Synthesis of mono-(p-dimethylamino)styryl-containing BOPHY dye for a turn-on pH sensor

Abstract

Mono-substitutional bis(difluoroboron)1,2-bis((1H-pyrrol-2-yl)methylene)hydrazine (BOPHY) 3a with a (p-dimethylamino)styryl group in the α-position was confirmed to be synthesized by the Knoevenagel-type condensation. Dimethylamino-containing BOPHY dye 3a is almost non-fluorescent by the ICT effect. Upon the protonation of the tertiary amine function of 3a, the strong fluorescence (Φ_f = 0.98) was released and the fluorescence intensity was dramatically increased by one thousand fold. BOPHY 3a can be used as a pH probe.

---

1. Introduction

In recent decades, 4,40-difluoro-4-bora-3a,4a-diaza-sindacene (abbreviated as BODIPY) dyes have received increased attention because of their high fluorescence quantum yields, high absorption coefficients, good solubility in organic solvents, and excellent thermal and photochemical stabilities.¹ Since the discovery of the excellent photochemical properties of BODIPY, various modifications on the BODIPY dye 1 (Fig. 1a) including the modification on the core or aza-BODIPY,² have been widely explored for sensors, laser dyes, light harvesters, organic light emitting diodes, and sensitizers for solar cells and so forth.³ Therefore, the BODIPY dyes have been attracting increasing interest in the design and synthesis of these novel fluorescent dyes to date.

Fig. 1 The core structure of BODIPY and BOPHY.

Ziegler et al. recently presented another type of unique pyrrole-BF₂-based fluorophore bis(difluoroboron)1,2-bis((1H-pyrrol-2-yl)methylene)hydrazine (BOPHY) 2 (Fig. 1a).⁴ The new fluorescent BOPHY dye 2 can be successfully obtained by the reaction of pyrrole-2-carboxaldehyde with hydrazine, and followed by complexation with Et₃N–BF₃·Et₂O, based on the traditional classical method (Fig. 1b).⁴ The aesthetic symmetric structure is composed of four rings at the same plane, including two BF₂ units in six-membered chelate rings in the center and two pyrrole units on the periphery (Fig. 1).⁴ The fluorescence quantum yield for the unmodified BOPHY 2 is so high to near to be 100%.⁴ Since the new BOPHY dye 2 has a grid structure, excellent optical properties and is similar to BODIPY 1, these urge us to investigate the BOPHY structural motif for a functionalization. Our recent research interest lies in the novel BODIPY/aza-BODIPY family of fluorescent dyes and their application.⁵ During preparation of this manuscript, modifications on BOPHY dyes, including BOPHYs 3a and 3b, were reported by Jiao and Hao group (Fig. 2).⁶ However, the synthesis of 3b was not shown, and no study of BOPHY as a pH fluorescent probe was explored either. Therefore, we herein communicate our studies on a (p-dimethylamino)styryl-containing BOPHY 3a as a turn-on fluorescent probe for pH (Fig. 2).

Fig. 2 Structure of BOPHY 3a and 3b.

2. Experimental section

2.1 General

¹H NMR spectra were recorded on a Bruker AVANCE III 500 MHz spectrometer. ¹H NMR chemical shifts (δ) are given in ppm downfield from Me₄Si, determined by chloroform (δ = 7.26 ppm). ¹³C NMR spectra were recorded on a Bruker AVANCE III 125 MHz spectrometer. ¹³C NMR chemical shifts (δ) are reported in ppm with the internal CDCl₃ at δ 77.0 ppm as standard. Toluene solvents were distilled over CaH₂. Merck silica gel 60 was used for the column chromatography. All pH measurements were performed with a PHS-3E pH meter.

Fluorescence spectra were recorded on FluoroSENS spectrophotometer. UV/Vis spectra were recorded on UV-2550 spectrophotometer at room temperature. The refractive index of the medium was measured by 2 W Abbe's refractometer at 20 °C. The fluorescence quantum yields (Φ_f) of the BOPHY system was calculated using the following relationship (eqn (1)):

Φ_f = Φ_refF_samplA_refn_sampl²/F_refA_sampln_ref² (1)

here, F denotes the integral of the corrected fluorescence spectrum, A is the absorbance at the excitation wavelength, ref and sampl denote parameters from the reference and unknown experimental samples, respectively. The reference systems used was rhodamine 6G in methanol as standard (Φ_f = 0.78, in air equilibrated water and deaerated solutions) for 3a–H⁺.

The MO calculations were performed at the DFT level, and the frontier molecular orbitals of BOPHY 3a and 3a–H⁺ at the MP2/6-31G* level with Gaussian 03.

2.2 Synthesis of BOPHY 3a

4-N,N-Dimethylbenzaldehyde (33 mg, 0.22 mmol), compound 4 (40 mg, 0.12 mmol) (see following Scheme 1), AcOH (0.5 mL), and piperidine (0.5 mL) were stirred for 24 h at 95 °C in dry toluene (6 mL) in the presence of a small amount of activated 4 Å molecular sieves. The mixture was cooled to room temperature, quenched with water, extracted with CH₂Cl₂, washed with brine, dried over Na₂SO₄, evaporated and purified by TLC to afford BODIHY 3a (15 mg, 28%) as red solids. ¹H NMR (500 MHz, CDCl₃): δ (ppm) 7.96 (s, 1H), 7.85 (s, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 12.5 Hz, 2H), 6.71 (d, J = 6.0 Hz, 2H), 6.68 (s, 1H), 6.17 (s, 1H), 3.03 (s, 6H), 2.49 (s, 3H), 2.35 (s, 3H), 2.33 (s, 3H). ¹³C NMR (125 MHz, CDCl₃): δ (ppm) 151.6, 151.0, 150.2, 140.7, 140.1, 137.7, 133.7, 132.4, 128.9, 124.4, 124.0, 123.3, 118.2, 114.5, 112.8, 111.9, 110.9, 40.2, 14.1, 11.2, 11.1. HRMS-MODAI (ESI) calcd for C₂₃H₂₅N₅B₂F₄ [M + H]⁺: 470.2305, found 470.2305.

Scheme 1 Synthesis of the BOPHY dye 3a.

3. Results and discussion

3.1 Synthesis and characterization of BOPHY 3a

BOPHY dyes 3a and 3b with a (p-dimethylamino)styryl group in α/β-position of the pyrrole (Fig. 2), were found to occur in the text and the ESI in Jiao and Hao's paper, respectively.⁶ It is illusive to which one was synthesized in the Knoevenagel-type condensation. Therefore, we curiously repeated this reaction from BOPHY 4 (ref. 4 and 6) via the Knoevenagel-type condensation (Scheme 1).⁷ We successfully synthesized the mono-dimethylaminostyryl BOPHY 3a (Scheme 1). Though the use of 10 eq. 4-dimethylaminobenzaldehyde, no bis-dimethylamino styryl BOPHY was observed.

The structure of dye 3a was confirmed by ¹H NMR spectrum. Due to the shield from the full effect of the applied field by their surrounding electrons,⁸ the chemical shift of H_α is well-known to be lower than that of H_β in ¹H NMR spectrum in dye 3a and 4 (Scheme 1). By the measure of the ¹H NMR spectrum of 3a using a Bruker AVANCE III 500 MHz spectrometer, three sets of distinct hydrogen signals of the methyl group (δ = 2.49 (s, 3H_c), 2.35 (s, 3H_a), 2.33 (s, 3H_b) ppm in CDCl₃) were showed (Fig. 3 and Scheme 1). Therefore, the logical structure was thought to be the dye 3a not 3b which should have two sets of hydrogen signals in the low field and a set in the high field (Scheme 1 and Fig. 2).

Fig. 3 Partial ¹NMR spectra of the BOPHY dye 3a.

3.2 A turn-on fluorescent probe for pH

The most popular strategy for pH-responsive fluorescent sensors takes advantage of intramolecular charge transfer (ICT) or the photoinduced electron transfer (PET).⁹ Dimethylamino group is one of the fragments frequently used for the purpose of ICT. The BODIPY is a family of very widely used fluorescent dye for potential applications and often designed for pH probes, wherein the excited state of the fluorophore can be quenched by the electron transfer from electron donating amine to the fluorophore.¹⁰ Upon recognition of a proton, the electron transfer is “switched off” and in turn the emission of fluorescence is “switched on”. To the best of our knowledge, a functionalization of BOPHY dye for a pH probes has not been documented. Therefore, we continue to explore the response of the dimethylamino-containing BOPHY dye 3a to pH value.

Photoimage of 3a were taken under normal room illumination and UV light, and notable changes of relatively vivid bright colors of 3a with the pH can be easily observed with naked eye (Fig. 4). The mechanism of BOPHY 3a to pH value is same to that of the above described BODIPY (Fig. 5). Upon addition of hydrochloric acid to BOPHY 3a with a –NMe₂ group as a pH-sensitive functionality, 3a was protonated at relatively low pH value.¹¹ A stepwise decrease of the absorption intensity was observed in the 543 nm band of 3a, and this peak disappeared completely at 1 M (Fig. 6). The formation of a new band at 504 nm was first observed in pH 3, and another new peak subsequently arose at 481 nm (Fig. 6). The absorption intensity of 3a–H⁺ reached the maximum when 4 M HCl was used, and the absorption intensity was subsequently decreased at 6 and 8 M HCl. The absorption band of 3a–H⁺ is blue-shifted by about 50 nm compared to that of 3a. The fluorescence quantum yield of 3a in CH₃CN–H₂O (1 : 1, v/v) is very low (Φ_f = 0.01 at pH 7) due to the ICT effect; however, with decreasing pH the twin emission maxima were shifted to 532, 573 nm (Fig. 7a). A dramatic increase in fluorescence intensity at 532 nm by 1200 folds (Φ_f = 0.98 when treated with HCl to 4 M) (Fig. 7b).

Fig. 4 Photograph of solutions of 5 μM BOPHY dye 3a at pH 0, 1, 1.5, 2, 2.5, 3, 3.5, 4 and 7 in CH₃CN–H₂O (1 : 1, v/v) under normal room illumination (upper row) and under UV irradiation (bottom row), respectively.

Fig. 5 Protonation of BOPHY 3a to generate 3a–H⁺ and release the strong fluorescence by switching off the ICT effect.

Fig. 6 Absorption spectra (pH 7, 5, 4, 3.5, 3, 2.8, 2.5, 2.2, 2.0, 1.5, 1 and 1 M, 2 M, 4 M, 6 M, 8 M of HCl) of 5 μM dye 3a in CH₃CN–H₂O (1 : 1, v/v) as a function of pH.

Fig. 7 (a) Corresponding fluorescence spectra (pH 7, 5, 4, 3.5, 3, 2.8, 2.5, 2.2, 2.0, 1.5, 1 and 1 M, 2 M, 4 M, 6 M, 8 M of HCl, λ_ex = 490 nm) of 5 μM dye 3a in CH₃CN–H₂O (1 : 1, v/v) as a function of pH. (b) Fluorescence titration responses of 3a towards HCl at 532 nm.

Moreover, the molecular geometries of BOPHY 3a and 3a–H⁺ were optimized using density functional theory (DFT) at the MP2/6-31G* level.¹² The calculated HOMO and LUMO orbital energy levels were summarized in Fig. 8. Upon excitation of the BOPHY fluorophore, an electron of the highest occupied molecular orbital (HOMO) is promoted to the lowest unoccupied molecular orbital (LUMO), which enables ICT from the donor (amino nitrogen atom) to that of the BOPHY fluorophore, causing fluorescence quenching of the latter. Upon the protonation of 3a, ICT is no longer possible, and the fluorescence intensity of the probes was enhanced. The protonation of BOPHY 3a (3a–H⁺: λ_abs = 481, 504 nm) resulted in a remarkable hypsochromic shift compared to that (λ_abs = 540 nm) of the BOPHY 3a. It is due to the increase in the HOMO–LUMO band gap (2.65 eV) for the lowest energy absorption bands observed for 3a–H⁺ relative to that (2.53 eV) of 3a by MO calculations (Fig. 8). However, the energy level of frontier orbitals in 3a–H⁺ was lower than that of 3a (Fig. 8).

Fig. 8 The frontier molecular orbitals (MOs) of BOPHY 3a and 3a–H⁺. The energy levels of the MOs are shown (eV). Calculations are based on ground state geometry by DFT at the MP2/6-31G* level with Gaussian 03. HOMO/LUMO (eV) = −4.81/−2.28 for 3a; HOMO/LUMO (eV) = −7.42/−4.77 for 3a–H⁺.

4. Conclusions

Mono-substitutional BOPHY 3a with a (p-dimethylamino)styryl group in α-position was confirmed to be synthesized by a Knoevenagel-type condensation, and no dis substitutional BOPHY with a (p-dimethylamino)styryl group was observed. BOPHY 3a is almost non-fluorescent by ICT effect. Upon the protonation of the tertiary amine function of 3a, the strong fluorescence (Φ_f = 0.98) was released and the fluorescence intensity was dramatically increased by 1200 folds. Based on MO calculations the increase in the HOMO–LUMO band gap (2.65 eV) for the lowest energy absorption bands observed for 3a–H⁺ relative to that (2.53 eV) of 3a, and the energy level of frontier orbitals in 3a–H⁺ was lower than that of 3a. BOPHY 3a as a pH probe can be used. Further efforts for development of probes for thiol^5b,g based on BOPHY dyes in biotechnology are ongoing in our lab.

Acknowledgements

This work was supported by National Training Programs of Innovation and Entrepreneurship for Undergraduates (201410149052, 20141049000052), the Public Research Foundation of Liaoning Province for the Cause of Science (2014003009), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and the start-up funds from Shenyang University of Chemical Technology.

Notes and references

1. (a) A. Loudet and K. Burgess, Chem. Rev., 2007, 107, 4891; (b) L. Yuan, W. Lin, K. Zheng, L. He and W. Huang, Chem. Soc. Rev., 2013, 42, 622; (c) R. Weissleder and M. J. Pittet, Nature, 2008, 452, 580; (d) L. Yuan, W. Lin, S. Zhao, W. Gao, B. Chen, L. He and S. Zhu, J. Am. Chem. Soc., 2012, 134, 13510; (e) Z. Zhang, B. Xu, J. Su, L. Shen, Y. Xie and H. Tian, Angew. Chem., Int. Ed., 2011, 50, 11654; (f) X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010, 39, 2120; (g) Y. Yang, Q. Zhao, W. Feng and F. Li, Chem. Rev., 2013, 113, 192; (h) N. Boens, V. Leen and W. Dehaen, Chem. Soc. Rev., 2012, 41, 1130; (i) H. Peng, W. Chen, Y. Cheng, L. Hakuna, R. M. Strongin and B. Wang, Sensors, 2012, 12, 15907; (j) L. Yuan, W. Lin, K. Zheng, L. He and W. Huang, Chem. Soc. Rev., 2013, 42, 622; (k) H. Jung, X. Chen, J. Kim and J. Yoon, Chem. Soc. Rev., 2013, 42, 6019; (l) G. Ulrich, A. Harriman and R. Ziessel, Angew. Chem., Int. Ed., 2008, 47, 1184; (m) A. Bessette and G. S. Hanan, Chem. Soc. Rev., 2014, 43, 3342; (n) H. Lu, J. Mack, Y. Yang and Z. Shen, Chem. Soc. Rev., 2014, 43, 4778.
2. (a) M. A. T. Rogers, J. Chem. Soc., 1943, 596; (b) E. B. Knott, J. Chem. Soc., 1947, 1196.
3. (a) M. A. T. Rogers, Nature, 1943, 151, 504; (b) M. A. T. Rogers, J. Chem. Soc., 1943, 590; (c) J. Killoran, L. Allen, J. Gallagher, W. Gallagher and D. F. O'Shea, Chem. Commun., 2002, 1862; (d) A. Gorman, J. Killoran, C. O'Shea, T. Kenna, W. M. Gallagher and D. F. O'Shea, J. Am. Chem. Soc., 2004, 126, 10619; (e) L. Jiao, Y. Wu, Y. Ding, S. Wang, P. Zhang, C. Yu, Y. Wei, X. Mu and E. Hao, Chem.–Asian J., 2014, 9, 805; (f) S. O. McDonnell, M. J. Hall, L. T. Allen, A. Byrne, W. M. Gallagher and D. F. O'Shea, J. Am. Chem. Soc., 2005, 127, 16360; (g) M. J. Hall, L. T. Allen and D. F. O'Shea, Org. Biomol. Chem., 2006, 4, 776; (h) J. Killoran and D. F. O'Shea, Chem. Commun., 2006, 1503; (i) H. Lu, S. Shimizu, J. Mack, Z. Shen and N. Kobayashi, Chem.–Asian J., 2011, 6, 1026; (j) V. F. Donyagina, S. Shimizu, N. Kobayashi and E. A. Lukyanets, Tetrahedron Lett., 2008, 49, 6152; (k) Y. Wu, C. Cheng, L. Jiao, C. Yu, S. Wang, Y. Wei, X. Mu and E. Hao, Org. Lett., 2014, 16, 748.
4. I.-S. Tamgho, A. Hasheminasab, J. T. Engle, V. N. Nemykin and C. J. Ziegler, J. Am. Chem. Soc., 2014, 136, 5623.
5. (a) X.-D. Jiang, J. Zhang, T. Furuyama and W. Zhao, Org. Lett., 2012, 14, 248; (b) X.-D. Jiang, J. Zhang, X. Shao and W. Zhao, Org. Biomol. Chem., 2012, 10, 1966; (c) X.-D. Jiang, H. Zhang, Y. Zhang and W. Zhao, Tetrahedron, 2012, 68, 9795; (d) X.-D. Jiang, Y. Fu, T. Zhang and W. Zhao, Tetrahedron Lett., 2012, 53, 5703; (e) X.-D. Jiang, R. Gao, Y. Yue, G.-T. Sun and W. Zhao, Org. Biomol. Chem., 2012, 10, 6861; (f) R. Kang, X. Shao, F. Peng, Y. Zhang, G.-T. Sun, W. Zhao and X.-D. Jiang, RSC Adv., 2013, 3, 21033; (g) J. Zhang, X.-D. Jiang, X. Shao, J. Zhao, Y. Su, D. Xi, H. Yu, S. Yue, L.-J. Xiao and W. Zhao, RSC Adv., 2014, 4, 54080; (h) X.-D. Jiang, D. Xi, J. Zhao, H. Yu, G.-T. Sun and L.-J. Xiao, RSC Adv., 2014, 4, 60970; (i) P. Shi, X.-D. Jiang, R. Gao, Y. Dou and W. Zhao, Chin. Chem. Lett., 2015 DOI:10.1016/j.cclet.2014.05.035.
6. C. Yu, L. Jiao, P. Zhang, Z. Feng, C. Cheng, Y. Wei, X. Mu and E. Hao, Org. Lett., 2014, 16, 1952.
7. (a) Q. Zheng, G. Xu and P. N. Prasad, Chem.–Eur. J., 2008, 14, 5812; (b) D. P. Wang, Y. Shiraishi and T. Hirai, Chem. Commun., 2011, 47, 2673; (c) T. Bura, R. Pascal, G. Ulrich and R. Ziessel, J. Org. Chem., 2011, 76, 1109; (d) S. Zhu, J. Zhang, G. Vegesna, F. Luo, S. A. Green and H. Liu, Org. Lett., 2011, 13, 438.
8. (a) H. R. Westover, J. Chem. Educ., 1967, 44, 486; (b) D. Parker, Chem. Rev., 1991, 91, 1441; (c) K. C. Wong, J. Chem. Educ., 2014, 91, 1103.
9. (a) Y. Chen, D. Qi, L. Zhao, W. Cao, C. Huang and J. Jiang, Chem.–Eur. J., 2013, 23, 7342; (b) C. Thivierge, J. Han, R. M. Jenkins and K. Burgess, J. Org. Chem., 2011, 76, 5219; (c) A. W. Czarnik, Acc. Chem. Res., 1994, 27, 302; (d) W. Rettig, Angew. Chem., Int. Ed., 1986, 25, 971; (e) B. Tang, X. Liu, K. Xu, H. Huang, G. Yang and L. An, Chem. Commun., 2007, 3726; (f) W. F. Jager, T. S. Hammink, O. van den Berg and F. C. Grozema, J. Org. Chem., 2010, 75, 2169.
10. (a) S. Hoogendoorn, K. L. Habets, S. Passemard, J. Kuiper, G. A. van der Marel, B. I. Florea and H. S. Overkleeft, Chem. Commun., 2011, 47, 9363; (b) P. Song, X. Chen, Y. Xiang, L. Huang, Z. Zhou, R. Wei and A. Tong, J. Mater. Chem., 2011, 21, 13470; (c) X. Gao, Y. Zhang and B. Wang, Org. Lett., 2003, 5, 4615; (d) B. Tang, F. Yu, P. Li, L. Tong, X. Duan, T. Xie and X. Wang, J. Am. Chem. Soc., 2009, 131, 3016; (e) X. Ma, Y. Wang, T. Zhao, Y. Li, L.-C. Su, Z. Wang, G. Huang, B. D. Sumer and J. Gao, J. Am. Chem. Soc., 2014, 136, 11085; (f) S. Grunder, P. L. McGrier, A. C. Whalley, M. M. Boyle, C. Stern and J. F. Stoddart, J. Am. Chem. Soc., 2013, 135, 17691; (g) A. Thottiparambil, P. R. Anil Kumar and L. Chakkumkumarath, RSC Adv., 2014, 4, 56063.
11. (a) S. O. McDonnell and D. F. O'Shea, Org. Lett., 2006, 8, 3493; (b) K. Rurack, M. Kollmannsberger and J. Daub, Angew. Chem., Int. Ed., 2001, 40, 385.
12. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, V. G. Zakrzewski, J. A. Montgomery Jr, R. E. Stratmann, J. C. Burant, S. Dapprich, J. M. Millam, A. D. Daniels, K. N. Kudin, M. C. Strain, O. Farkas, J. Tomasi, V. Barone, M. Cossi, R. Cammi, B. Mennucci, C. Pomelli, C. Adamo, S. Clifford, J. Ochterski, G. A. Petersson, P. Y. Ayala, Q. Cui, K. Morokuma, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. Cioslowski, J. V. Ortiz, A. D. Baboul, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. Gomperts, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, C. Gonzalez, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chem, M. W. Wong, J. L. Andres, C. Gonzalez, M. Head-Gordon, E. S. Replogle and J. A. Pople, Gaussian 03, Gaussian Inc., Pittsburgh, PA, 2003.

---

Footnote

† Electronic supplementary information (ESI) available: Experimental details and ¹H NMR spectra. See DOI: 10.1039/c4ra15914d

---