESI-MS, found (m/z), 730.31 [M + Na]⁺). The

free oligosaccharide was then dissolved in water (1 mL), and

2-chloro-1,3-dimethylimidazolinium chloride (DMC) (78 mg,

460 μmol) and Et₃N (128 μL, 920 μmol) were added. The

solution was stirred at 4 °C for 1 h. Then the reaction mixture was

subjected to gel filtration on a Sephadex G-10 column eluted with

0.05% aqueous Et₃N. The fractions containing the product were

combined and lyophilized to give oxazoline 4 (30 mg, 95%) as a

white powder. ESI-MS: calcd for C₂₆H₄₃NO₂₀, M = 689.24;

Found (m/z): 690.35 [M + H]⁺. MALDI-TOF HRMS: calcd for

1

[M + Na]⁺, 712.2276; found (m/z), 712.2258; H NMR (400

MHz, D₂O) δ 5.97 (d, 1H, J = 7.3 Hz), 5.08 (s, 1H), 4.79 (s, 1H),

4.40 (d, 1H, J = 7.8 Hz), 4.27 (m, 1H), 4.08 (m, 1H), 3.92 (m,

1H), 3.94À3.46 (m, 19H), 3.31 (m, 1H), 3.21 (m, 1H), 1.94 (s,

3H); ¹³C NMR (100 MHz, D₂O) δ 168.12, 104.60, 101.10,

99.93, 99.70, 82.25, 79.62, 74.07, 72.90, 72.76, 71.91, 71.02,

70.79, 70.66, 70.40, 69.96, 68.92, 66.91, 66.63, 65.60, 65.43,

61.93, 61.03, 60.67, 12.97.

Overproduction of Human IgG1-Fc in Chinese Hamster

Ovary (CHO) Cell Lines. The expression of human IgG1-Fc in

Pichia pastoris was performed following the procedure that we

have previously described.³⁶For producing recombinant IgG1-

Fc in Chinese Hamster Ovary (CHO) cell lines, the CHO cells

were transiently transfected with a mammalian expression plas-

mid pcDNA-Fc encoding the human Fc gene, using Fugene6

transfection reagent (Roche). After transfection, kifunensine was

added into the medium to a final concentration of 2 μg/mL. The

expression of fusion protein was periodically monitored using an

antihuman IgG1 Fc ELISA assay kit (Human IgG ELISA

Quantitation Kit, BETHYL Laboratories Inc., Montgomery

TX). The culture supernatant was collected 4 days after transfec-

tion. The Fc protein was purified by affinity chromatography

using a protein A-agarose resin (Pierce). The cell-free culture

supernatant was loaded on the protein A column that was pre-

equilibrated with IgG binding buffer (pH 8.0, Thermo Scientific,

Rockford, IL) After extensive wash with the same buffer, Fc

protein was eluted with IgG elution buffer (pH 3.0, Thermo

Fc-2: calcd M = 64229 Da; found (m/z), 64212; the attached

N-glycan (bisecting-LacNAc-Man3GlcNAc2): calcd for C₄₈H₈₁-

N₃O₃₆, M = 1275.46 Da; found (m/z), 1298.40 [M + Na]⁺.

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Fc-3: calcd M = 63822 Da; found (m/z), 63840; the attached

N-glycan (bisecting-Man-Man3GlcNAc2): calcd for C₄₀H₆₈N₂O₃₁,

M = 1072.38 Da; found (m/z), 1095.10 [M + Na]⁺.

Fc-4: calcd M = M = 63498 Da; found (m/z), 63506; the

attached N-glycan (Man2GlcGlcNAc2): calcd for C₃₄H₅₈N₂O₂₆,

M = 910.33 Da; found (m/z), 933.42 [M + Na]⁺.

Fc-5: calcd M = M = 63498 Da; found (m/z), 63534; the

attached N-glycan (Man3GlcNAc2): calcd for C₃₄H₅₈N₂O₂₆,

M = 910.33 Da; found (m/z), 933.80 [M + Na]⁺.

(7) Jeﬀeris, R. Biotechnol. Prog. 2005, 21, 11–16.

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’ ASSOCIATED CONTENT

S

Supporting Information. Complete author list of ref 32;

b

amino acid sequence of the recombinant IgG1-Fc; reaction

scheme and SDS-PAGE analysis for the transglycosylation of

GlcNAc-Fc with complex-type N-glycan oxazoline. This material

is available free of charge via the Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION

Corresponding Author

lwang@som.umaryland.edu

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’ ACKNOWLEDGMENT

We thank Prof. Kaoru Takegawa (Kyushu University) for

providing the pGEX-2T/Endo-A plasmid that was used for

expressing the recombinant Endo-A, and the Gliknik Inc. for

providing the mammalian expression vector pcDNA-Fc that was

used for expressing the high-mannose type glycoform. This work

was supported by the National Institutes of Health (NIH Grants

R01 GM096973 and R01 GM080374).

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Chemoenzymatic synthesis and Fcγ receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcγIIIa receptor

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