Fluorogenic probes for aldol reactions: Tuning of fluorescence using π-conjugation systems

Article abstract of DOI:10.1016/j.tetlet.2013.10.122

Fluorogenic aldehydes or probes for monitoring of the progress of aldol reactions have been developed. Fluorescence of benzaldehydes conjugated with aryl groups via a double or triple bond and of their aldol products was evaluated in aqueous solutions. Ba

Full text of DOI:10.1016/j.tetlet.2013.10.122

Tetrahedron Letters 55 (2014) 74–78
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Fluorogenic probes for aldol reactions: tuning of fluorescence using
p-conjugation systems
⇑
Isamu Katsuyama, Pandurang V. Chouthaiwale, Hiroyuki Akama, Hai-Lei Cui, Fujie Tanaka
Chemistry and Chemical Bioengineering Unit, Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna, Okinawa 904-0495, Japan
Chemistry and Chemical Bioengineering Unit, Okinawa Institute of Science and Technology Graduate University, Mikuruma, 448-5 Kajii, Kamigyo, Kyoto 602-0841, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorogenic aldehydes or probes for monitoring of the progress of aldol reactions have been developed.
Fluorescence of benzaldehydes conjugated with aryl groups via a double or triple bond and of their aldol
products was evaluated in aqueous solutions. Based on the fluorescence, fluorogenic aldol reaction sub-
strates and retro-aldol reaction substrates were identified. Use of the probe system with optimal fluores-
cence properties for aldol reactions was demonstrated in assays with purified protein catalysts and with
overproduced crude protein catalysts in cell lysates.
Received 6 September 2013
Revised 23 October 2013
Accepted 25 October 2013
Available online 4 November 2013
Keywords:
Aldol reaction
Ó 2013 Elsevier Ltd. All rights reserved.
Retro-aldol reaction
Fluorogenic aldehyde
Fluorescence-based assay
Fluorescence-based reaction monitoring systems are useful for
the development and characterization of catalysts and catalyzed
reactions.1–4 These systems are especially useful in screening of
protein catalysts and related biomolecular catalysts using li-
braries.1–4 Fluorescence-based assay systems using appropriate
fluorogenic substrates or probes can be used to monitor reaction
progress through an increase in fluorescence on a small scale.1–4
These systems can be very sensitive to detect low concentrations
of the product formed at the initial stages of the reactions.2,4
Although the aldol reaction is one of the most basic carbon–carbon
bond-forming reactions,2a–c,4,5 reported fluorogenic substrates for
aldol reactions have limitations; for example, steric bulk limits
the utility, many are insoluble in aqueous solutions, and fluores-
cence wavelengths and intensity of the probes are not optimal.4
Here, we report the development of fluorogenic probes for aldol
reactions and demonstrate their utility in assays with protein cat-
alysts and even in assays with cell lysates containing overproduced
catalysts. We searched the probes from a set of aldehydes designed
based on structures of benzaldehyde conjugated with aryl moieties
through a double bond or a triple bond (Scheme 1).
Scheme 1. Fluorogenic probes for aldol reactions evaluated in this study.
We have previously reported that aldehyde 1 is non-fluorescent
and aldol product 2 is highly fluorescent (Scheme 2).4a These fluo-
rescence features of 1 and 2 are very different from those of previ-
ously reported aldehyde 3 and aldol 4; aldol 4 has been used as a
fluorogenic retro-aldol substrate2a–c,3a (i.e., aldol 4 shows no fluo-
rescence) and aldehyde 3 is highly fluorescent in aqueous solutions
⇑
Corresponding author. Tel.: +81 98 966 1552; fax: +81 98 966 1064.
E-mail address: ftanaka@oist.jp (F. Tanaka).
Scheme 2. Previously reported fluorogenic probes for aldol and retro-aldol
reactions that show fluorescence as either aldol or aldehyde.
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.10.122

I. Katsuyama et al. / Tetrahedron Letters 55 (2014) 74–78
75
group than those on the simple benzaldehyde system.6 Thus, we
reasoned that by using the 4-(phenylethynyl)benzaldehyde and
4-(arylethynyl)benzaldehyde systems with various substituents
and aryl groups, various fluorogenic substrates that generate aldol
products with distinct fluorescent features and with other features
such as solubilities should be obtained. Reactivities of these sub-
strates should not differ significantly.
First, a series of 4-styrylbenzaldehydes 5 and their aldol prod-
ucts 6, and a series of 4-(phenylethynyl)benzaldehydes 7 and their
aldol products 8 (Fig. 1) were evaluated. Fluorescence of each alde-
hyde and aldol product was analyzed in aqueous buffer (pH 7.4).
Except compounds with R = NO2, both or either of the aldehyde
and the corresponding aldol product were fluorescent to some de-
gree. Excitation and emission wavelengths (kex and kem) that
showed a significant difference in fluorescence (at least 10-fold dif-
ference) between aldehyde and the corresponding aldol product
are summarized with the relative fluorescence intensities in Tables
1 and 2. Fluorescence spectra are shown in Figures 2 and 3.
For aldehyde 5 and aldol 6 with R = H, Me, or OMe, either the
aldehyde or the aldol was fluorescent depending on excitation
and emission wavelengths (Table 1, Fig. 2A, B, D, E, F, and D). For
5b and 6b (R = CN), we observed excitation and emission wave-
length conditions that aldehyde 5b showed fluorescence signifi-
cantly higher than the aldol 6b (Table 1, entry 3; Fig. 2C), but
wavelength conditions in which aldol 6b showed higher fluores-
cence than aldehyde 5b were not found. These results suggest that
aldehydes 5a, 5c, and 5d are fluorogenic probes for aldol reactions
and that aldols 6a–d are fluorogenic retro-aldol reaction sub-
strates. As expected, fluorescence excitation and emission wave-
lengths suitable for detection of either aldehyde 5 or aldol 6
depended on the substituent.
Figure 1. 4-Styrylbenzaldehydes, 4-(phenylethynyl)benzaldehydes, and their ald-
ols tested as fluorogenic substrates and their products.
Table 1
Fluorescence of aldehyde 5 and aldol 6a
Entry
kex
kem
R (in 5 and 6)
Aldehyde 5
Aldol 6
1
2
3
4
5
6
7
290
340
365
270
345
285
350
360
465
440
365
495
380
550
H (a)
H (a)
<10
1.2 Â 103
1.2 Â 103
2.1 Â 103
24
<10
CN (b)
Me (c)
Me (c)
OMe (d)
OMe (d)
71
4.1 Â 102
30
5.0 Â 102
<5
2.3 Â 102
13
3.2 Â 102
a
Recorded on a microplate spectrophotometer using 100
lL of a 5
lM solution in
5% DMSO/50 mM sodium phosphate, pH 7.4.
For aldehyde 7 and aldol 8, fluorescence excitation and emis-
sion wavelengths sets at which the aldol showed higher fluores-
cence than the corresponding aldehyde were identified (Table 2).
However, except for the 7a/8a (R = H) set (Table 2, entries 1 and
2), the aldehyde did not show higher fluorescence than the corre-
sponding aldol. Fluorescence of 8a (R = H), 8c (R = Me), and 8d
(R = OMe) were also low (Table 2, entries 5 and 6) compared to
fluorescence of 6a, 6c, and 6d. Only 8b among 8a–d showed
fluorescence at a useful level (Table 2, entries 2 and 3; Fig. 3C
and D). In addition, aldols 8a–d showed fluorescence at shorter
excitation and emission wavelengths than did the corresponding
aldols 6a–d. These results indicate that the styrylbenzene core is
highly fluorescent, but the 4-(phenylethynyl)benzene core alone
is only moderately fluorescent to selectively detect either the
aldehyde or the corresponding aldol under the conditions used.
These results further suggest that fluorogenic ability of the 4-
(arylethynyl)benzaldehydes toward the formation of the aldols
relies on the fluorescence of the aryl group attached to the
ethynylbenzaldehyde.
Next, aldehydes and aldol products 9–12 (Fig. 4) were synthe-
sized and their fluorescence was analyzed (Table 3, Fig. 5). In these
compounds, fluorescent moieties (i.e., quinoline and 1,8-naphthal-
imide) were attached to the ethynylbenzaldehyde system. For the
9/10 set, aldol 10 showed high fluorescence at excitation/emission
wavelengths at 275/390 nm or 310/390 nm, longer excitation and
emission wavelengths than those of the 5/6 and 7/8 systems for
the detection of aldol reactions. On the other hand, for the 11/12
set, both aldehyde 11 and aldol 12 were fluorescent and no wave-
length sets for selective detection of either the aldehyde or the al-
dol were found. Thus, from the fluorescence of the aldehydes and
the aldols, among the 5/6 systems, aldehyde 5a was the best fluo-
rogenic substrate, and among the 7/8, 9/10, and 11/12 systems,
aldehyde 9 was the best.
Table 2
Fluorescence of aldehyde 7 and aldol 8a
Entry
kex
kem
R (in 7 and 8)
Aldehyde 7
Aldol 8
1
2
3
275
320
250
305
280
275
325
445
380
380
330
355
H (a)
H (a)
CN (b)
CN (b)
Me (c)
OMe (d)
<5
51
46
4.6 Â 102
46
9.5 Â 102
2.6 Â 103
2.4 Â 102
2.3 Â 102
4
1.8 Â 102
5b
6b
<5
<5
a
Recorded on a microplate spectrophotometer using 100
5% DMSO/50 mM sodium phosphate, pH 7.4 except noted.
lL of a 5
l
M solution in
b
A 50 lM solution was used.
(Scheme 2). Based on these results, we hypothesized that linking of
benzaldehyde with appropriate aryl groups in -conjugation via a
double bond or triple bond would generate a series of fluorogenic
aldehydes. When an arylaldehyde group is attached to or
contained as a part of a
fluorescent and simultaneously when the arylaldehyde group
quenches the fluorescence of the conjugated system,4 but the
corresponding aldol does not, the aldehyde substrate should be a
fluorogenic probe for the aldol reaction. As observed for the alde-
hyde/aldol pairs 1/2 and 3/4, prediction of which arylaldehyde
group will quench which fluorescence of a conjugated system is
difficult. We reasoned that fluorogenic aldehyde substrates useful
for monitoring of aldol reactions should be found among various
4-(arylethynyl)benzaldehydes and related 4-styrylbenzaldehydes
by analysis of the fluorescence of these aldehydes and the corre-
sponding aldols.
p
p-conjugated system that is intrinsically
Substituents on the 4-(phenylethynyl)benzaldehyde system
have significantly less influence on the reactivity of the aldehyde
When aldol reactions of acetone and 9 were performed in aque-
ous buffer (pH 7.4) in the presence and absence of protein catalyst

76
I. Katsuyama et al. / Tetrahedron Letters 55 (2014) 74–78
Figure 2. Fluorescence emission spectra of aldehydes 5a–d and aldols 6a–d. Compound (5
aldol 6; circle: blank.
lM) in 5% DMSO/50 mM sodium phosphate, pH 7.4. Square: aldehyde 5; triangle:
RA61_I72D2a and the fluorescence was monitored at kex 310 nm/
kem 390 nm, an increase in fluorescence was observed for the reac-
tion with the catalyst, but not for the reaction without the catalyst
other compounds with absorption in this UV region. Thus, for eval-
uations of aldol reactions catalyzed by protein catalysts, analysis
using 9 at kex 310 nm/kem 390 nm has the advantage over the
use of previously reported fluorogenic aldehydes4 with excitation
at 250–260 nm. The assay system using 9 was also useful for eval-
uation of the protein catalyst overproduced in Escherichia coli
(Fig. 6A). Formation of less than 0.03 lM of 10 in a 100 lL-reaction
was able to be detected. Analyses at kex 290 nm/kem 360 nm for
the reaction with 5 and analyses at kex 275 nm/kem 390 nm for
the reaction with 9 were less optimal; fluorescence using excita-
tion at 6305 nm is often affected by presence of proteins7 and by
(Fig. 6B). A 250
lM of aldehyde 9 was able to be used in the assays
using cell lysates; the quinoline moiety of 9 may contribute to an

I. Katsuyama et al. / Tetrahedron Letters 55 (2014) 74–78
77
Figure 3. Fluorescence emission spectra of aldehydes 7a–d and aldols 8a–d. Compound (5
phosphate, pH 7.4. Square: aldehyde 7; triangle: aldol 8; circle: blank.
l
M for 7a, 7b, 8a, and 8b; 50
lM for 7c, 7d, 8c, and 8d) in 5% DMSO/50 mM sodium
Table 3
Fluorescence of aldehyde 9 and aldol 10a
Entry
kex
kem
Aldehyde 9
Aldol 10
1
2
275
310
390
390
3.1 Â 102
1.3 Â 104
2.9 Â 102
8.5 Â 103
a
Recorded on a microplate spectrophotometer using 100
5% DMSO/50 mM sodium phosphate, pH 7.4.
lL of a 5
l
M solution in
In summary, we have developed fluorogenic probes for aldol
reactions. We have demonstrated that structures of benzaldehydes
Figure 4. 4-(Arylethynyl)benzaldehydes and their aldols tested as fluoregenic
substrates and products.
with
p-conjugation systems are useful for the generation of
fluorogenic probes with appropriate fluorescence features and
other features. Assays using the developed probe, fluorogenic
aldehyde 9, should be useful for aiding the development and
characterization of protein catalysts and related catalysts.
enhanced solubility in aqueous solutions, which is necessary for
assays of protein catalysts.

78
I. Katsuyama et al. / Tetrahedron Letters 55 (2014) 74–78
Figure 5. Fluorescence emission spectra of aldehyde 9 and aldol 10 upon excitation at (A) 275 nm and (B) 310 nm. Compound (5
pH 7.4. Square: aldehyde 9; triangle: aldol 10; circle: blank.
lM) in 5% DMSO/50 mM sodium phosphate,
Chandro Roy and Dr. Alejandro Villar Briones, Research Support
Division, Okinawa Institute of Science and Technology Graduate
University for mass analyses. This study was supported by the Oki-
nawa Institute of Science and Technology Graduate University.
Supplementary data
Supplementary data associated (syntheses and characterization
of compounds) with this article can be found, in the online version,
at http://dx.doi.org/10.1016/j.tetlet.2013.10.122.
References and notes
1. Reymond, J.-L.; Fluxa, V. S.; Maillard, N. Chem. Commun. 2009, 34. and references
cited thererin. Evans, C. A.; Miller, S. J. Curr. Opin. Chem. Biol. 2002, 6, 333.
2. (a) Jiang, L.; Althoff, E. A.; Clemente, F. R.; Doyle, L.; Rothlisberger, D.;
Zanghellini, A.; Gallaher, J. L.; Betker, J. L.; Tanaka, F.; Barbas, C. F.; Hilvert, D.;
Houk, K. N.; Stoddard, B. L.; Baker, D. Science 2008, 319, 1387; (b) Tanaka, F.;
Fuller, R.; Barbas, C. F. Biochemistry 2005, 44, 7583; (c) Goger, L.; Caner, S.;
Obexer, R.; Kast, P.; Baker, D.; Ban, N.; Hilvert, D. Nat. Chem. Biol. 2013, 9, 494; (d)
Khare, S. D.; Kipnis, Y.; Greisen, P. J.; Takeuchi, R.; Ashani, Y.; Goldsmith, M.;
Song, Y.; Gallaher, J. L.; Silman, I.; Leader, H.; Sussman, J. L.; Stoddard, B. L.;
Tawfik, D. S.; Baker, D. Nat. Chem. 2012, 8, 294.
3. (a) List, B.; Barbas, C. F.; Lerner, R. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 15351;
(b) Jo, J.; Olasz, A.; Chen, C.-H.; Lee, D. J. Am. Chem. Soc. 2013, 135, 3620; (c) Kim,
G.-J.; Kim, H.-J. Tetrahedron Lett. 2010, 51, 4670; (d) Jo, J.; Lee, D. J. Am. Chem. Soc.
2009, 131, 16283; (e) Halim, M.; Tremblay, M. S.; Jockusch, S.; Turro, N. J.;
Sames, D. J. Am. Chem. Soc. 2007, 129, 7704; (f) Froemming, M. K.; Sames, D. J.
Am. Chem. Soc. 2007, 129, 14518; (g) Baba, R.; Hori, Y.; Mizukami, S.; Kikuchi, K. J.
Am. Chem. Soc. 2012, 134, 14310; (h) Mizukami, S.; Watanabe, S.; Akimoto, Y.;
Kikuchi, K. J. Am. Chem. Soc. 2012, 134, 1623; (i) Sakabe, M.; Asanuma, D.;
Kamiya, M.; Iwatate, R. J.; Hanaoka, K.; Terai, T.; Nagano, T.; Urano, Y. J. Am.
Chem. Soc. 2013, 135, 409; (j) Shi, W.; Ma, H. Chem. Commun. 2012, 8732.
4. (a) Guo, H.-M.; Tanaka, F. J. Org. Chem. 2009, 74, 2417; (b) Tanaka, F.;
Thayumanavan, R.; Barbas, C. F. J. Am. Chem. Soc. 2003, 125, 8523; (c) Tanaka,
F.; Mase, N.; Barbas, C. F. J. Am. Chem. Soc. 2004, 126, 3692; (d) Mase, N.; Takabe,
K.; Tanaka, F. Tetrahedron Lett. 2013, 54, 4306.
Figure 6. Fluorescence assay (kex 310 nm/kem 390 nm) of aldol reaction of acetone
and aldehyde 9 in the presence of (A) purified protein catalyst RA61_I72D and (B)
E. coli cell lysates containing overproduced protein catalyst RA61_I72D. Conditions
for (A): [catalyst] 10
50 mM sodium phosphate (pH 7.4); a (triangle): reaction with catalyst RA61_I72D;
(circle): reaction without catalyst (blank). Conditions for (B): [9] 250 M,
lM, [9] 12.5 lM, [acetone] 10% (v/v) (1.36 M), 2.5% DMSO-
b
l
5. (a) Revell, J. D.; Wennemers, H. Curr. Opin. Chem. Biol. 2007, 11, 269; (b)
Oberhuber, M.; Joyce, G. F. Angew. Chem., Int. Ed. 2005, 44, 7580; (c) Muller, M.
M.; Windsor, M. A.; Pomerantz, W. C.; Gellman, S. H.; Hilvert, D. Angew. Chem.,
Int. Ed. 2009, 48, 922; (d) Cheriyan, M.; Toone, E. J.; Fierke, C. A. Biochemistry
2012, 51, 1658; (e) Mahrwald, R. Modern Aldol Reactions; New York: Wiley-VCH,
2004.
[acetone] 5% (v/v) (680 mM), 10% DMSO-cell lysate in 50 mM sodium phosphate
(pH 7.4); a (triangle): with cell lysate containing overproduced catalyst RA61_I72D;
b (diamond): with cell lysate of blank control (from control vector; no catalyst
protein); c (circle): with cell lysate containing overproduced non-catalytic mutant
RA61_K176A.
6. Katsuyama, I.; Chouthaiwale, P. V.; Cui, H.-L.; Ito, Y.; Sando, A.; Tokiwa, H.;
Tanaka, F. Tetrahderon 2013, 69, 4098.
7. Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic:
New York, 1999.
Acknowledgments
We thank Professor David Baker, University of Washington for
useful discussions about protein catalyst RA61 and Dr. Michael