A new cyano-substituted fluorescamine superior to its original form as a fluorescent probe for amino acid detection

Article abstract of DOI:10.1016/j.tetlet.2018.02.011

Synthesis and spectral study of two new cyano-substituted fluorescamine as the fluorescent probes for amino acid detection have been carried out comparing with the original fluorescamine. Of the three compounds, the derivative with a cyano group at the meta-position on the 4-phenyl group was found to be superior to the original one in the reactivity toward some amino acids as well as the fluorescence intensity of the adducts. The fluorescent amino acid adducts were also applied to the peroxyoxalate chemiluminescence system as the fluorophores, in which the derivative described above was found to be more effective also in chemiluminescence than the original one.

Full text of DOI:10.1016/j.tetlet.2018.02.011

Tetrahedron Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A new cyano-substituted fluorescamine superior to its original form as a
fluorescent probe for amino acid detection
⇑
Jiro Motoyoshiya , Satoshi Tomioka, Daigo Kobayashi, Tetsuya Fujimoto
Course of Applied Molecular Chemistry, Department of Chemistry and Materials, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano
386-8567, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and spectral study of two new cyano-substituted fluorescamine as the fluorescent probes for
amino acid detection have been carried out comparing with the original fluorescamine. Of the three com-
pounds, the derivative with a cyano group at the meta-position on the 4-phenyl group was found to be
superior to the original one in the reactivity toward some amino acids as well as the fluorescence inten-
sity of the adducts. The fluorescent amino acid adducts were also applied to the peroxyoxalate chemilu-
minescence system as the fluorophores, in which the derivative described above was found to be more
effective also in chemiluminescence than the original one.
Received 30 November 2017
Revised 29 January 2018
Accepted 6 February 2018
Available online xxxx
Keywords:
Fluorescamine
Fluorescent probe
Cyano group
Ó 2018 Elsevier Ltd. All rights reserved.
Peroxyoxalate chemiluminescence
Fluorescamine (1), 4-phenylspiro[furan-2(3H),10-phtalan]-3,30-
dione, is a commercially available fluorescent probe for the detec-
tion of primary amines including biologically important com-
pounds, especially, for the analysis of amino acids and proteins.1
In spite of prosperous exploitation of various fluorescent probes
for amino acid detection,2 fluorescamine is still keeping much
importance and convenience in biochemical analysis.3 Non-fluo-
rescent 1 reacts with primary amines smoothly to produce the
adducts, the strongly fluorescent pyrrolinones (Scheme 1). Need-
less to say, the amino acid determination by HPLC using 1 is a rou-
tine technique in the field of biological chemistry, which enables
the detection of the picomole level of amino acids.4 Because of
its prominent utility, several fluorescamine derivatives with alkoxy
and halogen substituents on the 4-phenyl group have been pre-
pared and their properties as the fluorescent probe investigated
up to date,5 but more excellent one than the original fluorescamine
has not been reported yet. Due to expectation that the introduction
of the electron-withdrawing functionality on the 4-phenyl group
might improve the required property as the fluorescent probe,
we prepared two new fluorescamine derivatives 2 and 3 with
cyano groups on the 4-phenyl group and found that the derivative
with a cyano group on the meta-position of 4-phenyl groups was
superior to fluorescamine (1) in the reactivity toward some amino
acids as well as the fluorescence intensities of the amino acid
adducts.
The fluorescamine derivatives 2 (R = 300-CN) and 3 (R = 400-CN)
were synthesized according to the general method for the synthe-
sis of 1 using m- or p-cyanobenzaldehydes instead of benzalde-
hyde,6 respectively, as shown in Scheme 2. Starting from the
known 2-benzylidene-1,3-indandiones with the cyano sub-
stituents on the phenyl groups of the benzylidene moieties,7 the
sequence processes, an epoxidation with alkaline hydrogen perox-
ide and the ring cleavage affording the enol of the a-diketones fol-
lowed by construction of spirocompounds using N,N-
dimethylformamideacetal, finally gave the 300- and 400-cyano sub-
stituted fluorescamines 2 and 3. The new compounds 2 and 3 were
identified by IR, NMR, and HRMS as shown in the experimental
section (Supporting Information).
Upon the reactions of the compounds 2, 3, and fluorescamine
(1) with 3 equiv. of phenylalanine in an aqueous THF (THF:H2O =
1:1) under the neutral conditions, the solution became fluorescent
due to the formation of pyrrolinone adducts. The measurements of
the fluorescence spectral change of the reaction mixtures revealed
that the fluorescence intensity increased with the elapse of time in
all cases as shown in Fig. 1a–c. The emission maxima of the pheny-
lalanine adducts of 2 and 3 were observed at 464 nm and 467 nm,
respectively, which were blue-shifted by ca. 10 nm compared to
that of 1 emitting at 476 nm.8 In addition, the differences in the flu-
orescence intensity and the reactivity were also found, namely, the
fluorescence intensity of the adduct of 2 was stronger than that of
1, but 3 was weaker than 1 at first glance. The plots of the increas-
ing intensities against time gave the curves as shown in Fig. 1d. The
kinetics of the reaction of 1 and alanine has been previously
⇑
Corresponding author.
E-mail address: jmotoyo@shinshu-u.ac.jp (J. Motoyoshiya).
https://doi.org/10.1016/j.tetlet.2018.02.011
0040-4039/Ó 2018 Elsevier Ltd. All rights reserved.
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2
J. Motoyoshiya et al. / Tetrahedron Letters xxx (2018) xxx–xxx
and some amino acids and bovine serum albumin (BSA) was com-
pared to those of 1 (Fig. 2a), indicating that the adducts of 2 gave
stronger intensities than those of 1 in all cases using several amino
acids. The tendency of the fluorescence intensities of 2 and amino
acids adducts, however, did not agree with that reported for 1,
because the order of the intensity of 2 and three amino acids
was, glycine, phenylalanine, and alanine, whose relative fluores-
cence quantum yields were 1:1.13:0.79, respectively. On the other
hand, the reaction profile of 1 and 2 and glycine was described in
Fig. 2b by plotting the fluorescence intensity against the glycine
concentration, showing that the reaction of 2 completed with
almost 1 eqiv. glycine similarly to 1. As shown in Fig. 2b the sensi-
tivity for detection of glycine by using 2 is at micromole level
under the present conditions, but it will be possible to increase
sensitivity at much higher level because analysis with 1 at the
picomole level applying a column chromatography attached by a
fluoromicrophotometer was reported.4
To understand the observed different reactivities and fluores-
cent intensities of the adducts, the molecular orbital calculations
were made. Fig. 3 shows the LUMOs of the three fluorescamines,
in which the LUMOs are developing on the 3(2H)-furanone ring
in all compounds. The C-5s at which the Michael addition takes
place are covered by LUMOs, and the Mulliken atomic charge of
C-3 and C-5 slightly increases in the order of 1, 2, and 3, which is
in agreement with the observed reactivity toward glycine as
described above. The introduction of cyano groups affected the
electronic distribution of C-5 slightly greater than that of C-3 car-
bonyl carbon. In addition, the emission energies of the HOMO-
LUMO transitions for the 1, 2, and 3-MeNH2 adducts as the concise
models were calculated to support the observed difference in the
fluorescence properties, the calculations giving the energies as
3.39, 3.63, and 3.57 eV, respectively, which explains the above
observed blue-shift of 2- and 3-phenylalanine adducts compared
to the adduct of 1.
Fluorescamine (1)
non-fluorescent
strongly fluorescent
Scheme 1. Reaction of fluorescamine (1) with primary amines forming the
fluorescent adducts.
described as a sequential process,9 the Michael addition of the
amino acid producing the intermediate and the following recon-
struction affording the final fluorescent product, but the whole
reaction can be approximated as a bimolecular process if only
the initial stage of the reaction is cut off for comparison of the
kinetic rates, because the latter reaction process was reported to
be much faster than the former Michael addition. Therefore, the
curves in Fig. 1d were kinetically treated by means of the bimolec-
ular manner. As expected, the plots of the values containing the
ratios of the product and probe concentrations, determined from
each fluorescence intensities of the adducts described in Fig. 1d,
against time gave the almost linear lines, whose slopes led to esti-
mation of the reaction rate constants, namely, 0.87, 1.87, and 3.80
Mꢀ1 sꢀ1 for the reactions of phenylalanine with 1, 2, and 3, respec-
tively. The ratio of the rate constants for 1, 2, and 3 was calculated
to be approximately 1:2.15:4.37, respectively, indicating that the
introduction of the cyano groups, the electron-withdrawing sub-
stituents, enhanced the reactivity toward phenylalanine, in which
the cyano groups would be effective to reduce the electron density
at 5-position of 2 and 3, making the Michel addition of phenylala-
nine favorable.
Since the most required performance for a fluorescent probe is
the stronger fluorescence intensity in view of amino acid detection,
2 superior to the others as described above was used for the pre-
sent investigation hereafter. It is known that the fluorescence
intensity of the fluorescamine-amino acid adduct is dependent
on the kind of amino acids, and various fluorescence quantum
yields for several adducts of the amino acids were reported. For
example, the values for phenylalanine, glycine, and alanine were
0.11, 0.092, and 0.081, respectively, namely, the ratio is
1:0.84:0.74.10 The fluorescence intensities in the reaction of 2
Many fluorescent compounds can be used as the fluorophores
for the peroxyoxalate chemiluminescence (PO-CL), which takes
place by the interaction of the high-energy intermediate, generated
by the reaction of an active oxalate and hydrogen peroxide, and a
fluorescent compound as an energy acceptor.11 Therefore, the flu-
orescent adducts of fluorescamines and amino acids described
above are also applicable for this chemiluminescence system. In
spite of increasing investigations of the chemiluminescent detec-
tion of amino acids, the fluorescamine-amino acid adducts have
been, to our knowledge, never used for the chemiluminescence
Scheme 2. Synthetic scheme of the fluorescamine derivatives 2 and 3.
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J. Motoyoshiya et al. / Tetrahedron Letters xxx (2018) xxx–xxx
3
(a)
(b)
(c)
2 + phenylalanine
3 + phenylalanine
1 + phenylalanine
600
600
500
400
300
200
100
0
600
500
400
300
200
100
0
464 nm
500
476 nm
.
.
400
.
.
.
.
.
.
30
20
10
30
467 nm
300
.
20
30
10
20
0 min.
0 min.
200
10
0 min.
100
0
400
450
500
550
600
400
450
500
550
600
400
450
500
550
600
Wave Length /nm
Wave Length /nm
Wave Length /nm
(d)
(e)
2500
600
2
3
500
2000
1500
1000
500
0
1
2
400
300
200
100
1
3
0
0
20
40
60
0
500
1000
Time (sec.)
1500
Time (min.)
Fig. 1. Fluorescence spectral change in the reaction of 1, 2, and 3 with phenylalanine and kinetic analysis. (a), (b), and (c) for the reaction of 1, 2, and 3 with phenylalanine, [1]
= [2] = [3] = [phenylalanine] = 2 ꢁ 10ꢀ3M in THF/H2O (1:1). (c) Time course of spectral change measured by the fluorescence spectra of the adducts. (e) Kinetic curves by the
treatment as the bimolecular process of the reactions of phenylalanine and 1, 2, and 3. The rates were estimated as 0.87 Mꢀ1 sꢀ1, 1.87 Mꢀ1 sꢀ1, and 3.80 Mꢀ1 sꢀ1 for 1, 2, and 3,
respectively.
(a)
(b)
350
300
250
200
150
100
50
500
400
300
200
100
0
1
2
2
1
0
Phe
0
1
2
3
Gly
Ala
Val
BSA
[glycine] (mM)
Fig. 2. Comparison of fluorescence intensity in the reactions of 1 or 2 and some amino acids (a) and a reaction profile of 1 or 2 and glycine (b). (a) [2] = 1.0 mM, [amino acid] =
3.0 mM, [BSA] = 6 mg/ml in acetone-phosphate buffer (1:3) solution. (b) [1] = [2] = 1.0 mM, [glycine] = 0–3.0 mM in acetone-phosphate buffer (1:3) solution.
systems until now.12 The numbers of photons produced by adding
an aqueous solution of alkaline hydrogen peroxide (Solution A) to a
solution containing 1 or 2, amino acids, and bis(4-chlorophenyl)
oxalate in THF/water (1:1) (Solution B) were counted by a photo-
multiplier tube, and the chemiluminescence quantum yields
UCL) were estimated as shown in Fig. 4. The capability of the
amino acid detection by the chemiluminescence system was
(
demonstrated and distinct preference of 2 over 1 was also
Please cite this article in press as: Motoyoshiya J., et al. Tetrahedron Lett. (2018), https://doi.org/10.1016/j.tetlet.2018.02.011

4
J. Motoyoshiya et al. / Tetrahedron Letters xxx (2018) xxx–xxx
C-5
0.1003
0.1117
0.1119
0.3120
C-3
0.3185
0.3197
1
2
3
Fig. 3. LUMOs of fluorescamine 1 and its derivatives 2 and 3, and the Mulliken atomic charges at C-3 and C-5 calculated by Gaussian 09 at the B3LYP/6-31G level.
Consequently, a new cyano-substituted fluorescamine deriva-
tive (2) provides preferential properties, such as the higher reactiv-
ity toward amino acids and the stronger fluorescence intensities of
the adducts, comparing with the original fluorescamine (1). It is
expected that 2 will be used for the amino acid and protein analy-
1
2
ses in place of the original 1.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.tetlet.2018.02.011.
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