Target-specific chemical acylation of lectins by ligand-tethered DMAP catalysts

Article abstract of DOI:10.1021/ja075684q

Because sugar-binding proteins, so-called lectins, play important roles in many biological phenomena, the lectin-selective labeling should be useful for investigating biological processes involving lectins as well as providing molecular tools for analysis of saccharides and these derivatives. We describe herein a new strategy for lectin-selective labeling based on an acyl transfer reaction directed by ligand-tethered DMAP (4-dimethylaminopyridine). DMAP is an effective acyl transfer catalyst, which can activate an acyl ester for its transfer to a nucleophilic residue. To direct the acyl transfer reaction to a lectin of interest, we attached the DMAP to a saccharide ligand specific for the target lectin. It was clearly demonstrated by biochemical analyses that the target-selective labeling of Congerin II, an animal lectin having selective affinity for Lactose/LacNAc (N-acetyllactosamine), was achieved in the presence of Lactethered DMAPs and acyl donors containing probes such as fluorescent molecules or biotin. Conventional peptide mapping experiments using HPLC and tandem mass-mass analysis revealed that the acyl transfer reaction site-specifically occurred at Tyr 51 of Cong II. This strategy was successfully extended to other lectins by changing the ligand part of the ligand-tethered DMAP. We also demonstrated that this labeling method is applicable not only to purified lectin in test tubes, but also to crude mixtures such as E. coli lysates or homogenized animal tissue samples expressing Congerin.

Full text of DOI:10.1021/ja075684q

Published on Web 12/13/2007
Target-Specific Chemical Acylation of Lectins by
Ligand-Tethered DMAP Catalysts
Yoichiro Koshi,† Eiji Nakata,† Masayoshi Miyagawa,† Shinya Tsukiji,†
Tomohisa Ogawa,‡ and Itaru Hamachi*,†
Contribution from the Department of Synthetic Chemistry and Biological Chemistry, Graduate
School of Engineering, Kyoto UniVersity, Katsura, Kyoto 615-8510, Japan, and Department of
Biomolecular Sciences, Graduate School of Life Sciences, Tohoku UniVersity,
Sendai 981-8555, Japan
Received July 30, 2007; E-mail: ihamachi@sbchem.kyoto-u.ac.jp
Abstract: Because sugar-binding proteins, so-called lectins, play important roles in many biological
phenomena, the lectin-selective labeling should be useful for investigating biological processes involving
lectins as well as providing molecular tools for analysis of saccharides and these derivatives. We describe
herein a new strategy for lectin-selective labeling based on an acyl transfer reaction directed by ligand-
tethered DMAP (4-dimethylaminopyridine). DMAP is an effective acyl transfer catalyst, which can activate
an acyl ester for its transfer to a nucleophilic residue. To direct the acyl transfer reaction to a lectin of
interest, we attached the DMAP to a saccharide ligand specific for the target lectin. It was clearly
demonstrated by biochemical analyses that the target-selective labeling of Congerin II, an animal lectin
having selective affinity for Lactose/LacNAc (N-acetyllactosamine), was achieved in the presence of Lac-
tethered DMAPs and acyl donors containing probes such as fluorescent molecules or biotin. Conventional
peptide mapping experiments using HPLC and tandem mass-mass analysis revealed that the acyl transfer
reaction site-specifically occurred at Tyr 51 of Cong II. This strategy was successfully extended to other
lectins by changing the ligand part of the ligand-tethered DMAP. We also demonstrated that this labeling
method is applicable not only to purified lectin in test tubes, but also to crude mixtures such as E. coli
lysates or homogenized animal tissue samples expressing Congerin.
Introduction
example, employment of a conventional assay based on tight
antigen-antibody binding such as pull-down technique is not
Advanced glycobiology and glycochemistry techniques have
recently demonstrated that many saccharides and their deriva-
tives play pivotal roles in various biological events by interacting
with the corresponding sugar-binding proteins.1,2 Therefore,
characterization of these sugar-binding proteins, so-called lectins
or other relevant proteins, is important for understanding the
complicated biological phenomena involving sugars.3,4 Selective
labeling of lectins should be useful for investigating the
biological roles of these proteins in detail,5,6 as well as for
providing useful molecular tools for analyzing saccharides.7 For
applicable for the study of the sugar-lectin interactions because
the normal sugar-lectin interactions are not sufficiently strong.
In such cases, it is expected that selective covalent labeling of
lectins can be alternative to these assays, as well as a microarray
analysis.8,9
For protein labeling, bio-orthogonal organic chemistry toward
protein surfaces represents one of the key methodologies. In
(7) (a) Nagase, T.; Nakata, E.; Shinkai, S.; Hamachi, I. Chem.-Eur. J. 2003,
9, 3660-3669. (b) Hamachi, I.; Nagase, T.; Shinkai, S. J. Am. Chem. Soc.
2000, 122, 12065-12066. (c) Nakata, E.; Koshi, Y.; Koga, E.; Katayama,
Y.; Hamachi, I. J. Am. Chem. Soc. 2005, 127, 13253-13261. (d) Koshi,
Y.; Nakata, E.; Hamachi, I. ChemBioChem 2005, 6, 1349-1352.
(8) (a) Tomizaki, K.; Usui, K.; Mihara, H. ChemBioChem 2005, 6, 782-799.
(b) Shin, I.; Park, S.; Lee, M.-r. Chem.-Eur. J. 2005, 11, 2894-2901. (c)
Park, S.; Lee, M.-r.; Pyo, S.-J.; Shin, I. J. Am. Chem. Soc. 2004, 126, 4812.
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A.; Disney, M. D.; Seeberger, P. H. ChemBioChem 2006, 7, 421. (f) Diseny,
M. D.; Seeberger, P. H. Chem. Biol. 2004, 11, 1701. (g) Adams, E. W.;
Ratner, D. M.; Bokesch, H. R.; McMahon, J. B.; O’Keefe, B. R.; Seeberger,
P. H. Chem. Biol. 2004, 11, 875. (h) Blixt, O.; et al. Proc. Natl. Acad. Sci.
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Med. Chem. Lett. 2004, 14, 3185.
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2006, 128, 10413-10422. (b) Hsu, K.-L.; Pilobello, K. T.; Mahal, L. K.
Nat. Chem. Biol. 2006, 2, 153-157. (c) Kuno, A.; Uchiyama, N.; Koseki-
Kuno, S.; Ebe, Y.; Takashima, S.; Yamada, M.; Hirabayashi, J. Nat. Meth.
2005, 2, 851-856. (d) Zheng, T.; Peelen, D.; Smith, L. M. J. Am. Chem.
Soc. 2005, 127, 9982-9973. (e) Pilobello, K. T.; Krishnamoorthy, L.;
Slawek, D.; Mahal, L. K. ChemBioChem 2005, 6, 985-989. (f) Chen, S.;
LaRoche, T.; Hamelinck, D.; Bergsma, D.; Brenner, D.; Simeone, D.; Brand,
R. E.; Haab, B. B. Nat. Meth. 2007, 4, 437-444.
† Kyoto University.
‡ Tohoku University.
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9
10.1021/ja075684q CCC: $40.75 © 2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 245-251
245

A R T I C L E S
Koshi et al.
Scheme 1. Scheme of Acyl Transfer Reaction for Lectina
2 and Table 1). It is considered that hydrolysis of the activated
acyl intermediate is a side reaction highly competitive with the
acyl transfer, in aqueous solution. Indeed, we found that, in the
absence of Cong II, Lac-DMAP 1 induced a significant degree
of thioester hydrolysis of 5 (48%), but hydrolysis scarcely
occurred without 1 (Table S1). However, the labeling of Cong
II by the acyl transfer reaction was remarkably facilitated by
Lac-DMAP 1 to give the FL-Cong II in 35% yield. This can be
attributed to the active intermediate (acylated DMAP) being
produced predominantly in the vicinity of the lectin surface by
virtue of the saccharide-lectin interaction and its rapid reaction
with a nucleophilic amino acid residue located on the lectin
surface, with suppression of the nonproductive hydrolysis. This
is supported by the experimental result that the acyl transfer
did not occur (less than 1%) in the presence of excess saccharide
ligand (50 mM lactose), because of inhibition of the interaction
of Lac-DMAP 1 with Cong II.
This acyl transfer reaction directed by Lac-DMAP 1 was
optimized by careful selection of the acyl donor structure
particularly in the carboxylic ester (acyl group) part and the
thiol (leaving group) part. As shown in Table 1, acyl donor 7
containing benzyl thioester was not reactive enough to be
activated by DMAP (less than 1%), as compared to thiophenyl
esters. Among the thiophenyl esters, on the other hand, acyl
donor 6, which includes an R-amino acid structure, was much
too reactive, so that it caused a considerable amount of
nonspecific acylation (11%). We found that acyl donor 5,
containing the gamma-amino acid moiety, showed enhanced
specificity by reducing nonspecific reactions.15 The selectivity,
that is, the ratio of the yield catalyzed by Lac-DMAP 1 over
that of the noncatalyzed reaction, was 22 for the γ-amino
thiophenyl ester 5, a value 20-fold greater than that for the
R-amino thiophenyl ester 6. The labeling site for Cong II using
Lac-DMAP 1 and the optimal acyl donor 816 having a coumarin
fluorophore was identified by conventional peptide mapping
experiments with HPLC and MALDI-TOF analysis. It was
confirmed that a single peptide fragment was fluorescent among
the trypsin-digested peptides as monitored by HPLC (Figure
3a). The mass/mass experiments for the isolated fluorescent
peptide determined that the modified site was Tyr51. Thus, it
is clear that the OH group of Tyr51 was the main acyl acceptor
in the present labeling reaction. Crystallographic analysis told
us that this Tyr is located near the lactose binding pocket (Figure
3b). The basic treatment of the labeled Cong II gave hydrolytic
cleavage of the attached fluorophore, which is consistent with
the above conclusion (Figure S8).
a (a) Complex formation between lectin and sugar-tethered DMAP. (b)
The acyl transfer reaction catalyzed by DMAP. (c) Removing sugar-tethered
DMAP.
addition to classical bioconjugation reactions, several modern
reactions have been recently demonstrated to be valid even under
biological conditions.10 Francis and co-workers, for example,
developed several unique organometallic reactions and a
coenzyme-driven reaction that are elegantly applicable for
protein labeling.11 We previously demonstrated that ligand-
directed affinity labeling coupled with subsequent chemical
reactions is one of the powerful methods for targeting a specific
protein to be functionalized.12 Here, we propose a new strategy
for chemically labeling lectins in a target-selective manner using
an acyl transfer reaction catalyzed by ligand-tethered DMAP
(4-dimethylaminopyridine). Our idea is shown in Scheme 1.
DMAP is a well-established acyl transfer catalyst, which can
activate an acyl ester for transfer to a nucleophilic residue.13
To direct the acyl transfer to a lectin of interest, DMAP was
connected to a suitable saccharide ligand having a high affinity
to the target lectin. It is reasonably expected that the ligand
moiety brings the DMAP unit close to the sugar binding pocket
of the lectin, so that the acyl group activated by the anchored
DMAP is transferred to a residue of the target lectin on the
proximal lectin surface.
Results and Discussion
To test this idea, Congerin II (Cong II), an animal lectin
having lactose/LacNAc selectivity, was employed.14 A series
of sugar-tethered DMAPs and acyl donors (Figure 1) were
synthesized according to Scheme 2. The acceleration of the acyl
transfer from the acyl donor 5 to Cong II in the presence of
Lac-DMAP 1 was clearly shown by MALDI-TOF mass
spectroscopy of the reaction mixture and UV-vis spectroscopy
of the purified fluorescein-labeled Cong II (FL-Cong II) (Figure
Restoration of the saccharide binding capability of the
modified Cong II was clearly demonstrated by the fluorescent
binding assay for various saccharides. The bimolecular fluo-
rescence quenching and recovery technique (BFQR, Figure 4)9a
was used for fluorescein-labeled Cong II (FL-Cong II) prepared
by the Lac-DMAP method. Fluorescence of FL-Cong II was
initially quenched by dabcyl-appended lactose (Dab-Lac 10),
(10) (a) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13-21. (b)
Sieber, S. A.; Cravatt, B. F. Chem. Commun. 2006, 2311-2319.
(11) (a) Antos, J. M.; Francis, M. B. Curr. Opin. Chem. Biol. 2006, 10, 253-
262. (b) Tilley, S. D.; Francis, M. B. J. Am. Chem. Soc. 2006, 128, 1080-
1081. (c) McFarland, J. M.; Francis, M. B. J. Am. Chem. Soc. 2005, 127,
13490-13491. (d) Antos, J. M.; Francis, M. B. J. Am. Chem. Soc. 2004,
126, 10256-10257. (e) Joshi, N. S.; Whitaker, L. R.; Francis, M. B. J.
Am. Chem. Soc. 2004, 126, 15942-15943.
(12) Takaoka, Y.; Tsutsumi, H.; Kasagi, N.; Nakata, E.; Hamachi, I. J. Am.
Chem. Soc. 2006, 128, 3273-3280.
(13) (a) Hofle, G.; Steglich, W.; Vorbruggen, H. Angew. Chem., Int. Ed. Engl.
1978, 17, 569-583. (b) Bhattacharya, S.; Snehalatha, K. J. Chem. Soc.,
Perkins Trans. 1996, 2, 2021-2025.
(14) (a) Kamiya, H.; Muramoto, K.; Goto, R. DeV. Comp. Immunol. 1988, 12,
309-318. (b) Ogawa, T.; Ishi, C.; Suda, Y.; Kamiya, H. Biosci. Biotechnol.
Biochem. 2002, 66, 476-480. (c) Shirai, T.; Matsui, Y.; Shionyu-
Mitsuyama, C.; Yamane, T.; Kamiya, H.; Ishii, C.; Ogawa, T.; Muramoto,
K. J. Mol. Biol. 2002, 321, 879-889. (d) Shionyu-Mitsuyama, C.; Ito, Y.;
Konno, A.; Miwa, Y.; Ogawa, T.; Muramoto, K.; Shirai, T. J. Mol. Biol.
2005, 347, 385-397.
(15) The hydrolysis tests of acyl donors 5-7 were carried out to compare the
reactivity of the acyl donors (Table S1). The order of hydrolysis rate
without DMAP was in good agreement with the order of the nonspecific
modification yield of lectins. In particular, acyl donor 6 was so reactive
that it did not need the activation by DMAP in the hydrolysis under
the present conditions (95% with Lac-DMAP, 93% without Lac-DMAP).
This may cause a considerable amount of the nonspecific acylation for
lectin.
(16) MALDI-TOF mass analysis confirmed that selective acylation occurred
by acyl donors 8 and 9 like acyl donor 5 (see Supporting Information).
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246 J. AM. CHEM. SOC. VOL. 130, NO. 1, 2008

Target-Specific Chemical Acylation of Lectins
A R T I C L E S
Figure 1. Molecular structures of sugar-tethered DMAPs (1-4) and acyl donors (5-9).
Scheme 2. Synthetic Scheme of Lac-DMAP 1 and Acyl Donor 5
and then various saccharides were added to the quenched
solution. Fluorescence intensity was recovered by the addition
of lactose or galactose, which can bind to Cong II, indicating
that these sugars replace the Dab-Lac. In contrast, maltose and
mannose did not cause the fluorescence recovery because of
their lack of binding to Cong II. These results indicate that the
binding pocket of Cong II was open to retain almost the same
saccharide selectivity after labeling.
the ligand-tethered DMAP. For example, the acylation of wheat
germ agglutinin (WGA, GlcNAc/NeuNAc selective lectin) or
concanavalin A (Con A, Man/Glc selective lectin) was suc-
cessfully carried out using NAG3-DMAP 3 and Man4-DMAP
4, respectively (see Table 1). The high target-selectivity was
clearly confirmed by the experiments with a mixture including
three kinds of lectins in the presence of each saccharide-tethered
DMAP. As shown in the SDS-PAGE gel images of Figure 5,
the fluorescent band of Cong II was clearly observed in lane 3
by the acylation of the lectin mixture catalyzed by Lac4-DMAP
The present strategy has the advantage that one can flexibly
switch the target lectin by changing the saccharide structure of
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J. AM. CHEM. SOC. VOL. 130, NO. 1, 2008 247

A R T I C L E S
Koshi et al.
mucus of Conger eel contains two kinds of galectins (Congerin
I and II).14 The Congerin was labeled with biotinylated acyl
donor 9 in the presence of the catalyst Lac4-DMAP 2. SDS-
PAGE (Figure 6b) demonstrated that the naturally occurring
Congerin was labeled (lane 7), which was analyzed by blotting
and the subsequent diaminobenzidine (DAB) staining. In
contrast, no labeling occurred in the presence of other DMAP
derivatives such as Man4- or NAG3-DMAP (lanes 8, 9), in the
coexistence of Lac4-DMAP with excess of saccharide ligand
(lane 10), or in the absence of sugar-tethered DMAP (lane 11).
In conclusion, we have developed a novel method for lectin-
selective covalent labeling using ligand-directed acyl transfer
catalyzed by DMAP. The method was successfully applied not
only to a purified lectin, but also to crude mixtures, such as
lysates of recombinant bacterial cells or animal tissue expressing
the target lectin. Thus, it is expected that this method can be
used for profiling lectin-like proteins in cells or in tissues without
corresponding antibodies, and fluorescent lectins prepared by
this method are useful for the scaffold of biosensor to various
saccharides. This chemical strategy may be generally applicable
to receptor proteins other than lectin, by changing the ligand
part for targeting a protein of interest and/or by replacing the
catalyst part for other chemical reactions available. Such
investigations are now underway.
Experimental Section
General Comments for Synthesis. All chemical reagents were
purchased from commercial suppliers (Aldrich, TCI, or Wako) and used
without further purification. All air- or moisture-sensitive reactions were
performed with distilled solvents (DMF, dichloromethane, or MeOH)
under a nitrogen or argon atmosphere.
Synthesis. Lac-DMAP 1 and acyl donor 5 were synthesized as shown
in Scheme 2, and details are described below. Detailed synthetic
procedures and characterizations of other sugar-tethered DMAPs 2-4,
acyl donors 6-9, and Dab-lac 10 are described in the Supporting
Information.
Figure 2. (a) MALDI-TOF mass spectra of reaction mixture of Cong II
with sugar-tethered DMAP 1 and acyl donor 5 (O, native Cong II (Mw
15 330); *, FL-Cong II (Mw 15 770)). (b) Absorption spectra of fluorescein-
labeled Cong II in the presence (black line) and the absence (red dot line)
of sugar-tethered DMAP 1.
2, whereas the other two bands coming from other lectins were
not clearly observed. On the other hand, the fluorescent WGA
band (lane 2) or Con A band (lane 1) selectively appeared by
the fluorescence imager, where the NAG3- or Man4-tethered
DMAP was used as the catalyst, respectively.17
3-(Methyl-4-pyridylamino)propionic Acid (11). 3-(Methyl-4-py-
ridylamino)propionic acid methyl ester 10 was synthesized as previously
reported.13b To a solution of 10 (240 mg, 1.23 mmol) in 2 mL of
methanol was added 3 mL of 1 N NaOH solution. After being stirred
for 8 h at room temperature, the reaction mixture was neutralized with
HCl solution. The resulting solution was lyophilized to yield a white
solid. Methanol was added to dissolve the crude product, and the
insoluble salts were removed by filtration. The filtrate was evaporated
under vacuum to afford 3-(methyl-4-pyridylamino)propionic acid 11
as a white solid (280 mg, 1.09 mmol, 89%). 1H NMR (400 MHz, CD3-
OD): δ/ppm 2.49 (t, JH ) 7.2 Hz, 2H), 3.20 (s, 3H), 3.84 (t, JH ) 7.2
Hz, 2H), 7.00 (d, JH ) 7.2 Hz, 2H), 8.07 (t, JH ) 7.2 Hz, 2H).
Peracetyl 2-Bromoethyl Lactose (12). 2-Bromoethanol (1.1 mL,
15.0 mmol) and molecular sieves 3A (2.00 g) were added to a solution
of peracetyl lactose (5.11 g, 7.50 mmol) in 20 mL of CH2Cl2. After
being stirred for 30 min at room temperature, boron trifluoride-diethyl
ether (4.70 mL, 37.5 mmol) was added to the above solution. The
reaction mixture was then stirred for 12 h at room temperature and
diluted with 200 mL of ethyl acetate. The organic layer was washed
with deionized water and dried over anhydrous Na2SO4. The crude
residue was purified by flash column chromatography on silica gel
(elution: ethyl acetate/hexanes 3:2) to give peracetyl 2-bromoethyl
This method was also applied to crude mixtures such as cell
lysates of E. coli cells overexpressing Cong II.14b As shown in
the SDS-PAGE gel image (Figure 6a), only one band was
fluorescent, which is ascribed to the coumarin-labeled Cong II,
indicating that the labeling using the ligand-tethered DMAP is
selective even in the presence of many other proteins. Interest-
ingly, the in-gel fluorescence intensity was greater when
catalyzed by Lac4-DMAP 2, which has four lactose units (lane
3), than with the mono-valent Lac-DMAP 1 (lane 2), suggesting
selective labeling is facilitated by multivalent binding, in such
a crude sample.6a,18 Labeling of Cong II did not take place
significantly in the presence of excess amounts of saccharide
ligand (50 mM lactose, lane 4) or without DMAP ligands (lane
5).
More interestingly, we can selectively label a lectin in a
animal tissue lysate using this method. The lysate of the skin
1
lactose 12 as a white solid (2.17 g, 2.92 mmol, 39%). H NMR (400
(17) In these two lectins, we did saponification experiments as the case of Cong
II to investigate the acylation site. After the incubation at pH 12 for 1 day,
the fluorescent band WGA remained clear, whereas the bands for Cong II
and Con A almost disappeared (see Supporting Information Figure S8).
This suggests that the acylation site of Con A is probably Tyr as the case
of Cong II, whereas the acylation site of WGA is not Tyr, but Thr or Ser
(for the alkyl ester) or Lys (for the amide).
MHz, CDCl3): δ/ppm 1.98-2.15 (m, 21H), 3.42-3.46 (m, 2H),
(18) (a) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed.
1998, 37, 2754-2794. (b) Gestwick, J. E.; Cairo, C. W.; Strong, L. E.;
Oetjen, K. A.; Kiessling, L. L. J. Am. Chem. Soc. 2002, 124, 14922-
4933.
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Target-Specific Chemical Acylation of Lectins
A R T I C L E S
Table 1. Modification Yield of Lectins in the Presence or Absence of Sugar-Tethered DMAPsa
lectin
Cong II
WGA
5
Con A
5
acyl donor
5
6
7
modification
yield
with ST-DMAP
35 ( 2%
12%
<1%
62%
74%
without ST-DMAP
1.6 ( 0.2%
22
11%
1.1
<1%
2.2%
28
11%
6.7
ratio of yield with/without DMAP
a The yield of Cong II acylated by 5 was averaged by three individual experiments, and other yields were calculated by single experiment.
solid (880 mg, 2.14 mmol, 99%). 1H NMR (400 MHz, CD3OD): 3.39-
3.94 (m, 16H), 4.30 (d, JH ) 7.2 Hz, 1H), 4.34 (d, JH ) 7.2 Hz, 1H).
2-Aminoethyl Lactose (15). To a solution of 2-azidoethyl lactose
14 (880 mg, 2.14 mmol) in 100 mL of methanol was added palladium-
carbon (10%, 100 mg). Under H2 atmosphere, the reaction mixture was
stirred for 20 h at room temperature. After the palladium-carbon was
removed by filtration, the filtrate was evaporated under vacuum to afford
2-aminoethyl lactose 15 as a white solid (709 mg, 1.84 mmol, 86%).
1H NMR (400 MHz, CD3OD): 3.38-4.02 (m, 16H), 4.35-4.37 (m,
2H).
Lac-DMAP (1). To a solution of 2-aminoethyl lactose 15 (32 mg,
168 µmol) in 200 µL of DMF were added WSC (16 mg, 84 µmol) and
DIEA (20 µL, 168 µmol). A solution of 3-(methyl-4-pyridylamino)
propionic acid 11 (10 mg, 56 µmol) in 60 µL of distilled water was
added to the above solution. The reaction mixture was then stirred for
10 h at room temperature, and the solution was purified by reversed-
phase HPLC. The eluent was lyophilized and redissolved in distilled
water to obtain Lac-DMAP 1 as a 300 µL aqueous solution (9.88 mM,
2.96 µmol, 5%). The RP-HPLC was performed using an ODS-A column
(YMC, 250 × 10 mm) using a linear gradient of CH3CN and 10 mM
aqueous ammonium acetate from 2/98 to 10/90 over 30 min: flow
rate ) 3 mL/min, detection at 280 nm. HRMS (FAB) calcd for [M +
H+] (C23H38N3O12), 548.2450; found, 548.2455.
3-Carboxypropylaminocarbonyl Fluorescein tert-Butyl Ester (16).
5,6-Carboxyfluorescein was synthesized as previously reported.19 To
a solution of 5,6-carboxyfluorescein (100 mg, 266 µmol) in 5 mL of
DMF were added WSC (78.6 mg, 399 µmol), DIEA (153 µL, 931
µmol), and γ-aminobutyric acid tert-butyl ester (104 mg, 532 µmol).
The reaction mixture was stirred for 3 h at room temperature, evaporated
under vacuum, and diluted with 200 mL of ethyl acetate. The organic
layer was washed with 5% aqueous citric acid and brine and dried over
anhydrous MgSO4. The crude residue was purified by flash column
chromatography on silica gel (elution: ethyl acetate/hexanes 3:1) to
give 3-carboxypropylaminocarbonyl fluorescein tert-butyl ester 16 as
a yellow solid (72 mg, 139 µmol, 59%). 3-Carboxypropylaminocarbonyl
fluorescein tert-butyl ester 16 was obtained as a mixture of 5′ and 6′
Figure 3. (a) HPLC analysis of coumarin-labeled Cong II fragments
monitored by a fluorescence detector (λex ) 360 nm, λem ) 410 nm). (b)
Structural model of the complex of Cong II and lactose (PDB ID: 1is4).
The fragment isolated by HPLC is marked in yellow, and the modified
amino acid (Tyr 51) is indicated by stick style.
3.62-3.64 (m, 1H), 3.78-3.82 (m, 2H), 3.88 (t, JH ) 7.2 Hz, 1H),
4.08-4.13 (m, 4H), 4.48-4.54 (m, 2H), 4.90-4.98 (m, 3H), 5.12 (dd,
JH ) 7.2, 3.6 Hz, 1H), 5.23 (d, JH ) 7.2 Hz, 1H), 5.35 (d, JH ) 3.6
Hz, 1H).
1
isomers. H NMR (400 MHz, CD3OD): δ/ppm 1.37 (s, 9H(6′)), 1.44
(s, 9H(5′)), 1.79 (quintet, JH ) 7.2 Hz, 2H(6′)), 1.91 (quintet, JH ) 7.2
Hz, 2H(5′)), 2.25 (t, JH ) 7.2 Hz, 2H(6′)), 2.35 (t, JH ) 7.2 Hz, 2H-
(5′)), 3.33 (t, JH ) 7.2 Hz, 2H(6′)), 3.46 (t, JH ) 7.2 Hz, 2H(5′)), 6.51-
6.61 (m, 4H(5′, 6′)), 6.68-6.69 (m, 2H(5′, 6′)), 7.29 (d, JH ) 8.0 Hz,
1H(5′)), 7.60 (d, JH ) 2.0 Hz, 1H(6′)), 8.07 (d, JH ) 8.0 Hz, 1H(6′)),
8.12 (d, JH ) 8.0 Hz, 1H(6′)), 8.18 (d, JH ) 8.0, 1H(5′)), 8.42 (s, 1H-
(5′)).
Peracetyl 2-Azidoethyl Lactose (13). To a solution of peracetyl
2-bromoethyl lactose 12 (2.17 g, 2.92 mmol) in 80 mL of DMF was
added sodium azide (949 mg, 14.6 mmol). The reaction mixture was
stirred for 3 h at 80 °C. After filtration, the filtrate was evaporated
under vacuum and diluted with 200 mL of ethyl acetate. The organic
layer was washed with brine and dried over anhydrous Na2SO4. The
solution was filtered and concentrated under vacuum to afford peracetyl
2-azidoethyl lactose 13 as a white solid (2.06 g, 2.17 mmol, 100%).
1H NMR (400 MHz, CDCl3): δ/ppm 1.97-2.15 (m, 21H), 3.29-3.47
(m, 2H), 3.62-3.70 (m, 2H), 3.80-3.88 (m, 2H), 4.06-4.14 (m, 4H),
4.49-4.57 (m, 2H), 4.90-4.97 (m, 3H), 5.11 (dd, JH ) 7.2, 3.6 Hz,
1H), 5.21 (d, JH ) 7.2 Hz, 1H), 5.36 (d, JH ) 3.6 Hz, 1H).
2-Azidoethyl Lactose (14). To a solution of peracetyl 2-azidoethyl
lactose 13 (2.06 g, 2.17 mmol) in 12 mL of methanol was added 181
µL of methanol solution of sodium methoxide (217 µmol). After being
stirred for 4 h at room temperature, the reaction was quenched by adding
ion-exchange resin (Amberlite IRC-50, 2 g). After filtration, the filtrate
was removed under vacuum to afford 2-azidoethyl lactose 14 as a white
Acyl Donor (5). 3-Carboxypropylaminocarbonyl fluorescein tert-
butyl ester 16 was dissolved in 2 mL of CH2Cl2 containing 40%
trifluoroacetic acid. The reaction mixture was stirred for 1 h at room
temperature and evaporated under vacuum to afford 3-carboxypropy-
laminocarbonyl fluorescein as a yellow solid. To a solution of
fluorescein aminobutyric acid in 2 mL of DMF were added BOP (20.5
mg, 46.4 µmol), DIEA (6.3 µL, 46.4 µmol), and thiophenol (7.9 µL,
77.4 µmol). The reaction mixture was stirred for 1 h at room
temperature, evaporated under vacuum, and diluted with 200 mL of
ethyl acetate. The organic layer was washed with 5% aqueous citric
(19) Ueno, U.; Jiao, G.-S.; Burgess, K. Synthesis 2004, 15, 2591-2593.
9
J. AM. CHEM. SOC. VOL. 130, NO. 1, 2008 249

A R T I C L E S
Koshi et al.
Figure 4. (a) Schematic illustration of bimolecular fluorescence quenching and recovery (BFQR). (b) Molecular structure of Dab-lac 10. (c) Fluorescence
quenching process of FL-Cong II upon addition of 10 (0-4 µM). (d) Fluorescence recovery of FL-Cong II by the addition of lactose (0-10 mM) in the
presence of 10 (4 µM). (e) Fluorescence titration plot of the relative intensity (I/I0) of FL-Cong II versus the saccharide concentration (log[saccharide]).
Titration conditions: 0.2 µM FL-Cong II, pH 7.5, 20 °C, λex ) 488 nm.
donors 5-8 (5-7, ꢀ494nm ) 75 000 (pH 9); 8, ꢀ419nm ) 36 000 (in
MeCN))20 was determined spectrophotometrically.
Chemical Acylation of Lectins in Test Tubes. Acyl transfer
reactions were carried out under the following conditions: 10 µM lectin,
50 µM acyl donor, and 50 µM sugar-tethered DMAP in 50 mM HEPES
buffer (pH 8.0). The reaction was initiated by the addition of acyl donor
and incubated at 25 °C. For MALDI-TOF mass measurements shown
in Figure 2a, aliquots were taken at every hour, immediately purified
by ZipTip (Millipore), and mixed with a matrix solution (sinapinic acid).
After 3 h incubation, the reaction solution was subjected to gel filtration
(TOYOPEARL HW-40F) using 50 mM acetate buffer (pH 5.0), and
the eluent was dialyzed against 50 mM HEPES buffer (pH 7.5).
Modification yields shown in Table 1 were calculated by the absorbance
at 464 nm to that at 280 nm using the molar extinction coefficients of
acyl donors 5-7 (ꢀ464nm ) 26 000, ꢀ280nm ) 16 000 (pH 7.5)) and lectins.
Acyl transfer reactions in a lectin mixture were carried out in 50
mM HEPES buffer (pH 8.0) containing 0.1 mg/mL of each lectin (Cong
II, Con A, and WGA), 50 µM acyl donor 8, and 50 µM sugar-tethered
DMAPs 2-4 as described above. After 3 h incubation at 25 °C, the
reaction mixture was fractionated by 15% SDS-PAGE in a reducing
condition. The gel was analyzed by a fluorescent gel imager ChemiDoc
XRS (BioRad) and stained by Coomasie brilliant blue.
Figure 5. Selective acylation in lectin mixture. Protein modification was
detected by using SDS-PAGE: left, CBB staining; right, coumarin
fluorescence image. Lane M, protein marker; 1, Man4-DMAP 4; 2, NAG3-
DMAP 3; 3, Lac4-DMAP 2; 4, no DMAP. Reaction conditions: 0.1 mg/
mL lectins (Con A, WGA, and Cong II), 50 µM sugar-tethered DMAPs
2-4, 50 µM acyl donor 8, pH 8.0, 3 h, 25 °C.
Determination of Modification Sites. The coumarin-modified Cong
II prepared as described above was diluted in 50 mM HEPES buffer
(pH 8.0) containing 2 M urea. Trypsin was added to the solution to a
trypsin/substrate ratio of 1/10 (w/w), and the digestion was allowed to
proceed for 3 h at 37 °C. The digested peptides were separated by
reversed-phase HPLC using an ODS-A column (YMC, 250 × 4.6 mm),
and each fraction was analyzed by MALDI-TOF mass spectrometry.
The HPLC condition used was as follows: CH3CN/H2O (both contain-
ing 0.1% TFA) 5/95-50/50 (linear gradient over 45 min), flow rate )
1 mL/min, detection by UV absorption (220 nm) and fluorescence (λex
) 360 nm, λem ) 410 nm). The most fluorescent fraction was collected,
lyophilized, and subjected to mass-mass analysis (Shimadzu, AXIMA
QIT (MALDI-QIT-TOF)) to determine the modified amino acid.
acid and brine and dried over anhydrous MgSO4. The crude residue
was purified by flash column chromatography on silica gel (elution:
ethyl acetate/hexanes 3:1) to give acyl donor 5 as a yellow solid (17.3
mg, 31.3 µmol, 81%). Acyl donor 5 was obtained as a mixture of 5′
1
and 6′ isomers. H NMR (400 MHz, CD3OD): δ/ppm 1.91 (quintet,
JH ) 7.2 Hz, 2H(6′)), 2.06 (quintet, JH ) 7.2 Hz, 2H(5′)), 2.69 (t, JH
) 7.2 Hz, 2H(6′)), 2.81 (t, JH ) 7.2 Hz, 2H(5′)), 3.36 (t, JH ) 7.2 Hz,
2H(6′)), 3.49 (t, JH ) 7.2 Hz, 2H(5′)), 6.50-6.70 (m, 4H(5′, 6′)), 6.68-
6.69 (m, 2H(5′, 6′)), 7.26-7.40 (m, 5H(5′, 6′)+1H(5′)), 7.61 (d, JH
)
2.0 Hz, 1H(6′)), 8.06 (d, JH ) 8.0 Hz, 1H(6′)), 8.12 (d, JH ) 8.0 Hz,
1H(6′)), 8.18 (JH ) 8.0 Hz, 1H(5′)), 8.41 (s, 1H(5′)). HRMS (FAB)
calcd for [M + H+] (C31H24NO7S), 554.1268; found, 554.1285.
BFQR Measurement. Fluorescence spectra were measured by a
Perkin-Elmer LS55 fluorescence spectrometer. The slit widths for
excitation and emission were set at 15 and 10 nm, respectively. The
excitation wavelength was 488 nm. In fluorescence quenching pro-
cesses, FL-Cong II solution prepared as described above was diluted
General Comments for Biochemical Assays. Recombinant Con-
gerin II (Cong II) was expressed in E. coli strain JM109/pTV-Cong II
and purified as previously described.12b Concanavalin A (Con A) and
Wheat Germ Agglutinin (WGA) were purchased from Funakoshi and
used without further purification. The concentration of lectins (Cong
II, ꢀ280nm ) 11 500; Con A, ꢀ280nm ) 35 000; and WGA, ꢀ280nm ) 25 500/
M-1 cm-1), sugar-tethered DMAPs 1-4 (ꢀ280nm ) 18 000), and acyl
(20) Haugland, R. P. Handbook of Fluorescent Probe and Research Chemicals,
9th ed.; Molecular Probes, Inc.: Eugene, OR, 2002.
9
250 J. AM. CHEM. SOC. VOL. 130, NO. 1, 2008

Target-Specific Chemical Acylation of Lectins
A R T I C L E S
Figure 6. Selective acylation of Congerin in crude mixtures. Protein modification was detected by SDS-PAGE: CBB staining (lanes M, 1, 6); coumarin
fluorescence image (lanes 2-5); biotin blotting (lanes 7-11). (a) Coumarin labeling in cell lysates of E. coli overexpressing Cong II. (b) Biotin labeling in
tissue lysates of conger skin mucus. Lane M, protein marker; 1, whole proteins of E. coli; 2, Lac-DMAP 1; 3, Lac4-DMAP 2; 4, Lac4-DMAP 2 with 50 mM
lactose; 5, in the absence of DMAP; 6, whole proteins of conger skin mucus; 7, Lac4-DMAP 2; 8, Man4-DMAP 4; 9, NAG3-DMAP 3; 10, Lac4-DMAP
2 with 50 mM lactose; 11, in the absence of DMAP. Reaction conditions: 50 µM sugar-tethered DMAPs, 50 µM acyl donor, pH 8.0, 3 h, 25 °C.
to 0.2 µM with 50 mM HEPES buffer (pH 7.5), and Dab-lac 10 was
added to this solution. In fluorescence recovery processes, to a solution
of 0.2 µM FL-Cong II with 4 µM Dab-lac 10 were added various
saccharide solutions. The structure of saccharides used for this BFQR
study was shown in the Supporting Information.
Selective Acylation of Cong II in E. coli lysates. E. coli cells
expressing Cong II (JM109/pTV-Cong II) were cultured in 2 × YT
medium at 37 °C for 20 h, harvested, and lysed by sonication using a
Sonifier 450 (Branson). After the insoluble materials were removed
by centrifugation, the soluble fraction was dialyzed agains 50 mM
HEPES buffer (pH 8.0) containing 0.1 M NaCl and incubated with 1
mM N-maleoyl-â-alanine to mask free cysteine residues at 25 °C
overnight. To the lysate solution were added acyl donor 6 (50 µM)
and sugar-tethered DMAP 1 or 2 (50 µM). After 3 h incubation at 25
°C, the reaction mixtures were fractionated by 15% SDS-PAGE in a
reducing condition and analyzed as described above.
inhibitor) at 25 °C overnight. To the extract were added acyl donor 7
(50 µM) and each sugar-tethered DMAP 2-4 (50 µM). After 3 h
incubation at 25 °C, the reaction mixtures were fractionated by 15%
SDS-PAGE as described above and blotted onto a PVDF membrane.
The biotinylated products were detected with avidin-peroxidase
conjugate (Sigma) using diaminobenzidine (DAB) staining.
Acknowledgment. We thank Dr. M. Yamada and Shimadzu
Corp. for mass-mass analysis (AXIMA QIT (MALDI-QIT-
TOF)). Y.K. thanks JSPS for the DC scholarship.
Supporting Information Available: Synthesis of sugar-
tethered DMAPs (2-4) and acyl donors (6-9) and Dab-lac 10,
mass and absorption spectra of labeled lectins, mass-mass
spectrum of modified fragment, complete ref 8h, and molecular
structures of saccharides used in BFQR measurement. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Congerin Acylation in Skin Mucous Extracts. Skin mucous
extracts of conger myriaster were prepared according to the procedure
reported.14a The extracts were dialyzed against 50 mM HEPES buffer
(pH 8.0) containing 0.1 M NaCl and incubated with 1 mM N-maleoyl-
â-alanine and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (protease
JA075684Q
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