Expeditious chemoenzymatic synthesis of homogeneous N-glycoproteins carrying defined oligosaccharide ligands

Article abstract of DOI:10.1021/ja805044x

An efficient chemoenzymatic method for the construction of homogeneous N-glycoproteins was described that explores the transglycosylation activity of the endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) with synthetic sugar oxazolines as the donor substrates. First, an array of large oligosaccharide oxazolines were synthesized and evaluated as substrates for the Endo-A-catalyzed transglycosylation by use of ribonuclease B as a model system. The experimental results showed that Endo-A could tolerate modifications at the outer mannose residues of the Man₃GlcNAc-oxazoline core, thus allowing introduction of large oligosaccharide ligands into a protein and meanwhile preserving the natural, core N-pentasaccharide (Man ₃GlcNAc₂) structure in the resulting glycoprotein upon transglycosylation. In addition to ligands for galectins and mannose-binding lectins, azido functionality could be readily introduced at the N-pentasaccharide (Man₃GlcNAc₂) core by use of azido-containing Man₃GlcNAc oxazoline as the donor substrate. The introduction of azido functionality permits further site-specific modifications of the resulting glycoproteins, as demonstrated by the successful attachment of two copies of αGal epitopes to ribonuclease B. This study reveals a broad substrate specificity of Endo-A for transglycosylation, and the chemoenzymatic method described here points to a new avenue for quick access to various homogeneous N-glycoproteins for structure-activity relationship studies and for biomedical applications.

Full text of DOI:10.1021/ja805044x

Published on Web 09/20/2008
Expeditious Chemoenzymatic Synthesis of Homogeneous
N-Glycoproteins Carrying Defined Oligosaccharide Ligands
Hirofumi Ochiai, Wei Huang, and Lai-Xi Wang*
Institute of Human Virology, Department of Biochemistry & Molecular Biology, UniVersity of
Maryland School of Medicine, Baltimore, Maryland 21201
Received July 1, 2008; E-mail: LWang3@ihv.umaryland.edu
Abstract: An efficient chemoenzymatic method for the construction of homogeneous N-glycoproteins was
described that explores the transglycosylation activity of the endo-ꢀ-N-acetylglucosaminidase from
Arthrobacter protophormiae (Endo-A) with synthetic sugar oxazolines as the donor substrates. First, an
array of large oligosaccharide oxazolines were synthesized and evaluated as substrates for the Endo-A-
catalyzed transglycosylation by use of ribonuclease B as a model system. The experimental results showed
that Endo-A could tolerate modifications at the outer mannose residues of the Man3GlcNAc-oxazoline core,
thus allowing introduction of large oligosaccharide ligands into a protein and meanwhile preserving the
natural, core N-pentasaccharide (Man3GlcNAc2) structure in the resulting glycoprotein upon transglycosy-
lation. In addition to ligands for galectins and mannose-binding lectins, azido functionality could be readily
introduced at the N-pentasaccharide (Man3GlcNAc2) core by use of azido-containing Man3GlcNAc oxazoline
as the donor substrate. The introduction of azido functionality permits further site-specific modifications of
the resulting glycoproteins, as demonstrated by the successful attachment of two copies of RGal epitopes
to ribonuclease B. This study reveals a broad substrate specificity of Endo-A for transglycosylation, and
the chemoenzymatic method described here points to a new avenue for quick access to various
homogeneous N-glycoproteins for structure-activity relationship studies and for biomedical applications.
Introduction
studies and biomedical applications. The past decade has
witnessed tremendous progress in this field, and many chemical,
Protein glycosylation is a ubiquitous posttranslational modi-
fication capable of transforming a protein’s properties in
different ways. It is known that glycosylation can profoundly
impact a protein’s folding, stability, and intracellular trafficking.1
On the other hand, the covalently linked oligosaccharides of
glycoproteins can serve as specific ligands for lectins and
receptors on cell surface, thus directly participating in many
important cellular communication processes, such as cell
adhesion, cell differentiation, pathogen-host interaction, de-
velopment, and immune responses.2 However, a detailed
understanding of the functions of glycoproteins is often hindered
by the structural microheterogeneity caused by the diverse
patterns of glycosylation. In most cases, natural and recombinant
glycoproteins are produced as mixtures of glycoforms that have
the same polypeptide backbone but differ in the pendant sugar
chains. As pure glycoforms are extremely difficult to isolate
from natural and recombinant sources with current techniques,
synthetic natural and unnatural homogeneous glycopeptides and
glycoproteins are urgently needed for both structural/functional
enzymatic, and bioengineering methods have been explored in
order to overcome a series of technical obstacles in the road
toward the ultimate assembly of homogeneous glycoproteins
carrying defined oligosaccharides.3 In particular, the exploration
of various chemical and enzymatic ligation methods, including
native chemical ligation, auxiliary-assisted ligation, and ex-
pressed protein ligation, as well as the use of glycoenzymes
for sugar chain elongation in glycoprotein synthesis, have
4-7
significantly expanded our synthetic repertoire.
The chemoenzymatic method based on the transglycosylation
activity of endo-ꢀ-N-acetylglucosaminidase (ENGases) has
attracted much attention in this field in recent years.7 ENGases
are a class of hydrolytic enzymes that remove N-glycans from
glycoproteins by hydrolyzing the ꢀ-1,4-glycosidic bond in the
N,N′-diacetylchitobiose core. But a few ENGases, including the
(3) (a) Koeller, K. M.; Wong, C. H. Nat. Biotechnol. 2000, 18, 835–841.
(b) Seitz, O. ChemBioChem 2000, 1, 214–246. (c) Herzner, H.; Reipen,
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Marcaurelle, L. A.; Bertozzi, C. R. Annu. ReV. Biochem. 2002, 71,
593–634. (g) Wong, C. H. J. Org. Chem. 2005, 70, 4219–4225. (h)
Guo, Z.; Shao, N. Med. Res. ReV. 2005, 25, 655–678. (i) Pratt, M. R.;
Bertozzi, C. R. Chem. Soc. ReV. 2005, 34, 58–68. (j) Liu, L.; Bennett,
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643–649. (b) Sola, R. J.; Rodriguez-Martinez, J. A.; Griebenow, K.
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13790 J. AM. CHEM. SOC. 2008, 130, 13790–13803
10.1021/ja805044x CCC: $40.75
2008 American Chemical Society

Site-Specific Introduction of Ligands into Proteins
A R T I C L E S
Endo-M from Mucor hiemali8 and Endo-A from Arthrobactor
protophormiae, 9-11 possess transglycosylation activity, that is,
the ability to transfer the released oligosaccharide moiety to an
acceptor such as an N-acetylglucosamine (GlcNAc)-containing
peptide to form a new homogeneous glycopeptide. A big
advantage of the endoglycosidase-based method is its high
convergence, as the enzyme is able to attach a large intact
oligosaccharide to a GlcNAc-polypeptide in a single step and
in a regio- and stereospecific manner to form a homogeneous
glycopeptide or glycoprotein in natural glycosidic linkages,
without the need for any protecting groups.7 To address the
relatively low transglycosylation yield, the limitation of the use
of only natural N-glycans as donor substrate, and the problem
of product hydrolysis, we and other groups have recently
explored synthetic sugar oxazolines as donor substrates for
transglycosylation, with a focus on its application for glyco-
peptide synthesis.6,7,12,13 The highly activated synthetic sugar
oxazoline can be regarded as a mimic of the presumed
oxazolinium ion intermediate generated by a substrate-assisted
catalytic mechanism. It was demonstrated that oligosaccharide
oxazolines corresponding to the N-glycan core Man3GlcNAc,
that is, ManR1,3(ManR1,6)Manꢀ1,4GlcNAc and some of its
truncated and selectively modified forms, were able to serve as
donor substrates for transglycosylation. Interestingly, the result-
ing glycopeptides that carry truncated or modified N-glycans
were poor substrates for hydrolysis because of the structural
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Chem. Soc. 2004, 126, 6576–6578. (b) Macmillan, D.; Bertozzi, C. R.
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Y.; Nakahara, Y.; Ito, E.; Suzuki, Y.; Suzuki, M.; Suzuki, A. J. Am.
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J. D.; Chen, G.; Hua, Z.; Danishefsky, S. J. Angew. Chem., Int. Ed.
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Figure 1. Chemoenzymatic strategy for N-glycoprotein synthesis
modifications, resulting in accumulation of the glycopeptide
product. Moreover, we have also reported a preliminary study
showing that this approach could be extended to glycoprotein
synthesis and glycosylation engineering without loss of the
transglycosylation efficiency.6 In addition to the truncated or
modified N-glycans, full-size natural N-glycan such as
Man9GlcNAc2 might also be introduced into a GlcNAc-protein
by a novel glycosynthase, EndoM-N175A mutant, that we have
recently reported.14 EndoM-N175A can take the high-mannose
sugar oxazoline as substrate for transglycosylation but lacks the
ability to hydrolyze the resulting natural N-glycopeptide because
of the mutation. These studies implicate a great potential of the
endoglycosidase-based method for synthesizing both modified
and natural N-glycoproteins. We describe in this paper the
expansion of this chemoenzymatic approach to the synthesis
of an array of homogeneous N-glycoproteins carrying defined
oligosaccharide ligands potentially useful for biological recogni-
tion. This chemoenzymatic approach involves two key steps,
as depicted in Figure 1. The first step is to prepare a GlcNAc-
protein, which can be achieved by selective deglycosylation of
a natural or recombinant glycoprotein by an endoenzyme such
as Endo-H to remove the heterogeneous N-glycan, leaving only
the innermost GlcNAc residue attached to the Asn at the
glycosylation site. Alternatively, a GlcNAc-protein can be
synthesized by modern chemical protein synthesis techniques,
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A R T I C L E S
Ochiai et al.
Figure 2. Synthetic oligosaccharide oxazolines.
in which the stable Asn-linked GlcNAc can be incorporated at
any desired site(s) during the synthesis. The second step is the
regio- and stereospecific enzymatic ligation of a preassembled
oligosaccharide moiety (in the form of the activated oxazoline)
to the GlcNAc-protein by the endoglycosidase-catalyzed trans-
glycosylation. We have previously shown that Endo-A could
tolerate modifications on the outer mannose residues of the
Man3GlcNAc oxazoline.6,13 Moreover, unnatural modification
on the outer residues of the N-glycan core also retarded the
enzymatic hydrolysis of the glycoprotein product, thus permit-
ting accumulation of the transglycosylation product.6,13 There-
fore, to introduce specific oligosaccharide ligands into a protein,
we have decided to glue the ligands at the outer residues of the
Man3GlcNAc oxazoline core, so that a native core N-pentasac-
charide (Man3GlcNAc2) at the glycosylation site would be
generated after transglycosylation. The preservation of a natural
core N-glycan structure may be important for maintaining the
native conformations of the glycoproteins upon ligand introduc-
tion, as the N-glycan core, especially the innermost chitobiose
(GlcNAcꢀ1,4GlcNAc) moiety, plays a primary role in impacting
a protein’s conformations.15
lines modified with functional groups and/or large carbohydrate
ligands, we have designed three novel oligosaccharide oxazo-
lines 3-5 (Figure 2), which will allow the introduction of
specific carbohydrate ligands into proteins if proven as substrates
for ENGases. For oxazoline 3, an additional mannose residue
was added at the bisecting location of the N-glycan core
Man3GlcNAc. If this oxazoline is a substrate for Endo-A, then
a novel oligomannose ligand can be introduced into a protein,
which may be useful for targeting the protein to specific cells,
for example, macrophages and dendritic cells that express
mannose receptors or DC-SIGN.16 Oligosaccharide oxazoline
4 carries two lactose moieties ꢀ-1,6-linked to the R-mannoside
residues in the Man3GlcNAc core. Lactose moiety is known to
be recognized by various lectins such as PNA and galectins17
and can serve as a substrate for various glycosyltransferases
like R1,3-galactosyltransferase and sialyltransferases. The in-
corporation of two lactose moieties on the core N-glycan is
likely to enhance the avidity of the ligand to various lectins.18
In oligosaccharide oxazoline 5, two azido groups were intro-
duced at the 6-positions of the terminal mannose residues in
the Man3GlcNAc core. Azido group is one of the most
fascinating functional groups in bioconjugate chemistry because
of its small size and its chemoselective reactivity. Thus, once
it is introduced into a protein, a variety of functional groups
could be site-specifically and chemo-selectively attached through
orthogonal Staudinger ligation or the Huisgen 1,3-dipolar
cycloaddition.19
Results and Discussion
Design and Synthesis of Novel Oligosaccharide Oxazolines
as Donor Substrates. We have previously synthesized several
oligosaccharide oxazolines corresponding to the N-glycan core,
such as oxazolines 1 and 2 (Figure 2), and evaluated them as
substrates of Endo-A for transglycosylation.6,13 We have found
that Endo-A could tolerate certain modifications on the outer
mannose residues of Man3GlcNAc-oxazoline and even the
Manꢀ1,4GlcNAc-oxazoline core. These results imply a broad
substrate specificity for the oxazoline substrates and suggest that
the outer residues might tolerate various modifications, a
property ideal for introducing various carbohydrate ligands. To
further examine the substrate specificity of Endo-A for oxazo-
The synthesis of oxazoline 3 is summarized in Scheme 1.
Disaccharide 8 was prepared via direct ꢀ-mannosylation by the
method of Crich and Dudkin.20 The 2-azido-2-deoxy derivative
6
21 was chosen as the acceptor since it was demonstrated to be
a better glycosyl acceptor in the direct ꢀ-mannosylation reaction
than the corresponding 2-phthalimido or 2-N-acetyl derivative.20
(16) McGreal, E. P.; Miller, J. L.; Gordon, S. Curr. Opin. Immunol. 2005,
17, 18–24.
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1995, 92, 97–101. (c) Liang, R.; Andreotti, A. H.; Kahne, D. J. Am.
Chem. Soc. 1995, 117, 10395–10396. (d) O’Conner, S. E.; Imperiali,
B. Chem. Biol. 1998, 5, 427–437. (e) O’Connor, S. E.; Pohlmann, J.;
Imperiali, B.; Saskiawan, I.; Yamamoto, K. J. Am. Chem. Soc. 2001,
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2002, 12, 616–623.
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(19) Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi, C. R.
ACS Chem. Biol. 2006, 1, 644–648.
(20) Crich, D.; Dudkin, V. J. Am. Chem. Soc. 2001, 123, 6819–6825.
(21) Singh, L.; Seifert, J. Tetrahedron Lett. 2001, 42, 3133–3136.
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Site-Specific Introduction of Ligands into Proteins
A R T I C L E S
Scheme 1. Synthesis of Oligosaccharide Oxazoline 3a
a Reagents and conditions: (a) BSP, TTBP, Tf2O, CH2Cl2, 67%. (b) TFA, CH2Cl2, 85%. (c) TMSOTf, CH2Cl2, 79%. (d) AcSH, 86%. (e) (i) Pd(OH)2-C,
H2, MeOH; (ii) Ac2O, pyridine, 90% (2 steps). (f) TMSBr, BF3 ·OEt2, 2,4,6-collidine, CH2Cl2, 67%. (g) MeONa, MeOH, quant.
Thus, reaction of 6 and glycosyl donor 722 by use of a mixture
of 1-benzenesulfinyl piperidine (BSP)/2,4,6-tri-tert-butylpyri-
midine (TTBP)/trifluoromethanesulfonic (triflic) anhydride
(Tf2O) as the promoter gave the disaccharide 8 in 67% yield.
The 4,6-O-benzylidene group and the 3-O-p-methoxybenzyl
(PMB) group of 8 were removed by treatment with TFA to
give the triol derivative 9. Glycosylation of 9 with an excess (8
mol equiv) of mannosyl trichloroacetimidate (10)23 under the
catalysis of trimethylsilyl triflate (TMSOTf) gave the pentasac-
charide derivative 11 in 79% yield. The newly attached
mannosyl residues were confirmed to be in the desired R-O-
glycosidic linkages by NMR analysis. Next, the 2-azide group
in 11 was converted to an acetamido group by treatment with
thioacetic acid to give 12. The O-benzyl groups were then
removed by hydrogenation, and the resulting hydroxyl groups
were acetylated to provide the fully acetylated derivative 13.
Oxazoline ring formation was achieved by treatment with
trimethylsilyl bromide (TMSBr), BF3 ·OEt2, and 2,4,6-collidine
to provide the oxazoline derivative 14 in 67% yield. Finally,
de-O-acetylation with a catalytic amount of MeONa in MeOH
afforded the pentasaccharide oxazoline 3 in quantitative yield
(Scheme 1).
Selective removal of the two O-acetyl groups in 18 by mild
acidic hydrolysis (acetic chloride in MeOH and dichlo-
romethane) afforded the diol 19. This compound can serve as
a key intermediate for introducing various functional groups at
the 6-positions of the R-mannoside residues. To introduce two
lactose moieties at the N-glycan core, compound 19 was
glycosylated with the lactose glycosyl donor 20,26 giving the
octasaccharide derivative 21. Compound 21 was then changed
to the peracetylated derivative 23 through several steps of
protecting group manipulations. Oxazoline formation was carried
out by treatment of 23 with TMSBr and BF3 ·Et2O to give the
octasaccharide derivative 24 (35%). The relatively low yield
of 24 was mainly due to the slow reaction, as the starting
material 23 was partially recovered after reaction for 3 days.
De-O-acetylation of 24 with MeONa/MeOH gave the octasac-
charide oxazoline 4 in quantitative yield.
The synthesis of azido-containing oxazoline 5 was carried
out with the tetrasaccharide derivative 18 as the starting material
(Scheme 3). The 2-azido group of 18 was converted to an
acetamido group by treatment with thioacetic acid to give 25.
The 6-O-acetyl group was then selectively removed by mild
acidic hydrolysis without affecting the benzoyl groups to give
the diol 26. Tosylation of 26, followed by azide substitution,
provided compound 27 in 84% yield, with two azido moieties
attached at the 6-positions of the terminal R-mannosyl residues.
Removal of the benzoyl groups gave compound 28. Since there
was no efficient method to selectively deprotect the benzyl
groups without affecting the azido functionality in oligosac-
charide synthesis, we decided to apply a two-step conversion
to restore the azido functionality. First, the benzyl groups in 28
were removed by catalytic hydrogenation. Under this condition,
The synthesis of sugar oxazoline 4 is summarized in Scheme
2. Selective removal of the PMB group in disaccharide 8 with
2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), followed by re-
gioselective opening of the 4,6-O-benzylidene with Cu(OTf)2
and BH3 in tetrahydrofuran (THF),24 gave the diol 16 in 58%
yield in two steps. Double glycosylation of diol 16 with 6-O-
acetyl-2,3,4-tri-O-benzoyl-R-D-mannopyranosyl trichloroace-
timidate 1725 provided the tetrasaccharide 18 in excellent yield.
(22) Crich, D.; Li, W.; Li, H. J. Am. Chem. Soc. 2004, 126, 15081–15086.
(23) Ren, T.; Liu, D. Tetrahedron Lett. 1999, 40, 7621–7625.
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Hung, S. C. Angew. Chem., Int. Ed. 2005, 44, 1665–1668.
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(26) Boons, G. J. P. H.; van Delft, F. L.; van der Klein, P. A. M.; van der
Marel, G. A.; van Boom, J. H. Tetrahedron 1992, 48, 885–904.
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Scheme 2. Synthesis of Sugar Oxazoline 4a
a Reagents and conditions: (a) DDQ, CH2Cl2, H2O, 80%. (b) Cu(OTf)2, BH3 ·THF, THF, 73%. (c) TMSOTf, CH2Cl2, 96%. (d) AcCl, CH2Cl2, MeOH,
90%. (e) TMSOTf, CH2Cl2, 65%. (f) (i) MeONa, CH2Cl2, MeOH; (ii) Ac2O, pyridine; (iii) AcSH, pyridine, CHCl3, 79% (3 steps). (g) (i) Pd(OH)2-C, H2,
MeOH; (ii) Ac2O, pyridine, 77% (2 steps). (h) TMSBr, BF3 ·OEt2, 2,4,6-collidine, CH2ClCH2Cl, 35%. (i) MeONa, MeOH, quant.
Scheme 3. Synthesis of Sugar Oxazoline 5a
a Reagents and conditions: (a) AcSH, pyridine, CHCl3, 85%. (b) AcCl, CH2Cl2, MeOH, 72%. (c) (i) TsCl, pyridine; (ii) NaN3, DMF, 84% (2 steps). (d)
MeONa, MeOH, 85%; (e) Pd(OH)2-C, H2, MeOH; (f) TfN3, K2CO3, CuSO4, CH2Cl2, MeOH, H2O. (g) Ac2O, pyridine, 61% (3 steps). (h) TMSBr, BF3 ·OEt2,
2,4,6-collidine, CH2Cl2, 52%. (i) MeONa, MeOH, quant.
the azido groups were simultaneously reduced to amino groups
to give compound 29. Then the amino groups in 29 were
changed back to azido functionality by the copper-catalyzed
diazo transfer reaction27 to form the azido derivative 30. The
reaction was monitored by electrospray ionization mass spec-
trometry (ESI-MS) and the conversion proceeded efficiently to
give the diazido compound, which was isolated as the peracety-
lated derivative 31 in 61% yield in three steps. Treatment of
9
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Site-Specific Introduction of Ligands into Proteins
A R T I C L E S
Scheme 4. Enzymatic Transglycosylationa
a Reagents and conditions: (a) Endo-A, phosphate buffer (50 mM, pH 6.5), 82%. (b) Endo-A, phosphate buffer (50 mM, pH 6.5), 96%. (c) Endo-A,
phosphate buffer (50 mM, pH 6.5), 71%. (d) Endo-A, phosphate buffer (50 mM, pH 6.5), 38%. (e) Endo-A, phosphate buffer (50 mM, pH 6.5), 89%.
31 with TMSBr, BF3 ·OEt2, and 2,4,6-collidine gave the
oxazoline derivative 32 in 52% yield. Finally, de-O-acetylation
with MeONa/MeOH afforded the azido-containing sugar ox-
azoline 5 in quantitative yield (Scheme 3).
mannose at the 4-position of the core ꢀ-mannose residue, could
serve as a donor substrate for Endo-A to react with GlcNAc-
RB (33) to give a new, homogeneous glycoprotein 36, in which
a novel oligomannose glycan was introduced (Scheme 4). This
newly formed glycoprotein 36 was eluted slightly earlier than
GlcNAc-RB under appropriate reverse-phase HPLC (RP-HPLC)
conditions (see Experimental Section) and was purified by RP-
HPLC in 71% yield. The ESI mass spectrum of the glycoprotein
36 (Figure 3A) clearly indicates its homogeneity. Deconvolution
of the ESI mass spectrum of 36 gave a molecular mass of 14 740
Da, which is in good agreement with the calculated mass (14 745
Da) of glycoprotein 36.
The oxazoline 4 was also able to serve as a donor substrate
of Endo-A for transglycosylation, but the enzymatic reaction
proceeded in a much slower path than the enzymatic reactions
with oxazolines 1, 2, and 3. After 3 days of reaction, the newly
formed glycoprotein 37 was isolated by RP-HPLC in 38% yield,
and the unreacted starting material GlcNAc-RB (33) was
recovered. Deconvolution of the ESI mass spectrum of 37
(Figure 3B) gave a molecular mass of 15 229 Da, which agreed
well with the calculated mass (15 231 Da) of glycoprotein 37.
The relatively low reactivity of octasaccharide oxazoline 4 in
comparison with core tetrasaccharide oxazoline 1 and hexas-
accharide oxazoline 2 might be attributed to the unnatural
modification on the 6-positions of the R-mannosyl residues and
the relatively large size of the lactose moieties. Consistent with
this assumption, the sugar oxazoline 5, which has two small
azido groups attached at the 6-position of the outer mannose
residues, was found to be an excellent substrate for Endo-A.
Thus, transglycosylation of GlcNAc-RB (33) with sugar ox-
azoline 5 under the catalysis of Endo-A proceeded efficiently
Enzymatic Transglycosylation To Form Homogeneous
Glycoproteins. To examine the efficiency of the ENGase-
catalyzed transglycosylation for glycoprotein synthesis with
preassembled oligosaccharide oxazolines as the donor substrates,
we have chosen bovine ribonuclease B (RB) as a model system
that has been used by us and others for testing new synthetic
strategy.5,6,9 Bovine RB is a small natural glycoprotein that
consists of 124 amino acids. It carries heterogeneous high-
mannose-type oligosaccharides (Man5-9) attached at the single
glycosylation site, Asn-34. As demonstrated in our previous
work,6 the heterogeneous N-glycans on native RB were removed
by treatment with Endo-H to provide the homogeneous GlcNAc-
protein 33, in which only the innermost GlcNAc residue of the
N-glycan was left at the Asn-34 site. This GlcNAc-containing
protein (GlcNAc-RB) was used as the acceptor to examine the
transglycosylation with synthetic oligosaccharide oxazolines. We
have previously demonstrated that oxazoline 1, corresponding
to the N-glycan Man3GlcNAc core, and oxazoline 2, which has
two galactose residues attached at the 4-positions of the outer
mannose residues of the core, were excellent substrates for
Endo-A-catalyzed transglycosylation with GlcNAc-RB to form
the glycoproteins 34 and 35.6 Thus the Endo-A-catalyzed
transglycosylations with oxazolines 3-5 were examined in a
similar way in phosphate buffer (50 mM, pH 6.5) at 30 °C. It
was found that sugar oxazoline 3, which carries a bisecting
(27) Nyffeler, P. T.; Liang, C. H.; Koeller, K. M.; Wong, C. H. J. Am.
Chem. Soc. 2002, 124, 10773–10778.
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Ochiai et al.
Scheme 5. Synthesis of Alkyne-Functionalized RGal Epitopea
a Reagents and conditions: (a) NaHCO3, MeOH, MeCN, H2O, 79%.
a stable amide bond,28 and the azide-alkyne [3 + 2] cycload-
dition to form a stable triazole linkage under catalysis.29 To
demonstrate usefulness of the combination of the chemoenzy-
matic transglycosylation and the “Click chemistry”, we selected
RGal epitope as a ligand to introduce into RB via the
transglycosylation product glycoprotein 38. For the purpose, an
alkyne-functionalized RGal trisaccharide epitope 41 was pre-
pared with the amino-containing trisaccharide derivative 39 as
the starting material (Schemes 5 and 6), which we have
previously synthesized for HIV-1 immunotargeting purposes.30
To incorporate RGal epitopes into glycoprotein 38 that contains
two azido groups, we examined several catalytic methods for
the 1,3-dipolar cycloaddition. It was found that the cycloaddition
reaction between glycoprotein 38 and the alkyne-oligosaccharide
41 proceeded most efficiently under the catalysis of CuSO4/L-
ascorbic acid in the presence of bathophenanthroline disulfonic
acid (to serve as a ligand for the catalyst).31 Thus, when an
excess of 41 was used, an esentially quantitative conversion of
the azidoprotein 38 to the new glycoprotein 42 was fulfilled
after 12 h at room temperature, as monitored by HPLC and
ESI-MS. After the reaction, the resulting glycoprotein 42 was
easily isolated in 87% yield by RP-HPLC, in which two copies
of the RGal epitope were introduced through the 1,3-dipolar
cycloaddition. The purity and identity of the new glycoprotein
42 were revealed by SDS-PAGE and ESI-MS analysis (Figure
4). The SDS gel of 42 showed a single band at 18 kDa, which
is about 2 kDa larger than the glycoprotein 38, implicating the
introduction of two RGal epitopes (Figure 4A). Deconvolution
of its ESI-MS (Figure 4B) gave a molecular mass of 15 971
Da, which matched well with the calculated mass (M ) 15 976
Da) of glycoprotein 42. It should be pointed out that addition
of the bathophenanthroline ligand in the reaction mixture is
Figure 3. ESI mass spectra of synthetic glycoproteins: (A) glycoprotein
36, (B) glycoprotein 37, and (C) glycoprotein 38. Peaks are labeled
according to the corresponding charge states.
to give the glycoprotein 38 in 89% yield (Scheme 4). Again,
deconvolution of the ESI mass spectrum of 38 (Figure 3C) gave
a molecular mass of 14 630 Da, indicating the attachment of
the azidooligosaccharide moiety to the GlcNAc-RB to form
glycoprotein 38 (calculated molecular mass 14 619 Da).
(28) (a) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007–2010. (b) Lin,
F. L.; Hoyt, H. M.; van Halbeek, H.; Bergman, R. G.; Bertozzi, C. R.
J. Am. Chem. Soc. 2005, 127, 2686–2695.
Ligand Introduction via Huisgen 1,3-Dipolar Cycloaddi-
tion. The efficient chemoenzymatic introduction into proteins
of a core N-pentasaccharide carrying azido functionality opens
an avenue to further site-specific functionalization of the protein,
as the azido group can serve as a unique tag for introducing
various functional groups through highly chemoselective bioor-
thogonal reactions.19 These include the Staudinger ligation, in
which the azide reacts with a functionalized phosphine to form
(29) (a) Breinbauer, R.; Kohn, M. ChemBioChem 2003, 4, 1147–1149. (b)
Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.;
Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192–3193. (c) Link, A. J.;
Vink, M. K.; Tirrell, D. A. J. Am. Chem. Soc. 2004, 126, 10598–
10602. (d) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem.,
Int. Ed. 2001, 40, 2004–2021.
(30) Naicker, K. P.; Li, H.; Heredia, A.; Song, H.; Wang, L. X. Org. Biomol.
Chem. 2004, 2, 660–664.
(31) Sen Gupta, S.; Kuzelka, J.; Singh, P.; Lewis, W. G.; Manchester, M.;
Finn, M. G. Bioconjugate Chem. 2005, 16, 1572–1579.
9
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Site-Specific Introduction of Ligands into Proteins
A R T I C L E S
Scheme 6. Catalytic 1,3-Dipolar Cycloaddition To Introduce RGal Epitopea
a Reagents and conditions: (a) Tris buffer (0.1 M, pH 8.0), CuSO4, L-ascorbic acid, bathophenanthrolinedisulfonic acid, 87%.
essential for the success of cycloaddition, as an inefficient
reaction was observed in the absence of bathophenanthroline
disulfonic acid, giving ca. 28% of a monosubstituted glycopro-
tein and less than 5% of the double-substituted glycoprotein 42
after 24 h of reaction (confirmed by ESI-MS analysis). On the
other hand, we have also observed that the use of CuSO4/copper
wire-catalyzed reaction32 resulted in precipitation of the protein.
There are precedents that certain catalytic conditions for the
Huisgen 1,3-dipolar azide-alkyne cycloaddition would lead to
precipitation of protein substrates.33 Thus, choice of appropriate
cycloaddition conditions is critical for the success of ligation,
particularly when it is applied to protein-based reactants. The
recognition of the RGal epitopes (an array of oligosaccharides
with terminal GalR1,3Gal moieties) on cells of most animals
such as pigs by natural human anti-RGal antibodies is mainly
responsible for the hyperacute rejection in xenotransplantation.34
A successful introduction of RGal epitopes into proteins under
physiological pH and temperature in a site-specific manner may
find interesting applications such as for potential immunotar-
geting.30
that binds terminal R-mannosyl residues.35 Interestingly, gly-
coprotein 36 exhibited a slightly better affinity for ConA than
glycoprotein 34, implicating a positive contribution of the
additional bisecting mannose residue. But the enhancement was
not significant. In contrast, the ribonuclease A that shares the
same sequence as RB but lacks the N-glycan did not bind to
ConA at all (Figure 5A). For the terminal galactose-containing
glycoproteins, it was observed that the glycoproteins 35 and
37, which carried the Galꢀ1,4Man and Galꢀ1,4Glc (lactose)
moieties, respectively, could efficiently bind to PNA, a lectin
that recognizes terminal ꢀ-linked galactose moieties.35 The two
glycoproteins showed very similar affinity to lectin PNA.
However, the RGal-containing glycoprotein 42, as well as the
ribonuclease A that does not contain glycan, did not bind to
PNA (Figure 5B). These results confirm the importance of the
anomeric configuration of carbohydrate ligands in lectin rec-
ognition. To detect the binding of RGal-glycoprotein 42 to
human anti-RGal antibodies, the whole human IgG that contains
about 1-2% anti-RGal antibodies were immobilized on chips
and the antibody chips were used for probing the synthetic
glycoproteins. As expected, the RGal-containing glycoprotein
42 could be readily recognized by the immobilized whole human
IgG that contains a small fraction of anti-RGal antibodies,
whereas the lactose-containing glycoprotein 37 and ribonuclease
A did not react to the immobilized human IgG (Figure 5C).
Binding of the Synthetic Glycoproteins with Respective
Lectins. The introduction of specific oligosaccharide ligands into
a protein or glycoprotein would enable various useful applica-
tions on the basis of ligand-receptor interactions. Thus, we have
performed a preliminary study on the binding of the synthetic
ligand-containing glycoproteins with respective lectin and
antibody, using surface plasmon resonance (SPR) technology.
It was found that the oligomannose-containing glycoproteins
34 and 36 could be efficiently recognized by ConA, a lectin
Conclusion
An array of large oligosaccharide oxazolines were synthesized
and evaluated as substrates for the Endo-A-catalyzed transgly-
cosylation for the synthesis of homogeneous N-glycoproteins
carrying defined oligosaccharide ligands. The results suggest
that Endo-A could tolerate modifications at the outer mannose
residues of the Man3GlcNAc-oxazoline core. Thus a broad
substrate specificity of the enzyme was revealed, which allows
introduction of large oligosaccharide ligands into a protein and
(32) Deiters, A.; Cropp, T. A.; Mukherji, M.; Chin, J. W.; Anderson, J. C.;
Schultz, P. G. J. Am. Chem. Soc. 2003, 125, 11782–11783.
(33) (a) Kalia, J.; Raines, R. T. ChemBioChem 2006, 7, 1375–1383. (b)
Speers, A. E.; Adam, G. C.; Cravatt, B. F. J. Am. Chem. Soc. 2003,
125, 4686–4687.
(34) (a) Galili, U. Immunol. Today 1993, 14, 480–482. (b) Sandrin, M. S.;
Vaughan, H. A.; Dabkowski, P. L.; McKenzie, I. F. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 11391–11395. (c) Alwayn, I. P.; Basker, M.;
Buhler, L.; Cooper, D. K. Xenotransplantation 1999, 6, 157–168.
(35) Lis, H.; Sharon, N. Chem. ReV. 1998, 98, 637–674.
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Figure 4. SDS-PAGE and ESI-MS analysis of the RGal-incorporated
glycoprotein product (42) generated by “Click” chemistry. (A) Coomassie
blue-stained SDS-polyacrylamide gel (lane M is protein marker with sizes
on the left; lane 1 is the starting material, glycoprotein 38; and lane 2 is the
purified product, glycoprotein 42). (B) ESI mass spectrum of glycoprotein
42. Charge states are labeled.
meanwhile preserves the natural N-pentasaccharide (Man3-
GlcNAc2) core upon transglycosylation. In particular, the
incorporation of the azido functionality at the N-pentasaccharide
(Man3GlcNAc2) core permits further structural modifications of
the resulting glycoproteins, as demonstrated by the successful
ligation of two copies of RGal epitopes to ribonuclease B. This
novel chemoenzymatic approach opens a new avenue to various
homogeneous N-glycoproteins that will be useful for basic
research and biomedical applications.
Figure 5. SPR sensorgrams of the binding between respective synthetic
glycoproteins and immobilized lectin or human serum antibody. (A) Binding
to immobilized ConA; (B) binding to immobilized PNA; (C) binding to
immobilized whole IgG-type antibodies from human serum. For comparison,
respective glycoprotein or ribonuclease A was injected onto the sensor chip
surface at a concentration of 1.0 µM.
Experimental Section
performed on a Waters 626 HPLC instrument with a Symmetry300
C18 column (5.0 µm, 4.6 × 250 mm) at 40 °C. The column was
eluted with a linear gradient of 23-29% MeCN containing 0.1%
TFA for 30 min at a flow rate of 1.0 mL/min. Preparative HPLC
was performed with a Waters 600 HPLC instrument of a Waters
C18 column (Symmetry 300, 19 × 300 mm). The column was
eluted with a suitable gradient of MeCN-H2O containing 0.1% TFA
at a flow rate of 12 mL/min.
Benzyl 2-O-Benzyl-4,6-O-benzylidene-3-O-p-methoxybenzyl-
ꢀ-D-mannopyranosyl-(1f4)-2-azido-3,6-di-O-benzyl-2-deoxy-ꢀ-
D-glucopyranoside (8). A mixture of phenyl 2-O-benzyl-4,6-O-
benzylidene-3-O-p-methoxybenzyl-1-thio-R-D-mannopyranoside 722
Materials. Endo-ꢀ-N-acetylglucosaminidase from Arthrobacter
protophormiae (Endo-A) was overproduced in Escherichia coli
following the reported procedure.10 ConA, PNA, and IgG from
normal human serum were purchased from Sigma (St. Louis, MO).
General Procedures. Thin-layer chromatography (TLC) was
performed by use of silica gel on aluminum plates (Sigma-Aldrich).
Flash column chromatography was performed on silica gel 60
(230-400 mesh). SDS-PAGE was performed with an 18% (w/v)
gel. NMR spectra were recorded on a JEOL ECX 400 MHz
spectrometer. The chemical shifts were assigned in parts per million
(ppm). ESI-MS spectra were measured on a Micromass ZQ-4000
single-quadrupole mass spectrometer. Analytical RP-HPLC was
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Site-Specific Introduction of Ligands into Proteins
A R T I C L E S
(450 mg, 0.79 mmol), BSP (182 mg, 0.87 mmol), TTBP (329 mg,
1.58 mmol), and activated 3 Å molecular sieves (3.46 g) in CH2Cl2
(16 mL) was stirred for 30 min at -60 °C under an argon
atmosphere. Then Tf2O (159 µL, 0.95 mmol) was added at this
temperature. After 5 min, a solution of benzyl 2-azido-3,6-di-O-
benzyl-2-deoxy-ꢀ-D-glucopyranoside 621 (250 mg, 0.53 mmol) in
CH2Cl2 (2.6 mL) was added and the mixture was stirred at -60
°C for 2 h. 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 concentrated. The residue was subjected to silica gel
column chromatography (hexanes/EtOAc, 8:1) to afford 8 (329 mg,
2H), 4.51 (d, 1H, J ) 12.3 Hz), 4.46 (d, 1H, J ) 11.9 Hz), 4.46 (s,
1H), 4.34 (d, 1H, J ) 8.3 Hz), 4.30 (m, 1H), 4.19-4.13 (m, 2H),
4.08 (dd, 1H, J ) 11.9, 1.8 Hz), 4.00-3.86 (m, 5H), 3.84 (d, 1H,
J ) 2.8 Hz), 3.79-3.69 (m, 5H), 3.55 (t, 1H, J ) 9.0 Hz),
3.44-3.29 (m, 4H), 3.18 (m, 1H), 2.16, 2.12, 2.10, 2.10, 2.06, 2.04,
2.02, 2.01, 2.00, 1.98, 1.94, 1.89 (12s, 36 H); 13C NMR (100 MHz,
CDCl3) δ 170.71, 170.49, 170.28, 169.97, 169.83, 169.71, 169.69,
169.62, 169.56, 169.49, 138.18, 138.03, 137.62, 136.72, 128.76,
128.38, 128.32, 128.24, 128.05, 127.99, 127.91, 127.53, 127.33,
126.47, 100.44, 99.94, 99.36, 97.62, 83.19, 80.98, 78.02, 77.20,
76.03, 75.78, 74.78, 74.69, 73.83, 73.71, 70.71, 69.65, 69.58, 69.28,
69.17, 68.95, 68.74, 68.50, 68.42, 68.34, 68.27, 66.08, 65.95, 65.65,
65.43, 62.53, 62.48, 61.80, 20.84, 20.78, 20.73, 20.65, 20.61, 20.56.
1
67%) as a colorless syrup. H NMR (400 MHz, CDCl3, TMS) δ
7.49-6.83 (m, 29H), 5.51 (s, 1H), 5.05 (d, 1H, J ) 10.6 Hz), 4.92
(d, 1H, J ) 11.9 Hz), 4.84 (d, 1H, J ) 11.9 Hz), 4.77 (d, 1H, J )
11.9 Hz), 4.69 (d, 1H, J ) 11.9 Hz), 4.67 (d, 1H, J ) 11.9 Hz),
4.62 (d, 2H, J ) 11.4 Hz), 4.52 (d, 1H, J ) 11.9 Hz), 4.49 (s, 1H),
4.40 (d, 1H, J ) 11.9 Hz), 4.28 (d, 1H, J ) 8.2 Hz), 4.10-4.03
(m, 2H), 3.96 (t, 1H, J ) 9.4 Hz), 3.78 (s, 3H), 3.69 (br d, 1H, J
) 2.7 Hz), 3.63 (dd, 1H, J ) 11.3, 2.1 Hz), 3.56-3.45 (m, 3H),
3.40 (dd, 1H, J ) 9.9, 3.1 Hz), 3.32 (t, 1H, J ) 9.2 Hz), 3.26 (m,
1H), 3.08 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 159.10, 138.48,
138.42. 137.60, 137.57, 136.76, 130.50, 129.01, 128.80, 128.49,
128.43, 128.26, 128.11, 128.07, 127.97, 127.94, 127.89, 127.76,
127.49, 126.04, 113.67, 101.42, 101.28, 100.38, 81.47, 78.61, 77.90,
77.31, 77.00, 75.11, 75.04, 74.72, 73.54, 72.29, 70.79, 68.40, 68.29,
67.32, 65.68, 55.21.
Benzyl 2-O-Benzyl-ꢀ-D-mannopyranosyl-(1f4)-2-azido-3,6-
di-O-benzyl-2-deoxy-ꢀ-D-glucopyranoside (9). To a stirred solu-
tion of compound 8 (80 mg, 85 µmol) in CH2Cl2 (4 mL) was added
TFA (0.75 mL) at -20 °C. The mixture was stirred for 30 min at
this temperature and then for 1 h at 0 °C. MeOH (2 mL) and CH2Cl2
(20 mL) were added to the reaction mixture, and the mixture was
sequentially washed with saturated NaHCO3 and brine and then
dried over MgSO4. After removal of the solvent, the residue was
purified by chromatography on a silica gel column (hexanes/EtOAc,
2:3) to yield 9 (53 mg, 85%) as a white amorphous solid. 1H NMR
(400 MHz, CDCl3, TMS) δ 7.41-7.24 (m, 20H), 5.02 (d, 1H, J )
10.5 Hz), 4.94 (d, 1H, J ) 11.5 Hz), 4.94 (d, 1H, J ) 11.9 Hz),
4.71 (d, 1H, J ) 11.9 Hz), 4.71-4.65 (m, 2H), 4.54 (d, 1H, J )
11.5 Hz), 4.50-4.47 (m, 2H), 4.31 (d, 1H, J ) 8.3 Hz), 3.95 (t,
1H, J ) 9.4 Hz), 3.73 (dd, 1H, J ) 11.2, 2.0 Hz), 3.67 (dd, 1H, J
) 11.0, 3.6 Hz), 3.62 (m, 1H), 3.57 (br d, 1H, J ) 3.7 Hz),
3.52-3.48 (m, 2H), 3.39-3.30 (m, 3H), 3.19 (br s, 1H), 3.01 (m,
1H), 2.45 (br s, 1H), 2.30 (br s, 1H), 1.80 (br s, 1H); 13C NMR
(100 MHz, CDCl3) δ 138.21, 137.98, 137.39, 136.70, 128.55,
128.42, 128.27, 128.06, 128.02, 127.91, 127.82, 127.65, 127.41,
100.96, 100.31, 81.35, 77.95, 77.08, 75.56, 75.25, 75.03, 74.79,
73.84, 73.65, 70.76, 68.96, 68.12, 65.72, 62.16.
Benzyl 2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-(1f3)-
[2,3,4,6-tetra-O-acetyl-r-D-mannopyranosyl-(1f4)]-{[2,3,4,6-
tetra-O-acetyl-r-D-mannopyranosyl-(1f6)]}-2-O-benzyl-ꢀ-D-
mannopyranosyl-(1f4)-2-azido-3,6-di-O-benzyl-2-deoxy-ꢀ-D-
glucopyranoside (11). A solution of compound 9 (37 mg, 50 µmol)
and 2,3,4,6-tetra-O-acetyl-R-D-mannopyranosyl tricholoroacetimi-
date 1023 (199 mg, 0.40 mmol) in CH2Cl2 (4 mL) containing
activated 4 Å molecular sieves (240 mg) was stirred under an
atmosphere of argon at room temperature for 1 h. After the mixture
was cooled to -40 °C, a solution of TMSOTf in CH2Cl2 (1 M, 81
µL, 81 µmol) was added and the resulting mixture was stirred at
room temperature for 24 h. The mixture was filtered through a Celite
pad, 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 flash silica gel column chromatography (hexanes/EtOAc, 1:1)
to provide 11 (69 mg, 79%) as a white amorphous solid. 1H NMR
(400 MHz, CDCl3, TMS) δ 7.44-7.18 (m, 20 H), 5.32-5.19 (m,
8H), 5.14-5.10 (m, 3H), 4.96 (s, 1H), 4.94-4.90 (m, 2H), 4.88
(d, 1H, J ) 10.5 Hz), 4.78 (d, 1H, J ) 11.9 Hz), 4.70-4.64 (m,
Benzyl 2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-(1f3)-
[2,3,4,6-tetra-O-acetyl-r-D-mannopyranosyl-(1f4)]-{[2,3,4,6-
tetra-O-acetyl-r-D-mannopyranosyl-(1f6)]}-2-O-benzyl-ꢀ-D-
mannopyranosyl-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
ꢀ-D-glucopyranoside (12). Compound 11 (59 mg, 35 µmol) was
treated with thioacetic acid (2 mL) for 24 h at room temperature.
The reaction mixture was concentrated under reduced pressure and
the residue was purified by silica gel column chromatography
(hexanes/EtOAc, 2:3) to give 12 (52 mg, 86%) as a white
amorphous solid. 1H NMR (400 MHz, CDCl3, TMS) δ 7.43-7.18
(m, 20H), 6.06 (d, 1H, J ) 7.8 Hz), 5.35-5.17 (m, 9H), 5.13 (d,
1H, J ) 12.3 Hz), 5.09 (d, 1H, J ) 1.8 Hz), 4.99 (d, 1H, J ) 7.8
Hz), 4.90-4.84 (m, 3H), 4.77 (d, 1H, J ) 12.4 Hz), 4.59-4.52
(m, 4H), 4.43 (d, 1H, J ) 12.4 Hz), 4.32 (dd, 1H, J ) 12.3, 5.5
Hz), 4.27 (dd, 1H, J ) 12.9, 3.7 Hz), 4.09-3.96 (m, 6H), 3.92-3.57
(m, 10H), 3.48 (m, 1H), 3.31 (m, 1H), 3.21 (m, 1H), 2.16, 2.12,
2.09, 2.08, 2.05, 2.03, 2.02, 1.99, 1.97, 1.94, 1.90, 1.83 (12s, 39H);
13C NMR (100 MHz, CDCl3) δ 170.93, 170.38, 170.31, 170.27,
169.92, 169.80, 169.77, 169.70, 169.62, 169.55, 169.46, 138.56,
138.12, 137.84, 137.55, 128.73, 128.35, 128.28, 128.20, 128.07,
127.70, 127.60, 127.50, 127.25, 126.49, 99.86, 99.62, 99.43, 99.28,
97.00, 82.75, 77.87, 77.63, 77.20, 76.83, 75.99, 74.74, 74.31, 73.72,
73.68, 73.55, 70.71, 69.54, 69.52, 69.43, 69.39, 69.36, 68.97, 68.69,
68.45, 68.33, 67.57, 66.08, 66.04, 65.38, 62.56, 62.47, 62.18, 56.49,
23.34, 20.82, 20.77, 20.76, 20.71, 20.66, 20.63, 20.59, 20.56.
2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-(1f3)-[2,3,4,6-
tetra-O-acetyl-r-D-mannopyranosyl-(1f4)]-{[2,3,4,6-tetra-O-
acetyl-r-D-mannopyranosyl-(1f6)]}-2-O-acetyl-ꢀ-D-mannopy-
ranosyl-(1f4)-2-acetamido-1,3,6-tri-O-acetyl-2-deoxy-D-
glucopyranose (13). To a solution of 12 (52 mg, 30 µmol) in
MeOH (3 mL) was added 20% palladium(II) hydroxide on activated
carbon (30 mg). The mixture was vigorously stirred at room
temperature under a hydrogen atmosphere overnight. The mixture
was filtered through a Celite pad and the filtrate was concentrated
in vacuo. The residue was dissolved in pyridine (4 mL), and Ac2O
(4 mL) was added. The mixture was stirred at room temperature
overnight and then was concentrated. The residue was then
dissolved in CH2Cl2, and the solution was washed sequentially with
1 M HCl, saturated NaHCO3, and brine. The organic layer was
dried over MgSO4 and concentrated. Silica gel column chroma-
tography (EtOAc) of the residue afforded 13 (41 mg, 90%, R/ꢀ )
1
79/21) as a white amorphous solid. H NMR (400 MHz, CDCl3,
TMS, selected signals) δ 6.06 (d, 0.79H, J ) 3.2 Hz), 5.65 (d,
0.21H, J ) 8.3 Hz), 2.22-1.93 (m, 51H); 13C NMR (100 MHz,
CDCl3, selected signals) δ 171.07, 170.65, 170.62, 170.40, 170.24,
170.07, 170.03, 169.86, 169.77, 169.70, 169.62, 169.57, 169.48,
169.22, 169.05, 98.33, 97.17, 97.03, 96.30, 92.23, 90.64, 22.84,
22.66, 20.88, 20.83, 20.68, 20.61, 20.50, 20.44, 20.32. ESI-MS:
calcd for C64H88NO42 [M + H]+, 1542.48; found, 1542.58.
2-Methyl-[2,3,4,6-tetra-O-acetyl-r-D-mannopyranosyl-(1f3)-
[2,3,4,6-tetra-O-acetyl-r-D-mannopyranosyl-(1f4)]-{[2,3,4,6-
tetra-O-acetyl-r-D-mannopyranosyl-(1f6)]}-2-O-acetyl-ꢀ-D-man-
nopyranosyl-(1f4)-3,6-di-O-acetyl-1,2-dideoxy-r-D-glucopyrano]-
[2,1-d]-2-oxazoline (14). To a solution of 13 (214 mg, 0.14 mmol)
in CH2Cl2 (21 mL) mL) containing activated 4 Å molecular sieves
(1.7 g) were added 2,4,6-collidine (365 µL, 2.8 mmol), TMSBr
9
J. AM. CHEM. SOC. VOL. 130, NO. 41, 2008 13799

A R T I C L E S
Ochiai et al.
(357 µL, 2.8 mmol), and BF3 ·OEt2 (347 µL, 2.8 mmol). The
reaction mixture was stirred at room temperature for 24 h. The
mixture was diluted with CH2Cl2, filtered through a Celite pad,
washed sequentially with saturated NaHCO3 and brine, dried over
MgSO4, filtered, and concentrated. The residue was chromato-
graphed (hexanes/EtOAc, 1:1 to 1:4) to give 14 (137 mg, 67%) as
(m, 25H), 5.05 (d, 1H, J ) 10.5 Hz), 4.95-4.92 (m, 2H), 4.81 (d,
1H, J ) 11.0 Hz), 4.71-4.66 (m, 3H), 4.59 (d, 1H, J ) 11.4 Hz),
4.57 (d, 1H, J ) 11.0 Hz), 4.49 (d, 1H, J ) 12.4 Hz), 4.50 (s, 1H),
4.31 (d, 1H, J ) 8.2 Hz), 3.94 (t, 1H, J ) 9.4 Hz), 3.71 (dd, 1H,
J ) 11.0, 1.9 Hz), 3.66 (dd, 1H, J ) 11.0, 3.2 Hz), 3.65-3.57 (m,
2H), 3.52-3.42 (m, 3H), 3.38-3.30 (m, 3H), 3.05 (m, 1 H), 2.28
(m, 1 H), 1.59 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 138.35,
138.20, 138.05, 137.40, 136.73, 128.55, 128.50, 128.43, 128.38,
128.33, 128.05, 127.94, 127.89, 127.77, 127.65, 127.19, 100.97,
100.32, 81.37, 78.32, 77.17, 76.40, 75.25, 74.92, 74.82, 74.64,
74.07, 73.67, 70.77, 68.12, 65.80, 62.02.
1
a white amorphous solid. H NMR (400 MHz, CDCl3, TMS) δ
5.91 (d, 1H, J ) 7.4 Hz), 5.55 (d, 1H, J ) 2.3 Hz), 5.36-5.16 (m,
10H), 5.10 (d, 1H, J ) 1.0 Hz), 5.02 (s, 1H), 4.98 (d, 1H, J ) 0.9
Hz), 4.79 (s, 1H), 4.37-3.89 (m, 16H), 3.67 (m, 1H), 3.60 (d, 1H,
J ) 8.7 Hz), 3.41 (m, 1H), 2.20, 2.15, 2.12, 2.12, 2.11, 2.11, 2.10,
2.09, 2.09, 2.05, 2.04 (11s, 33H), 2.03 (d, 3H), 2.01, 1.98, 1.96,
1.96 (4s, 12H); 13C NMR (100 MHz, CDCl3) δ 170.56, 170.47,
170.34, 169.75, 169.70, 169.67, 169.64, 169.58, 169.52, 169.40,
165.86, 99.55, 99.05, 98.93, 98.11, 78.33, 77.20, 76.45, 74.71,
74.00, 70.18, 69.52, 69.49, 69.39, 69.32, 69.18, 69.05, 68.94, 68.49,
68.36, 67.81, 67.18, 65.98, 65.81, 65.58, 64.43, 63.39, 62.36, 62.15,
62.01, 20.87, 20.80, 20.70, 20.62, 20.58, 20.54, 13.49. ESI-MS:
calcd for C62H84NO40 [M + H]+, 1482.46; found, 1482.37.
Benzyl 6-O-Acetyl-2,3,4-tri-O-benzoyl-r-D-mannopyranosyl-
(1f3)-[6-O-acetyl-2,3,4-tri-O-benzoyl-r-D-mannopyranosyl-
(1f6)]-2,4-di-O-benzyl-ꢀ-D-mannopyranosyl-(1f4)-2-azido-3,6-
di-O-benzyl-2-deoxy-ꢀ-D-glucopyranoside (18). A solution of
compound 16 (76 mg, 93 µmol) and 2,3,4-tri-O-acetyl-6-O-benzoyl-
R-D-mannopyranosyl trichloroacetimidate 1725 (315 mg, 464 µmol)
in CH2Cl2 (3.9 mL) containing activated 4 Å molecular sieves (469
mg) was stirred under an atmosphere of argon at room temperature
for 15 min. After the mixture was cooled to -40 °C, a solution of
TMSOTf in CH2Cl2 (1 M, 46 µL, 46 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 flash silica
gel column chromatography (hexanes/EtOAc, 5:1 to 3:1) to provide
18 (165 mg, 96%) as a white amorphous solid. 1H NMR (400 MHz,
CDCl3, TMS) δ 8.11-7.13 (m, 55H), 5.98-5.87 (m, 3H),
5.84-5.79 (m, 2H), 5.54 (d, 1H, J ) 1.9 Hz), 5.37 (s, 1H), 5.20
(d, 1H, J ) 12.3 Hz), 5.85-5.06 (m, 2H), 4.99 (d, 1H, J ) 10.5
Hz), 4.92 (d, 1H, J ) 11.9 Hz), 4.77 (d, 1H, J ) 12.4 Hz),
4.73-4.64 (m, 3H), 4.60 (s, 1H), 4.52 (d, 1H, J ) 11.9 Hz), 4.35
(d, 1H, J ) 8.2 Hz), 4.29-4.23 (m, 2H), 4.18-4.11 (m, 3H),
4.06-4.00 (m, 2H), 3.94 (m, 1H), 3.85-3.61 (m, 6H), 3.44 (m,
1H), 3.40-3.26 (m, 3H), 2.06 (s, 3H), 2.00 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 170.69, 170.36, 165.45, 165.40, 165.33, 165.09,
164.97, 138.52, 138.47, 137.72, 137.45, 136.93, 133.58, 133.44,
133.38, 133.23, 133.19, 132.90, 129.82, 129.80, 129.76, 129.70,
129.44, 129.22, 129.14, 129.02, 128.77, 128.57, 128.54, 128.51,
128.46, 128.34, 128.29, 128.25, 128.12, 127.99, 127.93, 127.83,
127.46, 127.35, 126.98, 100.78, 100.34, 99.84, 97.89, 83.24, 81.02,
78.33, 76.14, 75.67, 75.49, 74.95, 74.65, 74.39, 74.30, 73.69, 70.85,
70.35, 70.01, 69.94, 69.67, 69.17, 68.94, 68.60, 68.25, 66.75, 66.41,
65.36, 62.78, 62.27, 20.67, 20.58.
Benzyl 2,3,4-Tri-O-benzoyl-r-D-mannopyranosyl-(1f3)-[2,3,4-
tri-O-benzoyl-r-D-mannopyranosyl-(1f6)]-2,4-di-O-benzyl-ꢀ-D-
mannopyranosyl-(1f4)-2-azido-3,6-di-O-benzyl-2-deoxy-ꢀ-D-
glucopyranoside (19). To a solution of 18 (124 mg, 67 µmol) in
1:2 (v/v) CH2Cl2-MeOH (12 mL) was added acetyl chloride (0.4
mL). After being stirred overnight, the mixture was concentrated
under reduced pressure, and the residue was purified by flash silica
gel column chromatography (hexanes/EtOAc, 3:1 to 1:1) to give
19 (107 mg, 90%) as a white amorphous solid. 1H NMR (400 MHz,
CDCl3, TMS) δ 8.08-7.12 (m, 55H), 6.04 (dd, 1H, J ) 10.1, 3.2
Hz), 5.89 (dd, 1H, J ) 10.1, 3.2 Hz), 5.82 (dd, 1H, J ) 3.5, 1.7
Hz), 5.79-5.73 (m, 2H), 5.58 (dd, 1H, J ) 3.2, 1.8 Hz), 5.39 (d,
1H, J ) 1.4 Hz), 5.15 (d, 1H, J ) 1.8 Hz), 5.13 (d, 1H, J ) 12.4
Hz), 5.07-5.01 (m, 2H), 4.95 (d, 1H, J ) 11.9 Hz), 4.77 (d, 1H,
J ) 11.0 Hz), 4.72 (d, 1H, J ) 9.6 Hz), 4.69-4.65 (m, 2H), 4.62
(s, 1H), 4.51 (d, 1H, J ) 12.4 Hz), 4.33 (d, 1H, J ) 8.2 Hz), 4.28
(t, 1H, J ) 9.4 Hz), 3.97 (m, 1H), 3.90-3.86 (m, 2H), 3.82 (t, 1H,
J ) 9.4 Hz), 3.73-3.54 (m, 8H), 3.48-3.27 (m, 5H), 2.85 (m,
1H), 2.40 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 166.39, 166.25,
165.36, 165.33, 165.20, 138.48, 138.44, 137.58, 137.49, 136.99,
133.65, 133.54, 133.33, 133.18, 132.87, 129.88, 129.83, 129.67,
129.34, 129.22, 129.15, 129.06, 128.93, 128.59, 128.55, 128.49,
128.44, 128.39, 128.36, 128.28, 128.24, 128.17, 128.07, 128.03,
127.95, 127.81, 127.75, 127.71, 127.42, 127.34, 127.21, 100.74,
2-Methyl-[r-D-mannopyranosyl-(1f3)-[r-D-mannopyranosyl-
(1f4)]-{[r-D-mannopyranosyl-(1f6)]}-ꢀ-D-mannopyranosyl-
(1f4)-1,2-dideoxy-r-D-glucopyrano]-[2,1-d]-2-oxazoline (3). To
a solution of 14 (98 mg, 66 µmol) in MeOH (6 mL) was added
MeONa in MeOH (0.5 M, 13.2 µL, 6.6 µmol). After being stirred
at room temperature overnight, the reaction mixture was concen-
trated to dryness. The residue was dissolved in water and lyophilized
to give the oxazoline 3 (56 mg, quantitative). 1H NMR (400 MHz,
D2O) δ 5.96 (d, 1H, J ) 7.4 Hz), 5.00 (d, 1H, J ) 1.4 Hz), 4.94
(s, 1H), 4.88 (d, 1H, J ) 1.9 Hz), 4.60 (s, 1H), 4.23 (dd, 1H, J )
3.0, 1.6 Hz), 4.08-4.05 (m, 2H), 3.91-3.48 (m, 26H), 3.27 (m,
1H), 1.94 (d, 3H, J ) 1.8 Hz); 13C NMR (100 MHz, D2O) δ 168.67,
102.92, 101.94, 101.02, 100.07, 99.97, 82.39, 77.92, 73.91, 73.75,
73.55, 72.86, 70.97, 70.54, 70.40, 70.20, 69.97, 69.17, 66.84, 66.50,
66.34, 65.25, 61.77, 61.09, 60.99, 60.94, 13.01. ESI-MS: calcd for
C32H54NO25 [M + H]+, 852.30; found, 852.40.
Benzyl 2-O-Benzyl-4,6-O-benzylidene-ꢀ-D-mannopyranosyl-
(1f4)-2-azido-3,6-di-O-benzyl-2-deoxy-ꢀ-D-glucopyranoside
(15). To a solution of 8 (2.08 g, 2.22 mmol) in a mixture of CH2Cl2/
H2O (17.5/1, 74 mL) was added DDQ (1.16 g, 5.11 mmol) at 0
°C. After 30 min, the reaction mixture was warmed to room
temperature and further stirred for 1 h. The reaction mixture was
diluted with CH2Cl2, washed with saturated NaHCO3 and brine,
and dried over MgSO4. Concentration and purification by column
chromatography on silica gel (hexanes/EtOAc, 10:1 to 3:1) provided
15 (1.46 g, 80%) as a white amorphous solid. 1H NMR (400 MHz,
CDCl3, TMS) δ 7.47-7.25 (m, 25H), 5.44 (s, 1H), 5.05 (d, 1H, J
) 10.1 Hz), 4.94 (d, 1H, J ) 11.9 Hz), 4.92 (d, 1H, J ) 11.4 Hz),
4.73 (d, 1H, J ) 12.3 Hz), 4.69 (d, 1H, J ) 12.4 Hz), 4.66-4.61
(m, 2H), 4.58 (s, 1H), 4.47 (d, 1H, J ) 11.9 Hz), 4.30 (d, 1H, J )
7.8 Hz), 4.07 (dd, 1H, J ) 10.3, 4.8 Hz), 4.01 (t, 1H, J ) 9.2 Hz),
3.73-3.64 (m, 4H), 3.56-3.44 (m, 3H), 3.35-3.30 (m, 2H), 3.08
(m, 1H), 2.32 (d, 1H, J ) 8.7 Hz); 13C NMR (100 MHz, CDCl3)
δ 138.36, 138.00, 137.46, 137.16, 136.71, 129.08, 128.59, 128.43,
128.21, 128.15, 128.12, 127.98, 127.94, 127.92, 127.86, 127.55,
126.25, 101.92, 101.57, 100.34, 81.50, 79.02, 78.84, 77.49, 75.77,
75.14, 74.75, 73.68, 70.87, 70.79, 68.35, 68.15, 66.88, 65.67.
Benzyl 2,4-Di-O-benzyl-ꢀ-D-mannopyranosyl-(1f4)-2-azido-
3,6-di-O-benzyl-2-deoxy-ꢀ-D-glucopyranoside (16). A 1 M solu-
tion of BH3 ·THF in THF (6 mL, 6 mmol) was added to compound
15 (980 mg, 1.20 mmol) at room temperature. The mixture was
stirred for 5 min, and copper(II) trifluoromethanesulfonate (43 mg,
0.119 mmol) was added to the solution. After being stiredg for
5 h, the mixture was cooled down to 0 °C, and the reaction was
quenched by sequential additions of Et3N (167 µL, 1.2 mmol) and
MeOH (2.2 mL). The resulting mixture was concentrated under
reduced pressure followed by coevaporation with MeOH. The
residue was purified by flash column chromatography (hexanes/
EtOAc, 3:1) on silica gel to give 16 (718 mg, 73%) as a white
amorphous solid. 1H NMR (400 MHz, CDCl3, TMS) δ 7.42-7.25
9
13800 J. AM. CHEM. SOC. VOL. 130, NO. 41, 2008

Site-Specific Introduction of Ligands into Proteins
A R T I C L E S
100.43, 99.86, 97.78, 82.62, 81.23, 78.06, 76.12, 75.67, 75.44,
74.80, 74.70, 74.38, 74.33, 73.79, 71.56, 71.23, 70.78, 70.53, 70.35,
69.80, 69.40, 68.11, 67.98, 67.26, 67.12, 65.32, 61.33, 61.04.
66.87, 66.49, 66.38, 65.83, 65.38, 61.79, 60.67, 60.51, 52.70, 23.04,
20.72, 20.54, 20.42.
2,3,4,6-Tetra-O-acetyl-ꢀ-D-galactopyranosyl-(1f4)-2,3,6-tri-
O-acetyl-ꢀ-D-glucopyranosyl-(1f6)-2,3,4-tri-O-acetyl-r-D-man-
nopyranosyl-(1f3)-[2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl-
(1f4)-2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl-(1f6)-2,3,4-tri-O-
acetyl-r-D-mannopyranosyl-(1f6)]-2,4-di-O-acetyl-ꢀ-D-
mannopyranosyl-(1f4)-2-acetamido-1,3,6-tri-O-acetyl-2-deoxy-
D-glucopyranose (23). To a solution of 22 (37 mg, 14 µmol) in
25:30:1 (v/v/v) CH2Cl2-MeOH-AcOH (1.2 mL) was added 20%
palladium(II) hydroxide on activated carbon (37 mg). The reaction
mixture was vigorously stirred at room temperature under hydrogen
atmosphere for 24 h. The mixture was filtered through Celite and
the filtrate was concentrated in vacuo. Pyridine (5 mL) and Ac2O
(5 mL) were added, and the mixture was stirred 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 MgSO4 and concentrated. Silica
gel column chromatography (CH2Cl2/MeOH, 40:1) of the residue
afforded 23 (26 mg, 77%, R/ꢀ ) 90/10) as a white amorphous
Benzyl 2,3,4,6-Tetra-O-benzoyl-ꢀ-D-galactopyranosyl-(1f4)-
2,3,6-tri-O-benzoyl-ꢀ-D-glucopyranosyl-(1f6)-2,3,4-tri-O-ben-
zoyl-r-D-mannopyranosyl-(1f3)-[2,3,4,6-tetra-O-benzoyl-ꢀ-D-
galactopyranosyl-(1f4)-2,3,6-tri-O-benzoyl-ꢀ-D-glucopyranosyl-
(1f6)-2,3,4-tri-O-benzoyl-r-D-mannopyranosyl-(1f6)]-2,4-di-
O-benzyl-ꢀ-D-mannopyranosyl-(1f4)-2-azido-3,6-di-O-benzyl-
2-deoxy-ꢀ-D-glucopyranoside (21). A mixture of compound 19
(24 mg, 14 µmol) and 2,3,4,6-tetra-O-benzoyl-ꢀ-D-galactopyranosyl-
(1f4)-2,3,6-tri-O-benzoyl-R-D-glucopyranosyl trichloroacetimidate
2026 (83 mg, 68 µmol) in CH2Cl2 (1.1 mL) containing activated 4
Å molecular sieves (132 mg) was stirred under an atmosphere of
argon at -40 °C for 30 min. A solution of TMSOTf in CH2Cl2 (1
M, 6.8 µL, 6.8 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 flash silica gel column chroma-
tography (hexanes/EtOAc, 3:1 to 1:1) to provide 21 (34 mg, 65%)
as a white amorphous. 1H NMR (400 MHz, CDCl3, TMS) δ
8.11-6.85 (m, 125H), 5.98-5.51 (m, 14H), 5.33-5.29 (m, 2H),
5.05-4.35 (m, 23H), 4.20-3.34 (m, 26H), 3.16 (m, 1H); 13C NMR
(100 MHz, CDCl3) δ 165.74, 165.67, 165.59, 165.47, 165.33,
165.27, 165.24, 165.16, 165.09, 164.99, 164.96, 164.68, 138.84,
138.81, 138.31, 137.81, 137.10, 133.44, 133.30, 133.22, 133.17,
133.00, 132.92, 132.84, 132.70, 129.91, 129.84, 129.75, 129.60,
129.53, 129.40, 129.34, 129.28, 129.21, 129.17, 129.12, 128.86,
128.82, 128.74, 128.68, 128.65, 128.49, 128.36, 128.32, 128.20,
128.16, 128.12, 128.10, 128.00, 127.95, 127.79, 127.71, 127.49,
127.38, 127.10, 127.04, 126.88, 102.09, 101.44, 100.93, 100.84,
100.31, 98.92, 97.47, 81.19, 80.53, 78.46, 77.20, 76.18, 75.85,
75.01, 74.93, 74.79, 74.44, 74.05, 73.28, 72.91, 72.80, 72.72, 71.80,
71.61, 71.54, 71.26, 70.93, 70.72, 70.13, 70.04, 69.76, 69.54, 69.34,
68.91, 68.32, 67.43, 67.17, 67.05, 66.96, 66.86, 65.61, 62.67, 62.36,
60.99, 60.37.
1
solid. H NMR (400 MHz, CDCl3, TMS, selected signals) δ 6.04
(d, 0.90H, J ) 3.6 Hz), 5.75 (d, 0.10H, J ) 9.2 Hz), 5.61 (d, 0.90H,
J ) 9.2 Hz), 5.56 (d, 0.10H, J ) 8.7 Hz), 2.18-1.91 (m, 78H);
13C NMR (100 MHz, CDCl3, for the major R-anomer) δ 171.17,
170.60, 170.12, 170.09, 170.00, 169.93, 169.82, 169.64, 169.49,
169.39, 169.29, 168.91, 168.79, 168.72, 101.18, 100.84, 100.73,
100.55, 97.05, 96.59, 96.26, 90.55, 75.95, 75.71, 72.58, 72.35,
72.28, 71.73, 71.07, 71.01, 70.79, 70.73, 70.39, 70.21, 69.73, 69.49,
69.34, 69.20, 68.91, 67.71, 67.29, 66.55, 66.39, 65.84, 65.37, 61.74,
61.52, 60.56, 60.37, 50.18, 22.70, 20.56, 20.39, 20.28. ESI-MS:
calcd for C100H136NO66 [M + H]+, 2406.73; found, 2307.06.
2-Methyl-[2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl-(1f4)-
2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl-(1f6)-2,3,4-tri-O-acetyl-
r-D-mannopyranosyl-(1f3)-[2,3,4,6-tetra-O-acetyl-ꢀ-D-galac-
topyranosyl-(1f4)-2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl-(1f6)-
2,3,4-tri-O-acetyl-r-D-mannopyranosyl-(1f6)]-2,4-di-O-benzyl-
ꢀ-D-mannopyranosyl-(1f4)-3,6-di-O-acetyl-1,2-dideoxy-r-D-
glucopyrano]-[2,1-d]-2-oxazoline (24). In a manner similar to the
preparation of the oxazoline derivative 14, compound 23 (10 mg,
4.3 µmol) was converted to 24 (3.5 mg, 35%). 1H NMR (400 MHz,
CDCl3, TMS) δ 5.92 (d, 1H, J ) 6.8 Hz), 5.55 (t, 1H, J ) 3.2
Hz), 5.36-4.88 (m, 20H), 4.83 (d, 1H, J ) 1.4 Hz), 4.52-4.44
(m, 5H), 4.38 (d, 1H, J ) 7.7 Hz), 4.26-3.43 (m, 26H), 3.37 (m,
1H), 2.22-1.94 (m, 75H); 13C NMR (100 MHz, CDCl3) δ 170.80,
170.53, 170.31, 170.11, 170.00, 169.91, 169.68, 169.63, 169.57,
169.50, 169.41, 169.04, 166.03, 101.00, 100.97, 100.90, 100.82,
99.67, 98.93, 97.81, 97.21, 76.17, 75.97, 74.19, 72.68, 72.57, 72.26,
71.29, 71.24, 70.96, 70.56, 70.18, 70.10, 69.93, 69.59, 69.24, 69.11,
69.03, 68.57, 68.45, 67.91, 67.73, 66.99, 66.54, 65.99, 64.83, 63.71,
61.84, 60.73, 60.60, 20.74, 20.57, 20.45, 13.80. ESI-MS: calcd for
C98H132NO64 [M + H]+, 2346.71; found, 2346.65.
Benzyl 2,3,4,6-Tetra-O-acetyl-ꢀ-D-galactopyranosyl-(1f4)-
2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl-(1f6)-2,3,4-tri-O-acetyl-
r-D-mannopyranosyl-(1f3)-[2,3,4,6-tetra-O-acetyl-ꢀ-D-galac-
topyranosyl-(1f4)-2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl-(1f6)-
2,3,4-tri-O-acetyl-r-D-mannopyranosyl-(1f6)]-2,4-di-O-benzyl-
ꢀ-D-mannopyranosyl-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
ꢀ-D-glucopyranoside (22). Compound 21 (148 mg, 38 µmol) was
dissolved in 1:1 (v/v) CH2Cl2-MeOH (8 mL), and a solution of 0.5
M MeONa in MeOH (0.4 mL, 0.2 mmol) was added. After being
stirred at room temperature overnight, the solution was neutralized
with Dowex 50W (H+), filtered, and concentrated. The residue was
dissolved in dry pyridine (5 mL) and treated with Ac2O (5 mL) at
room temperature overnight. The mixture was evaporated to dryness
under reduced pressure, and the residue was diluted with CH2Cl2,
washed sequentially with 1 M HCl, saturated NaHCO3 and brine,
dried over MgSO4, filtered, and concentrated. To the residue were
added CHCl3 (1 mL), pyridine (1 mL), and thioacetic acid (2 mL),
and the mixture was stirred at room temperature for 48 h.
Evaporation of the solvent under reduced pressure was followed
by silica gel column chromatography (hexanes/EtOAc, 2:5 to 1:3)
2-Methyl-[ꢀ-D-galactopyranosyl-(1f4)-ꢀ-D-glucopyranosyl-
(1f6)-r-D-mannopyranosyl-(1f3)-[ꢀ-D-galactopyranosyl-(1f4)-
ꢀ-D-glucopyranosyl-(1f6)-r-D-mannopyranosyl-(1f6)]-ꢀ-D-
mannopyranosyl-(1f4)-1,2-dideoxy-r-D-glucopyrano]-[2,1-d]-
2-oxazoline (4). In the same manner as described for the conversion
of 14 to 3, de-O-acetylation of compound 24 (3.5 mg, 1.5 µmol)
with a catalytic amount of MeONa in MeOH gave compound 4
1
to yield 22 (80 mg, 79%) as a white amorphous solid. H NMR
1
(2.0 mg) in quantitative yield. H NMR (400 MHz, D2O) δ 5.98
(400 MHz, CDCl3, TMS) δ 7.43-7.18 (m, 25H), 6.11 (d, 1H, J )
7.8 Hz), 5.35-4.33 (m, 38H), 4.13-3.41 (m, 26H), 3.35 (m, 1H),
3.25 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 170.25, 170.20,
170.15, 170.06, 169.96, 169.73, 169.65, 169.57, 169.55, 169.46,
168.92, 138.75, 138.30, 138.21, 137.64, 137.49, 128.33, 128.31,
128.17, 127.99, 127.94, 127.69, 127.61, 127.52, 127.46, 127.23,
101.21, 100.92, 100.62, 100.26, 99.64, 98.49, 97.12, 79.61, 78.44,
77.11, 76.65, 76.14, 76.06, 75.49, 75.37, 75.06, 74.96, 74.59, 73.19,
72.73, 72.60, 72.25, 72.16, 71.24, 71.05, 70.90, 70.85, 70.53, 70.49,
70.15, 69.73, 69.52, 69.46, 69.15, 69.00, 68.97, 68.73, 68.57, 67.30,
(d, 1H, J ) 7.3 Hz), 4.97 (s, 1H), 4.83 (s, 1H), 4.63 (s, 1H), 4.43
(d, 1H, J ) 7.7 Hz), 4.42 (d, 1H, J ) 7.8 Hz), 4.33 (d, 1H, J ) 7.8
Hz), 4.32 (d, 1H, J ) 7.3 Hz), 4.28 (m, 1H), 4.09-3.18 (m, 47H),
1.97 (s, 3H); 13C NMR (100 MHz, D2O) δ 168.62, 102.98, 102.67,
102.56, 102.54, 101.34, 99.92, 99.80, 81.12, 78.50, 78.42, 77.79,
75.39, 74.82, 74.79, 74.29, 74.23, 73.05, 72.86, 72.55, 72.27, 71.69,
70.99, 70.42, 70.24, 70.00, 69.85, 69.07, 68.78, 68.58, 66.51, 66.43,
65.83, 65.69, 65.12, 62.51, 61.89, 61.06, 60.98, 60.11, 13.10. ESI-
MS: calcd for C50H84NO40 [M + H]+, 1338.46; found, 1338.85.
9
J. AM. CHEM. SOC. VOL. 130, NO. 41, 2008 13801

A R T I C L E S
Ochiai et al.
Benzyl 6-O-Acetyl-2,3,4-tri-O-benzoyl-r-D-mannopyranosyl-
(1f3)-[6-O-acetyl-2,3,4-tri-O-benzoyl-r-D-mannopyranosyl-
(1f6)]-2,4-di-O-benzyl-ꢀ-D-mannopyranosyl-(1f4)-2-acetamido-
3,6-di-O-benzyl-2-deoxy-ꢀ-D-glucopyranoside (25). To a solution
of 18 (200 mg, 0.108 mmol) in CHCl3 (2 mL) were consecutively
added pyridine (2 mL) and thioacetic acid (2 mL) at room
temperature. After being stirred for 48 h, the mixture was
concentrated and purified by flash column chromatography on silica
gel column chromatography (hexanes/EtOAc, 2:1 to 1:1) of the
residue afforded 27 (261 mg, 84%) as a white amorphous solid.
1H NMR (400 MHz, CDCl3, TMS) δ 8.09-7.16 (m, 55H), 5.95
(dd, 1H, J ) 10.3, 3.5 Hz), 5.85-5.76 (m, 4H), 5.64 (s, 1H), 5.44
(d, 1H, J ) 7.8 Hz), 5.40 (s, 1H), 5.21-5.18 (m, 2H), 5.05 (d, 1H,
J ) 11.0 Hz), 5.00 (d, 1H, J ) 7.8 Hz), 4.92-4.86 (m, 2H), 4.87
(d, 1H, J ) 11.9 Hz), 4.76 (s, 1H), 4.71 (d, 1H, J ) 11.0 Hz),
4.64-4.59 (m, 2H), 4.53 (d, 1H, J ) 12.3 Hz), 4.49 (d, 1H, J )
11.9 Hz), 4.19-4.10 (m, 2H), 4.02 (d, 1H, J ) 2.8 Hz), 3.91-3.73
(m, 6H), 3.59-3.35 (m, 5H), 3.29-3.24 (m, 2H), 1.61-1.57 (m,
3H); 13C NMR (100 MHz, CDCl3) δ 170.00, 165.49, 165.47,
165.26, 165.10, 164.95, 138.88, 138.51, 138.05, 137.81, 137.45,
133.56, 133.51, 133.37, 133.14, 133.04, 129.82, 129.76, 129.63,
129.60, 129.32, 129.06, 128.94, 128.92, 128.81, 128.71, 128.54,
128.44, 128.24, 128.13, 128.11, 128.04, 127.99, 127.87, 127.77,
127.64, 127.56, 127.42, 127.26, 100.47, 99.70, 99.36, 97.43, 82.14,
78.06, 77.14, 76.69, 75.42, 75.04, 74.91, 74.37, 73.37, 73.00, 70.90,
70.79, 70.27, 70.02, 69.97, 69.93, 69.42, 69.20, 67.98, 67.56, 67.50,
55.96, 51.19, 50.88, 23.28.
1
gel to afford 25 (172 mg, 85%) as a white amorphous solid. H
NMR (400 MHz, CDCl3, TMS) δ 8.13-7.13 (m, 55H), 5.99-5.90
(m, 3H), 5.82-5.79 (m, 2H), 5.61 (t, 1H, J ) 2.3 Hz), 5.36 (d,
1H, J ) 0.9 Hz), 5.28-5.25 (m, 2H), 5.22 (d, 1H, J ) 12.8 Hz),
5.07 (d, 1H, J ) 11.0 Hz), 5.02 (d, 1H, J ) 7.8 Hz), 4.87 (d, 1H,
J ) 11.5 Hz), 4.82 (d, 1H, J ) 11.9 Hz), 4.76 (d, 1H, J ) 12.4
Hz), 4.70 (d, 1H, J ) 11.0 Hz), 4.66 (s, 1H), 4.65 (d, 1H, J ) 11.9
Hz), 4.55 (d, 1H, J ) 10.9 Hz), 4.51 (d, 1H, J ) 11.9 Hz), 4.46 (d,
1H, J ) 11.9 Hz), 4.27-4.08 (m, 8H), 3.94 (d, 1H, J ) 3.2 Hz),
3.85-3.72 (m, 4H), 3.71-3.66 (m, 2H), 3.47-3.38 (m, 2H), 3.35
(m, 1H), 2.09 (s, 3H), 1.99 (s, 3H), 1.58 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 170.75, 170.31, 170.04, 165.55, 165.44, 165.39,
165.32, 165.10, 164.77, 138.80, 138.54, 137.95, 137.73, 137.44,
133.55, 133.42, 133.37, 133.29, 133.15, 133.07, 129.81, 129.75,
129.68, 129.61, 129.20, 129.00, 128.96, 128.80, 128.75, 128.55,
128.50, 128.46, 128.39, 128.34, 128.30, 128.26, 128.19, 128.12,
128.08, 127.95, 127.78, 127.60, 127.50, 127.41, 127.31, 126.95,
100.28, 99.82, 99.32, 97.33, 83.11, 78.30, 77.20, 77.15, 75.50,
75.38, 74.82, 74.52, 74.26, 73.56, 73.21, 70.98, 70.34, 70.15, 70.10,
69.69, 69.19, 69.11, 68.80, 68.26, 66.70, 66.49, 62.82, 62.40, 56.62,
23.28, 20.67, 20.56.
Benzyl 6-Azido-r-D-mannopyranosyl-(1f3)-[6-Azido-r-D-
mannopyranosyl-(1f6)]-2,4-di-O-benzyl-ꢀ-D-mannopyranosyl-
(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-ꢀ-D-glucopyrano-
side (28). Compound 27 (262 mg, 0.143 µmol) was dissolved in
1:4 (v/v) CH2Cl2-MeOH (20 mL), and a solution of 0.5 M MeONa
in MeOH (0.8 mL, 0.4 mmol) was added. After being stirred at
room temperature for 2 h, the solution was neutralized with Dowex
50W (H+), filtered, and concentrated. The residue was subjected
to flash silica gel column chromatography (CH2Cl2/MeOH, 10:1)
1
to give 28 (146 mg, 85%) as a white amorphous solid. H NMR
(400 MHz, CD3OD, selected signals) δ 7.42-7.14 (m, 25H), 1.80
(s, 3H); 13C NMR (100 MHz, CD3OD) δ 173.40, 140.56, 140.29,
139.49, 139.44, 139.17, 129.77, 129.62, 129.45, 129.41, 129.33,
129.27, 129.22, 129.16, 129.09, 129.04, 128.95, 128.83, 128.75,
128.54, 128.42, 104.16, 101.83, 101.38, 82.54, 81.62, 80.23, 78.08,
76.69, 76.13, 76.10, 76.02, 75.97, 75.46, 74.86, 74.48, 73.54, 72.58,
72.48, 72.32, 71.99, 71.88, 69.90, 69.58, 69.47, 67.51, 56.45, 53.36,
52.79, 23.21. ESI-MS: calcd for C61H74N7O19 [M + H]+, 1208.50;
found, 1208.90.
2,3,4-Tri-O-acetyl-6-azido-r-D-mannopyranosyl-(1f3)-[2,3,4-
tri-O-acetyl-6-azido-r-D-mannopyranosyl-(1f6)]-2,4-di-O-acetyl-
ꢀ-D-mannopyranosyl-(1f4)-2-acetamido-1,3,6-tri-O-acetyl-2-
deoxy-D-glucopyranose (31). The title compound was prepared via
a three-step procedure:
(1) To a solution of 28 (100 mg, 83 µmol) in MeOH (5 mL)
was added 20% palladium(II) hydroxide on activated carbon (100
mg). The reaction mixture was vigorously stirred at room temper-
ature under hydrogen atmosphere for 7 h. After addition of H2O (5
mL), the reaction mixture was stirred overnight at room temperature
under hydrogen atmosphere. The mixture was filtered through a
Celite pad and the filtrate was concentrated in vacuo to give
6-amino-R-D-mannopyranosyl-(1f3)-[6-amino-R-D-mannopyrano-
syl-(1f6)]-ꢀ-D-mannopyranosyl-(1f4)-2-acetamido-2-deoxy-D-
glucopyranose 29: ESI-MS: calcd for C26H48N3O19 [M + H]+,
706.29; found, 706.40. Compound 29 was used in the next step
without further purification.
Benzyl 2,3,4-Tri-O-benzoyl-r-D-mannopyranosyl-(1f3)-[2,3,4-
tri-O-benzoyl-r-D-mannopyranosyl-(1f6)]-2,4-di-O-benzyl-ꢀ-D-
mannopyranosyl-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
ꢀ-D-glucopyranoside (26). The title compound was prepared from
25 (85 mg, 46 µmol) in a manner similar to that described for 19.
Flash silica gel column chromatography (hexanes/EtOAc, 1:1 to
2:3) of the crude product afforded 26 (59 mg, 72%) as a white
amorphous solid. 1H NMR (400 MHz, CDCl3, TMS) δ 8.13-7.08
(m, 55H), 6.05 (dd, 1H, J ) 10.1, 3.2 Hz), 5.92 (t, 1H, J ) 10.1
Hz), 5.85-5.81 (m, 3H), 5.61 (t, 1H, J ) 2.6 Hz), 5.41 (d, 1H, J
) 1.4 Hz), 5.37 (d, 1H, J ) 1.4 Hz), 5.14 (d, 1H, J ) 12.4 Hz),
5.04 (d, 1H, J ) 11.0 Hz), 5.03 (d, 1H, J ) 7.3 Hz), 4.90 (d, 1H,
J ) 11.9 Hz), 4.88 (d, 1H, J ) 11.0 Hz), 4.77 (d, 1H, J ) 12.4
Hz), 4.69-4.63 (m, 3H), 4.49 (d, 1H, J ) 10.9 Hz), 4.48 (d, 1H,
J ) 12.4 Hz), 4.47 (d, 1H, J ) 11.9 Hz), 4.31 (t, 1H, J ) 8.5 Hz),
4.21 (t, 1H, J ) 8.7 Hz), 4.07 (m, 1H), 3.97 (m, 1H), 3.88-3.70
(m, 7H), 3.65-3.55 (m, 4H), 3.49 (m, 1H), 3.37 (m, 1H), 3.30-3.21
(m, 2H), 2.38 (dd, 1H, J ) 7.8, 6.0 Hz), 1.45 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 170.16, 166.35, 165.76, 165.54, 165.39,
165.19, 164.84, 138.74, 138.35, 137.78, 137.55, 137.37, 133.63,
133.51, 133.17, 132.96, 129.84, 129.70, 129.64, 129.11, 129.02,
128.94, 128.56, 128.50, 128.42, 128.33, 128.26, 128.20, 127.98,
127.94, 127.80, 127.57, 127.45, 127.27, 100.44, 99.77, 99.08, 97.56,
82.29, 78.06, 76.67, 76.60, 75.96, 75.50, 74.83, 74.44, 74.25, 73.61,
73.34, 71.53, 71.47, 70.99, 70.49, 70.36, 70.23, 69.42, 68.74, 68.40,
66.99, 61.23, 60.92, 56.73, 23.18.
(2) Compound 29 was dissolved in H2O (4 mL) and treated with
K2CO3 (91 mg, 0.66 mmol) and CuSO4 (6.0 mg, 37 µmol). To the
solution was added MeOH (9.4 mL) and a freshly prepared solution
of trifluoromethanesulfonyl azide (0.53 mmol)36 in CH2Cl2 (4 mL).
After being stirred at room temperature for 24 h, the reaction
mixture was concentrated to afford crude 6-azido-R-D-mannopy-
ranosyl-(1f3)-[6-amino-R-D-mannopyranosyl-(1f6)]-ꢀ-D-man-
nopyranosyl-(1f4)-2-acetamido-2-deoxy-D-glucopyranose 30: ESI-
MS: calcd for C26H43KN7O19 [M + K]+ 796.23; found, 796.36.
Compound 30 was used directly in the next step.
Benzyl 6-Azido-2,3,4-tri-O-benzoyl-r-D-mannopyranosyl-
(1f3)-[6-azido-2,3,4-tri-O-benzoyl-r-D-mannopyranosyl-(1f6)]-
2,4-di-O-benzyl-ꢀ-D-mannopyranosyl-(1f4)-2-acetamido-3,6-di-
O-benzyl-2-deoxy-ꢀ-D-glucopyranoside (27). To a solution of 26
(304 mg, 0.171 mmol) in pyridine (2 mL) was added TsCl (195
mg, 1.02 mmol). The mixture was stirred overnight at room
temperature. The reaction mixture was then concentrated, diluted
with CH2Cl2, and washed sequentially with 1 M HCl, saturated
NaHCO3, and brine. The organic layer was dried over MgSO4. After
removal of solvent, the tosylated crude product was dissolved in
DMF (5 mL), and NaN3 (222 mg, 3.41 mmol) was added. The
reaction solution was stirred at 80 °C overnight. After removal of
DMF, the residue was poured into brine and extracted with EtOAc.
The organic layer was dried over MgSO4 and concentrated. Silica
(3) Compound 30 was acetylated with acetic anhydride (10 mL)
in pyridine (10 mL) at room temperature for overnight. The mixture
(36) Alper, P. B.; Hung, S. C.; Wong, C.-H. Tetrahedron Lett. 1996, 37,
6029–6032.
9
13802 J. AM. CHEM. SOC. VOL. 130, NO. 41, 2008

Site-Specific Introduction of Ligands into Proteins
A R T I C L E S
was concentrated, diluted with CH2Cl2, and washed sequentially
with 1 M HCl, saturated NaHCO3, and brine. The organic layer
was dried over MgSO4 and concentrated. Silica gel column
chromatography (CH2Cl2/MeOH, 60:1) of the residue afforded 31
(61 mg, 61% from 28, R/ꢀ ) 38/62) as a white amorphous solid.
1H NMR (400 MHz, CDCl3, TMS, selected signals) δ 6.07 (d,
0.38H, J ) 3.6 Hz), 5.96 (d, 0.62H, J ) 9.6 Hz), 5.73 (d, 0.38H,
J ) 9.1 Hz), 5.61 (d, 0.62H, J ) 8.7 Hz), 2.22-1.92 (m, 36H);
13C NMR (100 MHz, CDCl3, selected signals) δ 171.09, 170.65,
170.39, 170.36, 170.09, 170.03, 169.99, 169.88, 169.78, 169.61,
169.49, 169.34, 168.91, 168.85, 98.13, 97.65, 97.25, 96.63, 96.55,
96.19, 92.37, 90.40, 22.73, 22.64, 20.71, 20.58, 20.53, 20.48, 20.36.
ESI-MS: calcd for C48H66N7O30 [M + H]+, 1220.39; found,
1220.47.
2-Methyl-[2,3,4-tri-O-acetyl-6-azido-r-D-mannopyranosyl-
(1f3)-[2,3,4-tri-O-acetyl-6-azido-r-D-mannopyranosyl-(1f6)]-
2,4-di-O-acetyl-ꢀ-D-mannopyranosyl-(1f4)-3,6-di-O-acetyl-1,2-
dideoxy-r-D-glucopyrano]-[2,1-d]-2-oxazoline (32). Compound 32
(26 mg, 52%) was obtained from 31 (53 mg, 43 µmol) following
the procedure described for 14. 1H NMR (400 MHz, CDCl3, TMS)
δ 5.92 (d, 1H, J ) 6.8 Hz), 5.59 (m, 1H), 5.44 (d, 1H, J ) 3.2
Hz), 5.30-5.16 (m, 6H), 5.01-4.99 (m, 2H), 4.83 (s, 1H), 4.81 (s,
1H), 4.27-4.06 (m, 5H), 3.93-3.91 (m, 2H), 3.69-3.62 (m, 3H),
3.45-3.26 (m, 5H), 2.24 (s, 3H), 2.16-2.15 (m, 6H), 2.14, 2.11,
2.09, 2.06 (4s, 12H), 2.05-2.04 (m, 3H), 2.02, 1.99, 1.97 (3s, 9H);
13C NMR (100 MHz, CDCl3) δ 170.72, 170.55, 169.99, 169.87,
169.79, 169.59, 169.56, 169.51, 169.38, 165.96, 99.23, 98.52, 97.33,
76.44, 76.01, 72.66, 70.36, 69.86, 69.78, 69.70, 69.08, 68.69, 67.89,
67.77, 67.38, 66.87, 66.57, 64.57, 63.30, 50.76, 50.60, 20.86, 20.69,
20.53, 13.62. ESI-MS: calcd for C46H62N7O28 [M + H]+, 1160.36;
found 1160.41.
2-Methyl-[6-azido-r-D-mannopyranosyl-(1f3)-[6-azido-r-D-
mannopyranosyl-(1f6)]-ꢀ-D-mannopyranosyl-(1f4)-1,2-dideoxy-
r-D-glucopyrano]-[2,1-d]-2-oxazoline (5). Compound 32 (27 mg,
23 µmol) was de-O-acetylated in a manner similar to the preparation
of compound 3 to give the title compound (5) (17 mg, quantitative).
1H NMR (400 MHz, D2O) δ 5.97 (d, 1H, J ) 7.3 Hz), 4.96 (d,
1H, J ) 1.4 Hz), 4.83 (d, 1H, J ) 1.4 Hz), 4.64 (s, 1H), 4.27 (dd,
1H, J ) 3.0, 1.6 Hz), 4.08 (m, 1H), 4.04 (d, 1H, J ) 3.2 Hz), 3.96
(dd, 1H, J ) 3.4, 1.6 Hz), 3.91-3.87 (m, 2H), 3.82-3.39 (m, 17H),
3.28 (m, 1H), 1.95 (d, 3H, J ) 1.4 Hz); 13C NMR (100 MHz,
D2O) δ 168.68, 102.72, 101.44, 99.92, 99.68, 81.05, 77.86, 74.36,
72.20, 71.52, 71.01, 70.46, 70.37, 70.13, 69.97, 69.81, 69.05, 67.70,
67.53, 65.56, 65.11, 61.83, 51.37, 51.16, 13.01. ESI-MS: calcd for
C26H42N7O18 [M + H]+, 740.26; found, 740.46.
38%). ESI-MS of 37: calcd, M ) 15 231; found, 1693.04 [M +
9H]9+, 1523.86 [M + 10H]10+, 1385.46 [M + 11H]11+, 1270.13
[M + 12H]12+, 1172.57 [M + 13H]13+, and 1088.84 [M +
14H]14+
.
Glycoprotein 38. Glycoprotein 38 was prepared from 5 (0.64
mg, 0.86 µmol) and GlcNAc-RB 33 (1.0 mg, 0.072 µmol), as
described in the preparation of 36, to give glycoprotein 38 (0.0643
µmol, 0.94 mg, 89%). ESI-MS of 38: calcd, M ) 14 619; found,
1626.58 [M + 9H]9+, 1464.00 [M + 10H]10+, 1331.10 [M +
11H]11+, 1220.23 [M + 12H]12+, 1126.47 [M + 13H]13+, and
1046.13 [M + 14H]14+
.
2-[2-(4-Pentynamido)ethoxy]ethyl r-D-galactopyranosyl-(1f3)-
ꢀ-D-galactopyranosyl-(1f4)-ꢀ-D-glucopyranoside (41). To a
solution of 2-(2-aminoethoxy)-ethyl R-D-galactopyranosyl-(1f3)-
ꢀ-D-galactopyranosyl-(1f4)-ꢀ-D-glucopyranoside 3930 (5.0 mg, 8.5
µmol) in aqueous NaHCO3 (0.3 M, 250 µL) and MeOH (50 µL)
was added a solution of N-succinimido 4-pentynonate 4037 (2.5
mg, 13 µmol) in MeCN (250 µL). The residue was purified by
preparative HPLC (0-10% MeCN in 30 min) to give 41 (4.5 mg,
6.7 µmol, 79%). 1H NMR (400 MHz, D2O) δ 5.03 (d, 1H, J ) 3.5
Hz), 4.41-4.39 (m, 2H), 4.09-4.06 (m, 2H), 3.96-3.82 (m, 4H),
3.76-3.46 (m, 16H), 3.32-3.29 (m, 2H), 3.23 (t, 1H, J ) 8.3 Hz),
2.41-2.32 (m, 5H); ESI-MS: calcd for C27H46NO18 [M + H]+,
672.27; found, 672.45.
Glycoprotein 42. Compound 41 (37.6 µL, 20 mM in H2O),
CuSO4 (9.4 µL, 50 mM in H2O), bathophenanthrolinedisulfonic
acid (18.8 µL, 20 mM in H2O), and L-ascorbic acid (9.4 µL, 50
mM in H2O) were added to a solution of glycoprotein 38 (0.55
mg, 0.038 µmol) in Tris buffer (0.17 M, pH 8.0, 113 µL). The
reaction mixture was incubated at room temperature overnight. The
solution was subjected to preparative HPLC purification (23-29%
MeCN containing 0.1% TFA in 30 min) to afford the glycoprotein
42 (0.033 µmol, 0.52 mg, 87%). ESI-MS: calcd MS ) 15 976;
found, 1775.43 [M + 9H]9+, 1598.08 [M + 10H]10+, 1453.03 [M
+ 11H]11+, 1331.98 [M + 12H]12+, and 1229.71 [M + 13H]13+
.
Surface Plasmon Resonance Measurements. SPR measure-
ments were performed with a BIACore T100 instrument (GE
Healthcare). ConA, PNA, and IgG from human serum were
immobilized on CM5 sensor chips in an acetate buffer (10 mM,
pH 5.0) by use of the amine coupling kit provided by the
manufacturer. Approximately 4000 resonance units (RU) of protein
was immobilized. Analyses were performed at 25 °C with a flow
rate of 30 mL/min in either HBS-P+ buffer (10 mM HEPES, 150
mM NaCl, and 0.05% surfactant P20, pH 7.4), for PNA and IgG,
or HBS-P+ buffer containing 1 mM CaCl2 and 1 mM MnCl2, for
ConA. Injection times for glycoproteins were 2 min followed by
10 min of dissociation. Regeneration was performed by either a
1-min pulse of 100 mM glycine (pH 1.7) stripping buffer, for ConA
and IgG, or a 1-min pulse of 0.3 M lactose, for PNA.
Glycoprotein 36. A mixture of pentasaccharide oxazoline 3 (0.36
mg, 0.42 µmol) and GlcNAc-RB 336 (1.95 mg, 0.14 µmol) in a
phosphate buffer (50 mM, pH 6.5, 75 µL) was incubated at 30 °C
with Endo-A (20 milliunits). The reaction was monitored by
analytical HPLC. After 7 h, additional oxazoline 3 (0.36 mg, 0.42
µmol) and Endo-A (20 milliunits) were added to the solution, and
the mixture was incubated at 30 °C. This procedure was repeated
twice to push the reaction to completion. The product was then
purified by preparative HPLC (23-29% MeCN containing 0.1%
TFA in 30 min) to afford the glycoprotein 36 (0.099 µmol, 1.46
mg, 71%). The isolated glycoprotein product was quantified by UV
absorbance at 280 nm, with a standard solution (at an accurate molar
concentration) of GlcNAc-RB as the reference. ESI-MS of 36:
calcd, M ) 14 745; found, 1638.71 [M + 9H]9+, 1475.01 [M +
10H]10+, 1341.05 [M + 11H]11+, 1229.32 [M + 12H]12+, 1134.91
Acknowledgment. We thank Professor Kaoru Takegawa for
providing the pGEX-2T/Endo-A plasmid that was used for expres-
sion of the Endo-A enzyme, Dr. Bing Li for providing compound
35 that was included in the binding studies, and Dr. Cishan Li for
helpful discussions. This work was supported by the National
Institutes of Health (NIH Grant R01 GM080374).
Supporting Information Available: 1H and 13C NMR spectra
of key synthetic compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
[M + 13H]13+, and 1053.91 [M + 14H]14+
.
JA805044X
Glycoprotein 37. Incubation of oxazoline 4 (1.15 mg, 0.86
µmol), GlcNAc-RB 33 (1.0 mg, 0.072 µmol), and Endo-A (20
milliunits) was performed in the same way as described for the
preparation of 36 to give glycoprotein 37 (0.028 µmol, 0.42 mg,
(37) Salmain, M.; Vessieres, A.; Butler, I. S.; Jaouen, G. Bioconjugate
Chem. 1991, 2, 13–15.
9
J. AM. CHEM. SOC. VOL. 130, NO. 41, 2008 13803