A visible light excitable pyrene–naphthalene conjugate for ON fluorescence sensing of histidine in living cells

Abstract

Pyrene appended naphthalene derivative, NEDAP can selectively detect histidine via inter-molecular H-bond assisted molecular ring formation that imparts rigidity to the molecular assembly to inhibit the TICT (Twisted Intra-molecular Charge Transfer) process leading to fluorescence enhancement. ¹H NMR titration, mass spectra and DFT (Density Functional Theory) studies nicely demonstrate this sensing process. Visible light excitation favours the living cell imaging as low as 0.01 μM histidine.

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Introduction

Histidine is an essential amino acid for human growth¹ function as a neurotransmitter or neuromodulator in mammalian central nervous system.^2,3 The imidazole unit of histidine is the active site in metallo-proteins and enzymes.⁴ Histidine deficiency causes epilepsy, Parkinson's disease and normal erythropoiesis.⁵ On the other hand, excess accumulation of histidine causes histidinemia.⁶ Thus, trace level determination of histidine in the human body has immense research interest. Several methods viz. capillary electrophoresis^7,8 liquid chromatography^9,10 voltammetry¹¹ and spectrophotometry^12–16 are available for histidine determination. However, they require either sophisticated equipment or long analysis time or lack accuracy at sub-ppm levels for in vivo application. Fluorescence method overcomes these difficulties. Most of the available fluorescent probes for histidine^17–22 require UV-light excitation, and hence inappropriate for in vivo studies. Visible light excitation can minimize sample damage and native auto-fluorescence events associated with UV excitation. Our attempt towards development of low cost, visible light excitable fluorescence probes^23–26 for selective monitoring of in vivo histidine have resulted the probe NEDAP, in which pyrene is chosen as visible light excitable fluorophore. Intermolecular H-bonding assisted on-fluorescence sensing has become an important technique in recent years.^24b–e Well known basic N-center of imidazole unit of histidine has the required proton affinity²⁷ to form intermolecular H-bond with –NH part of NEDAP. On the other hand, the acidic proton of histidine forms H-bond with imine N-center of NEDAP; leading to the formation of a molecular ring.

Results and discussion

Synthesis and spectral characteristics

NEDAP is synthesized (Scheme 1) by a facile one step condensation between N¹-(naphthalen-1-yl)ethane-1,2-diamine hydrochloride with pyrene-1-carbaldehyde in methanol (details are in the ESI†).

Scheme 1 Synthesis of NEDAP.

A DMSO–water (7 : 3, v/v, pH 7.4) solution of NEDAP has absorbances at 393 nm, 360 nm, 340 nm, and 325 nm (Fig. 1).

Fig. 1 Changes in the UV-Vis spectra of NEDAP (10 μM, DMSO–water, 7 : 3, v/v, pH 7.4) upon gradual addition of histidine (1.0, 2.5, 4.0 5.5, 7.5, 9.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 500 μM). Black (bold) line is for free NEDAP.

Aqueous DMSO solution is chosen to compare the results with TD-DFT studies.²⁶ Gradual addition of histidine to NEDAP produces a new band at 435 nm with simultaneous disappearance of two other peaks viz. 393 nm and 360 nm. The absorbance at 340 nm and 325 nm enhances with increasing histidine concentration.

NEDAP in HEPES buffered solution exhibits very weak fluorescence at 458 nm (λ_Ex = 410 nm, Φ = 0.05). Gradual addition of histidine (up to 10 equivalent) causes a maximum of ∼15-fold fluorescence enhancement (Φ = 0.26) (Fig. 2). Plot of emission intensity as a function of the histidine concentration results a gradual increase of emission intensity up to almost 1 μM histidine after which no significant change in the emission intensity is observed (Fig. S1†).

Fig. 2 Changes in the emission spectra of NEDAP (1 μM, λ_Ex = 410 nm, λ_Em = 458 nm) in HEPES buffer (0.1 M, DMSO–water, 7 : 3, v/v, pH 7.4) upon gradual addition of histidine (0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 10.0 μM).

The linear part is useful to determine unknown histidine concentration (Fig. S1 inset, ESI†). Most of the bio-relevant cations and anions either do not affect or quench the emission intensity to some extent (Fig. 3). Except lysine and arginine (∼2 fold fluorescence enhancement), other naturally occurring amino acids do not show any significant change in the emission intensity of NEDAP in a binary mixture. Very weak fluorescence of NEDAP is attributed to the PET (Photo-induce Electron Transfer) process^23e,28 from imine N-center and associated TICT^24d,29 due to free rotation around imine (–C=N–) bond.

Fig. 3 Fluorescence spectra of NEDAP (λ_Ex = 410 nm, 1 μM) in HEPES buffered (0.1 M, DMSO–H₂O, 7 : 3, v/v; pH 7.4) solution in presence of common cations (Na⁺, K⁺, Ca²⁺, Fe³⁺, Fe²⁺, Cu²⁺, Zn²⁺, Co²⁺, Mg²⁺, Ni²⁺), anions as their sodium salt (F⁻, Cl⁻, Br⁻, I⁻, OCl⁻, N₃⁻, NCO⁻, NO₂⁻, NO₃⁻, CH₃COO⁻, ClO₄⁻, HPO₄²⁻, CrO₇²⁻, C₂O₄²⁻, SO₄²⁻, S²⁻ and H₂AsO₄⁻) and other 19 naturally occurring amino acids.

In presence of histidine, NEDAP forms a molecular ring through inter-molecular H-bonding between the basic N-atom of imidazole ring of histidine with –NH in NEDAP and the acid proton of histidine with the imine N-center (Fig. 4). Formation of molecular ring provides rigidity to the system, which inhibits the TICT process by restricting the free rotation around-C=N– bond. Due to involvement of imine N-center in H-bond formation, the PET process is also inhibited. Combination of these two processes is responsible for fluorescence enhancement. Except basic amino acids (viz. histidine, lysine and arginine), no other amino acid is capable to form such type of molecular ring with NEDAP due to the absence of basic N-center, and hence no fluorescence enhancement is observed. Again, amongst basic amino acids, the pK_a value of the basic N-center is lowest for histidine (histidine, 6.04; lysine, 10.79 and arginine, 12.48). Hence, the ability to form intermolecular H-bonding with –NH center of NEDAP is maximum for histidine. Combining these two facts, one can easily rationalize the fluorescence enhancement (∼15 fold) of NEDAP in presence of histidine. Fig. 5 shows the effect of other naturally occurring amino acids on the emission intensity of [NEDAP–His] adduct. No interference is observed. Moreover, addition of imidazole in a solution of NEDAP–amino acid mixture (excluding basic amino acids) causes no significant changes in the emission intensity. This fact strongly supports our proposed mechanism of H-bond assisted molecular ring forming.

Fig. 4 Plausible histidine sensing mechanism of NEDAP.

Fig. 5 Effect of other amino acids (5 μM) on the emission intensity of [NEDAP–His] molecular assembly (1 μM) in a ternary mixture in HEPES buffer (0.1 M) (DMSO–water, 7 : 3, v/v, pH 7.4).

Job's plot reveals the stoichiometry of the [NEDAP–His] adduct as 1 : 1 (NEDAP : His, mole ratio) (Fig. S2†) while its mass spectrum (Fig. S3†) have also supported the composition. Binding interaction of NEDAP with Histidine has been estimated using the modified Benesi–Hildebrand equation³⁰ as presented below:

(ΔF_max/ΔF) = 1 + (1/K[C]ⁿ) (i)

here, ΔF = (F_x − F₀) and ΔF_max = F_∞ − F₀, where F₀, F_x, and F_∞ are the emission intensities of NEDAP in the absence of His, at an intermediate His concentration, and at saturation (1 × 10⁻³ M), respectively. K is the association constant, C is the [His] and n is the number of His molecule bound per molecule of NEDAP (here, n = 1) (Fig. S4†). Plot of (F_∞ − F₀)/(F_x − F₀) vs. [His]⁻¹ have yielded, K = 1.93 × 10⁶ M⁻¹. However, another equation has also been used to calculate the association constant as given bellow:³¹

here, F is fluorescence intensity at any [His], F₀ is fluorescence intensity of free NEDAP, C_D and C_P are the concentration. of NEDAP and histidine respectively. The plot of [(F – F₀)/C_D] vs. C_P have yielded a sigmoidal curve (Fig. S5†). Association constant, K obtained from the plot is 1.214 × 10⁶ M⁻¹, which is in close agreement with the above value obtained from modified Benesi–Hildebrand equation.

The same plot has also been used to estimate the lowest detection limit of histidine as 0.01 μM, a value that falls within the normal histidine level in human beings (details in ESI†).

¹H NMR titration in DMSO-d₆ (Fig. 6) clearly demonstrates the interaction of NEDAP with histidine as significant spectral changes have been observed upon gradual addition of histidine to NEDAP. The imine (–CH = N–) proton at 9.36 ppm of free NEDAP has been downfield shifted to 9.41 ppm in presence of histidine. Similarly, the –NH proton at 8.986 ppm has also downfield shifted to 9.031 ppm. Although, no significant shift of aromatic protons has been observed, however, most of them behave differently in presence of histidine. This fact supports the restricted rotation of NEDAP in presence of histidine. Upon addition of histidine, the aliphatic –CH₂ protons of NEDAP have up-field shifted to an extent of ∼0.05 ppm (from 4.09 ppm→ 4.04 ppm and 3.56 ppm → 3.50 ppm respectively). Interestingly, significant higher shift of histidine protons have been observed, e.g., –CH proton (δ = 8.07 ppm), in between two N-centers of imidazole unit has experienced a large up-field shift ∼1.0 ppm (δ = 7.0 ppm) while the other aromatic proton (δ = 7.12 ppm) has also up-field shifted by 0.25 ppm (δ = 6.87 ppm). All these strongly suggest the formation of inter-molecular H-bonding.

Fig. 6 Changes in the ¹H NMR spectra of NEDAP upon gradual addition of histidine in DMSO-d₆. (I) Free NEDAP, (II) NEDAP + 0.5 equivalent histidine, (III) NEDAP + 1.0 equivalent histidine and (IV) free histidine.

The energy optimized geometries of NEDAP and its histidine adduct are presented in Fig. 7. In the adduct, N (68)-H (44) and N (9)-H (7) distances are 1.99 Å and 1.76 Å respectively, within the range of H-bond distance. Moreover, optimized geometry of the adduct also supports the formation of molecular ring. Theoretical UV-Vis spectra of NEDAP and its histidine adduct are generated from TDDFT calculation using CPCM formalism, which considers the solvent as a polarisable continuum but not as discrete solvent molecules for solvation of the solute.³² Theoretical predictions (Fig. 8) are in close agreement to the experimentally observed peaks. The absorption peak of NEDAP at 390 nm is mainly due to the pyrene unit whereas a broad shoulder near 450 nm in the spectrum of [NEDAP–His] adduct is due to the charge transfer from naphthalene to pyrene unit.

Fig. 7 Energy optimized geometries of NEDAP [A] and its histidine adduct [B] using B3LYP/6-31 G basis set.

Fig. 8 Energy gap between HOMO-1 and LUMO of NEDAP (left) and HOMO-LUMO for [NEDAP–His] adduct.

Histidine treated and untreated cells are observed under fluorescence microscope in presence of NEDAP. Fig. 9 indicates that the probe can easily permeate the cell membrane of living cell tested without destroying them as the cells remain alive even after 30 minutes exposure to 1 μM NEDAP.

Fig. 9 Bright field images of Bacillus sp. (a1) and Candida albicans (c1)in presence of NEDAP (1 μM in 15% DMSO–HEPES buffered solution) while their fluorescence microscope images are presented as (a2) and (c2). Images (b1) and (d1) are bright field images of Bacillus sp. and Candida albicans in presence of both NEDAP and histidine, while their respective fluorescence microscope images are presented as (b2) and (d2). Red bar stands for 10 μM. Blue filter is used.

Conclusions

A new low cost fluorescence probe NEDAP has been synthesized by a facile one step Schiff base condensation reaction between N¹-(naphthalen-1-yl)ethane-1,2-diamine hydrochloride with pyrene-1-carbaldehyde. Greater proton affinity of basic N-center of histidine allows to form a molecular ring via intermolecular H-bonding with NEDAP which imparts rigidity to the molecular assembly and inhibits the TICT process. Theoretical studies (DFT calculations) nicely support this fact substantiated from NMR titration and mass spectra. NEDAP can detect 0.01 μM histidine, below the level present in living systems (blood serium). Visible light excitation allows the probe in vivo studies, particularly intracellular histidine determination.

Notes and references

1. (a) J. D. Kopple and M. E. Swendseid, J. Clin. Invest., 1975, 55, 881; (b) S. E. Snyderman, A. Boyer, E. Roitman, L. E. Holt Jr and P. H. Prose, Pediatrics, 1963, 31, 786.
2. T. E. Creighton, Encyclopedia of Molecular Biology, Wiley, New York, 1999, vol. 2, p. 1147.
3. G. N. Chen, X. P. Wu, J. P. Duan and H. Q. Chen, Talanta, 1999, 49, 319.
4. Y. He, X. Wang, J. Zhu, S. Zhong and G. Song, Analyst, 2012, 137, 4005.
5. M. L. Rao, H. Stefan, C. Scheid, A. D. S. Kuttler and W. Froscher, Epilepsia, 1993, 34, 347.
6. P. M. Kovach and M. E. Meyerhoff, Anal. Chem., 1982, 54, 217.
7. L. Zhou, N. Yan, H. G. Zhang, X. M. Zhou, Q. S. Pu and Z. I. Hu, Talanta, 2010, 82, 72.
8. J. Meng, W. Zhang, C. X. Cao, L. Y. Fan, J. Wu and Q. L. Wang, Analyst, 2010, 135, 1592.
9. N. Tateda, K. Matsuhisa, K. Hasebe and T. Miura, Anal. Sci., 2001, 17, 775.
10. S. Wadud, M. M. Or-Rashid and R. Onodera, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2002, 767, 369.
11. M. Shahlaei, M. B. Gholivand and A. Pourhossein, Electroanalysis, 2009, 21, 2499.
12. M. A. Hortalá, L. Fabbrizzi, N. Marcotte, F. Stomeo and A. Taglietti, J. Am. Chem. Soc., 2003, 125, 20.
13. S. Y. Zhang, C. M. Yang, W. P. Zhu, B. B. Zeng, Y. J. Yang, Y. F. Xu and X. H. Qian, Org. Biomol. Chem., 2012, 10, 1653.
14. F. Pu, Z. Z. Huang, J. S. Ren and X. G. Qu, Anal. Chem., 2010, 82, 8211.
15. Z. Huang, J. Du, J. Zhang, X. Q. Yu and L. Pu, Chem. Commun., 2012, 48, 3412.
16. L. Fabbrizzi, G. Francese, M. Licchelli, A. Perotti and A. Taglietti, Chem. Commun., 1997, 581.
17. M. A. Hortala, L. Fabbrizzi, N. Marcotte, F. Stomeo and A. Taglietti, J. Am. Chem. Soc., 2003, 125, 20.
18. (a) J. F. Folmer-Andersen, V. M. Lynch and E. V. Anslyn, Chem.–Eur. J., 2005, 11, 5319; (b) S.-K. Sun, K.-X. Tu and X.-P. Yan, Analyst, 2012, 137, 2124.
19. G. Patel and S. Menon, Chem. Commun., 2009, 3563.
20. R. Yang, K. Wang, L. Long, D. Xiao, X. Yang and W. Tan, Anal. Chem., 2002, 74, 1088.
21. (a) Z. Huang, J. Du, J. Zhang, X.-Q. Yu and L. Pu, Chem. Commun., 2012, 48, 3412; (b) J. Du, Z. Huang, X.-Q. Yu and L. Pu, Chem. Commun., 2013, 49, 5399.
22. E. Oliveira, C. Santos, P. Poeta, J. L. Capelo and C. Lodeiro, Analyst, 2013, 138, 3642.
23. (a) A. Banerjee, A. Sahana, S. Guha, S. Lohar, I. Hauli, S. K. Mukhopadhyay, J. S. Matalobos and D. Das, Inorg. Chem., 2012, 51, 5699; (b) A. Banerjee, D. Karak, A. Sahana, S. Guha, S. Lohar and D. Das, J. Hazard. Mater., 2011, 186, 738; (c) A. Sahana, A. Banerjee, S. Das, S. Lohar, D. Karak, B. Sarkar, S. K. Mukhopadhyay, A. K. Mukherjee and D. Das, Org. Biomol. Chem., 2011, 9, 5523; (d) A. Sahana, A. Banerjee, S. Lohar, S. Guha, S. Das, S. K. Mukhopadhyay and D. Das, Analyst, 2012, 137, 3910; (e) S. Das, A. Sahana, A. Banerjee, S. Lohar, D. A. Safin, M. G. Babashkina, M. Bolte, Y. Garcia, I. Hauli, S. K. Mukhopadhyay and D. Das, Dalton Trans., 2013, 42, 4757; (f) A. Sahana, A. Banerjee, S. Lohar, B. Sarkar, S. K. Mukhopadhyay and D. Das, Inorg. Chem., 2013, 52, 3627.
24. (a) A. Banerjee, A. Sahana, S. Lohar, I. Hauli, S. K. Mukhopadhyay, D. A. Safin, M. G. Babashkina, M. Bolte, Y. Garcia and D. Das, Chem. Commun., 2013, 49, 2527; (b) A. Sahana, A. Banerjee, S. Guha, S. Lohar, A. Chattopadhyay, S. K. Mukhopadhyay and D. Das, Analyst, 2012, 137, 1544; (c) S. Lohar, A. Sahana, A. Banerjee, A. Banik, S. K. Mukhopadhyay, J. S. Matalobos and D. Das, Anal. Chem., 2013, 85, 1778; (d) A. Sahana, A. Banerjee, S. Lohar, S. Panja, S. K. Mukhopadhyay, J. S. Matalobos and D. Das, Chem. Commun., 2013, 49, 7231; (e) A. Sahana, A. Banerjee, S. Lohar, A. Chottapadhyay, S. K. Mukhopadhyay and D. Das, RSC Adv., 2013, 3, 14044; (f) A. Banerjee, A. Sahana, S. Lohar, S. Panja, S. K. Mukhopadhyay and D. Das, RSC Adv., 2014, 4, 3887,  10.1039/c3ra45362f.
25. S. Das, S. Guha, A. Banerjee, S. Lohar, A. Sahana and D. Das, Org. Biomol. Chem., 2011, 9, 7097.
26. A. Banerjee, A. Sahana, S. Lohar, B. Sarkar, S. K. Mukhopadhyay and D. Das, RSC Adv., 2013, 3, 14397.
27. BRS Biochemistry, Molecular Biology and Genetics, (Fifth edition): T. A. Swanson, S. I. Kim, M. J. Glucksman.
28. (a) S. Das, A. Sahana, A. Banerjee, S. Lohar, S. Guha, J. S. Matalobos and D. Das, Anal. Methods, 2012, 4, 2254; (b) H. Sharma, K. Narang, N. Singh and N. Kaur, Mater. Lett., 2012, 84; (c) R. Azadbakht and J. Khanabadi, Inorg. Chem. Commun., 2013, 30, 21; (d) U. Heinz, K. Hegetschweiler, P. Acklin, B. Faller, R. Lattmann and H. P. Schnebli, Angew. Chem., Int. Ed., 1999, 38, 2568.
29. (a) E. Lippert, W. Luder and H. Boos, in Advances in Molecular Spectroscopy, ed. A. Mangini, Pergamon, Oxford, 1962, p. 443; (b) K. Rotkiewicz, K. H. Grellmann and Z. R. Grabowski, Chem. Phys. Lett., 1973, 19, 315; (c) K. A. Zachariasse, M. Grobye and E. Tauer, Chem. Phys. Lett., 1997, 274, 372; (d) W. Schuddeboom, S. A. Jonker and J. M. Warman, J. Phys. Chem., 1992, 96, 10809; (e) A. L. Sobolewski, W. Sudholt and W. Domcke, J. Phys. Chem. A, 1998, 102, 2716; (f) D. I. Schuster, M. D. Goldstein and P. Bane, J. Am. Chem. Soc., 1977, 99, 187; (g) L. C. T. Soute, Chem. Phys. Lett., 1992, 195, 255; (h) P. R. Callis and R. W. Wilson, Chem. Phys. Lett., 1972, 13, 417; (i) M. Glasbeek and H. Zhang, Chem. Rev., 2004, 104, 1929; (j) J. A. Mondal, H. N. Ghosh, T. K. Ghanty, T. Mukherjee and D. K. Palit, J. Phys. Chem. A, 2006, 110, 3432.
30. (a) H. A. Benesi and J. H. Hildebrand, J. Am. Chem. Soc., 1949, 71, 2703; (b) A. Banerjee, A. Sahana, S. Das, S. Lohar, S. Guha, B. Sarkar, S. K. Mukhopadhyay, A. K. Mukherjee and D. Das, Analyst, 2012, 137, 2166.
31. T. Biver, M. Pulzonetti, F. Secco, M. Venturini and S. Yarmoluk, Biochem. Biophys., 2006, 451, 103.
32. (a) S. Miertus, E. Scrocco and J. Tomasi, Chem. Phys., 1981, 55, 117; (b) M. Cossi, V. Barone, R. Cammi and J. Tomasi, Chem. Phys. Lett., 1996, 255, 327; (c) M. Cossi, V. Barone and M. A. Robb, J. Chem. Phys., 1999, 111, 5295; (d) M. Cossi, G. Scalmani, N. Rega and V. Barone, J. Chem. Phys., 2002, 117, 43.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3ra46604c

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