One-pot and sequential organic chemistry on an enzyme surface to tether a fluorescent probe at the proximity of the active site with restoring enzyme activity

Article abstract of DOI:10.1021/ja057926x

A new and simple method to tether a functional molecule at the proximity of the active site of an enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the overall steps under mild conditions. The enzymatic activity assay demonstrated that the release of the affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy derivatives, so that it restored the enzymatic activity from the completely suppressed state of the labeled hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to conventional affinity labeling methods. The peptide mapping experiment revealed that the labeling reaction was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When the fluorescent probe was tethered using the present sequential chemistry, the engineered hCAII can act as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential reaction re-opens the active site cavity of the target enzyme.

Full text of DOI:10.1021/ja057926x

Published on Web 02/21/2006
One-Pot and Sequential Organic Chemistry on an Enzyme
Surface to Tether a Fluorescent Probe at the Proximity of the
Active Site with Restoring Enzyme Activity
Yousuke Takaoka,§ Hiroshi Tsutsumi,‡ Noriyuki Kasagi,§ Eiji Nakata,† and
Itaru Hamachi*,†,‡
Contribution from the Department of Synthetic Chemistry and Biological Chemistry,
Graduate School of Engineering, Kyoto UniVersity, Kyotodaigaku-katsura, Nishikyo-ku,
Kyoto 615-8510, Japan, PRESTO (Synthesis and Control, JST), and Department of
Chemistry and Biochemistry, Graduate School of Engineering, Kyushu UniVersity,
Fukuoka 812-8581, Japan
Received November 22, 2005; E-mail: ihamachi@sbchem.kyoto-u.ac.jp
Abstract: A new and simple method to tether a functional molecule at the proximity of the active site of an
enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was
conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the
subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the
overall steps under mild conditions. The enzymatic activity assay demonstrated that the release of the
affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy
derivatives, so that it restored the enzymatic activity from the completely suppressed state of the labeled
hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to
conventional affinity labeling methods. The peptide mapping experiment revealed that the labeling reaction
was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When
the fluorescent probe was tethered using the present sequential chemistry, the engineered hCAII can act
as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this
method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential
reaction re-opens the active site cavity of the target enzyme.
Introduction
they and other groups incorporated unnatural amino acids
containing azide or acetylene into proteins and used them as a
There is considerable desire for useful chemical methods that
can modify proteins under physiological conditions with high
site- and chemoselectivities in the field of protein engineering
and chemical biology.1 In the case of synthetic or semisynthetic
proteins, several selective reactions have been developed
recently using artificially incorporated bio-orthogonal reactive
tags. For example, the reaction of an unnatural amino acid
bearing a ketone with hydrazides or aminooxy compounds was
reported.2 Schultz et al. demonstrated that ketone amino acids
incorporated by a genetically modulated codon method3 could
be employed as a tag for chemical modification.4 More recently,
selective modification site for the Huisgen [3+2] cycloaddition
developed by Sharpless et al.5 and Finn et al.6 on a protein
surface.7 In addition, another azide-based chemistry such as
Staudinger ligation has been explored to modify proteins by
Bertozzi8 and Raines.9 It is undoubted that these bio-orthogonal
reactions have expanded chemical strategies for the selective
(3) (a) Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Science 2001, 292,
498-500. (b) Chin, J. W.; Santoro, S. W.; Martin, A. B.; King, D. S.;
Wang, L.; Schultz, P. G. J. Am. Chem. Soc. 2002, 124, 9026-9027. (c)
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A.; Cho, C.; Schultz, P. G. Biochemistry 2003, 42, 6735-6746.
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Taylor, P.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41,
1053-1057. (b) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless,
K. B. Angew. Chem., Int. Ed. 2002, 41, 2596-2599.
† Kyoto University.
‡ PRESTO (Synthesis and Control, JST).
§ Kyushu University.
(1) (a) Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nat. ReV. Mol.
Cell Biol. 2002, 3, 906-918. (b) Chen, I.; Ting, A. Y. Curr. Opin.
Biotechnol. 2005, 16, 35-40. (c) Miller, L. W.; Cornish, V. W. Curr. Opin.
Chem. Biol. 2005, 9, 56-61. (d) Johnsson, N.; Johnsson, K. ChemBioChem
2003, 4, 803-810. (e) Johnsson, N.; George, N.; Johnsson, K. ChemBio-
Chem 2005, 6, 47-52.
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M. G. Bioconjugate Chem. 2005, 16, 1572-1579. (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.
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J. AM. CHEM. SOC. 2006, 128, 3273-3280
3273

A R T I C L E S
Takaoka et al.
Scheme 1. P-ALM Scheme for the Selective Modification of a Proteina
a (a) Complex formation between hCAII and P-ALM reagent. (b) The affinity labeling reaction via the epoxide ring opening to generate the labeled
hCAII. (c) The step to produce the modified hCAII by the hydrazone/oxime exchange reaction.
Results and Discussion
labeling of various proteins although a genetic mutation is
required during the initial stage for these modifications in many
cases.
Modification of Human Carbonic Anhydrase II Using
Sequential Reaction. In the present P-ALM, an affinity labeling
reaction using epoxide12 and the subsequent hydrazone/oxime
exchange13 are combined as a designer sequential reaction on a
protein surface. Scheme 1 illustrates the strategy for the
sequential reaction on the surface of a target protein, that is,
the affinity labeling is directed to the modification site close to
the active site and the hydrazone linkage between the ligand
and the reaction site is subsequently replaced by an oxime bond.
These two reactions proceed in a sequential manner under mild
conditions, so that a one-pot modification can be carried out.
In a proof-of-principle experiment, human carbonic anhydrase
II (hCAII) was used as a model protein.14 In the design of the
P-ALM reagents, benzenesulfonamide, which is a typical
inhibitor of hCAII, and an epoxide moiety which was demon-
strated to be a suitable electrophile for hCAII by Sames,12 were
used for targeting the active site of hCAII and for reacting with
hCAII, respectively (Figure 1a, 1-4). These two moieties are
linked by a hydrazone unit with various spacer lengths. The
For a native protein as a target, on the other hand, limited
kinds of reactions have been developed which involve a covalent
bond formation between nucleophilic groups such as a cysteine
or a lysine on protein surfaces and external artificial reactive
groups. Although interesting tyrosine-directed azo-coupling and
Mannich reactions have very recently been reported by Francis
et al.,10 these are still rare examples. This is because it is difficult
to recognize a protein surface for site-selective reactions, in
addition to the poor development of bio-orthogonal reactions
conducted under physiological conditions.
We recently reported a new method (P-PALM method) based
on photoaffinity labeling to incorporate a unique chemoselective
tag proximal to the active site of a lectin, i.e., a saccharide-
binding protein.11 This is beneficial from the viewpoint that the
active site-directed introduction of a chemical tag to naturally
occurring proteins is potentially carried out without the use of
genetically engineered proteins. However, thiol chemistry on
the protein surface previously reported by us is not sufficiently
general because this method is not applicable to many proteins
which have cysteine residues. Another drawback of P-PALM
is that the tedious and time-consuming purification procedures
including several steps are needed to remove the reagents and
other byproducts. A simpler modification method is most
preferable.
During exploration of a general and efficient methodology,
we have developed a one-pot sequential chemistry (the so-called
Post-Affinity Labeling Modification: P-ALM) based on the
affinity labeling modification followed by the hydrazone/oxime
exchange reaction. Human carbonic anhydrase II (hCAII) was
labeled at two selective positions proximal to the active site
using epoxide-based P-ALM reagents having a suitable spacer
length, and subsequently, the masking ligand was cleaved off
using the hydrazone/oxime exchange under one-pot conditions
in over 90% yield. This exchange process restores the enzyme
activity of hCAII. More interestingly, we demonstrated that
several fluorophores were successfully attached to hCAII by
the present exchange reaction using fluorescent aminooxy
derivatives, and the resultant modified hCAIIs act as fluorescent
biosensors that can sense sulfonamide derivatives, a family of
hCAII inhibitors.
(8) (a) Kiick, K. L.; Saxon, E.; Tirrell, D. A.; Bertozzi, C. R. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 19-24. (b) Lemieux, G. A.; Graffenried, C. L.;
Bertozzi, C. R. J. Am. Chem. Soc. 2003, 125, 4708-4709. (c) Saxon, E.;
Bertozzi, C. R. Science 2000, 287, 2007-2010.
(9) (a) Soellner, M. B.; Dickson, K. A.; Nilsson, B. L.; Raines, R. T. J. Am.
Chem. Soc. 2003, 125, 11790-11791. (b) Nilsson, B. L.; Hondal, R. J.;
Soellner, M. B.; Raines, R. T. J. Am. Chem. Soc. 2003, 125, 5268-5269.
(10) (a) Hooker, J. M.; Kovacs, E. W.; Francis, M. B. J. Am. Chem. Soc. 2004,
126, 3718-3719. (b) Sclick, T. L.; Ernest, Z. D.; Kovacs, E. W.; Francis,
M. B. J. Am. Chem. Soc. 2005, 127, 3718-3723. (c) Joshi, N. S.; Whitaker,
L. R.; Francis, M. B. J. Am. Chem. Soc. 2004, 126, 15942-15943.
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12065-12066. (b) Nagase, T.; Shinkai, S.; Hamachi, I. Chem. Commun.
2001, 3, 229-230. (c) Nagase, T.; Nakata, E.; Shinkai, S.; Hamachi, I.
Chem. Eur. J. 2003, 9, 3660-3669. (d) Nakata, E.; Nagase, T.; Shinkai,
S.; Hamachi, I. J. Am. Chem. Soc. 2004, 126, 490-495. (e) Koshi, Y.;
Nakata, E.; Hamachi, I. ChemBioChem 2005, 6, 1349-1352. (f) Nakata,
E.; Koshi, Y.; Koga, E.; Katayama, Y.; Hamachi, I. J. Am. Chem. Soc.
2005, 127, 13253-13261. (g) Ojida, A.; Tsutsumi, H.; Kasagi, N.; Hamachi,
I. Tetrahedron Lett. 2005, 46, 3301-3305.
(12) Chen, G.; Heim, A.; Riether, D.; Yee, D.; Milgrom, Y.; Gawinowicz, M.
A.; Sames, D. J. Am. Chem, Soc. 2003, 125, 8130-8133.
(13) (a) Polyakov, V. A.; Nelen, M. I.; Nazarpack-Kandlousy, N.; Ryabov, A.
D.; Eliseev, A. V. J. Phys. Org. Chem. 1999, 12, 357-363. (b) Goral, V.;
Nelen, M. I.; Eliseev, A. V.; Lehn, J.-M. Proc. Natl. Acad. Soc. U.S.A.
2001, 98, 1347-1352. (c) Shao, H.; Crnogorac, M. M.; Kong, T.; Chen,
S.-Y.; Williams, J. M.; Tack, J. M.; Gueriguian, V.; Cagle, E. N.; Carnevali,
M.; Tumelty, D.; Paliard, X.; Miranda, L. P.; Bradburne, J. A.; Kochen-
doerfer, G. G. J. Am. Chem. Soc. 2005, 127, 1350-1351.
(14) (a) Roy, B. C.; Banerjee, A. L.; Swanson, M.; Jia, X. G.; Haldar, M. K.;
Mallik, S.; Srivastava, D. K. J. Am. Chem. Soc. 2004, 126, 13206-13207.
(b) Banerjee, A. L.; Swanson, M.; Roy, B. C.; Jia, X.; Haldar, M. K.; Mallik,
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Post-Affinity Labeling Modification
A R T I C L E S
Figure 1. Chemical structures of (a) P-ALM reagents and (b) aminooxy derivatives used in this study. (c) Structural model of hCAII.
Figure 2. (a) MALDI-TOF mass spectra of reaction mixture of hCAII
with 3 (*; native hCAII (Mw 29 057), O; 3-labeled-hCAII (Mw 29 514),
spectra changed at 0, 24, 48, and 72 h from bottom to top). (b) Time course
of the labeling reaction process of hCAII with 1 (O), 2 (4), 3 (0), and 4
(×), respectively. The detail reaction conditions were described in the
Experimental Section.
Figure 3. (a) MALDI-TOF mass spectra of reaction mixture of 3-labeled-
hCAII with 100 equiv of 5 at 45 °C, pH 5.5 (O; 3-labeled-hCAII (Mw
29 514), 4; CM-hCAII (Mw 29 308), spectra changed at 0, 5, and 24 h
from bottom to top). (b) Time course of the hydrazone/oxime exchange
reaction process of 3-labeled-hCAII in the presence of 100 equiv of 5 at
45 °C, pH 5.5 (b), pH 7.2 ([).
hydrazone can be reversibly replaced with other functionalities
such as an aminooxy or hydrazine group. After 3 was mixed
with hCAII in an aqueous buffer (pH 7.2), the mixture was
incubated at 25 °C for 48-72 h. The reaction progress was
monitored by MALDI-TOF mass spectroscopy (Figure 2a and
2b), showing the almost total completion of the labeling reaction
(3-labeled-hCAII) within 48 h (0). In the case of the P-ALM
reagent 4 ([) having the longer spacer, the labeling reaction
was completed within 48 h similar to 3. In contrast, the reaction
using 2 (4) less efficiently proceeded to only 50%, and 1 (O)
gave less than 10% of the 1-labeled-hCAII after the 72 h
incubation. It is clear that the yield in the labeling step
significantly depends on the methylene length between the
sulfonamide site and the reactive epoxide site of the P-ALM
reagent (Figure 1a,c). The two or more modifications of hCAII
were not observed in the MALDI-TOF data, indicating that the
single-site modification predominantly took place in this method.
As control experiments, neither reaction using an epoxide
lacking the sulfonamide part, nor the reaction with 3 in the
presence of 10 equiv of a strong inhibitor, 6-ethoxy-2-ben-
zothiazolesulfonamide (ET),15 afforded the labeled hCAII. These
results strongly suggest that the present labeling process is
effectively assisted by the ligand binding of 3 to the active site
pocket of hCAII.
After the labeling reaction (3-labeled hCAII), carboxymethyl-
oxyamine (CM) 5 was added to the labeled reaction mixture at
various concentrations, and then the reaction mixture was
incubated at various pHs and temperatures to determine the
optimal conditions. All modified hCAIIs were purified by gel-
filtration chromatography before analyzing the function (details
are described in the Experimental Section). The hydrazone/
oxime exchange reaction was again traced by MALDI-TOF MS,
showing that the exchange reaction at pH 5.5 was almost
completed within less than 24 h in the presence of 100 equiv
of 5 (Figure 3). For the reaction at neutral pH (pH 7.2), on the
other hand, the yield slightly decreased to 77% at 45 °C for 24
h or 73% (at 25 °C), and it significantly decreased to 43-30%
under more basic conditions (pH 9.0). Table 1 summarizes the
yields of the reaction under various conditions, indicating that
(i) the reaction efficiently proceeds under acidic pH conditions,
due to acceleration of the proton-assisted exchange reaction,
(ii) the excess amount of the aminooxy group is needed for the
good conversion, and (iii) the reaction yield is not very sensitive
to the reaction temperature. At the more acidic pH (pH ) 4)
and/or the higher temperature (at 50 °C), the isolation yield
(15) Casini, A.; Antel, J.; Abbate, F.; Scozzafava, A.; David, S.; Waldeck, H.;
Scha¨fer, S.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2003, 13, 841-845.
9
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A R T I C L E S
Takaoka et al.
connected to the active site (Figure 1c).17 Therefore, it is
reasonable that one of the two sites was selectively labeled by
the nucleophilic ring opening of the epoxide when the labeling
reagent 3 was bound to the active site of hCAII. In addition,
this result was in good agreement with the detailed molecular
modeling study reported by Srivastava et al.14b,18 On the other
hand, Sames et al. reported that His-64 of hCAII was selectively
labeled using an epoxide-based affinity labeling reagent having
a fluorescein unit. Since the hydrophobic fluorescein part is
favorably bound to the hydrophobic crevasse close to the active
site, the epoxide part might be juxtaposed toward the rather
buried His-64, so that His-64 was selectively labeled in their
case.12 Another minor peak (Rt ) 30 min, Figure S8b) was
assigned to the dehydrogenated L1-fragment conjugated with
EDANS because the mass peak was observed at m/z ) 1510,
two molecular weights less than the calculated mass of the
EDANS-modified L1 peptide. This may suggest that some
oxidation occurred as a side reaction during or after the P-ALM
reaction processes.
Restoring Enzyme Activity by Hydrazone/Oxime Ex-
change Reaction. The enzyme activities of the labeled and
modified hCAIIs were examined by the conventional ester
hydrolysis reaction using p-nitrophenyl acetate as the substrate.19
The initial rates of the ester hydrolysis were measured at various
substrate concentrations, and the kinetic parameters of the
labeled or modified hCAIIs were determined (Figure 5 and Table
2). For the 3-labeled-hCAII ([), the enzymatic activity was
almost perfectly suppressed so that the catalytic parameters could
not be determined. This is because the benzenesulfonamide part
of the P-ALM reagent efficiently masked the active site. In
contrast, CM-hCAII (b) showed an activity having a kcat value
(7.0 s-1) almost identical with that of the native hCAII (O, 6.2
s-1), clearly indicating that the closed active site was re-opened
by the hydrazone/oxime exchange reaction. In the case of
EDANS-hCAII (0) and FITC-hCAII ([), the suppressed
activity was partially recovered. There is not a significant
decrease in the kcat values of EDANS- (3.8 s-1) and FITC-hCAII
(4.5 s-1) relative to the native hCAII (6.2 s-1), whereas the
Table 1. Yields of the One-Pot Sequential Reaction under Various
Conditions for 24 h
yield (%)
aminooxy
temp
C)
quantity
(equiv)
derivatives
(
°
pH 5.5
pH 7.2
pH 9.0
25
45
45
45
45
100
20
100
20
83a
84a
73a
43a
77a
c
c
30a
43a
c
CM
>95a
77b
EDANS
FITC
20
78b
c
c
a Yield determined by MALDI-TOF MS analysis. b Isolation yield
determined by UV-visible spectroscopy. c Not determined.
decreased, probably due to the partial denaturation of hCAII
under the rather severe reaction conditions. When the fluorescent
aminooxy derivatives, such as EDANS (6) or FITC (7), were
employed in the second step, a fluorescent probe was success-
fully introduced to hCAII in a similar manner (the isolation yield
is 77% for EDANS- and 78% for FITC-hCAII at 45 °C, pH
5.5, for 72 h). These results have demonstrated that various
artificial molecules can be incorporated into natural hCAII via
the aminooxy tag using the hydrazone/oxime exchange modi-
fication with good yield under mild conditions.
Structural Characterization of Labeled/Modified hCAII.
The labeled hCAII in the first step of the present P-ALM was
characterized by MALDI-TOF mass spectroscopy, and the
modified hCAII in the second step was characterized by
MALDI-TOF MS, UV-visible, and fluorescence spectroscopies.
The mass peaks (m/z) were observed at 29 432, 29 489, 29 517,
and 29 545 for the 1-, 2-, 3-, and 4-labeled-hCAII, respectively.
The mass peaks of the modified hCAIIs appeared at 29 308 for
CM-hCAII, at 29 556 for EDANS-hCAII, and at 29 739 for
FITC-hCAII, respectively. These values agree well with the sum
of the Mw’s of the native hCAII and the corresponding tagged
molecules. In the UV-visible spectra of EDANS-hCAII, the
absorbance at 336 nm is characteristic of the EDANS unit that
appeared together with 280 nm due to hCAII. In addition, the
emission maximum at 470 nm and the excitation maximum at
336 nm due to the EDANS fluorophore were clearly observed
in EDANS-hCAII. Similarly, in the case of FITC-hCAII, the
494 nm absorption in the UV-vis spectrum and the 520 nm
fluorescence, both due to FITC, appeared for FITC-hCAII.
The labeling site of EDANS-hCAII prepared from the
3-labeled hCAII was determined by the conventional peptide
mapping experiment using Lysyl endopeptidase (LEP) which
selectively hydrolyzes peptide bonds at the C terminal site of
the lysine residues.16 Figure 4 shows the reversed-phase HPLC
trace of the digested peptides for EDANS-hCAII monitored by
the UV (Figure 4b) and fluorescence detector, compared to that
of the native hCAII (Figure 4a). In the HPLC chart of the
fluorescence mode (Figure 4c), two major peaks (retention time
(Rt) ) 42, 44 min) and one minor peak (Rt ) 30 min) mainly
appeared. The MALDI-TOF MS analysis indicates that the two
major peaks had identical mass, both of which can be assigned
to the EDANS modified N-terminal peptide (the N-terminal L1
fragment plus one molecule of the EDANS unit, i.e., [M + H]+
) 1512). The subsequent tandem mass-mass analysis clarified
that the labeling site was His-3 (Rt ) 44 min, Figure 4d) or
His-4 (Rt ) 42 min, Figure S8a). On the 3D-structure of hCAII,
His-3 and His-4 are exposed on the protein surface closely
substrate binding affinity (1/Km) of EDANS- (4.85 × 102 M-1
)
and FITC-hCAII (4.55 × 102 M-1) was reduced to 22 and 20%
relative to that of the native hCAII (2.25 × 103 M-1),
respectively. The CM-hCAII tethering the simple aminooxy
acetic acid, which is much smaller than the EDANS- and FITC-
group, showed an intermediate 1/Km value (9.15 × 102 M-1
)
between the native hCAII and EDANS-/FITC-hCAII. In addi-
tion, the specificity constants (kcat/Km) of CM- (6.45 × 103 s-1
M-1), EDANS- (1.85 × 103 s-1 M-1), and FITC-hCAII (2.05
× 103 s-1 M-1) were 46, 13, and 14% relative to that of the
native hCAII (1.45 × 104 s-1 M-1), respectively. These speci-
ficity constants were mainly affected by the binding affinity,
not by the catalytic efficiency. As demonstrated by the fluor-
escent anisotropy experiment described in the next section, these
(17) The X-ray crystallographic structure of recombinant hCAII in the presence
of 4-fluorobenzenesulfonamide has been known to atomic resolution. Kim,
C.-Y.; Christianson, D. W. PDB ID: 1IF4.
(18) Upon performing the energy minimization of P-ALM reagent 1, 2, 3, and
4, it was noted that the distance between the NH2 nitrogen of benzene-
sulfonamide and the epoxide ring was 14, 16, 20, and 23 Å for 1, 2, 3, and
4, respectively. By the way, the linear distance between the zinc atom of
the active site and the Nꢀ2 nitrogen of the surface-exposed histidine for
His-64, His-4, and His-3 was determined to be 12, 15, and 24 Å,
respectively.14b
(16) Shikata, Y.; Kuwada, M.; Hayashi, Y.; Hashimoto, A.; Koide, A.; Asakawa,
(19) (a) Elleby, B.; Sjoblom, B.; Lindskog, S. Eur. J. Biochem. 1999, 262, 516-
N. Anal. Chim. Acta, 1998, 365, 241-247.
521. (b) Pocker, Y.; Stone, J. T. Biochemistry 1967, 6, 668-678.
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Post-Affinity Labeling Modification
A R T I C L E S
Figure 4. (a) HPLC analysis of native hCAII fragments monitored by UV detector. EDANS-hCAII fragments monitored by (b) UV detector and (c)
fluorescence detector (λex ) 340 nm, λem ) 490 nm). (d) Tandem mass-mass analysis of the EDANS-labeled fragment (Rt ) 44 min). The full amino acid
sequence of native hCAII was shown in Figure S7. L1-L11 are fragments of native and EDANS-hCAII hydrolyzed by lysyl endopeptidase.
fluorophores should locate in the hydrophobic pocket proximal
to the substrate binding site.20 Therefore, it is plausible that the
bulky fluorophore tethering to the hCAII surface proximal to
the active site may partially block the substrate entry.
which was weakly bound to the hydrophobic environment of
the active site in the absence of SA, was turned out upon SA
binding.11b, 21 This mechanism is consistently supported by the
fluorescence anisotropy measurement with or without the SA
binding (Figure S10). That is, in the absence of SA, the
anisotropy parameter (r) of EDANS-hCAII was 0.158, and it
decreased to be 0.063 with the addition of SA in the saturation
manner. The fluorescence titration curve also showed the typical
saturation behavior, producing the binding constant of 8.85 ×
105 M-1 for SA. As shown in Figure 6b, similar titration curves
were obtained by the addition of the sulfonamide family (>106
M-1 for ET, >106 M-1 for NB, 3.65 × 105 M-1 for BS, 1.25
× 103 M-1 for SA1. The molecular structures are summarized
in Figure 6c), whereas SA2 never induced the fluorescence
change (the binding constant was too low to be determined). A
similar spectral change was observed in the case of FITC-hCAII
Function of a Modified hCAII as a Fluorescent Biosensor.
As already-mentioned, the present P-ALM method can maintain
the active site of hCAII while a fluorescent probe is attached
to the proximity of the active site. This unique advantage was
utilized for the development of a fluorescent biosensor on the
basis of the hCAII scaffold. Figure 6a shows the typical
fluorescence spectral change in EDANS-hCAII by the addition
of 4-sulfamoylbenzoic Acid (SA). The emission maximum at
470 nm due to the EDANS fluorophore was slightly red-shifted
to 477 nm with the concurrent decrease in the emission intensity.
Such a spectral change clearly implies that the EDANS moiety,
(20) (a) Pocker, Y.; Storm, D. R. Biochemistry 1968, 7, 1202-1212. (b)
Cheˆnevert, R.; Rhlid, R. B.; Le´tourneau, M.; Gagnon, R.; D’Astous, L.
Tetrahedron: Asymmetry 1993, 4, 1137-1140. (c) Gru¨neberg, S.; Stubbs,
M. T.; Klebe, G. J. Med. Chem. 2002, 45, 3588-3602.
(21) Enander, K.; Dolphin, G. T.; Liedbreg, B.; Lundstrom, I.; Baltzer, L. Chem.
Eur. J. 2004, 10, 2375-2385.
9
J. AM. CHEM. SOC. VOL. 128, NO. 10, 2006 3277

A R T I C L E S
Takaoka et al.
drazone/oxime exchange reaction so as to restore the enzyme
activity. Such an activity recovery upon the sequential modifica-
tion is advantageous over the conventional affinity labeling
methods, so that one can use (or visualize) the modified enzyme
bearing the original function. As a proof-of-principle example,
a fluorescent biosensor based on the enzyme scaffold was
successfully produced by P-ALM. These features potentially
expand the present chemical method to various proteins includ-
ing the disulfide bond or cysteine-containing proteins, and thus
we might apply this method not only in a test tube but also
inside or on the surface of a living cell with appropriate
improvement.
Experimental Section
Materials and Methods. Human carbonic anhydrase II (isozyme)
and lysyl endopeptidase were purchased from Sigma and Wako Pure
Chemical Industries, Ltd., respectively. Other chemicals and solvents
were purchased from Tokyo Kasei Kogyo, Co. Ltd., or Aldrich and
used without further purification. Matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF MS) was
recorded on PE Voyager DE-RP or Bruker Daltonics autoflex, where
sinapic acid and R-cyano-4-hydroxycinnamic acid were used as a
matrix. HPLC was carried out on a reversed-phase column (Hitachi
L-7100 HPLC system). UV-vis spectra were recorded on a Shimadzu
UV-visible 2550 spectrometer. Fluorescence spectra were recorded
on a Perkin-Elmer LS55 spectrometer equipped with polarizer.
Figure 5. Michaelis-Menten for hydrolysis of p-nitrophenyl acetate
catalyzed by native hCAII (O), 3-labeled-hCAII (×), CM-hCAII (b),
EDANS-hCAII (0) and FITC-hCAII ([), respectively. The concentration
of hCAIIs was fixed at 1.0 µM. The concentration of p-nitrophenyl acetate
was varied from 0.1 to 20 mM. The lines were obtained by least-squares
curve-fitting analysis using the Michaelis-Menten equation.
Table 2. Kinetic Parameters of Native hCAII, 3-labeled-hCAII,
CM-hCAII, EDANS-hCAII, and FITC-hCAII for the Hydrolysis of
p-Nitrophenyl Acetate
Synthesis of the P-ALM Reagent 3. To a suspension of 4-sulfa-
moylbenzoic acid (1.00 g, 5.00 mmol) in 10 mL of dry dimethylfor-
mamide solution containing WSC HCl (1.15 g, 6.00 mmol), HOBt‚
H2O (913 mg, 6.00 mmol), and DIPEA (2.17 mL, 12.5 mmol) was
added methyl 4-aminobutyrate hydrochloride (917 mg, 6.00 mmol).
After the reaction mixture was stirred at room temperature for 3 h, the
solution was diluted with 50 mL of water. The aqueous solution was
extracted with ethyl acetate (3×30 mL), and the extract was washed
with brine, dried over anhydrous MgSO4, and concentrated under
reduced pressure. The oily residue was precipitated with CHCl3 to give
4-(4-sulfamoylbenzoylamino)butyric acid methyl ester as a white solid
kinetic parameters
1
1
1
hCAII derivatives
k
cat (s-
)
1/Km (M-
)
k
cat/Km (s-1 M-
)
native
3-labeled
CM-modified
EDANS-modified
FITC-modified
6.2
2.2 × 103
9.1 × 102
4.8 × 102
4.5 × 102
1.4 × 104
6.4 × 103
1.8 × 103
2.0 × 103
n.d.a
7.0
n.d.a
n.d.a
3.8
4.5
a n.d. ) not determined due to the low reactivity.
1
(1.19 g, 80%). H NMR (400 MHz, CD3OD): δ/ppm 7.95 (m, 4H),
(Figure S9), and Table 3 summarizes the association constants
of the modified hCAII for various benzenesulfonamide deriva-
tives, together with those of the native hCAII.15,22 It is clear
that the orders of both the inhibitor affinity and the affinity value
were practically identical with that of native hCAII. As a control
experiment, we prepared a randomly modified hCAII with the
dansyl group at some lysine residues. No significant changes
in the fluorescence intensity and wavelength were induced by
the addition of SA, undoubtedly indicating that the active-site-
directed tethering of the fluorescent probe is essential for the
fluorescence sensing of the sulfonamide derivatives in EDANS-
or FITC-hCAII.
3.42 (m, 2H), 2.41 (m, 2H), 1.93 (m, 2H).
To a suspension of 4-(4-sulfamoylbenzoylamino)butyric acid methyl
ester (500 mg, 1.7 mmol) in 5 mL of dry dimethylformamide was added
hydrazine monohydrate (83 mg, 2.0 mmol), and the reaction mixture
was stirred at room temperature for 12 h. The solution was evaporated,
and then the residue was precipitated by ethyl acetate to give N-(3-
hydrazinocarbonyl-propyl)-4-sulfamoylbenzamide as a white solid (450
1
mg, 90%). H NMR (400 MHz, CD3OD): δ/ppm 7.96 (m, 4H), 3.41
(m, 2H), 2.25 (m, 2H), 1.93 (m, 2H).
4-Oxiranylmethoxybenzaldehyde (40 mg, 0.22 mmol) which was
prepared according to the literature,23 was mixed with N-(3-hydrazi-
nocarbonylpropyl)-4-sulfamoylbenzamide (67 mg, 0.22 mmol) in 0.5
mL of dry dimethylformamide. The reaction mixture was stirred at room
temperature for 12 h, and the solution was evaporated. The oily residue
was precipitated with cooled methanol to give the P-ALM reagent 3
as a white solid (80 mg, 78%). 1H NMR (400 MHz, DMSO-d6): δ/ppm
8.68 (m, 1H), 8.01 (d, JH ) 8.4 Hz, 2H), 7.89 (d, JH ) 8.4 Hz, 2H),
7.45 (d, JH ) 8.8 Hz, 2H), 7.00 (d, JH ) 8.8 Hz, 2H), 4.38 (m, 1H),
3.85 (m, 1H), 3.41 (m, 2H), 3.35 (m, 1H), 2.84 (m, 1H), 2.69 (m, 1H),
2.66 (m, 1H), 2.25 (m, 2H), 1.85 (m, 2H). HRMS (FAB+) calculated
for C21H25N4O6S m/z 461.1495, observed m/z 461.1508
Conclusion
In summary, we successfully developed a unique chemistry-
based method to tether a fluorophore at the proximity of the
active site of a naturally occurring enzyme. The one-pot se-
quential reaction conducted on the enzyme surface has sig-
nificantly simplified the tedious modification procedure with-
out any genetic techniques. The removal of the ligand part
from the active site was concurrently achieved with the hy-
Elementary analysis: calculated for C21H24N4O6S: C, 54.77; H, 5.25;
N, 12.17. Found: C, 54.62; H, 5.25; N, 12.07.
(22) (a) Taylor, P. W.; King, R. W.; Burgen, A. S. V. Biochemistry 1970, 9,
2638-2645. (b) Innocenti, A.; Zimmerman, S.; Ferry, J. G.; Scozzafova,
A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2004, 14, 4563-4567.
(23) Kitaori, K.; Furukawa, Y.; Yoshimoto, H.; Otera, J. Tetrahedron 1999,
55, 14381-14390.
9
3278 J. AM. CHEM. SOC. VOL. 128, NO. 10, 2006

Post-Affinity Labeling Modification
A R T I C L E S
Figure 6. (a) Fluorescence spectral change of EDANS-hCAII upon the addition of SA: [SA] ) 0, 0.5, 1.0, 1.5, 2.0, 2.5, 5, 10, 20 µM (from top to bottom).
(Inset) Titration curve of EDANS-hCAII with SA. I and I0 represent the fluorescent intensity at various inhibitor concentrations and the initial fluorescent
intensity, respectively. The solid line was obtained with the nonlinear curve-fitting analysis. (b) Fluorescence titration profile of the relative intensity change
(I/I0 - 1) versus the inhibitor concentration for EDANS-hCAII with ET(O), NB(2), SA([), BS(0), SA1(∇), PB(b). (c) Chemical structure of the inhibitors
for hCAII used in this study. The abbreviations are ET: 6-ethoxy-2-benzothiazolesulfonamidel; NB: 4-nitrobenzenesulfonamide; SA: 4-sulfamoylbenzoic
acid; BS: benzenesulfonamide; SA1: sulfamoic acid; PB: phenylboronic acid; SA2: sulfanilic acid.
1
Table 3. Association Constants of EDANS-hCAII, FITC-hCAII,
and Native hCAII for Various Inhibitors
4: yield: 30 mg (34%), H NMR (400 MHz, DMSO-d6): δ/ppm
8.62 (br, 1H), 8.08 (br, 1H), 7.96 (d, JH ) 8.4 Hz, 2H), 7.87 (d, JH
)
1
Ka/M-
8.4 Hz, 2H), 7.57 (d, JH ) 8.8 Hz, 2H), 6.98 (d, JH ) 8.8 Hz, 2H),
4.43-3.86 (br, 3H), 3.32 (m, 1H), 3.28 (m, 2H), 2.84, 2.71 (m, 2H),
2.59 (m, 2H), 1.56 (m, 4H), 1.37 (m, 2H). HRMS (FAB+) calculated
for C23H29N4O6S m/z 489.1808, observed m/z 489.1805.
inhibitors
EDANS-hCAIIa
FITC-hCAIIa
native hCAIIb
ET
NB
SA
BS
SA1
PB
>106
>107c
1.3 × 108
1.6 × 107
3.7 × 106
6.5 × 105
4.7 × 103
9.4 × 101
f
>106
1.4 × 107d
4.5 × 105
2.7 × 105
1.2 × 103
8.3 × 101
e
8.8 × 105
3.6 × 105
1.2 × 103
2.4 × 102
e
Detailed experimental procedures of EDANS-ONH2 and FITC-ONH2
are in Supporting Information.
EDANS-ONH2 (6); yield: 10 mg (77%), 1H NMR (400 MHz,
DMSO-d6): δ/ppm 8.38 (m, 1H), 8.13 (d, JH ) 8.8 Hz, 1H), 8.07 (d,
JH ) 8.4 Hz, 1H), 7.92 (d, JH ) 7.2 Hz, 1H), 7.31 (dd, JH ) 7.2 Hz,
JH ) 8.4 Hz, 1H), 7.26 (dd, JH ) 7.6 Hz, JH ) 8.4 Hz, 1H), 6.57 (d,
JH ) 8.0 Hz, 1H), 4.42 (s, 2H), 3.45 (t, JH ) 6.0 Hz, 2H), 3.31 (t, JH
) 6.0 Hz, 2H). HRMS (FAB+): calculated for C14H17N3O5S m/z
340.089; observed m/z 340.100.
SA2
a Determined by the nonlinear curve-fitting analysis. b Reported values
determined by the enzyme kinetics.14,20 c Condition at [FITC-hCAII] )
0.1 µM. d Condition at [FITC-hCAII] ) 0.5 µM. e Not determined due to
the low affinity. f There is no literature value.
FITC-ONH2 (7); yield: 35 mg (83%), 1H NMR (400 MHz, DMSO-
d6): δ/ppm 7.95 (s, 1H), 7.73 (br, 1H), 7.12 (d, JH ) 8.8 Hz, 1H), 6.65
(d, JH ) 8.8 Hz, 2H), 6.52 (s, 2H), 6.48 (d, JH ) 8.8 Hz, 2H), 3.95 (s,
2H), 3.61 (s, 2H), 3.34 (br, 2H). HRMS (FAB+): calculated for
C25H22N4O7S m/z 523.13; observed m/z 523.13.
P-ALM reagent 1, 2, and 4 were synthesized in the same manner.
Detailed experimental procedures for these compounds are supplied in
Supporting Information.
1
1: yield: 23 mg (30%), H NMR (400 MHz, DMSO-d6): δ/ppm
8.63 (s, 1H), 8.05 (d, JH ) 8.4 Hz, 2H), 7.93 (d, JH ) 8.4 Hz, 2H),
7.80 (d, JH ) 8.8 Hz, 2H), 7.07 (d, JH ) 8.8 Hz, 2H), 4.42-3.90 (m,
2H), 3.41 (m, 1H), 2.85, 2.72 (m, 2H). HRMS (FAB+) calculated for
C17H18N3O5S m/z 376.0967, observed m/z 376.0974.
Post-affinity Labeling Modification of hCAII (One-Pot P-ALM).
hCAII (3.5 mg) was dissolved in 50 mM HEPES buffer (1.4 mL, pH
7.2). The concentration of hCAII was determined by absorbance at 280
nm using the molar extinction coefficient (54 000 M-1 cm-1). The
labeling reagent 3 (0.15 mg, 0.30 µmol, 2.5 equiv to hCAII) in 1.6 µL
of DMSO was slowly added to the hCAII solution, and the reaction
mixture was incubated at 25 °C. After 3 days, carboxymethyl-oxyamine
(CM) 5 (20-100 equiv to hCAII) was slowly added to the mixture,
and it was incubated again for 24 h at 45 °C. In the case of the
aminooxy-fluorophores, 1.4 mg aminooxy-EDANS 6 (2.4 µmol, 20
1
2: yield: 21 mg (33%), H NMR (400 MHz, DMSO-d6): δ/ppm
9.06 (br, 1H), 8.89 (br, 1H), 8.17 (br, 1H), 8.05 (d, JH ) 8.4 Hz, 2H),
7.93 (d, JH ) 8.4 Hz, 2H), 7.65 (d, JH ) 8.8 Hz, 2H), 7.03 (d, JH
)
8.8 Hz, 2H), 4.40-3.89 (m, 3H), 3.86 (d-d, JH ) 6.4 Hz, 1H), 3.35
(m, 1H), 2.85, 2.72 (m, 2H). HRMS (FAB+) calculated for C19H21N4O6S
m/z 433.1182, observed m/z 433.1181.
9
J. AM. CHEM. SOC. VOL. 128, NO. 10, 2006 3279

A R T I C L E S
Takaoka et al.
equiv to hCAII) or 1.3 mg of aminooxy-FITC 7 (2.4 µmol, 20 equiv
to hCAII) was used. The yield for the labeling reaction and the exchange
reaction were determined by MALDI-TOF mass spectra, where the
reaction percentage was calculated by dividing the target peak intensity
by the total mass peak intensity due to hCAII’s. Excess amount of the
labeling reagent and the aminooxy-fluorophore were removed by gel
filtration chromatography (TOYO PEARL: 258 cm) eluted with 50
mM HEPES buffer pH 7.2 and the labeled/modified hCAII mixture
was collected.
of the pair of FITC-hCAII and ET). The corresponding benzensul-
fonamide analogue was added dropwise to the modified hCAII solution,
and emission spectra were measured (λex ) 336 nm for EDANS-hCAII
and λex ) 490 nm for FITC-hCAII): [ET] ) 0-1.5 µM; [NB] ) 0-10
µM; [SA] ) 0-20 µM; [BS] ) 0-20 µM; [SA1] ) 0-1.5 mM; [PB]
) 0-40 mM; [SA2] ) 0-40 mM. An average value of three
measurements was plotted for each point. Fluorescence titration curves
(λem ) 477 nm for EDANS-hCAII and 520 nm for FITC-hCAII) were
analyzed with the nonlinear curve-fitting analysis or the Benesi-
Hildebrand relationship to evaluate Ka values.
Preparation of the Randomly Dansyl-Modified hCAII. To a
solution of hCAII (3.0 mg) in 3.0 mL of Tris HCl buffer (25 mM, pH
8.5) was added a solution of dansyl chloride (0.81 mg, 3 µmol, 30
equiv to hCAII) in 40 µL of acetone. The mixture was incubated at 4
°C for 48 h in the dark and then dialyzed against 50 mM HEPES pH
7.2 to yield the randomly dansyl-modified hCAII. It was estimated that
one hCAII was modified with approximately 1.5 dansyl unit on the
average by absorbance at 280 and 336 nm using molar extinction
coefficient (ꢀ280 ) 54 000 M-1 cm-1 and ꢀ336 ) 5700 M-1 cm-1).
Enzyme Activity Assay.19 The hydrolytic activity assay of hCAIIs
was performed on UV-vis spectrum at 25 °C in 50 mM HEPES buffer
(pH 7.2) using a quartz cell with 1.0 cm path length. Initial rates of
p-nitrophenyl acetate hydrolysis were determined by the increase of
the absorbance at 348 nm (∆ꢀ ) 1090 M-1 cm-1) for the released
p-nitrophenolate as a product. The concentration of all hCAIIs was
fixed at 1.0 µM. The concentrations of 3-labeled-hCAII and CM-hCAII
were corrected by absorbance at 280 nm using the molar extinction
coefficient, 65 700 M-1 cm-1 and 71 000 M-1 cm-1, respectively. In
the case of EDANS-hCAII and FITC-hCAII, the labeled/modified
hCAII mixture solution was used, and the concentrations of EDANS-
hCAII and FITC-hCAII were corrected by absorbance at 280 and 336
nm/490 nm using the molar extinction coefficient: ꢀ280 ) 72 000 M-1
cm-1 and ꢀ336 ) 5700 M-1 cm-1 for EDANS-hCAII, ꢀ280 ) 94 000
M-1 cm-1 and ꢀ494 ) 68 000 M-1 cm-1 for FITC-hCAII, respectively.
Substrate concentrations were varied from 0.2 to 20 mM, and initial
rates were plotted against substrate concentrations. The stock solution
of p-nitrophenyl acetate (100 and 500 mM) was prepared with
acetonitrile. Kinetic parameters were obtained by the least-squares
curve-fitting analysis with Michaelis-Menten equation (eq 1) using
Kaleida Graph (Synergy Software).
Fluorescence anisotropy values (r) of EDANS-hCAII and FITC-
hCAII were evaluated from the following equation: r ) (IV - G×IH)/
(IV + 2G×IH), where IV and IH are the fluorescence intensities (λem
)
480 nm) observed through polarizers parallel and perpendicular to the
polarization of the exciting light (λex ) 330 nm), respectively, and G
is a correction factor to account for instrumental differences in detecting
emitted compounds.24
Peptide Mapping Experiment and Sequencing.11c The EDANS-
hCAII (including labeled hCAII) solution was diluted with 100 mM
(NH4)2CO3, pH 8.5 containing 2 M urea. Lysyl endopeptidase (LEP)
was added so that the LEP/substrate ratio was 1/50 (w/w), and the
digestion was allowed to proceed at 37 °C overnight. The digested
peptides were separated by HPLC using YMC-Pack ODS-A column
(4.6φ × 250 mm). HPLC solvents employed were acetonitrile contain-
ing 0.1% trifluoroacetic acid (TFA) (solvent A) and water containing
0.1% TFA (solvent B). HPLC analysis was carried out using a 5-55%
linear gradient in solvent A over 30 min (flow rate ) 1.0 mL/min).
The HPLC peak was monitored by both UV absorbance at 220 nm
and fluorescence at 490 nm (λex ) 340 nm), and each fraction was
analyzed by MALDI-TOF MS. Among them, the fluorescent peptide
peaks (Rt: 30, 42, 44 min) were conducted with tandem mass-mass
analysis (Bruker, PSD (Post Source Decay) mode) to determine the
sequence by PSD method using Biotools software.
Acknowledgment. We gratefully thank Mr. Hiroya Hokazono
for the analysis of MALDI-TOF mass spectroscopy. N.K and
E.N. are JSPS fellows for Japanese Junior Scientists.
Supporting Information Available: Synthesis of P-ALM
reagent 1, 2, 4, aminooxy derivative 6, 7, UV-visible and
fluorescence spectra of EDANS-/FITC-hCAII, the full amino
acid sequence of native hCAII, MS-MS analysis data of
EDANS-hCAII, fluorescence titration profile of the relative
intensity change (I/I0 - 1) versus the inhibitor concentration
for FITC-hCAII, fluorescence anisotropy data of EDANS-/
FITC-hCAII. This material is available free of charge via the
Internet at http://pubs.acs.org.
V0 ) kcat‚[E]0[S]/([S] + Km)
(1)
where V0 represents initial rate of hydrolysis, kcat and Km represent first-
order rate constant from the catalyst-substrate complex and Michaelis-
Menten constant, respectively. [E]0 and [S] represent the initial
concentrations of hCAIIs and substrate, respectively.
Fluorescence Titration Experiment. Fluorescence titration experi-
ments of benzensulfonamide analogues were performed at 25 °C in 50
mM HEPES buffer (pH 7.2) using a quartz cell. The concentrations of
EDANS-hCAII and FITC-hCAII were fixed at 1.0, 0.5 (only in the
case of the pair of FITC-hCAII and NB), or 0.1 µM (only in the case
JA057926X
(24) Vinson, V. K.; Cruz, E. M.; Higgs, H. N.; Pollard, T. D. Biochemistry
1998, 37, 10871-10880.
9
3280 J. AM. CHEM. SOC. VOL. 128, NO. 10, 2006