Photophysical properties of 3-[2-cyano-4-(dimethylamino)phenyl]alanine: A highly fluorescent and environment-sensitive amino acid with small molecular size

Article abstract of DOI:10.1246/cl.2006.620

A new nonnatural amino acid 3-[2-cyano-4-(dimethylamino)phenyl]alanine (CDAPA) is virtually nonfluorescent in water but shows significant enhancement of fluorescence intensity and lifetime when the molecule partitions to nonaqueous environments, suggestin

Full text of DOI:10.1246/cl.2006.620

620
Chemistry Letters Vol.35, No.6 (2006)
Photophysical Properties of 3-[2-Cyano-4-(dimethylamino)phenyl]alanine:
A Highly Fluorescent and Environment-sensitive Amino Acid with Small Molecular Size
Juro Oshima, Hitomi Tsujimoto, Toshitada Yoshihara, Keiichi Yamada, Ryoichi Katakai, and Seiji Tobitaꢀ
Department of Chemistry, Gunma University, Kiryu 376-8515
(Received March 20, 2006; CL-060338; E-mail: tobita@chem.gunma-u.ac.jp)
A new nonnatural amino acid 3-[2-cyano-4-(dimethyl-
Abs.
Fluo.
(a)
(b)
amino)phenyl]alanine (CDAPA) is virtually nonfluorescent in
water but shows significant enhancement of fluorescence inten-
sity and lifetime when the molecule partitions to nonaqueous
environments, suggesting that this molecule is a good candidate
as small fluorescent probe for proteins.
Fluorescent probes are powerful tools for studying the struc-
ture and dynamics of biomolecular systems such as proteins,
membranes, and DNA. To achieve adequate detection of pro-
teins, nonnatural fluorescent amino acids, whose emission spec-
tra and quantum yields are sensitive to surrounding environ-
ments, have been developed.1–5 In the course of studies on excit-
ed-state proton-transfer reactions of aniline and its derivatives,6
we found that the fluorescence properties of m-cyanoaniline and
its N-alkylated derivatives exhibit remarkable sensitivity to
aqueous environment, e.g. the fluorescence quantum yield and
lifetime of m-cyanoaniline in ethanol (0.19 and 6.2 ns, respec-
tively) are reduced drastically in water (0.0019 and 45 ps, re-
spectively).7 Since the molecular size of the m-cyanoaniline
and its N-alkylated derivatives is comparable to that of fluores-
cent natural amino acids (tryptophan, tyrosine, and phenylala-
nine), we expected that they can be used successfully in labeling
proteins keeping the conformation intact.
We report here the synthesis of a fluorescent nonnatural
amino acid, Boc-3-[2-cyano-4-(dimethylamino)phenyl]alanine
(Boc-CDAPA; Boc = (tert-butoxycarbonyl) (Supporting Infor-
mation),10 and the photophysical properties of Boc-CDAPA
and its chromophoric moiety, 3-cyano-4-methyl-N,N-dimethyl-
aniline (CMDA) (Scheme 1), in aqueous and nonaqueous solu-
tions. Fluorescence lifetimes (ꢀf) in a picosecond range were
obtained using a time-correlated single-photon counting fluorim-
eter using the third harmonic (266 nm) of a femtosecond mode-
locked Ti:sapphire laser as an excitation source (Supporting
Information).10
(c)
(d)
300
400
500
600
Wavenumber / nm
Figure 1. Absorption and fluorescence spectra of CMDA
(broken line) and Boc-CDAPA (solid line) in (a) CH, (b) MeCN,
(c) EtOH, and (d) H2O at 293 K (The data for Boc-CDAPA in
CH was not obtained because of the low solubility to CH).
that only small perturbation due to the alanyl group to the elec-
tronic structure of the aromatic moiety is involved in Boc-
CDAPA. The first absorption band of CMDA is red-shifted with
changing the solvent from nonpolar CH to polar MeCN and
EtOH. In contrast, it is noticed that in water the first absorption
band is shifted to the blue. This can be attributed to hydrogen-
bonding interactions between the amino lone-pair electrons in
CMDA and water molecules. The molar extinction coefficient
of CMDA in EtOH at the first absorption maximum (345 nm)
was determined to be 2770 Mꢁ1 cmꢁ1. Although tryptophan pos-
sesses a larger extinction coefficient (5350 Mꢁ1 cmꢁ1)8 at its first
absorption maximum (277 nm) in water (pH 7.3), the wave-
length is much shorter as compared with Boc-CDAPA. The
fluorescence of Boc-CDAPA appears in the blue region and
the spectral position depends strongly on the solvent polarity,
suggesting that Boc-CDAPA can be used as micropolarity probe.
The absorption (ꢁambasx) and fluorescence (ꢁfmluaox) maxima, and
Figure 1 illustrates the absorption and fluorescence spectra
of CMDA and Boc-CDAPA in cyclohexane (CH), acetonitrile
(MeCN), ethanol (EtOH), and water (H2O) at 293 K. The ab-
sorption and fluorescence spectra of both compounds are almost
identical with each other except that a slight blue-shift is seen for
the fluorescence spectrum of Boc-CDAPA in water. This implies
CH3
N
CH3
N
H3C
COOH
H3C
ꢁ
Stokes shifts (ꢀꢂ) of CMDA and Boc-CDAPA are listed
in Table 1. The extremely large Stokes shift in H2O suggests
significant variation of solute–solvent interactions in water upon
electronic excitation.
Figure 2 displays the fluorescence decay profiles of Boc-
CDAPA in MeCN, EtOH, and H2O (in phosphate buffer solu-
NHBoc
CH3
CN
Boc-CDAPA
CN
CMDA
Scheme 1.
Copyright Ó 2006 The Chemical Society of Japan

Chemistry Letters Vol.35, No.6 (2006)
Table 1. Spectral properties and photophysical parameters of CMDA and Boc-CDAPA in solution at 293 K
621
3
ꢁ1
ꢁ
Compound
CMDA
Solvent
ꢁambasx/nm
ꢁfmluaox/nm
ꢀꢂ=10 cm
ꢂf
ꢀf/ns
5.80
29.4
23.3
0.163
26.9
22.5
0.274 (55%)
0.536 (45%)
kf=107 sꢁ1
knr=108 sꢁ1
CH
338
346
345
324
345
343
325
377
415
416
442
415
414
438
3.06
4.81
4.95
8.24
4.89
5.00
7.94
0.15
0.48
0.42
0.0040
0.54
0.48
2.7
1.6
1.8
2.5
2.0
2.1
1.4
0.59
1.5
0.18
0.25
61
0.17
0.23
MeCN
EtOH
H2O
MeCN
EtOH
H2Oa
Boc-CDAPA
0.0070
36
19
aPhosphate buffer solution, pH ¼ 6:88.
in MeCN and EtOH remains almost constant (ꢂ2:0 ꢃ 107 sꢁ1
)
4
0
and is not different appreciably from those of CMDA, whereas
in H2O the kf values for Boc-CDAPA are slightly smaller than
that of CMDA. In contrast to the kf values, the knr values of
CMDA and Boc-CDAPA is influenced strongly by solvent prop-
erties. In water the knr values are much larger than those in non-
aqueous solvents, indicating that fast radiationless processes are
induced in water. The nanosecond laser photolysis experiments
of Boc-CDAPA and CMDA using a XeCl excimer laser showed
no transient absorption signal due to the triplet state. This sug-
gests that the fast radiationless process of CMDA and Boc-CDA-
PA in water can be ascribed to internal conversion, as in the case
of m-cyanoaniline.7 The rapid S1 ! S0 internal conversion is a
favorable deactivation mode for environment-sensitive fluores-
cent probes, because photochemical reactions that can occur
from the S1 and/or T1 states are inhibited. In spite of the relative-
ly small molecular size, Boc-CDAPA gives the first absorption
band at much longer wavelength compared with tryptophan,
and the strong blue fluorescence in nonaqueous environment is
significantly quenched on exposure to water. These characteris-
tic fluorescence properties of Boc-CDAPA demonstrate that
Boc-CDAPA can be a candidate for novel fluorescent probe with
small molecular size.
(a)
–4
4
0
(b)
(c)
–4
4
0
–4
Time / ns
0
20
40
60
80
100
103
102
101
100
(a)
(b)
(d)
(c)
0
1
2
3
Time / ns
Figure 2. Fluorescence decay for Boc-CDAPA in (a) MeCN,
(b) EtOH, and (c) H2O (pH 6.88), excited at (a) 345, (b) 343,
and (c) 266 nm and monitored at their fluorescence maximum
wavelengths at 293 K, and (d) instrument response function
(FWHM: ꢂ25 ps).
The authors are grateful to Dr. Hiroaki Ozaki for help in
measuring mass spectra and useful discussions.
tion, pH ¼ 6:88). The fluorescence decay curves of Boc-
CDAPA in MeCN and EtOH follow single-exponential kinetics
with relatively long lifetimes, 26.9 and 22.5 ns, respectively. It
is noteworthy that the fluorescence decay rate in H2O increases
dramatically, and the decay profile no longer conforms to the
References and Notes
1
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single-exponential kinetics. The solid line represents the
1
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þ
3
4
5
A2eꢁt=ꢀ ) superimposed on the experimental data points, using
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identical with that of CMDA, and a substantial difference is seen
in water: the fluorescence decay profile of CMDA in H2O can be
analyzed in terms of a single-exponential term with a lifetime of
163 ps (ꢃ2 ¼ 1:017). Although the origin of the two-exponential
kinetics of Boc-CDAPA in water has not yet been clarified, one
possible explanation is given in terms of different rotamers or
conformers of the alanyl side chain of Boc-CDAPA, as has been
reported for the similar behavior of tryptophan.9
2
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8
9
The radiative (kf) and nonradiative (knr) rate constants of
CMDA and Boc-CDAPA in each solvent were calculated from
ꢀf and ꢂf. As shown in Table 1, the kf value for Boc-CDAPA
10 Supporting Information is available electronically on the CSJ-
Journal web site.
Published on the web (Advance View) May 13, 2006; doi:10.1246/cl.2006.620