Fluorescence detection of serum albumin with a turnover-based sensor utilizing Kemp elimination reaction

Article abstract of DOI:10.1016/j.bmcl.2017.05.076

The Kemp elimination reaction is a well-known chemical reaction that is facilitated on a protein surface microenvironment, and in particular is highly accelerated in a unique binding pocket of serum albumin. We have designed and synthesized a fluorescently activatable coumarin derivative with a benzisoxazole scaffold to enable monitoring of the Kemp elimination reaction in terms of fluorescence change for the first time. We show that this fluorescent sensor can sensitively and selectively quantitate serum albumin in blood samples. It also works in a dry-chemistry format.

Full text of DOI:10.1016/j.bmcl.2017.05.076

Accepted Manuscript
Fluorescence detection of serum albumin with a turnover-based sensor utilizing
Kemp elimination reaction
Shingo Sakamoto, Toru Komatsu, Tasuku Ueno, Kenjiro Hanaoka, Yasuteru
Urano
PII:
S0960-894X(17)30573-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.05.076
BMCL 25021
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
18 April 2017
23 May 2017
25 May 2017
Please cite this article as: Sakamoto, S., Komatsu, T., Ueno, T., Hanaoka, K., Urano, Y., Fluorescence detection of
serum albumin with a turnover-based sensor utilizing Kemp elimination reaction, Bioorganic & Medicinal
Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.05.076
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Fluorescence detection of serum albumin
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Shingo Sakamoto, Toru Komatsu, Tasuku Ueno, Kenjiro Hanaoka, and Yasuteru Urano

Bioorganic & Medicinal Chemistry Letters
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Fluorescence detection of serum albumin with a turnover-based sensor utilizing
Kemp elimination reaction
Shingo Sakamotoa, Toru Komatsua, ∗, Tasuku Uenoa, Kenjiro Hanaokaa, and Yasuteru Uranoa, b, c∗
aGraduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
bGraduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
cCore Research for Evolutional Science and Technology (CREST) Investigator, Japan Agency for Medical Research and Development (AMED), 1-7-1 Otemachi,
Chiyoda-ku, Tokyo 100-0004, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
The Kemp elimination reaction is a well-known chemical reaction that is facilitated on a protein
surface microenvironment, and in particular is highly accelerated in a unique binding pocket of
serum albumin. We have designed and synthesized a fluorescently activatable coumarin
derivative with a benzisoxazole scaffold to enable monitoring of the Kemp elimination reaction
in terms of fluorescence change for the first time. We show that this fluorescent sensor can
sensitively and selectively quantitate serum albumin in blood samples. It also works in a dry-
chemistry format.
Revised
Accepted
Available online
Keywords:
Chemical biology
Fluorescent probes
Proteins
2009 Elsevier Ltd. All rights reserved.
Enzymes
Kemp elimination
Protein surfaces provide unique microenvironments due to the
arrangement of reactive side chains, e.g., at enzyme active sites.
Various chemical reactions are accelerated on protein surface
sites, and among them, the Kemp elimination reaction has
attracted many researchers as a model of enzymatic catalysis[1].
The reaction involves formation of salicylonitriles from
benzisoxazoles via deprotonation of a carbon by a general base in
a cooperative E2 reaction[2]. The reaction is accelerated under
basic conditions and in hydrophobic environments (such as on
protein surfaces), and much work has been done to develop
catalytic antibodies or other protein surface structures suitable to
serve as Kemp eliminases for studies of the basic mechanism of
sensitivity and wider applicability, being especially advantageous
for use with bio-samples[5]. We aimed to develop a fluorescent
substrate to monitor Kemp elimination by combining
a
fluorescent 7-hydroxycoumarin (umbelliferone) moiety with a
benzisoxazole scaffold. We confirmed that the synthesized
substrate works as a sensitive platform for detection of the Kemp
elimination reaction. In particular, we found that this sensor can
quantify serum albumin concentration in blood samples with high
specificity and sensitivity. Changes of serum albumin
concentration are associated with various pathophysiological
states, as well as affecting free drug concentration; thus, we
believe this sensor has potential for clinical application,
especially since it also works in a dry-chemistry format.
enzymatic catalysis by transition state stabilization[1, 3]
. It has
also been reported that certain naturally occurring proteins, such
as serum albumin, catalyze the reaction very efficiently due to
their unique binding properties for small molecules[4].
In designing the Kemp elimination substrate, we required a
strategy for employing the reaction to trigger fluorescence
activation. Since the Kemp elimination reaction generates
phenolate as the end product, we considered that this could be
utilized to switch the fluorescence of umbelliferone, because the
ether or ester form of umbelliferone is known to exhibit
absorbance/fluorescence at shorter wavelengths than the
phenolate form, and this has been employed as a basis for
fluorescence switching in various sensors for ester or ether
Here, we describe the design, synthesis and characterization of
the first fluorescent Kemp reaction substrate, with the aim of
developing a highly sensitive tool to study this interesting
enzyme-like reaction in biological samples. Currently, Kemp
elimination reactions are mainly studied by using colorimetric
substrates such as 5-nitrobenzisoxazole[4]. However, compared
to colorimetric assays, fluorescence assays offer greater
———
cleavage reactions[5b, 6]
.
∗ Toru Komatsu. Tel.: +81-3-5841-1075; fax: +81-3-5841-4855; e-mail: tkomatsu@mol.f.u-tokyo.ac.jp
∗ Yasuteru Urano. Tel.: +81-3-5841-3601; fax: +81-3-5841-3563; e-mail: uranokun@m.u-tokyo.ac.jp

acids. Many therapeutic drugs also bind to albumin, which
consequently influences their efficacy by reducing the free
(effective) drug concentration[10]. Since albumin is produced in
the liver, its blood concentration is often altered in liver
disease[11], and therefore monitoring of changes in serum albumin
concentration is important for clinical management of patients.
At present, colorimetric assays that utilize the binding of organic
dyes (e.g. bromocresol green) to albumin are widely used to
measure blood albumin levels, but these assays have limited
sensitivity and selectivity[12]. We expected that a fluorescent
sensor would offer superior performance.
Figure 1. Design of fluorescent Kemp elimination substrate KEMp-1.
Thus, we planned to obtain a fluorescent Kemp elimination
substrate, KEMp-1 (1) (Figure 1), by introducing a formyl group
into 7-hydroxy-4-methylcoumarin with the Duff reaction[7], and
then forming a benzisoxazole ring by oxime formation and
oxidative condensation (Scheme S1). The expected elimination
product (2) was also prepared by treatment of benzisoxazole in
mild basic conditions.
KEMp-1 (1) showed an absorbance maximum at 320 nm, like
other umbelliferone derivatives with protection of the 7-OH
group (Figure 2a). Unexpectedly, it did not exhibit fluorescence
on excitation at 320 nm (ΦFL < 0.01). In contrast, the hydrolysis
product, 8-cyano-7-hydroxy-4-methylcoumarin (2), had an
absorbance maximum at around 380 nm, and it exhibited strong
fluorescence (ΦFL = 0.59). Therefore, the Kemp elimination
reaction can be monitored in terms of fluorescence activation
under 380 nm excitation. Indeed, after the addition of bovine
serum albumin (BSA), a protein known to have high catalytic
activity toward benzisoxazoles[4a], a dramatic absorbance change
was observed at pH 7.4, and the reaction resulted in more than
80-fold fluorescence activation (Figure 2b). LC-MS analysis
confirmed clean conversion of benzisoxazole to the
salicylonitrile derivative (Figure S1).
Figure 2. (a) Absorbance spectra of KEMp-1 (10 µM) before and after
addition of bovine serum albumin (BSA; 1 mg/mL) in phosphate buffer
(100 mM, pH 7.4) and incubation for 18 hr. (b) Fluorescence spectra of
KEMp-1 (10 µM) after addition of bovine serum albumin (BSA; 1
mg/mL) in phosphate buffer (100 mM, pH 7.4). (c) Fluorescence
increase rates of KEMp-1 (10 µM) after addition of various proteins (1
mg/mL or 0.1 mg/mL). n = 4. Error bars represent S.D.. (d)
Normalized fluorescence increase rates of KEMp-1 (10 µM) after
addition of human serum albumin (HSA; 1 mg/mL) modified with or
without fluorescein isothiocyanate (FITC) in phosphate buffer (100 mM,
pH 7.4). n = 4. Error bars represent S.D..
Next, we examined the reactivity of the KEMp-1 with various
proteins. Interestingly, the fluorescence increase was highly
selective for serum albumin, especially for human serum albumin
(Figure 2c). Addition of glutathione had no effect on the
reaction, suggesting that KEMp-1 binds specifically to the
surface of albumin for the catalytic elimination. The kcat/Km
value was not high compared to those of general enzymatic
reactions (Table 1). However, the reaction of conventional
benzisoxazoles[4b] with serum albumin is blocked by the
modification of key lysine residues [8], and the modification of
lysine residues with fluorescein isothiocyanate (FITC) was
confirmed to dramatically reduce the reactivity of KEMp-1
(Figure 2d). Therefore, the reaction of KEMp-1 may proceed via
a similar mechanism to that of conventional benzisoxazoles.
Aiming to take advantage of the high sensitivity of fluorometric
assay with turnover-based signal amplification, we next
examined whether this system could be applied to monitor Kemp
elimination reaction catalyzed by serum albumin in blood
samples.
Table 1. Kinetic parameters of KEMp-1 reactivity with human
serum albumin (HSA) and bovine serum albumin (BSA).
kcat (s-1)
Km (M)
kcat/Km (M-1 s-1)
BSA
HSA
8.5 × 10-6
2.4 × 10-5
7.2 × 10-6
7.0 × 10-6
1.2
3.4
Indeed, KEMp-1 was able to detect human serum albumin
(HSA) at a concentration as low as 0.02 mg/mL (Figure 3a); this
sensitivity is comparable to that of conventional Kemp
elimination substrates, and is greater than that of a commercial
albumin detection kit, which failed to detect HSA at
concentrations below 0.04 mg/mL (Figure S2). We were able to
detect a fluorescence increase even in the human serum diluted to
an albumin concentration of 0.04% (Figure S3). Our serum
sample contained 75 mg/mL of proteins (determined by Bradford
assay using HSA as a standard), of which approximately 60-70%
Serum albumin is a major blood protein[9], serving as a carrier
of hydrophobic compounds such as steroids, hemins, and fatty

was considered to be albumin, as judged from SDS-PAGE
analysis (Figure 3b, S4). We found that calibration curves
prepared with diluted HSA solution and with diluted serum
calculated to contain equivalent amounts of HSA coincided very
closely, suggesting that most of the fluorescence increase in the
latter case originated from albumin in blood (Figure 3b). We
also examined a HeLa cell lysate, which contained no albumin.
The cell lysate showed dramatically reduced Kemp elimination
activity (about 14% of that of serum), supporting the idea that the
fluorescence increase observed with human serum is
predominantly due to albumin (Figure S5).
optimized, structural modifications should enable further
improvement of the specificity and sensitivity, and it may also be
possible to synthesize derivatives targeting other proteins. Such
sensors could be useful for detection of various small molecule-
protein engagements in complex biological systems, since
turnover-based detection enables dramatic signal amplification;
for example, turnover-based strategies are often employed to
detect even very low levels of enzyme activity[5b, 13]. We wish to
emphasize that KEMp-1 can specifically quantify serum albumin
concentration in blood samples with high sensitivity, and is also
applicable in a dry-chemistry format. Therefore, we believe this
sensor has potential to be developed for clinical application.
Further studies are ongoing in our laboratory.
In chronic hepatic failure, albumin concentration is known to
decrease from 3.8-5.3 g/dL to 2-3 g/dL. Therefore, we prepared
model samples by mixing human serum (5 g/dL HSA) and HeLa
cell lysate (0 g/dL HSA) having the same protein content in
different ratios to obtain final HSA concentrations of 0, 1, 2, 3, 4,
and 5 g/dL. These solutions were clearly differentiated by the
fluorometric assay using 100-fold diluted samples, and we
obtained a good linear relationship between fluorescence increase
and HSA concentration (Figure S6).
Acknowledgments
This work was financially supported by MEXT (24655147,
15H05371, and 15K14937 to T.K., and 16H05099, 16H00823 to
K.H.), JST (T.K. and K.H.), AMED (Y.U.), and JSPS (Core-to-
Core Program, A. Advanced Research Networks). T.K. was
supported by Naito Foundation and Mochida Memorial
Foundation for Medical and Pharmaceutical Research. Authors
would like to thank Yugo Kuriki for assistance with the dry-
chemistry experiments.
Finally, in order to test the potential utility of this system as a
dry-chemistry platform for on-site detection of serum albumin in
blood samples in the clinical context, we applied a solution of
KEMp-1 to a filter paper and dried it. Application of a 5 µL
serum sample to the dried paper induced a dramatic fluorescence
increase, which was easily detectable with the naked eye under
excitation with a handy UV lamp (Figure 3c).
References and notes
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DeChancie, J. Betker, J. L. Gallaher, E. A. Althoff, A.
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Baker, Nature 2008, 453, 190-195. (b) W. Cullen, M. C.
Misuraca, C. A. Hunter, N. H. Williams and M. D. Ward, Nat
Chem 2016, 8, 231-236.
2. (a) M. L. Casey, D. S. Kemp, K. G. Paul and D. D. Cox, J. Org.
Chem. 1973, 38, 2294-2301; (b) D. S. Kemp and M. L. Casey, J.
Am. Chem. Soc. 1973, 95, 6670-6680; (c) D. S. Kemp and K. G.
Paul, J. Am. Chem. Soc. 1975, 97, 7305-7312; (d) D. S. Kemp, D.
D. Cox and K. G. Paul, J. Am. Chem. Soc. 1975, 97, 7312-7318.
3. (a) S. N. Thorn, R. G. Daniels, M. T. Auditor and D. Hilvert,
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J. Am. Chem. Soc. 1996, 118, 6462-6471.
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Hilvert, J. Am. Chem. Soc. 2000, 122, 1022-1029; b) Y. Hu, K. N.
Houk, K. Kikuchi, K. Hotta and D. Hilvert, J. Am. Chem. Soc.
2004, 126, 8197-8205.
5. (a) T. Komatsu and Y. Urano, Anal. Sci. 2015, 31, 257-265; (b) T.
Komatsu, K. Hanaoka, A. Adibekian, K. Yoshioka, T. Terai, T.
Ueno, M. Kawaguchi, B. F. Cravatt and T. Nagano, J. Am. Chem.
Soc. 2013, 135, 6002-6005; (c) H. Onoyama, M. Kamiya, Y.
Kuriki, T. Komatsu, H. Abe, Y. Tsuji, K. Yagi, Y. Yamagata, S.
Aikou, M. Nishida, K. Mori, H. Yamashita, M. Fujishiro, S.
Nomura, N. Shimizu, M. Fukayama, K. Koike, Y. Urano and Y.
Seto, Sci. Rep. 2016, 6, 26399.
6. A. Aitio, Anal. Biochem. 1978, 85, 488-491.
7. A. Garg, D. M. Manidhar, M. Gokara, C. Malleda, C. Suresh
Reddy and R. Subramanyam, PLoS One 2013, 8, e63805.
8. R. P. Taylor, J. Am. Chem. Soc. 1976, 98, 2684-2686.
9. B. T. Doumas and T. Peters, Jr., Clin. Chim. Acta 1997, 258, 3-20.
10. (a) D. Rudman, T. J. Bixler, 2nd and A. E. Del Rio, J. Pharmacol.
Exp. Ther. 1971, 176, 261-272; (b) I. Sjoholm and T. Sjodin,
Biochem. Pharmacol. 1972, 21, 3041-3052.
Figure 3. (a) Fluorescence increase rate of KEMp-1 (10 µM) after
addition of HSA in phosphate buffer (100 mM, pH 7.4) and incubation
for 60 min. n = 4. Error bars represent S.D.. (b) (Left) Fluorescence
increase rate of KEMp-1 (10 µM) after addition of HSA (black) or
serum (red) in phosphate buffer (100 mM, pH 7.4) and incubation for 60
min. n = 3. Error bars represent S.D.. (Right) SDS-PAGE analysis of
human serum (0.2%) and HSA solution (0.1 mg/mL). (c) Photo of dried
filter paper with KEMp-1 (50 pmol per spot) incubated with HSA (10-50
mg/mL), human serum, or phosphate buffer for 20 min. Illumination
was done with a handy UV lamp (365 nm).
11. R. Sallie, J. M. Tredger and R. Williams, Biopharm. Drug.
Dispos. 1991, 12, 251-259.
12. D. Webster, Clin. Chim. Acta 1974, 53, 109-115.
13. (a) J. Onagi, T. Komatsu, Y. Ichihashi, Y. Kuriki, M. Kamiya, T.
Terai, T. Ueno, K. Hanaoka, H. Matsuzaki, K. Hata, T. Watanabe,
T. Nagano and Y. Urano, J. Am. Chem. Soc. 2017, 139, 3465-
3472; (b) Y. Kimura, T. Komatsu, K. Yanagi, K. Hanaoka, T.
Ueno, T. Terai, H. Kojima, T. Okabe, T. Nagano and Y. Urano,
Angew. Chem. Int. Ed. Engl. 2017, 56, 153-157; (c) Y. Rondelez,
G. Tresset, K. V. Tabata, H. Arata, H. Fujita, S. Takeuchi and H.
Noji, Nat. Biotechnol. 2005, 23, 361-365.
In conclusion, we have developed a Kemp elimination
substrate that enables fluorescence-based visualization of the
reaction in bio-samples. Like conventional benzisoxazoles, it
showed high selectivity for serum albumin, and it also provided
very high sensitivity. While the current molecule is not

Synthesis and characterization of KEMp-1, methods, and
supplementary data.
Supplementary material
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(EndNote references)
[1] a) D. Rothlisberger, O. Khersonsky, A. M. Wollacott, L. Jiang, J.
DeChancie, J. Betker, J. L. Gallaher, E. A. Althoff, A. Zanghellini,
O. Dym, S. Albeck, K. N. Houk, D. S. Tawfik and D. Baker, Nature
2008, 453, 190-195; b) W. Cullen, M. C. Misuraca, C. A. Hunter, N.
H. Williams and M. D. Ward, Nat Chem 2016, 8, 231-236.
[2] a) M. L. Casey, D. S. Kemp, K. G. Paul and D. D. Cox, Journal
of Organic Chemistry 1973, 38, 2294-2301; b) D. S. Kemp and M.
L. Casey, Journal of the American Chemical Society 1973, 95, 6670-
6680; c) D. S. Kemp and K. G. Paul, Journal of the American
Chemical Society 1975, 97, 7305-7312; d) D. S. Kemp, D. D. Cox
and K. G. Paul, Journal of the American Chemical Society 1975, 97,
7312-7318.
[3] a) S. N. Thorn, R. G. Daniels, M. T. Auditor and D. Hilvert,
Nature 1995, 373, 228-230; b) J. Na, K. N. Houk and D. Hilvert,
Journal of the American Chemical Society 1996, 118, 6462-6471.
[4] a) F. Hollfelder, A. J. Kirby, D. S. Tawfik, K. Kikuchi and D.
Hilvert, Journal of the American Chemical Society 2000, 122, 1022-
1029; b) Y. Hu, K. N. Houk, K. Kikuchi, K. Hotta and D. Hilvert, J
Am Chem Soc 2004, 126, 8197-8205.
[5] a) T. Komatsu and Y. Urano, Anal Sci 2015, 31, 257-265; b) T.
Komatsu, K. Hanaoka, A. Adibekian, K. Yoshioka, T. Terai, T.
Ueno, M. Kawaguchi, B. F. Cravatt and T. Nagano, J Am Chem Soc
2013, 135, 6002-6005; c) H. Onoyama, M. Kamiya, Y. Kuriki, T.
Komatsu, H. Abe, Y. Tsuji, K. Yagi, Y. Yamagata, S. Aikou, M.
Nishida, K. Mori, H. Yamashita, M. Fujishiro, S. Nomura, N.
Shimizu, M. Fukayama, K. Koike, Y. Urano and Y. Seto, Sci Rep
2016, 6, 26399.
[6] A. Aitio, Anal Biochem 1978, 85, 488-491.
[7] A. Garg, D. M. Manidhar, M. Gokara, C. Malleda, C. Suresh
Reddy and R. Subramanyam, PLoS One 2013, 8, e63805.
[8] R. P. Taylor, J Am Chem Soc 1976, 98, 2684-2686.
[9] B. T. Doumas and T. Peters, Jr., Clin Chim Acta 1997, 258, 3-20.
[10] a) D. Rudman, T. J. Bixler, 2nd and A. E. Del Rio, J Pharmacol
Exp Ther 1971, 176, 261-272; b) I. Sjoholm and T. Sjodin, Biochem
Pharmacol 1972, 21, 3041-3052.
[11] R. Sallie, J. M. Tredger and R. Williams, Biopharm Drug
Dispos 1991, 12, 251-259.
[12] D. Webster, Clin Chim Acta 1974, 53, 109-115.
[13] a) J. Onagi, T. Komatsu, Y. Ichihashi, Y. Kuriki, M. Kamiya, T.
Terai, T. Ueno, K. Hanaoka, H. Matsuzaki, K. Hata, T. Watanabe, T.
Nagano and Y. Urano, J Am Chem Soc 2017, 139, 3465-3472; b) Y.
Kimura, T. Komatsu, K. Yanagi, K. Hanaoka, T. Ueno, T. Terai, H.
Kojima, T. Okabe, T. Nagano and Y. Urano, Angew Chem Int Ed
Engl 2016; c) Y. Rondelez, G. Tresset, K. V. Tabata, H. Arata, H.
Fujita, S. Takeuchi and H. Noji, Nat Biotechnol 2005, 23, 361-365.