Crystal structure and spectral studies of green fluorescent protein (GFP) chromophore analogue ethyl 2-[(4Z)-(6-hydroxy naphthalen-2-yl) methylene)-2-methyl-5-oxo-4,5-di hydro-1H-imidazol-1-yl] acetate European Journal of Chemistry 10 (2) (2019) 175-179 European Journal of Chemistry View Journal Online View Article Online Crystal structure and spectral studies of green fluorescent protein (GFP) chromophore analogue ethyl 2-[(4Z)-(6-hydroxy naphthalen-2-yl) methylene)- 2-methyl-5-oxo-4,5-di hydro-1H-imidazol-1-yl] acetate Anisha Puthuvakkal and Kochunnoonny Manoj * Photosciences and Photonics, Chemical Sciences and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Thiruvananthapuram 695019, India anishap371@gmail.com (A.P.), k.manoj.chem@gmail.com (K.M.) * Corresponding author at: Photosciences and Photonics, Chemical Sciences and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Thiruvananthapuram 695019, India. Tel: +91.471.2515408 Fax: +91.471.2491712 e-mail: k.manoj.chem@gmail.com (K. Manoj). 10.5155/eurjchem.10.2.175-179.1869 Received: 10 April 2019 Received in revised form: 19 May 2019 Accepted: 20 May 2019 Published online: 30 June 2019 Printed: 30 June 2019 Synthetically modified green fluorescent protein chromophore derivative was prepared, its crystal structure and spectral properties were studied. Crystal data for C19H18N2O4: triclinic, space group P-1 (no. 2), a = 8.2506(17) Å, b = 11.934(2) Å, c = 17.461(4) Å, α = 102.89(3)°, β = 94.62(3)°, γ = 96.68(3)°, V = 1654.5(6) Å3, Z = 4, T = 173(2) K, μ(MoKα) = 0.096 mm-1, Dcalc = 1.358 g/cm3, 7227 reflections measured (4.722° ≤ 2Θ ≤ 53.996°), 7227 unique (Rint = 0.0453, Rsigma = 0.0662) which were used in all calculations. The final R1 was 0.0561 (I > 2σ(I)) and wR2 was 0.1658 (all data). The single crystal structure showed, the benzylidine moiety adopts Z-conformation in solid state and the molecules were associated by various O−H···O and C−H···O non-covalent interactions. The UV absorption-emission spectral analysis indicated that a significant red shift of emission observed at alkaline pH indicating its utility for live cell imaging applications. Red shift Emission UV absorption Crystal structure Fluorescent protein Intermolecular Interactions Cite this: Eur. J. Chem. 2019, 10(2), 175-179 Journal website: www.eurjchem.com 1. Introduction Discovery of green fluorescent protein (GFP) from jellyfish Aequorea victoria provided a powerful tool in the cellular imaging technique due to its wide range of emission colors, photo stability and a low background noise upon UV excitation [1-6]. A number of fluorescence turn-on sensors for various pH, human serum albumin (HSA) and ribonucleic acid (RNA) have been developed using synthetically modified GFP molecules, which exhibit selective high fluorescence, wide range of spectral tunability, high environmental sensitivity and a very low toxicity [7-10]. The high fluorescence quantum yield of the GFP chromophore can be attributed to the non- radiative relaxation of benzylidine imidazolinone (BI) double bond and the exact mechanism of light emission from the GFP proteins have been anticipated to involves a variety of processes such as E-Z isomerization, tautomer formation, excited state proton transfer (ESPT), triplet formation, hula- twisting, etc. [11-13]. Although, majority of the literature suggests that GFP chromophore and its analogues exhibit high fluorescence due to the tautomerization as well as restricted Z- conformation (Scheme 1) of benzylidene imidazolinone (BI) moiety and it undergo excited state E-Z twisting in solutions that triggers internal conversion making them weakly emissive [14,15]. The concept of inhibiting the free rotation of the aryl- alkene bond of GFP chromophore for efficient fluorescence, various synthetic strategies has been explored. Baldridge et al. investigated a reversible locking of BI moiety by pyridyl substitution, which produces selective ‘turn on’ fluorescence in the presence of Zn2+ or Cd2+ ions [16]. Wu and Burgess deve- loped another approach by using a Lewis acid (BF2 group), which connect the benzylidine and imidazolinone ring to restrain the twisting, resulted a strong fluorescence in solutions [17]. Chen et al. synthesized various ortho-hydroxy- lated GFP derivatives, which gave high emission in the solid state through intramolecular excited-state proton transfer (ESPT) pathways [18]. Tolbert et al. reported hydrophobic derivatives of the GFP chromophore that exhibits fluorescence in the solid due to the aggregation induced emission [19]. ABSTRACT RESEARCH ARTICLE KEYWORDS European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2019 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. http://dx.doi.org/10.5155/eurjchem.10.2.175-179.1869 http://dx.doi.org/10.5155/eurjchem.10.2.175-179.1869 https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.10.2.175-179.1869&domain=pdf&date_stamp=2019-06-30 http://www.eurjchem.com/ http://dx.doi.org/10.5155/eurjchem.10.2.175-179.1869 mailto:anishap371@gmail.com mailto:k.manoj.chem@gmail.com mailto:k.manoj.chem@gmail.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.10.2.175-179.1869&domain=pdf&date_stamp=2019-06-30� 176 Puthuvakkal and Manoj / European Journal of Chemistry 10 (2) (2019) 175-179 Scheme 1 N N O HO O O O HO H O N O CH3 H3C H3C O NH CH3H3CO CH3 O H2N + NIEO N O CH3 H3C H3C + Scheme 2 Chou et al. come up with a double locking of GFP chromophore to its Z-form by five-membered ring cyclization, from which the excited-state intramolecular proton transfer takes place, produced a very high quantum yield and has been successfully applied to fabricate a yellow organic light emitting diodes (OLED) [20]. In order to achieve the emissive form of BI, we used a new design strategy that involved aromatic ring expansion leading to the preparation of ethyl 2-[(4Z)-(6- hydroxy naphthalen-2-yl) methylene)-2-methyl-5-oxo-4,5-di hydro-1H-imidazol-1-yl] acetate (NIE). 2. Experimental All chemicals and reagents were purchased from commercial sources and used without further purification. NMR spectra were measured on a 500 MHz Bruker Avance DPX NMR instrument and IR spectra recorded on a Shimadzu IR Prestige-21 spectrophotometer with sample on KBr. High resolution mass spectra (HR-MS) were measured on a Bruker microTOF II instrument using electron spray ionization (ESI). UV-VIS spectra were recorded with a Varian Cary 100 spectrophotometer. The fluorescence spectra were performed on a Hitachi F-4500 fluorescence spectrophotometer. 2.1. Synthesis Methyl glycinate hydrochloride (5 mmol, 625 mg) and K2CO3 (5 mmol, 691 mg) were suspended in di ethyl ether (125 mL), followed by addition of water (20 mL) then addition of ethyl acetimidate hydrochloride (5 mmol, 698 mg). The mixture was shaken for 6 min then the ether was decant off. An additional portion of di ethyl ether (75 mL) was added, the mixture was shaken for 6 min then the di ethyl ether was decant off. The combined organic portions were dried over anhydrous MgSO4 and the solvent was removed in vacuo to give imidate, which further mix with the Schiff base of 6- hydroxy 2-napthaldehyde (5 mmol, 860 mg) at room temperature for 12 h (Scheme 2). The residue obtained was finally purified by flash column chromatography on a silica gel (230-400 mesh) using ethyl acetate-hexane (1:2) mixture. Ethyl 2-[(4Z)-((6-hydroxy naphthalen-2-yl) methylene)-2- methyl-5-oxo-4,5-di hydro-1H-imidazol-1-yl] acetate (NIE): Yield: 912 mg, 54%. M.p.: 206-207 °C. FT-IR (KBr, ν, cm-1): 1697, 1741 (C=O), 3319 (OH). 1H NMR (500 MHz, CDCl3, δ, ppm): 1.31 (t, 3H, J = 7.9 Hz, CH3), 2.38 (s, 3H, Im-CH3), 4.23- 4.28 (q, 2H, J = 4.2 Hz, CH2), 4.41 (s, 2H, CH2), 5.41 (s, 1H, Ar- OH), 7.10-7.11 (d, 1H, J = 11.4 Hz, Ar-H), 7.13 (d, 1H, J = 2.2 Hz, Ar-H), 7.27 (s, 1H, CH), 7.68 (d, 1H, J = 9.1 Hz, Ar-H), 7.81 (d, 1H, J = 8.8 Hz, Ar-H), 8.38 (t, 2H, J = 9.5 Hz, Ar-H). 13C NMR (125 MHz, CDCl3, δ, ppm): 14.13, 15.59, 41.45, 55.96, 56.31, 56.47, 61.99, 96.02, 109.72, 114.93, 115.20, 122.43, 135.42, 143.30, 152.63, 155.60, 158.99, 167.78, 170.07. HRMS (EI, m/z) calcd. for C19H18N2O4: 338.13; found: 339.13 [NIE+H+]. 2.2. Crystallographic details X-ray intensity data were collected on a Bruker SMART APEX II CCD Diffractometer in omega and phi scan mode, λMoKα = 0.71073 Å at low temperature (173 K) using OXFORD LN2 cryosystem. All the intensities were corrected for Lorentz- polarisation and absorption effects using Bruker’s SAINT and SADABS programs [21]. The crystal structures were solved by Direct methods using program SHELXT-2014 [22]; the full- matrix least squares refinements on F2 were carried out by using SHELXL-2014 [23]. Hydrogen atoms were included in the refinement as per the riding model. Table 1 summarizes the crystallographic data for NIE. 3. Results and discussion 3.1. Crystal structure Yellow crystals of NIE were obtained by dissolving the compounds in dichloromethane:methanol (3:1, v:v) mixture and slow evaporation at room temperature. Suitable needle crystals of NIE were selected for the single X-ray diffraction studies. The single crystal XRD analysis indicated that the NIE crystals belong to triclinic, P−1 and the asymmetric unit containing two molecules that are labeled with A & B numbering scheme (Figure 1). 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.2.175-179.1869 Puthuvakkal and Manoj / European Journal of Chemistry 10 (2) (2019) 175-179 177 Table 1. Summary of crystallographic data. Parameters NIE CCDC No. 1902393 Empirical formula C19H18N2O4 Formula weight 338.35 Temperature (K) 173 Crystal system Triclinic Space group P−1 a (Å) 8.2506(17) b (Å) 11.934(2) c (Å) 17.461(4) α (°) 102.89(3) β (°) 94.62(3) γ (°) 96.68(3) Volume (Å3) 1654.5(6) Z 4 ρcalc (g/cm3) 1.358 μ (mm-1) 0.96 F(000) 712.0 Crystal size (mm3) 0.25 × 0.10 × 0.05 Radiation MoKα (λ = 0.71073) 2Θ range for data collection (°) 4.722 to 53.996 Index ranges −10 ≤ h ≤ 10, −15 ≤ k ≤ 14, −22 ≤ l ≤ 22 Reflections collected 7227 Independent reflections 5031 [Rint = 0.066] Data/restraints/parameters 7227/0/456 Goodness-of-fit on F2 0.995 Final R indexes [I≥2σ (I)] R1 = 0.0561, wR2 = 0.1390 Final R indexes [all data] R1 = 0.0947, wR2 = 0.1658 Largest diff. peak/hole (e.Å-3) 0.28/-0.32 (a) (b) (c) (d) Figure 1. (a)Molecular structure of the compound showing the atom-numbering scheme, (b) Molecular layers of molecule A, (c) Molecular layers of molecule B and (d) crystal packing of NIE crystal. 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.2.175-179.1869 178 Puthuvakkal and Manoj / European Journal of Chemistry 10 (2) (2019) 175-179 Table 2. Geometrical parametersof hydrogen bonding interaction in crystals of NIE. D−H⋅⋅⋅A H⋅⋅⋅A (Å) D⋅⋅⋅A (Å) D−H⋅⋅⋅A (°) Symmetry code C3A−H3A⋅⋅⋅O4A 2.62 3.438(4) 147 x+1, y-1, z C3B−H3B⋅⋅⋅O3B 2.53 3.331(4) 145 -x, -y-1, -z C15A−H15A⋅⋅⋅O3B 2.52 3.366(4) 148 x-1, y+1, z C15B−H15D⋅⋅⋅O3B 2.60 3.244(5) 125 x, y, z C16B−H16D⋅⋅⋅O3A 2.51 3.151(4) 123 x+1, y-1, z C18B−H18D⋅⋅⋅O1A 2.56 3.389(5) 144 x, y, z O1A−H1A1⋅⋅⋅O2A 1.92 2.718(3) 166 x, y-1, z O1B−H1B1⋅⋅⋅O2B 1.97 2.785(4) 178 x, y-1, z Figure 2. Absorption (line) and emission spectra (dotted line) of NIE in solution. Color codes: Blue = MeOH, Red = Methanol acidified with trifluoro acetic acid and Green = Methanol solution with butyl amine. The benzylidine and imidazole moiety of both molecules was in the same plane according to Z-conformation (Figure 1a), which is the fluorescent emissive form of Green Fluorescent Protein (GFP) chromophore as discussed earlier. The molecules of A were translated to form a chain like arrangement via C3A−H3A⋅⋅⋅O4A and C16A−H16B⋅⋅⋅O1A interactions [24-26] diagonal to ab-plane (Figure 1b). Such two chains extended to form a double layer (red in Figure 1d) along the c-axis using the hydrogen bonds O1A−H1A1⋅⋅⋅O2A (Table 2). Similarly, the other asymmetric molecules of B associated to form a chain like arrangement using O1B−H1B1⋅⋅⋅O2B hydrogen bonds along the b-axis (Figure 1c). Two such chains further extended to form double layer (blue color in Figure 1d) via weak non-covalent interactions C3B−H3B⋅⋅⋅O3B and C16B−H16D⋅⋅⋅O2A diagonal to ac-plane (Table 2). These molecular layers together to form a ladder like close packing, via C15A−H15A⋅⋅⋅O3B and C16B−H16D⋅⋅⋅O3A (Figure 1d and Table 2). 3.2. Spectral studies The absorption emission spectral variation observed with the NIE and the effect of pH studied (Figure 2). In methanol solution, NIE has absorption maximum of 390 nm and fluorescence emission maximum at 484nm resulting green emission. Upon acidification with trifluoro acetic acid (pH = 2.5), the protonated NIE species have similar absorption maximum (390 nm), whereas the emission maximum shifted to 521 nm with yellowish green color. However, in alkaline condition (pH= 10.8, using butyl amine addition), absorption maximum changed to 445 nm due to the deprotonation of NIE hydroxyl group and its respective emission shifted to orange fluorescence (621 nm). It is interesting to note that depending on the pH, the fluorescence emission of NIE changed from green to orange can be used for the pH sensor application of live cell imaging. 4. Conclusion We have synthesized a new GFP analogue, NIE and carried out its spectral characterization and crystal structure. The single crystal structure analysis of NIE indicated that the molecule adopts Z-conformation in its crystal lattice, which is the fluorescent emissive form of the GFP chromophore that are associated by non-covalent interactions of O−H⋅⋅⋅O and C−H⋅⋅⋅O. The fluorescence emission of NIE was found to be significantly red shifted under alkaline conditions, which can be used as pH sensor applications for live cell imaging and studies are exploring further in this direction. Acknowledgements We thank Kerala State Council for Science, Technology and Environment (KSCSTE), Thiruvananthapuram, India for the financial support under the Varghese Kurien Young Scientist Scheme (Ref. No. 004/YSS/CS/KM/2013/KSCSTE). We gratefully acknowledge Dr. A. Ajayagosh, Director, CSIR-NIIST for his encouragement and support. We acknowledge Prof. K. M. Sureshan and Mr. Alex for the Single Crystal XRD data collection at IISER-Thiruvananthapuram. Supporting information CCDC-1902393 contains the supplementary crystal- lographic data for this paper. These data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/, or by e-mailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Author contributions: All authors contributed equally to this work. 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.2.175-179.1869 https://www.ccdc.cam.ac.uk/structures/ mailto:data_request@ccdc.cam.ac.uk Puthuvakkal and Manoj / European Journal of Chemistry 10 (2) (2019) 175-179 179 Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. Funding Kerala State Council for Science, Technology and Environment http://dx.doi.org/10.13039/501100006144 ORCID Anisha Puthuvakkal http://orcid.org/0000-0001-6385-3113 Kochunnoonny Manoj http://orcid.org/0000-0002-1511-1091 References [1]. Shimomura, FEBS Lett. 1979, 104, 220-222. [2]. Zimmer, M. Chem. Rev. 2002, 102, 759-782. [3]. Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science 2006, 312, 217-224. [4]. Chalfie, M. Angew. Chem. Int. Ed. 2009, 48, 5603-5611. [5]. Chudakov, D. M.; Matz, M. V.; Lukyanov, S.; Lukyanov, K. A. Physiol. Rev. 2010, 90, 1103-1163. [6]. Chang, J.; Chen, X.; Glass, Z.; Gao, F.; Mao, L.; Wang, M.; Xu, Q. Acc. Chem. Res. 2019, 52, 665-675. [7]. Kneen, M.; Farinas, J.; Li, Y.; Verkman, A. S. Biophys. J. 1998, 74, 1591- 1599. [8]. Tamura, T.; Hamachi, I. ACS Chem. Biol. 2014, 9, 2708-2717. [9]. Walker, C. L.; Konstantin A Lukyanov, K. A.; Yampolsky, I. 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This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.2.175-179.1869 http://dx.doi.org/10.13039/501100006144 http://orcid.org/0000-0001-6385-3113 http://orcid.org/0000-0002-1511-1091 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Synthesis 2.2. Crystallographic details 3. Results and discussion 3.1. Crystal structure 3.2. Spectral studies 4. Conclusion Acknowledgements Supporting information Disclosure statement Funding ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: