Glycoinsulins: Dendritic sialyloligosaccharide-displaying insulins showing a prolonged blood-sugar-lowering activity

Article abstract of DOI:10.1021/ja046426l

Mono-, di-, and trisialyloligosaccharides were introduced to mutant insulins through enzymatic reactions. Sugar chains were sialylated by α2,6-sialyltransferase (α2,6-SiaT) via an accessible glutamine residue at the N-terminus of the B-chain attached by t

Full text of DOI:10.1021/ja046426l

Published on Web 10/07/2004
Glycoinsulins: Dendritic Sialyloligosaccharide-Displaying
Insulins Showing a Prolonged Blood-Sugar-Lowering Activity
Masaaki Sato,† Tetsuya Furuike,‡ Reiko Sadamoto,§ Naoki Fujitani,†
Taku Nakahara,† Kenichi Niikura,† Kenji Monde,† Hirosato Kondo,| and
Shin-Ichiro Nishimura*,†,
Contribution from the DiVision of Biological Sciences, Graduate School of Science,
Frontier Research Center for Post-Genomic Science and Technology,
Hokkaido UniVersity, Kita 21 Nishi 11, Sapporo 001-0021, Japan, DiVision of Bio-science,
Graduate School of EnVironmental Earth Science, Hokkaido UniVersity, Kita 10 Nishi 5,
Sapporo 060-0810, Japan, Shionogi Laboratory of Biomolecular Chemistry,
DiVision of Biological Science, Graduate School of Science,
Frontier Research Center for Post-Genomic Science and Technology, Hokkaido UniVersity,
Kita 21 Nishi 11, Sapporo 001-0021, Japan, DiscoVery Research Laboratories,
Shionogi & Company, Ltd., Osaka 553-0002, Japan, and
National Institute of AdVanced Industrial Science and Technology (AIST),
Sapporo 062-8517, Japan
Received June 17, 2004; E-mail: shin@glyco.sci.hokudai.ac.jp
Abstract: Mono-, di-, and trisialyloligosaccharides were introduced to mutant insulins through enzymatic
reactions. Sugar chains were sialylated by R2,6-sialyltransferase (R2,6-SiaT) via an accessible glutamine
residue at the N-terminus of the B-chain attached by transglutaminase (TGase). Sia2,6-di-LacNAc-Ins(B-
F1Q) and Sia2,6-tri-LacNAc-Ins(B-F1Q), displaying two and three sialyl-N-acetyllactosamines, respectively,
were administered to hyperglycemic mice. Both branched glycoinsulins showed prolonged glucose-lowering
effects compared to native or lactose-carrying insulins, showing that sialic acid is important in obtaining a
prolonged effect. Sia2,6-tri-LacNAc-Ins(B-F1Q), in particular, induced a significant delay in the recovery of
glucose levels.
in controlling circulating glucose levels,10 and in the risk of
anaphylactoid reactions.11-13 Soluble long-acting derivatives,
Introduction
Insulin, which is composed of two polypeptide chains (A and
B) joined by disulfide bridges, is the most important protein
for the treatment of virtually all type 1 and many type 2 diabetic
patients.1-4 Its greatest weakness, however, is its short half-
life in the circulatory system.5 Commercial long-acting insulin
formulations are designed to be less soluble in physiological
solutions and thereby are able to dissolve more slowly at the
injection site.6-8 The disadvantages of these long-acting insulins
are in the complex administration procedure,9 in the difficulty
such as Co3+-insulin,14 fatty acid-acylated insulin,9 PEG-
conjugated insulin,15 and so on, have been reported as potential
candidates to overcome these shortcomings.10
In the case of other widely used protein-based drugs, such
as erythropoietin, the introduction of sialic acid-containing
carbohydrates into the protein has been reported to extend their
half-life in vivo.16,17
In many cases, glycosylation of targeted proteins was carried
out using an expression system in mammalian cells. However,
glycosylation in mammalian cells cannot be controlled in terms
of the structure of the glyco chain, glycosylation site, and
number. Therefore, the preparation of controlled site-specific
glycosylation may benefit from a chemical or chemoenzymatic
† Graduate School of Science, Hokkaido University.
‡ Graduate School of Environmental Earth Science, Hokkaido University.
§ Shionogi Laboratory of Biomolecular Chemistry, Graduate School of
Science, Hokkaido University.
| Shionogi & Co., Ltd.
AIST.
(10) Gershonov, E.; Goldwaser, I.; Fridkin, M.; Shechter, Y. J. Med. Chem.
2000, 43, 2530-2537.
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1999, 68, 71-83.
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J. Circulation 1984, 70, 788-792.
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(4) DeWitt, D. E.; Hirsch, I. B. J. Am. Med. Assoc. 2003, 289, 2254-2264.
(5) Gershonov, E.; Shechter, Y.; Fridkin, M. Diabetes 1999, 48, 1437-1442.
(6) Hallas-Moller, K. Diabetes 1956, 5, 7-14.
(12) Ellerhorst, J. A.; Comstock, J. P.; Nell, L. J. Am. J. Med. Sci. 1990, 299,
298-301.
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2003, 31, 290-299.
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I. Diabetologia 1996, 39, 281-288.
9
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J. AM. CHEM. SOC. 2004, 126, 14013-14022
14013

A R T I C L E S
Sato et al.
Scheme 1. Chemoenzymatic Syntheses of Sialooligosaccharide-Displaying Insulins
in vitro approach. Recently, Davis et al. have explored a
chemical approach to creating a “glycodendriprotein” by the
chemical attachment of galactose derivatives onto the inserted
cysteine residue of target proteins.18 Introduction of a sugar
moiety to the glutamine (Q) residues using transglutaminase
(TGase) was reported to be a very effective method for obtaining
glycoconjugate peptides and proteins.19-21
and synthesized a single-sialyllactose-displaying insulin, Sia2,6-
Lac-Ins(B-F1Q), in which sialyllactose was enzymatically
transferred to the glutamine residues (Q1 or Q4) of the insulin
B-chain (Scheme 1). Sia2,6-Lac-Ins(B-F1Q) had increased
solubility and prolonged biological potency. On the contrary,
lactose-displaying insulin (Lac-Ins(B-F1Q)) showed glucose
depression potencies similar to those of normal insulin (Ins-
(WT)). We speculated that the sialic acid moiety of Sia2,6-
Lac-Ins(B-F1Q) is an important factor in construction of long-
acting insulins by protecting the insulin from decomposition
and/or clearance in vivo.
We have reported that a glycosylated insulin using TGase
could serve as a soluble long-acting option.22 We had designed
(18) Rendle, P. M.; Seger, A.; Rodrigues, J.; Oldham, N. J.; Bott, R. R.; Jones,
J. B.; Cowan, M. M.; Davis, B. G. J. Am. Chem. Soc. 2004, 126, 4750-
4751.
(19) Ramos, D.; Rollin, P.; Klaffke, W. J. Org. Chem. 2001, 66, 2948-2956.
(20) Sato, H.; Hayashi, E.; Yamada, N.; Yatagai, M.; Takahara, Y. Bioconjugate
Chem. 2001, 12, 701-710.
(21) Ohta, T.; Miura, N.; Fujitani, N.; Nakajima, F.; Niikura, K.; Sadamoto,
R.; Guo, C. T.; Suzuki, T.; Suzuki, Y.; Monde, K.; Nishimura, S. Angew.
Chem., Int. Ed. 2003, 42, 5186-5189.
In this study, aiming to obtain a further prolonged effect, we
created divalent- and trivalent-sialyloligosaccharide-displaying
insulins using an enzymatic approach. The glycosylation method
(22) Sato, M.; Sadamoto, R.; Niikura, K.; Monde, K.; Kondo, H.; Nishimura,
S. Angew. Chem., Int. Ed. 2004, 43, 1516-1520.
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14014 J. AM. CHEM. SOC. VOL. 126, NO. 43, 2004

Glycoinsulins
A R T I C L E S
Figure 1. Glycosylation sites on the insulin structure: the 3D structure of
human insulin in the T-state. This model is based on the crystal structure
of Smith et al. (PDB ID 1G7A23,25). The A-chain is colored green, and the
B-chain is colored cyan. The proposed receptor interaction sites (Gly A1,
Ile A2, Val A3, Gln A5, Tyr A19, Asn A21, Val B12, Tyr B16, Gly B23,
Phe B24, Phe B25, and Tyr B26) are colored red. The glycosylation sites
are indicated by arrows: 1, N-terminus of the A-chain; 2, C-terminus of
the A-chain; 3, N-terminus of the B-chain. This figure was prepared by
PyMOL.24
Figure 2. A single-step conversion from His-PI(B-F1Q) to Ins(B-F1Q).
The His-tag and insulin C-chain are released concomitantly by trypsin and
carboxypeptidase B.
through which the synthesized branched-sugar compounds were
introduced to Ins(B-F1Q) was performed using TGase, and the
transference of sialic acids from CMP-sialic acids to branched-
sugar termini was performed by R2,6-sialyltransferase (R2,6-
SiaT). Biological activities of these sugar-displaying insulins
were described.
2. Genetic Expression of Mutant Insulins. Mutagenic
insulins Ins(B-F1Q), having a glutamine (Q) residue instead of
phenylalanine (F) at the N-terminus of the insulin B-chain, Ins-
(A-G1Q), having a Q residue instead of glycine (G) at the
N-terminus of the insulin A-chain, and Ins(A-N21Q), having a
Gln residue instead of asparagine (N) at the C-terminus of the
A-chain, were expressed as proinsulin, which is suitable for
refolding after expression as an inclusion body in Escherichia
coli.26,27 In 1996, a single-step conversion method from IgG-
binding domain (ZZ)-tagged proinsulin (ZZ-proinsulin) to
insulin was reported by Per Jonasson et al.28 This method was
applied to the removal of the polyhistidine (His) tag fused to
the N-terminus of proinsulins; the expressed His-fused proin-
sulins His-PI(WT), His-PI(B-F1Q), His-PI(A-G1Q), and
His-PI(A-N21Q) had an arginine (R) residue between the His-
tag and the proinsulin (Figure 2). It was revealed that trypsin
cleaved at basic amino acid residues located in the C-terminus
region during proinsulin processing, however, did not cleave
at Arg-B22 and Lys-B29.29 A single-step conversion from His-
PI to insulin was carried out using trypsin and carboxypeptidase
B in quantitative yield, and purification was performed by RP-
HPLC.
Results and Discussion
A. Expression of Mutant Insulins Having an Accessible
Glutamine Residue for Use as a Glycosylation Site. 1. Design
of Sugar-Displaying Insulins. Our previous research22 showed
that native insulin was not a substrate of transglutaminase
although it has three glutamine (Gln) residues. Therefore,
accessible Gln residues for use as glycosylation sites needed to
be inserted by genetic mutation. Although the details and
mechanism of the insulin-insulin receptor (IR) interaction are
still to be investigated, some residues on the insulin surface are
known to be important to its interaction with the IR. The
“classical binding surface” includes Gly A1, Gln A5, Tyr A19,
Asn A21, Val B12, Tyr B16, Gly B23, Phe B24, Phe B25, and
Tyr B26.23 Additionally, internal residues Ile A2 and Val A3
are also believed to interact with the IR. These residues cluster
on one side of the 3D structure of the insulin (Figure 1).24,25
We prepared three mutant insulins with Gln substitutions on
either the N-terminus of the A- or B-chains or the C-terminus
of the A-chain. The addition of sugar chains to the Gln residues
on either terminus of the insulin A-chain would be expected to
weaken the interaction between the insulin and the IR, resulting
in a slowing of the degradation rate of the insulin molecules.
The addition of a sugar chain to the Gln residues on the
N-terminus of the B-chain would be expected to have less effect
on the insulin-IR interaction.
B. Synthesis of Branched Oligosaccharides. The syntheses
of divalent and trivalent N-acetyllactosaminyl (LacNAc) deriva-
tives 1 and 2 are outlined in Schemes 2 and 3, respectively.
First, Nꢀ-(benzyloxycarbonyl)-L-lysine (Lys(Z)) was acylated
with succinic anhydride to afford dicarboxyl peptide 3 in 86%
(26) Williams, D. C.; Van Frank, R. M.; Muth, W. L.; Burnett, J. P. Science
1982, 215, 687-689.
(27) Winter, J.; Neubauer, P.; Glockshuber, R.; Rudolph, R. J. Biotechnol. 2001,
84, 175-185.
(23) De Meyts, P.; Whittaker, J. Nat. ReV. Drug DiscoVery 2002, 1, 769-783.
(24) Smith, G. D.; Pangborn, W. A.; Blessing, R. H. Acta Crystallogr. 2001,
D57, 1091-1100.
(28) Jonasson, P.; Nilsson, J.; Samuelsson, E.; Moks, T.; Stahl, S.; Uhlen, M.
Eur. J. Biochem. 1996, 236, 656-661.
(29) Wang, M.; Scott, W. A.; Rao, K. R.; Udey, J.; Conner, G. E.; Brew, K. J.
Biol. Chem. 1989, 264, 21116-21121.
(25) DeLano, W. L. http://www.pymol.org, 2002.
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A R T I C L E S
Scheme 2 a
Sato et al.
a Reagents and conditions: (i) succinic anhydride, methanol, 40 °C, 12 h, 86%; (ii) pentafluorophenyl trifluoroacetate, pyridine, DMF, room temperature
(rt), 1 h, 77%; (iii) 3-aminopropyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyranoside, diisopropylethylamine, DMF, rt, 17 h, 84%; (iv) triethylamine,
MeOH-H2O, rt, 4 h, 90%; (v) UDP-galactose, â-1,4-galactosyltransferase, 50 mM HEPES buffer containing 10 mM MnCl2 (pH 6.0), 37 °C, 48 h, 83%;
(vi) H2, 10% Pd-C, AcOH, MeOH-H2O, rt, 6 h, 92%.
Scheme 3 a
a Reagents and conditions: (i) N-succinimidylsuccinic acid allyl ester, triethylamine, DMF, rt, 8 h, 98%; (ii) L-glutamic acid diallyl ester,
N-hydroxysuccinimide, dicyclohexylcarbodiimide, DMF, rt, 15 h, 86%; (iii) (Ph3P)4Pd, N-methylaniline, THF, rt, 14 h, 74%; (iv) pentafluorophenyl
trifluoroacetate, pyridine, DMF, rt, 1 h, 79%; (v) 3-aminopropyl 2-acetamido-3,4,6-tri-O-acetyl- 2-deoxy-â-D-glucopyranoside, diisopropylethylamine, DMF,
rt, 17 h, 85%; (vi) triethylamine, MeOH-H2O, rt, 4 h, quantitative yield; (vii) UDP-galactose, â-1,4-galactosyltransferase, 50 mM HEPES buffer containing
10 mM MnCl2 (pH 6.0), 37 °C, 48 h, 96%; (viii) H2, 10% Pd-C, AcOH, MeOH-H2O, rt, 6 h, 84%.
yield. The coupling reaction of 3-aminopropyl 2-acetamido-
3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyranoside (Ac3-GlcNAc-
propNH2)30 with the peptide 3 was carried out via pentafluo-
rophenyl ester31 4 to give glycopeptide 5 in 84% yield. After
de-O-acetylation of 5 by treatment with triethylamine, enzymatic
elongation of the divalent GlcNAc derivative 6 was carried out
using â1,4-galactosyltransferase with UDP-Gal in a HEPES
buffer (50 mM, pH 6.0) for 48 h according to the conditions
reported previously32 to give divalent LacNAc peptide 7 in 83%
yield. Finally, hydrogenation of 7 gave the desired glycopeptide
1 in 92% yield. On the other hand, glutamic acid was chosen
as the branch point for the synthesis of trivalent LacNAc peptide
(Scheme 3). Lys(Z) was treated with N-succinimidylsuccinic
acid allyl ester to afford 8 in 98% yield. Condensation of the
monocarboxyl derivative 8 with L-glutamic acid diallyl ester
was carried out by DCC coupling in the presence of HOSu and
gave triallyl ester 9 in 86% yield. Deallylation of 9 by
N-methylaniline in THF catalyzed by (Ph3P)4Pd(0)33 then gave
the tricarboxyl derivative 10 in 74% yield. Intermediate 10 was
activated by the pentafluorophenyl groups and condensed with
Ac3-GlcNAc-propNH2. After de-O-acetylation, enzymatic ga-
(30) Nishimura, S.-I.; Furuike, T.; Matsuoka, K.; Maruyama, K.; Nagata, K.;
Kurita, K.; Nishi, N.; Tokura, S. Macromolecules 1994, 27, 4876-4880.
(31) LaBell, R. Y.; Jacobsen, N. E.; Gervay-Hague, J.; O’Brien, D. F.
Bioconjugate Chem. 2002, 13, 143-149.
(32) Furuike, T.; Yamada, K.; Ohta, T.; Monde, K.; Nishimura, S.-I. Tetrahedron
2003, 59, 5105-5113.
(33) Holm, B.; Broddefalk, J.; Flodell, S.; Wellner, E.; Kihlberg, J. Tetrahedron
2000, 56, 1579-1586.
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Glycoinsulins
A R T I C L E S
lactosylation of the trivalent GlcNAc derivative 13 proceeded
smoothly to give trivalent LacNAc peptide 14 in 96% yield.
Finally, hydrogenation of 14 gave the desired glycopeptide 2
in 84% yield. All spectral data (1H and 13C NMR) are described
in the Experimental Section.
C. Glycoslylation of Mutant Insulins. Purified Ins(B-F1Q),
Ins(A-G1Q), and Ins(A-N21Q) were tested for glycosylation
with lactose derivatives 15 and 16 (Scheme 1). Ins(B-F1Q) and
Ins(A-G1Q) were reacted with 15 to produce the monolactose-
attached insulin in 50% yield, but Ins(A-N21Q) did not react
with 15 (Scheme 1). Compound 16, which has a shorter spacer
compared to that of 15, did not attach to any mutant insulins.
The glycosylation site of Ins(B-F1Q) was determined to be at
Q1 or Q4 of the insulin B-chain by MALDI-TOF analysis of
the B-chain degraded from the N-terminus by aminopeptidase
1
M.22 The H NMR spectrum of Ins(B-F1Q) showed that the
chemical shift of the N-terminal region (B2-B5) of the insulin
B-chain was changed (data not shown), suggesting the local
structure of the insulin B-chain was changed. This change may
explain why TGase glycosylation occurred at Q4 of the B-chain
of Ins(B-F1Q). Since insulin is known to form stable hexamers
in the presence of Zn2+ 6
, the introduction of carbohydrate
residues to this region may influence the formation of the insulin
self-assembly.
Surprisingly, these divalent and trivalent sugar derivatives
(2 and 3) were also effectively introduced to Ins(B-F1Q) using
TGase. Sialylation of the lactose-attached insulins was almost
quantitively carried out in the presence of sialyltransferase and
CMP-sialic acid (Scheme 1). Synthesized divalent-sugar-
displaying insulin (Sia2,6-di-LacNAc-Ins(B-F1Q)) and trivalent-
sugar-displaying insulin (Sia2,6-tri-LacNAc-Ins(B-F1Q)) were
purified by RP-HPLC, and their molecular weights were
analyzed by MALDI-TOF mass spectroscopy (Figure 3). These
mass spectra demonstrate that the sialyl groups were transferred
by R2,6-SiaT to all termini of the branched sugars.
Figure 3. MALDI-TOF mass spectra of (a) Sia2,6-di-LacNAc-Ins(B-F1Q)
and (b) Sia2,6-tri-LacNAc-Ins(B-F1Q).
To investigate whether changes in the secondary/tertiary
structure of the insulin molecule occurred because of mutation
and glycosylation, far-UV CD spectra of the insulin derivatives
were compared to that of Ins(WT) (Figure 4). The CD spectrum
of Ins(A-G1Q) alone was different from that of Ins(WT),
suggesting the R-helix structure of the N-terminal region of the
insulin A-chain was changed by point mutation. The spectra of
all insulin derivatives except for Ins(A-G1Q) were similar to
that of Ins(WT).15,34-37 Therefore, F1Q mutation and glycosy-
lation at the N-terminus of the insulin B-chain had no effect on
their overall structures on the basis of the CD spectra.
D. Biological Activity of Sugar-Displaying Insulins. 1.
Blood-Glucose-Lowering Effects Were Measured Using
Streptozotocin (STZ)-Mice. STZ is a drug that selectively
destroys insulin-producing pancreatic â-cells, providing a model
of type 1 diabetes.38 STZ-mice are an appropriate model for
testing the prolonged action of insulin derivatives.5 Figure 5
shows the monitored blood-glucose levels of STZ-mice after
subcutaneous administrations of Ins(WT), Ins(B-F1Q), Ins(A-
G1Q), Lac-Ins(B-F1Q), Sia2,6-Lac-Ins(B-F1Q), Sia2,6-di-Lac-
Figure 4. CD spectra of insulins and glycoinsulins.
NAc-Ins(B-F1Q), and Sia2,6-tri-LacNAc-Ins(B-F1Q). Three
types of sialyloligosaccharide-displaying insulins showed an
apparent prolonged glucose-lowering effect compared to the
control Ins(WT). T1/2 values of these compounds are listed in
Table 1. Monovalent and divalent-sialyloligosaccharide-display-
ing insulin showed minor differences in glucose-lowering ability.
(34) Goldman, J.; Carpenter, F. H. Biochemistry 1974, 13, 4566-4574.
(35) Markussen, J.; Volund, A. Int. J. Pept. Protein Res. 1975, 7, 47-56.
(36) Uchio, T.; Baudys, M.; Liu, F.; Song, S. C.; Kim, S. W. AdV. Drug DeliVery
ReV. 1999, 35, 289-306.
(37) Weiss, M. A.; Hua, Q. X.; Jia, W.; Chu, Y. C.; Wang, R. Y.; Katsoyannis,
P. G. Biochemistry 2000, 39, 15429-15440.
(38) Ranhotra, H. S.; Sharma, R. Mech. Ageing DeV. 2000, 119, 15-24.
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A R T I C L E S
Sato et al.
Table 2. Insulin Receptor Binding Potencya
sample
binding potency (%)
Ins(WT)
100
Ins(B-F1Q)
100.5
100.9
89.9
74.4
Sia2,6-Lac-In s(B-F1Q)
Sia2,6-di-La cNAc-Ins(B-F1Q)
Sia2,6-tri-La cNAc-Ins(B-F1Q)
a The rate of displacement of [125I]insulin was estimated from adipocytes.
The binding potency of Ins(WT) is represented as 100%.
was similar to that of Sia2,6-Lac-Ins(B-F1Q) (receptor binding
capacity 100.9%) as there was little loss of binding capacity.
The loss of binding affinity of the sterically hindered Sia2,6-
tri-LacNAc-Ins(B-F1Q) on the receptor may explain why it has
a low glucose-lowering effect, and this seems to cause the delay
in the recovery of the glucose level.5,40
Conclusion
We synthesized mono-, di-, and trisialyloligosaccharide-
displaying insulins using a chemoenzymatic approach. These
sialic acid-displaying insulins showed prolonged blood-glucose-
lowering activity in STZ-mice compared to native insulin. The
glucose-lowering effect of Sia2,6-tri-LacNAc-Ins(B-F1Q) was
mild and continued for a longer period than that of the other
insulins tested. This suggests that the trivalent-sialyloligosac-
charide-displaying insulin is useful in treating diabetes in that
it may prevent insulin shock, which results from a sudden drop
in glucose level after the injection of insulin. The prolonged
effect may be due to the multivalent effects of the sialooli-
gosaccharides on the stability of insulin in the blood stream
and low affinity for the insulin receptor. This strategy can be
widely applicable to the synthesis of various sugar-displaying
protein drugs with increased half-lives in the circulatory system,
since large amounts of nonnatural oligosaccharide structures can
also be prepared by the combined chemical and enzymatic
strategy described herein as well as partial structures of naturally
occurring N- and O-glycan chains.
Figure 5. Blood-glucose levels in STZ-mice following subcutaneous
injection (1 U/mouse) of insulins and glycoinsulins. Data are means ( SE,
n ) 5.
Table 1. Half-Life Values (T1/2) of Ins(WT) and Glycoinsulins in
Vivo
sample
T1 2 (h) (±SE)
/
Ins(WT)
Ins(B-F1Q)
Ins(A-G1Q)
3.22 (0.27)
2.98 (0.24)
1.83 (0.28)
3.31 (0.12)
3.82 (0.22)
3.62 (0.21)
3.59 (0.25)
Lac-Ins(B-F1 Q)
Sia2,6-Lac-Ins(B-F1Q)
Sia2,6-di-LacNAc-Ins(B-F1Q)
Sia2,6-tri-LacNAc-Ins(B-F1Q)
The glucose-lowering effect of Sia2,6-tri-LacNAc-Ins(B-F1Q)
was weak, but the recovery of glucose levels was slower than
those of the other insulin preparations. On the contrary, lactose-
displaying insulin, Lac-Ins(B-F1Q), showed rapid recovery of
glucose levels. This might be due to the binding to the lactose
receptor on the liver.
The behavior of Ins(B-F1Q) was quite close to that of Ins-
(WT), but the glucose depression activity of Ins(A-G1Q) was
significantly lower (Figure 5). The reason for the decreased
activity was thought to be that the structure of Ins(A-G1Q) was
disturbed by genetic mutation as supported by the CD study
(Figure 4).
2. Evaluation of the Insulin Receptor Binding Capacities
of the Glycoinsulins. The insulin receptor binding capacities
of the glycoinsulins were analyzed by the procedure reported
by Shechter et al.5,39 Briefly, [125I]insulin was used as a tracer,
and the binding capacity of glycoinsulins was determined by
calculation of the rate of displacement of [125I]insulin from
mouse adipocytes (Table 2). The binding capacity of Ins(WT)
was taken as 100%. The binding capacities of Sia2,6-di-
LacNAc-Ins(B-F1Q) and Sia2,6-tri-LacNAc-Ins(B-F1Q) were
89.9% and 74.4%, respectively. The N-terminal region of the
insulin B-chain was located on the opposite site of the receptor
binding domain.23 However, the sugar-chain moieties of Sia2,6-
di-LacNAc-Ins(B-F1Q) and, especially, Sia2,6-tri-LacNAc-Ins-
(B-F1Q) were large, thereby inhibiting insulin receptor binding.
The glucose depression curve of Sia2,6-di-LacNAc-Ins(B-F1Q)
Experimental Section
General Methods. Unless otherwise stated, all commercially avail-
able solvents and reagents were used without further purification.
Optical rotations were determined with a Perkin-Elmer 241 polarimeter
1
for samples in a 10 cm cell at ambient temperature (22 ( 2 °C). H
and proton-decoupled carbon NMR spectra were recorded at 400 and
100.4 MHz, respectively, on a JEOL JNM-lambda-400 FT-NMR
spectrometer. Ring-proton assignments in NMR were made by first-
order analysis of the spectra and supported by HH-COSY experiments.
Elemental analyses were performed with a Yanako CHN recorder MT-
6. FAB, ESI, and MALDI-TOF mass spectra were obtained with a
JEOL JMS-HX110 mass spectrometer, a JEOL JMS-700TZ mass
spectrometer, and a Bruker REFLEX III mass spectrometer, respec-
tively. Reactions were monitored by thin-layer chromatography (TLC)
on a precoated plate of silica gel 60F254 (layer thickness 0.25 mm; E.
Merck, Darmstadt, Germany) and/or RP-HPLC (Hitachi HPLC system
equipped with a D-7000 series interface). Flash chromatography was
performed on silica gel (silica gel 60N, 40-50 µm; Kanto Chemical
Industries Co. Ltd., Japan, and Iatro-beads, Iatron Laboratories Inc.).
Nꢀ-(Benzyloxycarbonyl]-L-lysine was purchased from Nova Biochem.
Nr-(3-Carboxy-1-oxopropyl)-NE-(benzyloxycarbonyl)-L-lysine Di-
ammonium Salt (3). To a suspension of Nꢀ-(benzyloxycarbonyl)-L-
lysine (2.00 g, 7.13 mmol) in MeOH (80 mL) was added succinic
(39) Shechter, Y.; Maron, R.; Elias, D.; Cohen, I. R. Science 1982, 216, 542-
(40) Duckworth, W. C.; Bennett, R. G.; Hamel, F. G. Endocr. ReV. 1998, 19,
608-624.
545.
9
14018 J. AM. CHEM. SOC. VOL. 126, NO. 43, 2004

Glycoinsulins
A R T I C L E S
anhydride (2.14 g, 21.4 mmol). The mixture was stirred for 12 h at 40
°C and concentrated. The residual syrup was dissolved in water and
chromatographed on a DEAE-Sephacel column with 0.01 M NH4HCO3
as the eluant to give 3 (2.54 g, 86%): [R]D +20.4° (c 0.264, MeOH);
1H NMR (D2O) δ 7.48-7.39 (m, 5H, aromatic), 5.10 (s, 2H, PhCH2),
4.13 (dd, 1H, Lys-CH(R)), 3.12 (t, 2H, Lys-CH2(ꢀ)), 2.55-2.46 (br,
4H, COCH2CH2CO), 1.77 (m, 1H, Lys-CH2(â)), 1.66 (m 1H, Lys-
CH2(â)), 1.48 (m, 2H, Lys-CH2(δ)), 1.34 (m 2H, Lys-CH2(γ)). Anal.
Calcd for C18H22N2O7‚(NH4)2: C, 52.16; H, 7.30; N,13.52. Found: C,
52.64; H, 7.09; N, 13.03.
NMR (D2O) δ 177.4, 177.1, 176.8, 161.0, 139.2, 131.4, 131.0, 130.2,
103.6, 78.5, 76.5, 72.6, 70.4, 70.3, 69.4, 63.4, 58.2, 56.7, 42.7, 38.9,
38.8, 33.6, 33.5, 33.3, 31.0, 24.8. Anal. Calcd for C40H64N6O17‚5H2O:
C, 48.48; H, 7.52; N, 8.47. Found: C, 48.26; H, 7.14; N, 6.89.
N-{3-[2-Acetamido-2-deoxy-4-O-(â-D-galactopyranosyl)-â-D-glu-
copyranosyloxyl]propyl}-Nr-{8-[2-acetamido-2-deoxy-4-O-(â-D-ga-
lactopyranosyl)-â-D-glucopyranosyloxyl]-5-aza-1,4-dioxooctyl}-NE-
(benzyloxycarbonyl)-L-lysinamide (7). To a solution of 6 (30.0 mg,
33.3 µmol) and UDP-galactose (48.8 mg, 79.9 µmol) in 50 mM
HEPES buffer (pH 6.0, 0.50 mL) (containing 10 mM manganese
chloride) was added â-1,4-galactosyltransferase (1 U), and the reaction
mixture was incubated for 48 h at 37 °C. The protein was removed by
centrifugation in a 4 mL Microsep concentrator (10000 MW cutoff).
The filtrate was evapolated in vacuo and subjected to a DEAE-Sephacel
column (0.01 M NH4HCO3) for the removal of the nucleosides. The
obtained residue was purified by gel filtration using Sephadex G-25
with water as the eluant to give 7 (34.0 mg, 83%): [R]D -29.0° (c
0.172, H2O); 1H NMR (D2O) δ 7.50-7.40 (m, 5H, aromatic), 5.14 (s,
2H, PhCH2), 4.54 (d, 2H, J1,2 ) 7.2 Hz, H-1), 4.50 (d, 2H, J1,2 ) 7.9
Hz, H-1′), 4.18 (br t, 1H, Lys-CH(R)), 3.91 (br d, 2H, H-6a), 3.86 (d,
2H, H-4′), 3.83 (m, 2H, OCH2), 3.76 (dd, 2H, J5,6b ) 4.9 Hz and J6a,6b
) 12.0 Hz, H-6b), 3.80-3.65 (m, 16H, H-2, H-3, H-4, H-5, H-3′, H-5′,
H-6′a, H-6′b), 3.63-3.55 (m, 4H, OCH2, H-2′), 3.36-3.20 (m, 4H,
NHCH2), 3.18 (br, 2H, Lys-CH2(ꢀ)), 2.57 (m, 2H, COCH2), 2.54 (m,
2H, COCH2), 2.06 (s, 6H, Ac), 1.80-1.75 (m, 5H, -CH2-, Lys-
CH2(â)) 1.72 (m, 1H, Lys-CH2(â)) 1.53 (m, 2H, Lys-CH2(δ)) 1.37 (m,
2H, Lys-CH2(γ)); 13C NMR (D2O) δ 177.3, 177.0, 176.7, 164.4, 161.0,
139.2, 131.4, 130.9, 130.2, 105.5, 103.6, 81.1, 77.9, 77.3, 75.1, 75.0,
73.5, 71.1, 70.3, 70.2, 69.3, 63.6, 62.7, 57.7, 56.6, 42.6, 38.8, 38.7,
33.5, 33.4, 33.2, 30.9, 24.8; HRMS-FAB m/z calcd for C52H85N6O27
[M + H]+ 1225.5462, found 1225.5440.
Nr-(3-Carboxy-1-oxopropyl)-NE-(benzyloxycarbonyl)-L-lysine Bis-
(pentafluorophenyl) Ester (4). To a solution of 3 (300 mg, 0.724
mmol) in DMF (5.0 mL) were added pyridine (140 µL, 1.74 mmol)
and pentafluorophenyl trifluoroacetate (318 µL, 1.81 mmol). The
reaction mixture was stirred for 1 h at room temperature and poured
into ethyl acetate. The organic layer was successively washed with
water, 0.1 N HCl(aq), water, 5% NaHCO3(aq), and water, then dried,
and concentrated. The obtained oil was purified by column chroma-
tography with 1:1 (v/v) hexanes-ethyl acetate as the eluant to give 4
1
(440 mg, 77%): [R]D -7.8° (c 0.155, CHCl3); H NMR (CDCl3) δ
7.36-7.26 (m, 5H, aromatic), 7.04 (d, 1H, NH(R)), 5.20 (brt, 1H, NH-
(ꢀ)), 5.08 (s, 2H, PhCH2), 4.84 (dd, 1H, Lys-CH(R)), 4.20 (br, 2H,
Lys-CH2(ꢀ)), 3.01 (m, 2H, COCH2), 2.66 (m, 2H, COCH2), 1.95 (m,
2H, Lys-CH2(â)), 1.52 (m, 4H, Lys-CH2(δ, γ)). Anal. Calcd for
C30F10H22N2O7: C, 50.57; H, 3.11; N, 3.93. Found: C, 50.84; H, 3.45;
N, 4.08.
N-[3-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyrano-
syloxyl)propyl]-Nr-[8-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-
glucopyranosyloxyl)-5-aza-1,4-dioxooctyl]-NE-(benzyloxycarbonyl)-
L-lysinamide (5). To a solution of 3-aminopropyl 2-acetamido-3,4,6-
tri-O-acetyl-2-deoxy-â-D-glucopyranoside (195 mg, 0.420 mmol) in
DMF (5.0 mL) were added 4 (100 mg, 0.140 mmol) and diisopropy-
lethylamine (73.2 µL, 0.420 mmol). The mixture was stirred for 17 h
at room temperature and concentrated. The residual syrup was chro-
matographed on Iatro-beads with 8:1 (v/v) CHCl3-MeOH as the eluant
to give 5 (136 mg, 84%): [R]D -11.0° (c 0.197, DMF); 1H NMR
(DMSO-d6) δ 7.92 (dd, 1H, NH(R)), 7.89 (t, 2H, NH), 7.80 (dd, 1H,
NH), 7.75 (dd, 1H, NH), 7.38-7.28 (m, 5H, aromatic), 7.19 (t, 1H,
NH(ꢀ)), 5.06 (t, 2H, J2,3 ) 10.0 Hz, H-3), 4.99 (s, 2H, PhCH2), 4.82 (t,
2H, J3,4 ) 10.5 Hz, H-4), 4.59(d, 1H, J1,2 ) 8.2 Hz, H-1), 4.58(d, 1H,
J1,2 ) 8.4 Hz, H-1), 4.18 (dd, 2H, J5,6a ) 4.7 Hz and J6a,6b ) 12.4 Hz,
H-6a), 4.10 (m, 1H, Lys-CH(R)), 4.00 (dt, 2H, H-6b), 3.82 (m, 2H,
H-5), 3.75-3.68 (m, 4H, OCH2, H-2), 3.43 (m, 2H, OCH2), 3.07 (m,
2H, NHCH2), 3.02 (m, 2H, NHCH2), 2.95 (m, 2H, Lys-CH2(ꢀ)), 2.38-
2.28 (m, 4H, COCH2), 2.01, 2.01, 1.96, 1.90, 1.77, 1.76 (all s, 24H,
Ac), 1.66-1.55 (m, 5H, -CH2-, Lys-CH2(â)) 1.46 (m, 1H, Lys-
CH2(â)) 1.36 (m, 2H, Lys-CH2(δ)) 1.27 (m, 1H, Lys-CH2(γ)) 1.20 (m,
1H, Lys-γ). Anal. Calcd for C52H76N6O23‚3H2O: C, 51.73; H, 6.85;
N, 6.96. Found: C, 51.76; H, 6.65; N, 6.89.
N-{3-[2-acetamido-2-deoxy-4-O-(â-D-galactopyranosyl)-â-D-glu-
copyranosyloxyl]propyl}-Nr-{8-[2-acetamido-2-deoxy-4-O-(â-D-ga-
lactopyranosyl)-â-D-glucopyranosyloxyl]-5-aza-1,4-dioxooctyl}-L-
lysinamide, Monoacetic Acid (1). To a solution of 7 (20 mg, 16.32
µmol) in MeOH-water (7.0 mL, 5:2 (v/v)) were added 10% Pd-C
(10 mg) and AcOH (2.81 µL, 48.96 µmol). The suspension was stirred
for 6 h under a hydrogen atmosphere and filtrated. After concentration,
the residue was subjected to a CM-Sephadex C-25 column with 0.01-
0.10 M NH4OAc as the eluant. The fractions containing the product
were purified by column chromatography using Sephadex G-25 with
H2O as the eluant and lyophilized to give 1 (17.5 mg, 92%): [R]D
1
-28.2° (c 0.294, H2O); H NMR (D2O) δ 4.52 (d, 1H, J1,2 ) 7.6 Hz,
H-1), 4.51 (d, 1H, J1,2 ) 7.8 Hz, H-1), 4.48 (d, 2H, J1,2 ) 7.7 Hz,
H-1′), 4.19 (dd, 1H, Lys-CH2(R)), 3.96 (br d, 2H, J6a,6b ) 12.2 Hz,
H-6a), 3.93 (d, 2H, J3,4 ) 3.5 Hz, H-4′), 3.91 (m, 2H, OCH2), 3.84
(dd, 2H, J5,6b ) 5.2 Hz and J6a,6b ) 12.0 Hz, H-6b), 3.80-3.68 (m,
14H, H-2, H-3, H-4, H-5, H-5′, H-6′a, H-6′b), 3.67 (dd, 2H, J2,3 ) 9.9
Hz, H-3′), 3.61 (br, 2H, OCH2), 3.54 (dd, 2H, J2,3 ) 10.1 Hz, H-2′),
3.30-3.15 (m, 4H, NHCH2), 3.00 (t, 2H, Lys-CH2(ꢀ)), 2.60 (m, 2H,
COCH2), 2.54 (m, 2H, COCH2), 2.04 (s, 6H, Ac), 1.93 (s, 3H, AcOH),
1.76 (m, 4H, -CH2-), 1.69 (m, 4H, Lys-CH2(â,δ)) 1.55-1.36 (m,
2H, Lys-CH2(γ)); HRMS-ESI m/z calcd for C44H78N6O25Na [M + Na]+
1113.4914, found 1113.4910.
Nr-(3-Carboxy-1-oxopropyl)-NE-(benzyloxycarbonyl)-L-lysine Al-
lyl Ester (8). To a suspension of Nꢀ-[(phenylmethoxy)carbonyl]-L-lysine
(1.30 g, 4.64 mmol) in DMF (80 mL) were added N-succinimidylsuc-
cinic acid allyl ester (1.29 mL, 9.28 mmol) and triethylamine (1.29
mL, 9.28 mmol), and the mixture was stirred for 8 h at room
temperature. After concentration, the residual oil was chromatographed
on Iatro-beads with 40:1 (v/v) CHCl3-MeOH as the eluant to give 8
(1.92 g, 98%): [R]D +8.7° (c 0.282, MeOH); 1H NMR (CDCl3) δ 7.33
(br s, 5H, aromatic), 6.79 (br d, 1H, NH(R)), 5.85 (m, 1H, CH2dCH-
), 5.29-5.11 (m, 3H, CH2dCH-, NH(ꢀ)), 5.07 (s, 2H, PhCH2), 4.60
(m, 1H, Lys-CH(R)),), 4.55 (d, 2H, -CH2O), 3.18 (m, 2H, Lys-CH2(ꢀ)),
2.73 (m, 2H, COCH2), 2.54 (m, 2H, COCH2), 1.88 (m, 1H, Lys-CH2-
N-[3-(2-Acetamido-2-deoxy-â-D-glucopyranosyloxyl)propyl]-Nr-
[8-(2-acetamido-2-deoxy-â-D-glucopyranosyloxyl)-5-aza-1,4-dioxooc-
tyl]-NE-(benzyloxycarbonyl)-L-lysinamide (6). To a solution of 5 (40
mg, 34.7 µmol) in MeOH-water (3.0 mL, 2:1) was added triethylamine
(232 µL, 1.66 mmol), and the mixture was stirred for 4 h at room
temperature. After concentration, the residue was purified by gel
filtration using Sephadex G-25 with H2O as the eluant. The fraction
was lyophilized to give 6 (28.0 mg, 90%): [R]D -28.0° (c 0.314, H2O);
1H NMR (D2O) δ 7.45-7.35 (m, 5H, aromatic), 5.03 (s, 2H, PhCH2),
4.48 (d, 1H, J1,2 ) 8.5 Hz, H-1), 4.47 (d, 1H, J1,2 ) 8.8 Hz, H-1), 4.15
(br, 1H, Lys-CH(R)), 3.92 (dd, 2H, J5,6a ) 4.2 Hz and J6a,6b ) 13.4
Hz, H-6a), 3.88 (m, 2H, OCH2), 3.75 (dd, 2H, J5,6b ) 3.7 Hz, H-5),
3.70 (dd, 2H, J2,3 ) 10.1 Hz, H-2), 3.59 (m, 2H, OCH2), 3.56 (m, 2H,
H-3), 3.45 (m, 4H, H-4, H-6b), 3.30-3.15 (m, 4H, NHCH2), 2.95 (t,
2H, Lys-CH2(ꢀ)), 2.55 (m, 2H, COCH2), 2.50 (m, 2H, COCH2), 1.93
(s, 6H, Ac), 1.83-1.73 (m, 5H, -CH2-, Lys-CH2(â)) 1.67 (m, 1H,
Lys-CH2(â)) 1.48 (m, 2H, Lys-CH2(δ)) 1.33 (m, 2H, Lys-CH2(γ)); 13
C
9
J. AM. CHEM. SOC. VOL. 126, NO. 43, 2004 14019

A R T I C L E S
Sato et al.
(â)), 1.73 (m 1H, Lys-CH2(â)), 1.50 (m, 2H, Lys-CH2(δ)), 1.38 (m
2H, Lys-CH2(γ)). Anal. Calcd for C21H28N2O7: C, 59.99; H, 6.71; N,
6.66. Found: C, 59.60; H, 6.81; N, 6.68.
syrup was chromatographed on Iatro-beads with 7:1 (v/v) CHCl3-
MeOH as the eluant to give 12 (140 mg, 85%): [R]D -14.4° (c 0.323,
DMF); 1H NMR (DMSO-d6) δ 8.14 (br d, 1H, NH(R)), 7.96-7.88 (m,
4H, NH), 7.77 (m, 2H, NH), 7.53 (br t, 1H, NH), 7.38-7.28 (m, 5H,
aromatic), 7.21 (t, 1H, NH(Lys-ꢀ)), 5.07 (t, 3H, J2,3 ) 10.4 Hz and J3,4
) 9.7 Hz, H-3), 5.00 (s, 2H, PhCH2), 4.82 (t, 3H, J4,5 ) 9.8 Hz, H-4),
4.58(d, 3H, J1,2 ) 8.5 Hz, H-1), 4.21 (dd, 3H, J5,6a ) 7.2 Hz and J6a,6b
) 11.8 Hz, H-6a), 4.09 (m, 2H, Lys-CH(R), Glu-CH(R)), 4.01 (br d,
3H, H-6b), 3.82 (m, 3H, H-5), 3.77-3.64 (m, 6H, OCH2, H-2), 3.43
(m, 3H, OCH2), 3.15-2.92 (m, 8H, NHCH2, Lys-CH2(ꢀ)), 2.50 (m,
2H, Glu-CH2(γ)), 2.35 (m, 4H, COCH2), 2.10 (m, 1H, Glu-CH2(â)),
2.01, 2.01, 1.96, 1.91, 1.77, 1.77 (all s, 36H, Ac), 1.79 (m, 1H, Glu-
CH2(â)), 1.66-1.55 (m, 6H, -CH2-, Lys-CH2(â)), 1.39 (m, 2H, Lys-
CH2(δ)), 1.29 (m, 2H, Lys-CH2(γ)). Anal. Calcd for C74H109N9O34‚
2H2O: C, 52.13; H, 6.68; N, 7.39. Found: C, 51.93; H, 6.54; N, 7.00.
Nr-(3-Carboxy-1-oxopropyl)-NE-(benzyloxycarbonyl)-L-lysyl-L-
glutamic Acid Triallyl Ester (9). A mixture of 8 (1.00 g, 2.38 mmol),
N-hydroxysuccinimide (410 mg, 3.57 mmol), and dicyclohexylcarbo-
diimide (589 mg, 2.86 mmol) in DMF (10 mL) was stirred for 30 min
at 0 °C. L-Glutamic acid diallyl ester (763 mg, 2.86 mmol) in DMF
(5.0 mL) was then added to the reaction mixture. After being stirred
for 15 h at room temperature, the solution was filtrated, diluted in ethyl
acetate, washed with 1 N HCl(aq) and brine, then dried, and
concentrated. The obtained residue was purified by column chroma-
tography on silica gel with 80:1 (v/v) CHCl3-MeOH as the eluant to
give 9 (1.29 g, 86%): [R]D -7.1° (c 0.272, CHCl3); 1H NMR (CDCl3)
δ 7.36-7.26 (br, 6H, aromatic, NH(Glu)), 6.76 (br d, 1H, NH(Lys-
CH2(R)), 5.88 (m, 3H, CH2dCH- × 3), 5.29-5.11 (m, 7H, CH2dCH-,
NH(Lys-ꢀ)), 5.06 (s, 2H, PhCH2), 4.60-4.48 (d, 8H, -CH2O, Lys-
CH(R), Glu-CH(R)), 3.17 (m, 2H, Lys-CH2(ꢀ)), 2.67 (m, 2H, COCH2),
2.49 (m, 2H, COCH2), 2.42 (m, 2H, Glu-CH2(γ)), 2.20 (m, 1H, Glu-
CH2(â)), 2.01 (m, 1H, Glu-CH2(â)), 1.85 (m, 1H, Lys-CH2(â)), 1.66
(m 1H, Lys-CH2(â)), 1.50 (m, 2H, Lys-CH2(δ)), 1.39 (m 2H, Lys-
CH2(γ)). Anal. Calcd for C32H43N3O10‚0.5H2O: C, 60.17; H, 7.05; N,
6.58. Found: C, 60.52; H, 6.89; N, 6.89.
Nr-[8-(2-Acetamido-2-deoxy-â-D-glucopyranosyloxyl)-5-aza-1,4-
dioxooctyl]-NE-(benzyloxycarbonyl)-L-lysyl-N-bis[3-(2-acetamido-2-
deoxy-â-D-glucopyranosyloxyl)propyl]-L-glutamamide (13). To a
solution of 12 (50 mg, 30.0 µmol) in MeOH-water (6.0 mL, 2:1) was
added triethylamine (150 µL, 1.08 mmol), and the mixture was stirred
for 4 h at room temperature. After concentration, the residue was
purified by gel filtration using Sephadex G-25 with H2O as the eluant.
The fraction was lyophilized to give 13 (38.7 mg, 100%): [R]D -40.8°
1
Nr-(3-Carboxy-1-oxopropyl)-NE-(benzyloxycarbonyl)-L-lysyl-L-
glutamic Acid (10). Tetrakis(triphenylphosphine)palladium(0) (138 mg,
0.119 mmol) and N-methylaniline (1.29 mL, 11.9 mmol) were added
to a solution of 9 (500 mg, 0.704 mmol) in THF (10 mL), and the
resulting solution was stirred for 14 h in the dark under a nitrogen
atmosphere. After concentration, the residue was subjected to an ion
exchange column (Dowex 50W-X8, H+-form) and purified by a column
of Iatro-beads with 15:1 (v/v) CHCl3-MeOH as the eluant to give 10
(c 0.159, H2O); H NMR (D2O) δ 7.47-7.39 (m, 5H, aromatic), 5.11
(s, 2H, PhCH2), 4.50 (d, 1H, J1,2 ) 8.8 Hz, H-1), 4.49 (d, 1H, J1,2
)
8.3 Hz, H-1), 4.47 (d, 1H, J1,2 ) 8.6 Hz, H-1), 4.21 (br, 2H, Lys-
CH(R), Glu-CH(R)), 3.92 (br d, 3H, H-6a), 3.88 (m, 3H, OCH2), 3.74
(dd, 3H, J6a,6b ) 9.7 Hz J5,6b ) 4.2 Hz, H-5), 3.69 (t, 3H, J2,3 ) 10.2
Hz, H-2), 3.60 (m, 3H, OCH2), 3.54 (m, 3H, H-3), 3.44 (m, 6H, H-4,
H-6b), 3.30-3.10 (m, 8H, NHCH2, Lys-CH2(ꢀ)), 2.54 (m, 4H, COCH2),
2.29 (m, 2H, Glu-CH2(γ)), 2.10 (m, 1H, Glu-CH2(â)), 2.04 (s, 9H, Ac),
1.98 (m, 1H, Glu-CH2(â)), 1.74 (br, 8H, -CH2-, Lys-CH2(â)), 1.51
(m, 2H, Lys-CH2(δ)), 1.37 (br, 2H, Lys-CH2(γ)); 13C NMR (D2O) δ
178.2, 177.8, 177.4, 177.3, 177.2, 175.8, 175.7, 161.2, 139.4, 131.6,
131.1, 130.3, 103.9, 78.7, 76.6, 72.7, 70.5, 70.3, 63.6, 58.4, 56.9, 56.1,
42.8, 39.1, 39.1, 39.0, 34.8, 33.6, 33.5, 33.2, 31.2, 31.1, 29.8, 25.0.
Anal. Calcd for C56H91N9O25‚3H2O: C, 50.03; H, 7.27; N, 9.37.
Found: C, 49.73; H, 6.82; N, 9.38.
Nr-{8-[2-Acetamido-2-deoxy-4-O-(â-D-galactopyranosyl)-â-D-glu-
copyranosyloxyl]-5-aza-1,4-dioxooctyl}-NE-(benzyloxycarbonyl)-L-
lysyl-N-bis{3-[2-acetamido-2-deoxy-4-O-(â-D-galactopyranosyl)-â-
D-glucopyranosyloxyl]propyl}-L-glutamamide (14). To a solution of
13 (28 mg, 21.7µmol) and UDP-galactose (47.7 mg, 78.1 µmol) in
50 mM HEPES buffer (pH 6.0, 0.50 mL) containing 10 mM manganese
chloride was added â-1,4-galactosyltransferase (1 U), and the reaction
mixture was incubated for 48 h at 37 °C. The reaction mixture was
treated as described for the preparation of 7 and gave 14 (37.0 mg,
96%): [R]D -27.0° (c 0.166, H2O); 1H NMR (D2O) δ 7.47-7.39 (m,
5H, aromatic), 5.11 (s, 2H, PhCH2), 4.50 (br d, 3H, J1,2 ) 7.0 Hz,
H-1), 4.47 (d, 3H, J1,2 ) 7.8 Hz, H-1′), 4.21 (m, 2H, Lys-CH(R), Glu-
CH(R)), 3.98 (dd, 3H, J6a,6b ) 12.2 Hz, J5,6a ) 2.2 Hz, H-6a), 3.93 (d,
1
(300 mg, 74%): [R]D -7.4° (c 0.262, MeOH); H NMR (CD3OD) δ
7.34-7.25 (br, 5H, aromatic), 5.05 (s, 2H, PhCH2), 4.42 (dd, 1H, Glu-
CH(R)), 4.34 (dd, 1H, Lys-CH(R)), 3.11 (t, 2H, Lys-CH2(ꢀ)), 2.60 (m,
2H, COCH2), 2.51 (m, 2H, COCH2), 2.40 (t, 2H, Glu-CH2(γ)), 2.19
(m, 1H, Glu-CH2(â)), 1.97 (m, 1H, Glu-CH2(â)), 1.83 (m, 1H, Lys-
CH2(â)), 1.66 (m 1H, Lys-CH2(â)), 1.51 (m, 2H, Lys-CH2(δ)), 1.42
(m 2H, Lys-CH2(γ)). Anal. Calcd for C23H31N3O10: C, 54.22; H, 6.13;
N, 8.25. Found: C, 53.98; H, 6.35; N, 8.13.
Nr-(3-Carboxy-1-oxopropyl)-NE-(benzyloxycarbonyl)-L-lysyl-L-
glutamic Acid Tris(pentafluorophenyl) Ester (11). To a solution of
10 (270 mg, 0.530 mmol) in DMF (5.0 mL) were added pyridine (141
µL, 1.75 mmol) and pentafluorophenyl trifluoroacetate (321 µL, 1.83
mmol). The reaction mixture was stirred for 1 h at room temperature
and poured into ethyl acetate. The organic layer was successively
washed with water, 0.1 N HCl(aq), water, 5% NaHCO3(aq), and water,
then dried, and concentrated. The obtained oil was purified by column
chromatography with 1:1 (v/v) hexanes-ethyl acetate as the eluant to
give 11 (420 mg, 79%): [R]D -10.6° (c 0.254, CHCl3); 1H NMR
(CDCl3) δ 7.49 (dd, 1H, NH(Glu)), 7.33-7.26 (br, 5H, aromatic), 6.86
(dd, 1H, NH(Lys-R)), 5.06 (s, 2H, PhCH2), 5.05 (m, 1H, NH(Lys-ꢀ)),
4.96 (m, 1H, Glu-CH(R)), 4.52 (dd, 1H, Lys-CH(R)), 3.16 (br, 2H,
Lys-CH2(ꢀ)), 3.10-2.90 (m, 2H, COCH2), 2.84 (m, 2H, Glu-CH2(γ)),
2.63 (m, 2H, COCH2), 2.47 (m, 1H, Glu-CH2(â)), 2.22 (m, 1H, Glu-
CH2(â)), 1.92 (m, 1H, Lys-CH2(â)), 1.74 (m 1H, Lys-CH2(â)), 1.52
(m, 2H, Lys-CH2(δ)), 1.41 (m 2H, Lys-CH2(γ)). Anal. Calcd for
C41F15H28N3O10: C, 48.87; H, 2.80; N, 4.17. Found: C, 49.15; H, 2.96;
N, 4.04.
Nr-[8-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyrano-
syloxyl)-5-aza-1,4-dioxooctyl]-NE-(benzyloxycarbonyl)-L-lysyl-N-bis-
[3-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyranosyloxyl)-
propyl]-L-glutamamide (12). To a solution of 3-aminopropyl
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyranoside (207 mg,
0.446 mmol) in DMF (5.0 mL) were added 11 (100 mg, 99.2 µmol)
and diisopropylethylamine (156 µL, 0.893 mmol), and the mixture was
stirred for 17 h at room temperature. After concentration, the residual
3H, J3′,4′ ) 3.4 Hz, H-4′), 3.89 (m, 3H, OCH2), 3.83 (dd, 3H, J5,6b
4.9 Hz, H-6b), 3.80-3.68 (m, 21H, H-2, H-3, H-4, H-5, H-5′, H-6′a,
H-6′b), 3.67 (dd, 3H, H-3′), 3.58 (m, 3H, OCH2), 3.55 (dd, 3H, J2′,3′
)
)
9.9 Hz, H-2′), 3.30-3.10 (m, 8H, NHCH2, Lys-CH2(ꢀ)), 2.55 (br, 4H,
COCH2), 2.29 (m, 2H, Glu-CH2(γ)), 2.10 (m, 1H, Glu-CH2(â)), 2.03
(s, 9H, Ac), 1.95 (m, 1H, Glu-CH2(â)), 1.74 (br, 8H, -CH2-, Lys-
CH2(â)), 1.51 (m, 2H, Lys-CH2(δ)), 1.37 (br, 2H, Lys-CH2(γ)); 13C
NMR (D2O) δ 178.0, 177.7, 177.4, 177.1, 175.7, 161.1, 139.4, 131.5,
131.1, 130.3, 105.7, 103.8, 81.6, 78.1, 77.5, 75.4, 75.2, 73.8, 71.3, 70.6,
70.3, 69.5, 63.7, 63.0, 57.9, 56.8, 56.1, 42.9, 39.1, 39.1, 39.0, 34.8,
33.7, 33.6, 33.2, 31.2, 29.8, 25.0; HRMS-FAB m/z calcd for
C74H121N9O40Na [M + Na]+ 1798.7609, found 1798.7600.
Nr-{8-[2-Acetamido-2-deoxy-4-O-(â-D-galactopyranosyl)-â-D-glu-
copyranosyloxyl]-5-aza-1,4-dioxooctyl}-L-lysyl-N-bis{3-[2-acetamido-
2-deoxy-4-O-(â-D-galactopyranosyl)-â-D-glucopyranosyloxyl]propyl}-
9
14020 J. AM. CHEM. SOC. VOL. 126, NO. 43, 2004

Glycoinsulins
A R T I C L E S
L-glutamamide, Monoacetic Acid (2). The title compound (16.2 mg,
84%) was obtained by hydrogenation of 14 (20 mg, 11.26 µmol), as
described for the preparation of 8: [R]D -30.0° (c 0.282, H2O); H
Proinsulin Refolding. The eluted proteins were precipitated by
dialysis against H2O. The sediment was dissolved and sulfonated in
10 mL/g (wet weight sediment) buffer D (20 mM Tris-HCl, pH 8.0,
100 mM Na2SO3, 10 mM Na2S4O6, 8 M urea) at room temperature for
3 h.43 This reaction was stopped by dialysis against 10 mM HCl, and
the sulfonated proteins were collected as a white precipitate. The
precipitate was dissolved in 50 mM glycine/NaOH, pH 10.5, at a final
concentration of 0.1 mg/mL. Refolding was performed by adding
2-mercaptoethanol (1.5 mol/mol of cysteine) at 4 °C for 16 h.43,44 To
stop the reaction, the pH was adjusted to 3.0. Refolded products were
concentrated and isolated by reversed-phase HPLC (RP-HPLC) using
a semipreparative Vydac C4 column (10 × 250 mm). Elution conditions
consisted of a linear gradient of acetonitrile (ACN) concentration from
25% to 45% containing 0.1% trifluoroacetic acid (TFA) for 25 min at
a flow rate of 4 mL min-1. The isolated products were then lyophilized.
Insulin Purification. The lyophilized His-PI fusion proteins were
dissolved in buffer E (100 mM Tris/H3PO4, pH 7.5, 0.1% Tween 20)
at a concentration of 2 mg/mL. Trypsin (sequencing grade, Roche) and
carboxypeptidase B (sequencing grade, Roche) were added to the fusion
protein at a ratio of 1:2:2000 (by mass).28 The reaction was carried out
at 37 °C for 30 min, and stopped by decreasing the pH to 3.0 by adding
AcOH. The digestion products were purified by RP-HPLC using a
semipreparative Vydac C4 column and analyzed by RP-HPLC using
an analytical Vydac C4 column (4.6 × 250 mm). Elution conditions
consisted of a linear gradient of ACN concentration from 25% to 35%
containing 0.1% TFA for 25 min at a flow rate of 4 mL min-1
(semipreparative) or 1 mL min-1 (analytical).
Insulin Glycosylation. For synthesis of glycoinsulin, TGase (Oriental
Yeast Co.) was used. A solution of Ins(B-F1Q) (0.6 mg, 104 nmol) in
20 mM Tris buffer (pH 7.5, 1 mL) containing 0.9 mM 1 (2, 8, or 16),
5 mM CaCl2, and 4 U of TGase was incubated for 30 min at 37 °C.45,46
An aliquot (20 µL) of the reaction mixture was withdrawn for analytical
RP-HPLC. The reaction was stopped by decreasing the pH to 3.0. The
mixture was applied to a semipreparative Vydac C4 column and eluted
with a linear gradient of ACN from 25% to 30% for 30 min at a flow
rate of 4 mL min-1. The eluted products were nonreacted Ins(B-F1Q)
(∼50%) and glycosylated Ins(B-F1Q), Lac-Ins(B-F1Q), di-Lac-Ins(B-
F1Q), or tri-Lac-Ins(B-F1Q) (∼50%).
1
NMR (D2O) δ 4.52 (d, 2H, J1,2 ) 7.3 Hz, H-1), 4.51 (d, 1H, J1,2 ) 7.5
Hz, H-1), 4.48 (d, 3H, J1′,2′ ) 7.8 Hz, H-1′), 4.23 (m, 2H, Lys-CH(R),
Glu-CH(R)), 3.99 (br d, 3H, H-6a), 3.93 (d, 3H, J3,4 ) 3.4 Hz, H-4′),
3.91 (m, 3H, OCH2), 3.84 (dd, 3H, J5,6b ) 5.2 Hz and J6a,6b ) 12.4 Hz,
H-6b), 3.80-3.65 (m, 24H, H-2, H-3, H-4, H-5, H-3′, H-5′, H-6′a,
H-6′b), 3.60 (br, 3H, OCH2), 3.54 (dd, 3H, J2,3 ) 10.1 Hz, H-2′), 3.30-
3.12 (m, 6H, NHCH2), 3.01 (t, 2H, Lys-CH2(ꢀ)), 2.57 (m, 4H, COCH2),
2.30 (m, 2H, Glu-CH2(γ)), 2.12 (m, 1H, Glu-CH2(â)), 2.04 (3s, 9H,
Ac) 2.01 (m, 1H, Glu-CH2(â)), 1.92 (s, 3H, AcOH), 1.76 (m, 6H,
-CH2-), 1.71 (m, 4H, Lys-CH2(â,δ)), 1.43 (m, 2H, Lys-CH2(γ));
HRMS-ESI m/z calcd for C66H115N9O38Na [M + Na]+ 1664.7241, found
1664.7279.
DNA Construction. The plasmid containing the human insulin gene
(YG-HG294) was donated by the Japan Health Science Research
Resources Bank. The normal human proinsulin (PI(WT)) gene41 was
amplified using primers PI, 5′-ACG GAT CCG GGT GGC CGC TTT
GTG AAC CAA CAC-3′, and PI-R, 5′-CAG GTC GAC CTA GTT
GCA GTA GTT CTC-3′. PCR was carried out using KOD-Plus
polymerase (TOYOBO). The amplified PCR product was digested with
restriction enzymes BamHI and SalI (TOYOBO), and inserted into the
His-tag fusion protein expression vector pQE31 (Qiagen) previously
digested with the same enzymes, resulting in plasmid His-PI(WT)/
pQE31. Furthermore, a mutation of the insulin B-chain (from F1 to Q)
was performed using the QuikChange mutagenesis kit (Stratagene) and
primers B-F1Q, 5′-GGA TCC GGG TGG CCG CCA AGT GAA CCA
ACA CCT C-3′, and B-F1Q-R, 5′-CAG GTG TTG GTT CAC TTG
GCG GCC ACC CGG ATC C-3′, resulting in plasmid His-PI(B-F1Q)/
pQE31. The primers used for the mutation of the insulin A-chain (from
G1 to Q and from N21 to Q) were A-G1Q, 5′-GTC CCT GCA GAA
GCG TCA AAT TGT GGA AGA ATG CTG-3′, A-G1Q-R, 5′-CAG
CAT TGT TCC ACA ATT TGA CGC TTC TGC AGG GAC-3′,
A-N21Q, 5′-GGA GAA CTA CTG CCA ATA GGT CGA CCT GC-
3′, and A-N21Q-R, GCA GGT CGA CCT ATT GGC AGT AGT TCT
CC-3′, resulting in plasmids His-PI(A-G1Q)/pQE31 and His-PI(A-
N21Q)/pQE31, respectively.
Sugar Elongation (Sialylation) Reaction of Lac-, Di-LacNAc-,
or Tri-LacNAc-Ins(B-F1Q). A solution of Lac-, di-LacNAc-, or tri-
LacNAc-Ins(B-F1Q) (80.5 nmol) in a 50 mM sodium cacodylate buffer
(pH 7.5, 1 mL) containing 0.6 mM CMP-NeuAc, 0.2% BSA, 1.6 mM
MnCl2, 0.2% Triton CF54, 20 U of CIAP, and 150 mU of R2,6-SiaT
was incubated for 2 h (Lac-Ins(B-F1Q)) or 30 min (di- or tri-LacNAc-
Ins(B-F1Q)) at 37 °C.47-49 The reaction procedure was checked by
MALDI-TOF mass analysis. After the reaction was completed, purifica-
tion of the products was performed by RP-HPLC as described above.
Circular Dichroism (CD) Studies. All insulin samples were
dissolved in PBS (pH 7.4), and the protein concentrations were adjusted
to 40 µM by UV absorption at 280 nm. CD measurements were
performed on a Jasco-820 CD spectropolarimeter at 25 °C with a cell
length of 0.1 cm. For the far-UV CD spectra, samples were scanned
from 200 to 250 nm and accumulated 10 times at a resolution of 1.0
nm with a scanning speed of 100 nm/min. All the CD data were
expressed as mean residue ellipticity.50,51
Proinsulin Expression. E. coli cells (M15 bacteria) harboring His-
PI(WT)/pQE31, His-PI(B-F1Q)/pQE31, His-PI(A-G1Q)/pQE31, or
His-PI(A-N21Q)/pQE31 were grown overnight at 37 °C in 60 mL of
LB medium containing ampicillin (100 µg/mL) and kanamycin (25 µg/
mL). The overnight cultures were diluted 100-fold to 6 L in a
Jarfermenter (BMS-10PI, ABLE) with the same medium and grown at
37 °C until OD600 reached approximately 0.7-0.8.42 Protein expression
was induced by the addition of isopropyl â-D-thiogalactopyranoside
(IPTG) to a final concentration of 1 mM, and the culture was grown
for an additional 5 h. Cells were harvested by centrifugation at 5000g
for 10 min at 4 °C, and the bacterial pellet was resuspended in 300
mL of buffer A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM
imidazole). Cellular membranes were disrupted by probe sonication
(two cycles of 15 s on and 15 s off), and insoluble proteins were
obtained by centrifugation at 5000g for 10 min at 4 °C.43 The pellet
was resuspended in 150 mL of buffer B (20 mM Tris-HCl, pH 8.0,
500 mM NaCl, 20 mM imidazole, 8 M urea, 1 mM 2-mercaptoethanol)
and shaken at room temperature for 30 min. This solution was then
centrifuged at 10000g for 20 min at 4 °C, and the pellet was discarded.
The supernatant was applied to a Superflow Ni-NTA resin column
(bed volume 20 mL, Qiagen) equilibrated with buffer B at 4 °C and
washed with buffer B, and then the bound proteins were eluted with
buffer C (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 250 mM imidazole,
8 M urea, 1 mM 2-mercaptoethanol).
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J. AM. CHEM. SOC. VOL. 126, NO. 43, 2004 14021

A R T I C L E S
Sato et al.
Induction of Diabetes. We purchased C57BL/6J male mice at 5
weeks of age from Clea Japan Inc., and started experiments at 7-8
weeks of age.52,53 The mice were fasted for 18-20 h and then injected
intraperitoneally with a single dose of STZ (200 mg/kg of body mass,
Wako) freshly dissolved in 0.05 M citrate buffer (pH 4.5).54,55 At 48 h
after STZ injection, blood samples were obtained from the tail vein,
and blood-glucose levels were measured with a Glucocard (Kyoto
Daiichi Kagaku Co.).56 Mice with blood-glucose levels above 400 mg/
dL were used as diabetic mice.57
Administration of Insulin Preparations. The dosage of insulin
preparations was determined as 1 U by the absorbance at 280 nm.
Solutions of insulin preparations in PBS (pH 7.4) were administered
subcutaneously to STZ-treated diabetic mice (STZ-mice) using a
disposable syringe (TERUMO). Blood sampling for glucose analysis
was performed before administration and at 1, 2, 3, 4, and 5 h after
administration. Groups consisted of five mice, and the data were
presented as means ( SE.10
adipocytes, cells were exposed to fresh medium (2 mL) containing 10%
FCS, 0.5 mM isobutylmethylxanthine, 0.25 µM dexamethasone, and 1
µg/mL insulin. After 2 days, the medium was replaced with DMEM
containing 10% FCS and insulin, and the cells were cultured for an
additional 6 days.58-60
Insulin Binding Assay. Differentiated cells were washed three times
with the binding buffer (100 mM Hepes, 120 mM NaCl, 1.2 mM MgCl2,
5 mM KCl, 25 mM glucose, 10 mg/mL BSA, and 2 mg/mL
Bacitracin)60 and assayed for the displacement of 125I-labeled insulin
(Amersham) by glycoinsulins as described by Shechter et al.5,38
Unbound insulins were removed, and the cells were washed three times
with ice-cold buffer and lysed with 0.1% Triton X-100.62 the radioactiv-
ity of the harvested cell was determined by γ-counter.
Acknowledgment. This work was supported partly by a grant
for “Research and Development on Glycocluster Controlling
Biomolecules” from the New Energy and Industrial Technology
Development Organization (NEDO). M.S. thanks Mr. A. Toda,
Dr. K. Yamada, Dr. K. Iwata, Dr. T. Ohta, and Dr. H. Nakagawa
for helpful discussion.
Cell Culture. 3T3-L1 fatty fibroblasts (Dainippon Seiyaku Co.) were
cultured to confluence in 35 mm dishes in DMEM (Gibco) containing
10% fetal calf serum (FCS, Gibco) at 37 °C in a 5% CO2 environment
and were fed every 2 days. For differentiation of 3T3-L1 cells from
JA046426L
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