Selective fluorescence and naked eye detection of histidine in aqueous medium via hydrogen bonding assisted Schiff base condensation

Article abstract of DOI:10.1016/j.tetlet.2013.10.144

Visible light excitable rhodamine B derivative (TARDHD) has been developed for fluorescence and naked eye detection of histidine in aqueous medium. TARDHD shows 45 fold fluorescence enhancement in the presence of histidine. It forms Schiff base with histidine and stabilizes via intra-molecular H-bonding. TARDHD can efficiently detect intracellular histidine.

Full text of DOI:10.1016/j.tetlet.2013.10.144

Tetrahedron Letters 55 (2014) 174–176
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Selective fluorescence and naked eye detection of histidine
in aqueous medium via hydrogen bonding assisted
Schiff base condensation
Sisir Lohar a, Arnab Banerjee a, Animesh Sahana a, Sukanya Panja b, Ipsit Hauli b,
Subhra Kanti Mukhopadhyay b, Debasis Das a,
⇑
a Department of Chemistry, The University of Burdwan, Burdwan 713104, West Bengal, India
b Department of Microbiology, The University of Burdwan, Burdwan 713104, West Bengal, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Visible light excitable rhodamine B derivative (TARDHD) has been developed for fluorescence and naked
eye detection of histidine in aqueous medium. TARDHD shows 45 fold fluorescence enhancement in the
presence of histidine. It forms Schiff base with histidine and stabilizes via intra-molecular H-bonding.
TARDHD can efficiently detect intracellular histidine.
Received 11 September 2013
Revised 27 October 2013
Accepted 29 October 2013
Available online 6 November 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Visible light excitation
Naked eye
Histidine
Cell imaging
DFT
Among several essential amino acids, histidine is actively
involved in human growth, and acts as a neurotransmitter in the
central nervous system.1,2 Deviation from its normal level leads
to different diseases including chronic kidney problems having
impaired nutritional state.3,4 Recently, several probes for selective
detection of amino acid viz. cysteine and homocysteine,5 lysine,6
arginine (Arg),7 phenylalanine (Phe)8 and tryptophan9 have been
reported. Among a few histidine (His)10,11 selective fluorescence
sensors, mostly suffer interferences either from cysteine, lysine,
arginine12 or any other amino acids.13 Lin Pu group have used
terpyridine based Cu(II)14 and Zn(II)15 complexes as fluorescence
sensors for histidine. However, the former suffer interference from
cysteine and the latter lacks in quantum yield, lowest detection
limit, cell imaging studies and theoretical support. Most impor-
tantly, both the sensors require excitation with undesirable UV
light and hence, unworthy for intracellular studies. Interestingly,
till date, naked eye detection of histidine remains unexplored.
We have overcome these limitations and report a very simple rho-
damine-based visible light excitable ON type fluorescence probe,
TARDHD for selective, naked eye determination of histidine under
physiological condition. Rhodamine-based fluorescent probes are
chosen for recognition of amino acids5a for their excellent
spectroscopic properties, viz. high molar extinction coefficient (
e)
and fluorescence quantum yield ( ). Facile synthesis of TARDHD
U
has been achieved by two-step reaction using rhodamine B and
phthalaldehyde as shown in Scheme S1 (Supplementary data,
hereafter ESI). First, rhodamine B is refluxed with hydrazine hy-
drate for 3 h in ethanol to afford rhodamine–hydrazine adduct, 2
in 97% yield following a literature procedure.16 Then, 2 is reacted
with phthalaldehyde to get TARDHD in 75% yield. It is character-
ized by 1H NMR, QTOF–MS, FTIR spectra and elemental (CHN) anal-
ysis (Figs. S1, S2, and S3, ESI). The colourless non-fluorescent state
of TARDHD indicates its spirolactam ring structure.
TARDHD (10 lM) undergoes 45 fold fluorescence enhancement
upon addition of 100 equivalent of histidine in HEPES buffered
(0.1 M) solution (ethanol/water, 1:9, v/v, pH 7.3, kEx = 560 nm,
kEm = 601 nm). Changes in the emission profile of TARDHD upon
gradual addition of histidine are presented in Figure S4 (ESI). The
results clearly indicate that the spirolactam ring opens with the
formation of a highly conjugated structure (Fig. 1). The fluores-
cence quantum yields (UF) of TARDHD and its histidine adduct
are 0.007 and 0.12, respectively where rhodamine B is used as
standard (UF = 0.65 in ethanol).17 Job’s plot (Fig. S5, ESI) shows
1:1 (mole ratio) stoichiometry of the TARDHD–histidine adduct.
ESI-TOF (+) mass spectrum (Fig. S6, ESI) also supports the forma-
tion of TARDHD–histidine Schiff base having m/z at 710.47 [TARD-
HD + histidine ꢀ H2O + H+]. FTIR spectrum (Fig. S2, ESI) of free
⇑
Corresponding author. Tel.: +91 342 2533913; fax: +91 342 2530452.
E-mail address: ddas100in@yahoo.com (D. Das).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.10.144

S. Lohar et al. / Tetrahedron Letters 55 (2014) 174–176
175
Figure 1. Proposed mechanism of histidine sensing with TARDHD.
TARDHD shows peaks at 1730, 1691 and 1615 cmꢀ1 corresponding
to carbonyl stretching from aldehyde, ketone and imine (CH@N)
functionalities.
Figure 3. 1H NMR spectral changes of TARDHD upon gradual addition of histidine:
(A) free TARDHD, (B) TARDHD in MeOD + 0.5 equiv histidine in D2O, (C) TARDHD in
MeOD + 1 equiv histidine in D2O, (D) TARDHD in MeOD + 2 equiv histidine in D2O.
In Schiff base adduct (Fig. S7, ESI), 1730 cmꢀ1 peak disappears
with the appearance of a new broad peak at 3386 cmꢀ1, assigned
to imidazole N–H stretching of histidine residue.
The binding constant of TARDHD for histidine is 3.7 ꢁ 104 Mꢀ1
(Fig. S8, ESI), as estimated using the following Benesi–Hildebrand
equation:18
and Fig. S12, ESI). This indicates that neither hydrogen bonding
nor Schiff base formation is individually responsible for fluores-
cence enhancement. Their combined effect is essential for turning
on the fluorescence via ring opening of TARDHD.
À
Á
n
Interaction between TARDHD and histidine has been monitored
by 1H NMR titration in D2O/CD3OD = 1:9 (v/v) (Fig. 3). The alde-
hyde proton at 9.85 ppm of free TARDHD disappears upon addition
of 1 equiv histidine indicating Schiff base formation. Imine proton
(CH@N) of free TARDHD have shifted from 9.59 to 9.23 ppm and
merges to the new imine proton formed due to Schiff base forma-
tion with histidine. Imidazole ring N–H proton, exchangeable with
D2O does not appear, however, imidazole ring –CH2 and –N@CH
protons which generally appear at 7.12 ppm and 8.08 ppm in free
histidine have been shifted to 7.23 and 8.35 ppm upon reaction
with TARDHD.
Density functional theoretical studies (DFT, B3LYP/6-311G basis
set)19 provide additional support to the histidine sensing mecha-
nism. Figure 4 reveals that HOMO–LUMO energy gap in free TARD-
HD and its histidine adduct are 3.107 and 2.695 eV respectively.
Moreover, in HOMO of TARDHD, most of the charge resides in
the rhodamine moiety but in LUMO, it is on the phthalaldehyde
unit. In TARDHD–histidine adduct, most of the charges relocate
on pthalaldehyde unit in both HOMO and LUMO. Most impor-
tantly, it indicates a strong H-bond (2.436 Å) between ketone
oxygen of rhodamine moiety with imidazole N–H from histidine
residue (Fig. 5).
Flim ꢀ F0=FX ꢀ F0 ¼ 1 þ 1=K½Cꢂ
ð1Þ
where F0, Fx and Flim are the emission intensities of TARDHD in ab-
sence of histidine, at an intermediate histidine concentration, and at
histidine concentration of complete interaction respectively. K is
the binding constant, C is the concentration of histidine and n is
the number of histidine molecules bound per TARDHD molecule
(here n = 1). Lowest detection limit for histidine is 1.1 ꢁ 10ꢀ8
M
(Fig. S9, ESI). Figure 2 reveals that TARDHD has no absorbance in
the visible region. In presence of histidine, a new absorption peak
appears at 560 nm, the intensity of which increases with increasing
histidine concentration. This allows naked eye detection and color-
imetric determination of histidine. Figure S10 (ESI) shows UV–vis
titration of TARDHD with histidine which provides the binding con-
stant, 2.8 ꢁ 104 Mꢀ1, in close agreement to the value derived from
fluorescence titration.
The sensing of mechanism is attributed to the formation of
TARDHD–histidine Schiff base followed by its stabilization through
H-bonding involving carbonyl oxygen of rhodamine unit and imid-
azole N–H of histidine residue.
Free imidazole fails to show fluorescence enhancement of
TARDHD. Similarly, other amino acids do not interfere (Figs. S11
Figure 2. Changes of absorbance of TARDHD (10
1, 5, 10, 15, 20, 40, 50, 80, 100, 150, 200, 250, 300, 400, 500, 700, 800, 1000, 2000
and 5000 M) in HEPES buffered (0.1 M) solution (ethanol/water = 1:9, v/v, pH 7.3).
lM) upon addition of histidine (0,
Figure 4. Energy optimized structure and HOMO–LUMO energy gap of TARDHD
and its Schiff base with histidine.
l

176
S. Lohar et al. / Tetrahedron Letters 55 (2014) 174–176
4–9, it is suitable for histidine sensing. However, pH 7.3, being
physiological pH is chosen for the entire studies.
Figure 6 and Figure S14 (ESI) clearly show that the probe is eas-
ily permeable to the tested living cells and harmless (as the cells
remain alive even after a considerable time of exposure to the
probe).
A very simple visible light excitable fluorescent probe for naked
eye detection of histidine in aqueous medium is reported. The probe
has a very good lowest LOD and capable to detect intracellular
histidine without any interference.
Acknowledgments
S.L, A.B. and A.S. are grateful to CSIR, New Delhi for providing
fellowship. We sincerely thank USIC, Burdwan University for
providing fluorescence facilities.
Figure 5. Stereoscopic view of the optimized H-bonded structure of TARDHD + his-
tidine adduct.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.
10.144.
References and notes
1. (a) Kopple, J. D.; Swendseid, M. E. J. Clin. Invest. 1975, 55, 881; (b) Snyderman, S.
E.; Boyer, A.; Roitman, E.; Holt, L. E., Jr.; Prose, P. H. Pediatrics 1963, 31, 786.
2. Kusakari, Y.; Nishikawa, S.; Ishiguro, S.-i.; Tamai, M. Curr. Eye Res. 1997, 16, 600.
3. Watanabe, M.; Suliman, M. E.; Qureshi, A. R.; Garcia-Lopez, E.; Bárány, P.;
Heimbürger, O.; Stenvinkel, P.; Lindholm, B. Am. J. Clin. Nutr. 2008, 87, 1860.
4. (a) Jones, A. L.; Hulett, M. D.; Parish, C. R. Immunol. Cell Biol. 2005, 83, 106; (b)
Sullivan, D. J., Jr.; Gluzman, I. Y.; Goldberg, D. E. Science 1996, 271, 219; (c)
Leebeek, F. W. G.; Kluft, C.; Knot, E. A. R.; De Maat, M. P. M. J. Lab. Clin. Med.
1989, 113, 493; (d) Saito, H.; Goodnough, L. T.; Boyle, J. M.; Heimburger, N. Am.
J. Med. 1982, 73, 179.
5. (a) Yang, Y.-K.; Shim, S.; Tae, J. Chem. Commun. 2010, 7766; (b) Hao, W.;
McBride, A.; McBride, S.; Gao, J. P.; Wang, Z. Y. J. Mater. Chem. 2011, 21, 1040;
(c) Chen, X.; Zhou, Y.; Peng, X.; Yoon, J. Chem. Soc. Rev. 2010, 39, 2120.
6. (a) Wehner, M.; Schrader, T.; Finocchiaro, P.; Failla, S.; Consiglio, G. Org. Lett.
2000, 2, 605; (b) Sasaki, S.; Hashizume, A.; Citterio, D.; Fujii, E.; Suzuki, K.
Tetrahedron Lett. 2002, 43, 7243; (c) Secor, K.; Plante, J.; Avetta, C.; Glass, T. J.
Mater. Chem. 2005, 15, 4073; (d) Mandl, C. P.; Konig, B. J. Org. Chem. 2005, 70,
670.
7. Liu, K.; He, L.; He, X.; Guo, Y.; Shao, S.; Jiang, S. Tetrahedron Lett. 2007, 48, 4275.
8. Mitra, A.; Chinta, J. P.; Rao, C. P. Tetrahedron Lett. 2010, 51, 139.
9. Hariharan, M.; Karunakaran, S. C.; Ramaiah, D. Org. Lett. 2007, 9, 417.
10. Hortala, M. A.; Fabbrizzi, L.; Marcotte, N.; Stomeo, F.; Taglietti, A. J. Am. Chem.
Soc. 2003, 125, 20.
11. (a) Folmer-Andersen, J. F.; Lynch, V. M.; Anslyn, E. V. Chem.-Eur. J. 2005, 11,
5319; (b) Sun, S.-K.; Tu, K.-X.; Yan, X.-P. Analyst 2012, 137, 2124.
12. Patel, G.; Menon, S. Chem. Commun. 2009, 3563.
13. Yang, R.; Wang, K.; Long, L.; Xiao, D.; Yang, X.; Tan, W. Anal. Chem. 2002, 74,
1088.
14. (a) Huang, Z.; Du, J.; Zhang, J.; Yu, X.-Q.; Pu, L. Chem. Commun. 2012, 3412; (b)
Du, J.; Huang, Z.; Yu, X.-Q.; Pu, L. Chem. Commun. 2013, 5399.
15. Oliveira, E.; Santos, C.; Poeta, P.; Capelo, J. L.; Lodeiro, C. Analyst 2013, 138,
3642.
Figure 6. Bright field and fluorescence microscope images of pollen grains from
Tecomastans. Top row (bright field): cells incubated with histidine (5 mM) for 1 h
(A); cells treated with histidine and TARDHD (B). Bottom row (fluorescence image):
cells incubated only with histidine (C) and cells treated with histidine and TARDHD
(D).
Figure S13 (ESI) reveals that at low pH (below 4), the emission
intensities of TARDHD and its histidine adduct are high, and the
difference is very small, probably due to spirolactam ring opening.
Higher pH (above 9) favours the closed ring spirolactam form. The
difference of emission intensities being maximum in the pH range
16. Chen, X.; Wang, X.; Wang, S.; Shi, W.; Wang, K.; Ma, H. Chem. Eur. J. 2008, 14,
4719.
17. Kubin, R. J. Lumin. 1983, 27, 455.
18. Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703.
19. Gaussian 03, Rev. C.02; Gaussian Inc.: Wallingford, CT, 2004.