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Theoretically obtained insight into the effect of basic amino acids on Cypridina bioluminescence
Journal of Photochemistry & Photobiology, A: Chemistry 406 (2021) 113000
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Theoretically obtained insight into the effect of basic amino acids on Cypridina bioluminescence Chun-Xia Liu a, b, Qing-Bo Liu a, b, Kun Dong a, b, Shao-Jun Huang c, Xi-Kun Yang a, b, Ai-Min Ren d, Chun-Gang Min a, b, *, Gang Liu e, ** a
Research Center for Analysis and Measurement, Kunming University of Science and Technology, Kunming 650093 PR China Analysis and Test Center of Yunnan Province, Kunming 650093 PR China Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650093, PR China d Institute of Theoretical Chemistry, Jilin University, Changchun 130023, PR China e Institute of Chemical and Industrial Bioengineering, Jilin Engineering Normal University, Changchun 130052, PR China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Cypridina bioluminescence TDDFT Basic amino acid Hydrogen bond Solvent polarity
Because of high bioluminescent quantum yields, independent cofactors, high signal-to-noise ratio, Cypridina bioluminescence has been successfully applied to gene reporter assays, enzyme immunoassays, immunohistology and imaging. Although the chemical reaction that transforms Cypridina luciferin into Cypridina oxyluciferin is clearly defined, the environmental effects on the spectrochemistry of Cypridina oxyluciferin continue to be a particularly intriguing aspect. In this paper, the solvent effect on the fluorescent spectrum, the basicity of amino acids and the degree of covalent character of hydrogen bond were studied by a theoretical Density Functional Theory (DFT) and Time Dependent Density Functional Theory (TDDFT) approach. Our results demonstrate that the emission wavelengths varied little with solvent polarity. The compositive effects of basic amino acid and polarity of the environment only slightly affected the structure and spectroscopic properties. The fluorescent properties of Cypridina oxyluciferin can be modulated by the covalent character of the hydrogen bond.
1. Introduction The luminescence of the Cypridina is produced by the oxidation of Cypridina luciferin (C-LH2), catalyzed by luciferase to form Cypridina oxyluciferin (C-OxyLH2) in the first excited state (Fig. 1) [1,2]. Because of high bioluminescent quantum yields, independent cofactors, high signal-to-noise ratio [3–6], Cypridina bioluminescence has been suc cessfully applied to gene reporter assays [7], enzyme immunoassays [8, 9], immunohistology [10] and imaging [11]. The chemical reaction that transforms C-LH2 into C-OxyLH2 is clearly defined (Fig. 1) [12–14]. However, the environmental effects on the spectrochemistry of C-OxyLH2 continue to be a particularly intriguing aspect. C-OxyLH2 is usually thought to exist as acylamino or amide anion both in experimental and theoretical studies [12,13]. However, at least another three chemical forms may exist based on the chemical nature of the emitter: acylamino + NH+ 2 (protonation on N1 atom of 3-(1-guani dino) propyl, leading to a cation), acylimino (imide, neutral), amide + NH+ 2 (protonation on N1 atom of 3-(1-guanidino) propyl and
amide ion, leading to a neutral zwitterions), when the exact side chains were included (Fig. 2) [14]. As we known, the fluorescent spectra of C-OxyLH2 and its analogs are affected by pH value, solvent polarity and the presence of additives, which were extensively explored [15–21]. In 2012, Naumov et al. investigated the environmental effects on Cypridina bioluminescence [15]. The results found that the probable structure and emission spectrum of C-OxyLH2were strongly affected by polarity and basicity of the environment. In that paper, they recorded the absorption and emission spectra of C-OxyLH2 in 16 solvents of varying polarity, either without addition or with excess of 1,8-diazabicyclo[5.4.0]unde c-7-ene (DBU), which was used to simulate the presence of basic amino acids around the C-OxyLH2. However, DBU is different with basic amino acids. Moreover, the degree of covalent character of hydrogen bond between C-OxyLH2 and basic amino acid was not considered. In order to assess the solvent effect on the fluorescent spectrum, we studied the emission spectra of acylamino + NH+ 2 , acylamino, acylimino, amide + NH+ 2 and amide ion in chloroform, acetone, DMSO and water, respectively. For the polarization effect of the microenvironment of
* Corresponding author at: Research Center for Analysis and Measurement, Kunming University of Science and Technology, Kunming 650093 PR China. ** Corresponding author. E-mail addresses: [email protected] (C.-G. Min), [email protected] (G. Liu). https://doi.org/10.1016/j.jphotochem.2020.113000 Received 15 July 2020; Received in revised form 21 October 2020; Accepted 22 October 2020 Available online 27 October 2020 1010-6030/© 2020 Elsevier B.V. All rights reserved.
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Fig. 1. The reaction mechanism of the conversion of Cypridina luciferin to Cypridina oxyluciferin (X =H or none).
Fig. 2. Chemical structures of C-OxyLH2, the value in bracket is the total charge with various protonation states.
+ + Fig. 3. The models for acylamino + NH+ 2 +His, acylamino + NH2 +Arg and acylamino + NH2 +Lys and the formation of N1-H2…N3 hydrogen bond.
2
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Journal of Photochemistry & Photobiology, A: Chemistry 406 (2021) 113000
Fig. 4. The models for acylamino+His, acylamino + Arg and acylamino + Lys and the formation of N4-H5…N6 hydrogen bond.
luciferase and the degree of covalent character of hydrogen bonds, because the crystal structure of Cypridina luciferase was not described, it cannot be evaluated. On the other hand, both Cypridina and firefly are endowed with the ability to emit light. The reaction mechanisms are similar. Cypridina/firefly luciferin interacts with molecular oxygen to form a high energetic intermediate, Cypridina/firefly dioxetanone. The decomposition of Cypridina/firefly dioxetanone generates Cypridina/ firefly oxyluciferin, in a singlet excited state, which subsequently decays to the ground state with multicolor light. Moreover, under the effect of luciferase, both Cypridina and firefly oxyluciferin may exist many pro tonation states [14,22]. We guess basic amino acids but not neutral amino acids appear to work in the luciferase. For firefly biolumines cence, removal of the positive charge of Arginine (Arg)218 and Histidine (His)245 on the crystal structure of the thermostable luciferase of the Japanese firefly (2D1R) resulted in a red-shift in firefly bioluminescence [23–25], our computational results were also agree with the conclusions of experimental studies [26]. Base on these literature reviews, we think Arg, His and Lysine (Lys), may be present in the active sites of Cypridina luciferase, and play an important role in spectroscopic properties. Moreover, because only N1 atom of 3-(1-guanidino) propyl group or N4 atom of amide may join with Arg, Lys, His to form N1-H2⋯N3 or N4-H5⋯N6 hydrogen bond, Arg, Lys, His were simply added near N1 atom or N4 atom to construct six models, acylamino + NH+ 2 +His, acy + lamino + NH+ 2 +Arg, acylamino + NH2 +Lys, acylamino+His, acyla mino + Arg and acylamino + Lys (Figs. 3 and 4 ).
Table 1 Predicted emission maxima (nm) and oscillator strengths (f) for all possible emitters and acylamino-NH+ 2 /acylamino under the respective effects of His, Arg, and Lys in the gas phase, chloroform, acetone, DMSO and water at the TD wb97xd/6-31+G(d)// TD wb97xd/6-31+G(d) levels.
acylamino + NH+ 2 acylamino acylimino amide + NH+ 2 amide ion acylaminoNH+ 2 +His acylaminoNH+ 2 +Arg acylaminoNH+ 2 +Lys acylamino+His acylamino + Arg acylamino + Lys
gas phase
chloroform
acetone
DMSO
water
475 (0.0008) 352 (0.3311) 425 (0.1866) 933 (0.0004) 429 (0.1001) 358 (0.3061) 354 (0.1818) 366 (0.3017) 352 (0.3364) 355 (0.3333) 358 (0.3120)
376 (0.5764) 373 (0.6204) 444 (0.3885) 607 (0.0119) 415 (0.3526) 375 (0.5739) 377 (0.5847) 378 (0.6150) 371 (0.6229) 373 (0.6026) 374 (0.6248)
383 (0.6760) 383 (0.7195) 453 (0.4772) 549 (0.3376) 424 (0.4714) 386 (0.6870) 386 (0.6987) 385 (0.7050) 382 (0.7320) 385 (0.7064) 383 (0.7182)
385 (0.6965) 385 (0.7372) 455 (0.4963) 552 (0.3526) 428 (0.4777) 388 (0.7021) 388 (0.7196) 388 (0.7267) 383 (0.7367) 387 (0.7193) 384 (0.7358)
383 (0.7311) 386 (0.7593) 472 (0.5014) 536 (0.4824) 419 (0.5885) 390 (0.7366) 390 (0.7511) 391 (0.7587) 384 (0.7834) 392 (0.7888) 387 (0.7897)
2. Theoretical methods were relaxed during optimizations. The excited state optimizations were obtained by TD wB97XD/6− 31+G(d). Based on the optimized geome tries, the emission spectra were calculated by the same method.
All theoretical predictions were made with the Gaussian09 program package [27] suite using the wB97XD/6− 31+G(d) method [28]. This method was able to describe relatively accurate structure parameters, emission spectra for C-OxyLH2 [14] and Coelenteramide [29,30]. First, in order to consider the solvent effect on the fluorescent spectra of C-OxyLH2, the excited-state geometries of acylamino-NH+ 2 , acyla mino, acylimino, amide + NH+ 2 and amide ion were optimized by TD wB97XD/6− 31+G(d) method in vacuo, chloroform, acetone, DMSO and water using SMD model (universal salvation model based on solute electron density) [31]. Based on the optimized structure, the emission spectra were computed by the same method. Second, the compositive effects of basic amino acid and polarity of the environment surrounding acylamino + NH+ 2 and acylamino were mimicked by the simple models, acylamino + NH+ 2 +His, acyla mino + NH+ acylamino + NH+ acylamino+His, acyla 2 +Arg, 2 +Lys, mino + Arg and acylamino + Lys in vacuo, chloroform, acetone, DMSO and water using SMD model. TD wB97XD/6− 31+G(d) method were also used to optimize the excited-state structure and compute the emission spectra. At last, to simulate the different degree of covalent character of the N1-H2 and N4-H5 bonds, we constrained bond lengths of N1-H2 and N4H5, and increased it step by step. That is, for a given N1-H2 and N4-H5 distance ranging from 1.2 to 2.0 Å, all the remaining degrees of freedom
3. Results and discussion 3.1. The effect of solvent polarity and basic amino acids on the Cypridina bioluminescence To assess the effects of solvent polarity on the fluorescent spectra, the emission spectra, oscillator strengths (f) and main transition configu rations for all the possible emitters were calculated with TD wb97xd/ 6− 31+G (d) method in vacuo, chloroform, acetone, DMSO and aqueous based on the first excited-state optimized geometries. The data are listed in Table 1 and Table S1 (Supporting Information). Fitted curves by Gaussian function are shown in Fig. 5. The half peak width is set to 15 nm in the simulation. Obviously, all these emission peaks arise from S1→S0 electron transition corresponds dominated by HOMO (the highest occupied molecular orbital) →LUMO (the lowest unoccupied molecular orbital) (>90.4 %) except for acylamino and amide ion in gas phase. For acylamino in gas phase, the electronic transition is from HOMO-1 → LUMO (8.1 %) and HOMO → LUMO (80.8 %). For amide in gas phase, the electronic transition is from HOMO → LUMO (8.1 %), HOMO → LUMO + 1 (27.8 %) and HOMO → LUMO + 2 (60.0 %). For all the 3
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Journal of Photochemistry & Photobiology, A: Chemistry 406 (2021) 113000
+ Fig. 5. The simulated emission spectra of acylamino + NH+ 2 (a), acylamino (b), acylimino (c), amide + NH2 (d) and amide ion (e) in the gas phase, chloroform, acetone, DMSO and water.
+ + Fig. 6. The simulated emission spectra of acylamino-NH+ 2 +His (a), acylamino-NH2 +Arg (b), acylamino-NH2 +Lys (c), acylamino+His (d) acylamino + Arg (e) and acylamino + Lys (f) in the gas phase, chloroform, acetone, DMSO and water.
possible emitters, the corresponding oscillator strengths increase by increasing the polarity of the solvent. This indicates that the higher the polarity of the solvent, the stronger the luminescence. For the emission spectra of acylamino and acylimino, the same trends are shown as for the oscillator strengths discussed above. For acylamino-NH+ 2, + amide + NH+ 2 in gas phase and amide + NH2 in chloroform, the oscil lator strengths of electronic transition are almost zero, which can be considered as a forbidden transition. In polar medium (acetone, DMSO and water), the emission wavelengths varied little with solvent polarity (1− 17 nm). Fig. 6 Next, taking acylamino-NH+ 2 and acylamino as an example, we dis cussed the compositive effects of solvent polarity and basic amino acids on the Cypridina bioluminescence. Their excited state geometries were
optimized with SMD model by TD wb97xd/6− 31+G(d) method. The results of N1-H2 and N4-H5 bond length are shown in Table 2. The N1-H2 + bond lengths of acylamino-NH+ 2 +His, acylamino-NH2 +Arg, acylamino+ NH2 +Lys are slightly longer (0.004− 0.030 Å) than in sole acylamino+ NH+ 2 for the same solvent. The emission maxima of acylamino-NH2 on the effect of His, Arg, and Lys should be close to each other because of the similar structures. As shown in Table 1, Table S1 (Supporting In formation) and Fig. 6, All these emission peaks arise from S1 → S0 electron transition corresponds all exclusively to the promotion of an electron from HOMO to LUMO (89.1 %). For acylamino-NH+ 2 +His, + acylamino-NH+ 2 +Arg and acylamino-NH2 +Lys, the emission maxima are shifted to the red in the same solvent, which is comparable to the computed results for sole acylamino-NH+ 2 , the exception being 4
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covalent character of N1-H2 bond decrease, the acylamino-NH+ 2 geom etry evolves toward acylamino. At R(N1-H2) = 1.8 Å, acylamino is dominant. However, the emission wavelength and oscillator strength only have a little change along this transformation, because the values of emission wavelength and oscillator strength of acylamino-NH+ 2 and acylamino are close to each other under the effect of basic amino acids. At last, the covalent characters of the N4-H5 bond in acylamino+His were discussed (see Table 4). The covalent character of the N4-H5 bond was changed by enlarging the bond length of N4-H5 step by step. When the N4-H5 bond length increases, the N6-H5 bond length decreases, the negative charge on amide ion part increases, in contrast, the positive charge on His+H5 part increases. It showed that the strength of the covalent character of N4-H5 bond decrease, the acylamino geometry evolves toward to amide ion. The emission wavelength was gradually shifted to red along this transformation, while the oscillator strength decreased (f = 0.2717 to 0.0015). The calculated results determine that the less covalent the N4-H5 bond, the larger the emission wavelength, namely, the spectroscopic properties of acylamino are modulated by the covalent character of the N4-H5 bond. With N4-H5 bond length increasing, the weight of amide ion in the complex increase, the emis sion spectra shift to red. At R(N4-H5) = 1.7 Å, amide ion is dominant, the emission wavelength is equal to our previously calculation (see Table 1). It means amide ion is a free anion, that is to say, amide ion nearly forms a free ion-couple with His+H+ 5 . The currently computational results confirm that the emission spectrum of acyamino can be modulated by the covalent character of the N4-H5 bond. Furthermore, the fact that the formation of the amide ion is associated with f values close to zero, in dicates that this species should not be taken into consideration as a possible emitter.
Table 2 The N1-H2 and N4-H5 bond lengths of the first excited state acylamino-NH+ 2/ acylamino under the respective effects of His, Arg, and Lys in the gas phase, chloroform, acetone, DMSO and water predicted by TD wB97xd/6-31+G(d) method (unit: Å). acylamino-NH+ 2 acylamino-NH+ 2 +His acylamino-NH+ 2 +Arg acylamino-NH+ 2 +Lys acylamino acylamino+His acylamino + Arg acylamino + Lys
gas phase
chloroform
acetone
DMSO
water
1.009 1.030 1.022 1.039 1.015 1.029 1.036 1.031
1.011 1.027 1.026 1.038 1.016 1.031 1.040 1.039
1.011 1.026 1.028 1.039 1.017 1.032 1.041 1.042
1.011 1.026 1.028 1.040 1.017 1.031 1.041 1.042
1.012 1.016 1.030 1.039 1.014 1.026 1.036 1.041
acylamino-NH+ 2 +His in chloroform. For acylamino+His in chloroform, the emission maximum is slightly shifted to the blue, comparable to the computed results for sole acylamino-NH+ 2 His (1 nm). Due to the strong hydrogen bond typical of water adducts, we would focus on the emission properties of the adducts with His, Arg and Lys in water. According to Table 1, the predicted emission spectrum for acylamino-NH+ 2 +His is 390 nm which is shifted to the red compared to sole acylamino-NH+ 2. When Arg and Lys were included, the maxima shifted to 390 and 391 nm, respectively. The three red-shifts are due to N1-H2⋯N3 hydrogen bonds between the N1, H2 atom of the acylamino-NH+ 2 and N3 atom of His/Arg/Lys. Moreover, when the effect of basic amino acids was considered, the oscillator strengths also increased. To explain this, the Kohn-Sham frontier orbitals of acylamino + NH+ 2 in the gas phase (a) and chloro form (b), acylamino-NH+ 2 +His in the gas phase (c) and chloroform (d) are shown in Fig. 7. As shown in Fig. 7, the electronic clouds in HOMO of acylamino + NH+ 2 in the gas phase are mainly on the (S)-2-butyl and acetamidopyrazin moieties, while those acylamino + NH+ 2 in chloro form, acylamino-NH+ 2 +His in the gas phase and chloroform are delo calized over the 3-indolyl and acetamidopyrazin moieties. For LUMO, the electronic clouds of all cases are mainly on the acetamidopyrazin moiety. In a word, the blue shift and the change of oscillator strengths can be attributed to the different electron distribution. The same trends are shown for acylamino under the compositive effects of solvent polarity and basic amino acids. The N4-H5 bond lengths of acylamino+His, acylamino + Arg, acylamino + Lys are slightly longer (0.014− 0.027 Å) than in sole acylamino for the same solvent. The emission maxima of acylamino under the effect of His, Arg, and Lys are also close to those of sole acylamino in gas phase and solvents. It is concluded that the basicity of amino acid and polarity of the environ ment should only slightly affect the structure and spectroscopic prop erties of the acylamino-NH+ 2 and acylamino in the solvents.
4. Conclusion The solvent effect, the basic of amino acids and the degree of cova lent character of hydrogen bond on the spectrochemistry of Cypridina oxyluciferin were studied by a DFT and TD-DFT method. First, the solvent effect on the fluorescent spectra for all the possible emitters was calculated in vacuo, chloroform, acetone, DMSO and aqueous. For all the possible emitters, the corresponding oscillator strengths increase by increasing the polarity of the solvent. In polar medium (acetone, DMSO and water), the emission wavelengths varied little with solvent polarity. Second, the compositive effects of solvent polarity and basic amino acids on the Cypridina bioluminescence were considered. The emission maxima of acylamino-NH+ 2 and acylamino on the effect of His, Arg, and Lys are slightly shifted to the red in the same solvent. Moreover, the + emission spectra of acylamino-NH+ 2 +His, acylamino-NH2 +Arg and acylamino-NH+ 2 +Lys caused strong blue shift of 109− 121 nm compared with that of sole acylamino-NH+ 2 in the gas phase because of the different electron distribution. Third, the covalent character of hydrogen bond was considered by acylamino-NH+ 2 +His and acylamino+His models. For acylaminoNH+ 2 +His, when the N1-H2 bond length increases, the weight of acyla mino moiety increases. However, the emission wavelength and oscil lator strength only have a little change along this transformation. For acylamino+His, when the N4-H5 bond length increases, the negative charge on amide ion part increases. The emission wavelength was gradually shifted to red along this transformation, while the oscillator strength decreased. Furthermore, because f values of the formation of the amide ion is close to zero, it indicates that this species should not be taken into consideration as a possible emitter. The calculation and discussion in the paper offer reference for the design of Cypridina oxyluciferin analogues. More importantly, they offer help to Cypridina bioluminescence for better apply to bioanalysis, bio sensors, and biological imaging and other relevant areas.
3.2. Effect of covalent character of N1-H2 and N4-H5 bonds In order to see if the basic amino acids and polarity of the environ ment surrounding of acylamino-NH+ 2 and acylamino mainly change the covalent character of the N1-H2 and N4-H5 bonds, subsequently affect the fluorescent properties of acylamino-NH+ 2 and acylamino, the cova lent characters of the N1-H2 and N4-H5 bonds were discussed. Because the emission wavelengths of acylamino-NH+ 2 +His, acylamino+ NH+ 2 +Arg, acylamino-NH2 +Lys are close to each other, the fluorescent properties of acylamino+His, acylamino + Arg, acylamino + Lys are also close to each other, acylamino-NH+ 2 +His and acylamino+His are taken as an example to discuss the effect of changing the covalent character of the N1-H2 and N4-H5 bond. As shown in Table 3, the co valent character of the N1-H2 bond was changed by enlarging the bond length of N1-H2 step by step. When the N1-H2 bond length increases, the N3-H2 bond length decreases, at the same time, the mulliken charge on the acylamino part is close to zero. In contrast, the positive charge on His+H2 part is close to +1. It is indicated that as the strength of the 5
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+ + Fig. 7. Kohn-Sham frontier orbitals for acylamino + NH+ 2 in the gas phase (a), acylamino + NH2 in chloroform (b), acylamino-NH2 +His in the gas phase (c), + acylamino-NH2 +His in chloroform (d).
Table 3 The partially optimized main geometrical parameters (Å), the mulliken charge on acylamino part, the emission spectrum (nm) and oscillator strength (f) of acylamino-NH+ 2 +His are obtained by TD wb97xd/6-31+G(d) method.
Table 4 The partially optimized main geometrical parameters (Å), the mulliken charge on amide ion part, the emission spectrum (nm) and oscillator strength (f) of acylamino+His are obtained by TD wb97xd/6-31+G(d) method.
N1-H2
N3-H2
qacylamino
wavelengths
f
N4-H5
N6-H5
qamide
1.200 1.400 1.600 1.800 2.000
1.559 1.201 1.099 1.058 1.038
0.409 0.216 0.128 0.069 0.000
357 355 354 354 353
0.3094 0.3156 0.3176 0.3182 0.3206
1.200 1.400 1.600 1.700 1.800 2.000
1.678 1.193 1.087 1.062 1.042 1.027
− − − − − −
6
0.576 0.753 0.848 0.904 0.995 0.984
wavelengths
f
370 395 416 429 519 560
0.2717 0.1880 0.1096 0.0713 0.0017 0.0015
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CRediT authorship contribution statement
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Farkas, J. G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, o B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09.. Revision A. 02, Gaussian Inc., Wallingford CT, 2009. [28] J.D.M. Chai, M. Head-Gordon, Long-range corrected hybrid density functionals with damped atom-atom dispersion corrections, Phys. Chem. Chem. Phys. 10 (2008) 6615–6620. [29] C.G. Min, L. Pinto da Silva, J.C.G. Esteves da Silva, X.K. Yang, S.J. Huang, A. M. Ren, Y.Q. Zhu, Computational investigation of the fluorescent and chemiluminescent states equilibrium constants for coelenteramide, ChemPhysChem. 18 (2017) 117–123. [30] C.G. Min, P.J.O. Ferreira, L. Pinto da Silva, Theoretically obtained insight into the mechanism and dioxetanone species responsible for the singlet chemiexcitation of Coelenterazine, J. Photochem. Photobiol. B Biol. 174 (2017) 18–26. [31] A.V. Marenich, C.J. Cramer, D.G. 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Chun-Xia Liu: Data curation, Writing - original draft. Qing-Bo Liu: . Kun Dong: . Shao-Jun Huang: Conceptualization, Methodology, Soft ware. Xi-Kun Yang: Supervision. Ai-Min Ren: Software, Validation. Chun-Gang Min: Writing - review & editing. Gang Liu: Visualization, Investigation. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgements This work was supported by National Natural Science Foundation of China [grant numbers 11764026, 21473071]; Science and Technology Planning Project of Yunnan Province (grant number 202001AT070080); National Key Basic Research Program (grant number 2013CB834801, 973); Special Funding to Basic Scientific Research Projects for Central Colleges. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jphotochem.2020. 113000. References [1] O. Shimomura, F.H. Johnson, T. Masugi, Cypridina bioluminescence: light-emitting oxyluciferin-luciferase complex, Science 164 (1969) 1299–1300. [2] O. Shimomura, The discovery of aequorin and green fluorescent protein, J. Microsc. 217 (2005) 3–15. [3] O. Shimomura, Bioluminescence: Chemical Principles and Methods, World Scientific Publishing Co. Pte. Ltd., Singapore, 2006. [4] J. Vieira, L. Pinto da Silva, J.C.G. Esteves da Silva, Advances in the knowledge of light emission by firefly luciferin and oxyluciferin, J. Photochem. Photobiol. B 117 (2012) 33–39. [5] A. Roda, M. Guardigli, Analytical chemiluminescence and bioluminescence: latest achievements and new horizons, Anal. Bioanal. Chem. 402 (2012) 69–76. [6] A. Roda, M. Guardigli, P. Pasini, M. Mirasoli, E. Michelini, M. Musiani, Bio- and chemiluminescence imaging in analytical chemistry, Anal. Chim. Acta 541 (2005) 25–36. [7] Y. Yamada, S.Y. Nishide, Y. Nkajima, T. Watanabe, Y. Ohmiya, K. Honma, S. Honma, Monitoring circadian time in rat plasma using a secreted Cypridina luciferase reporter, Anal. Biochem. 439 (2013) 80–87. [8] C. Wu, K. Kawasaki, Y. Ogawa, Y. Yoshida, S. Ohgiya, Y. Ohmiya, Preparation of biotinylated Cypridina luciferase and its use in bioluminescent enzyme immunoassay, Anal. Chem. 79 (2007) 1634–1638. [9] C. Wu, S. Irie, S. Yamamoto, Y. Ohmiya, A bioluminescent enzyme immunoassay for prostaglandin E(2) using Cypridina luciferase, Luminescence 24 (2009) 131–133. [10] C. Wu, K.Y. Wang, X. Guo, M. Sato, M. Ozaki, S. Shimajiri, Y. Ohmiya, Y. Sasaguri, Rapid methods of detecting the target molecule in immunohistology using a bioluminescence probe, Luminescence 28 (2013) 38–43. [11] C. Wu, K. Mino, H. Akimoto, M. Kawabata, K. Nakamura, M. Ozaki, Y. Ohmiya, In vivo far-red luminescence imaging of a biomarker based on BRET from Cypridina bioluminescence to an organic dye, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 15599–15603. [12] B.W. Ding, P. Naumov, Y.J. Liu, Mechanistic insight into marine bioluminescence: photochemistry of the chemiexcited Cypridina (sea Firefly) lumophore, J. Chem. Theory Comput. 11 (2015) 591–599. [13] L. Pinto da Silva, R.F.J. Pereira, C.M. Magalh˜ aes, J.C.G. Esteves da Silva, Mechanistic insight into Cypridina bioluminescence with a combined experimental and theoretical chemiluminescent approach, J. Phys. Chem. B 121 (2017) 7862–7871.
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Journal of Photochemistry & Photobiology, A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem
Theoretically obtained insight into the effect of basic amino acids on Cypridina bioluminescence Chun-Xia Liu a, b, Qing-Bo Liu a, b, Kun Dong a, b, Shao-Jun Huang c, Xi-Kun Yang a, b, Ai-Min Ren d, Chun-Gang Min a, b, *, Gang Liu e, ** a
Research Center for Analysis and Measurement, Kunming University of Science and Technology, Kunming 650093 PR China Analysis and Test Center of Yunnan Province, Kunming 650093 PR China Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650093, PR China d Institute of Theoretical Chemistry, Jilin University, Changchun 130023, PR China e Institute of Chemical and Industrial Bioengineering, Jilin Engineering Normal University, Changchun 130052, PR China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Cypridina bioluminescence TDDFT Basic amino acid Hydrogen bond Solvent polarity
Because of high bioluminescent quantum yields, independent cofactors, high signal-to-noise ratio, Cypridina bioluminescence has been successfully applied to gene reporter assays, enzyme immunoassays, immunohistology and imaging. Although the chemical reaction that transforms Cypridina luciferin into Cypridina oxyluciferin is clearly defined, the environmental effects on the spectrochemistry of Cypridina oxyluciferin continue to be a particularly intriguing aspect. In this paper, the solvent effect on the fluorescent spectrum, the basicity of amino acids and the degree of covalent character of hydrogen bond were studied by a theoretical Density Functional Theory (DFT) and Time Dependent Density Functional Theory (TDDFT) approach. Our results demonstrate that the emission wavelengths varied little with solvent polarity. The compositive effects of basic amino acid and polarity of the environment only slightly affected the structure and spectroscopic properties. The fluorescent properties of Cypridina oxyluciferin can be modulated by the covalent character of the hydrogen bond.
1. Introduction The luminescence of the Cypridina is produced by the oxidation of Cypridina luciferin (C-LH2), catalyzed by luciferase to form Cypridina oxyluciferin (C-OxyLH2) in the first excited state (Fig. 1) [1,2]. Because of high bioluminescent quantum yields, independent cofactors, high signal-to-noise ratio [3–6], Cypridina bioluminescence has been suc cessfully applied to gene reporter assays [7], enzyme immunoassays [8, 9], immunohistology [10] and imaging [11]. The chemical reaction that transforms C-LH2 into C-OxyLH2 is clearly defined (Fig. 1) [12–14]. However, the environmental effects on the spectrochemistry of C-OxyLH2 continue to be a particularly intriguing aspect. C-OxyLH2 is usually thought to exist as acylamino or amide anion both in experimental and theoretical studies [12,13]. However, at least another three chemical forms may exist based on the chemical nature of the emitter: acylamino + NH+ 2 (protonation on N1 atom of 3-(1-guani dino) propyl, leading to a cation), acylimino (imide, neutral), amide + NH+ 2 (protonation on N1 atom of 3-(1-guanidino) propyl and
amide ion, leading to a neutral zwitterions), when the exact side chains were included (Fig. 2) [14]. As we known, the fluorescent spectra of C-OxyLH2 and its analogs are affected by pH value, solvent polarity and the presence of additives, which were extensively explored [15–21]. In 2012, Naumov et al. investigated the environmental effects on Cypridina bioluminescence [15]. The results found that the probable structure and emission spectrum of C-OxyLH2were strongly affected by polarity and basicity of the environment. In that paper, they recorded the absorption and emission spectra of C-OxyLH2 in 16 solvents of varying polarity, either without addition or with excess of 1,8-diazabicyclo[5.4.0]unde c-7-ene (DBU), which was used to simulate the presence of basic amino acids around the C-OxyLH2. However, DBU is different with basic amino acids. Moreover, the degree of covalent character of hydrogen bond between C-OxyLH2 and basic amino acid was not considered. In order to assess the solvent effect on the fluorescent spectrum, we studied the emission spectra of acylamino + NH+ 2 , acylamino, acylimino, amide + NH+ 2 and amide ion in chloroform, acetone, DMSO and water, respectively. For the polarization effect of the microenvironment of
* Corresponding author at: Research Center for Analysis and Measurement, Kunming University of Science and Technology, Kunming 650093 PR China. ** Corresponding author. E-mail addresses: [email protected] (C.-G. Min), [email protected] (G. Liu). https://doi.org/10.1016/j.jphotochem.2020.113000 Received 15 July 2020; Received in revised form 21 October 2020; Accepted 22 October 2020 Available online 27 October 2020 1010-6030/© 2020 Elsevier B.V. All rights reserved.
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Fig. 1. The reaction mechanism of the conversion of Cypridina luciferin to Cypridina oxyluciferin (X =H or none).
Fig. 2. Chemical structures of C-OxyLH2, the value in bracket is the total charge with various protonation states.
+ + Fig. 3. The models for acylamino + NH+ 2 +His, acylamino + NH2 +Arg and acylamino + NH2 +Lys and the formation of N1-H2…N3 hydrogen bond.
2
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Fig. 4. The models for acylamino+His, acylamino + Arg and acylamino + Lys and the formation of N4-H5…N6 hydrogen bond.
luciferase and the degree of covalent character of hydrogen bonds, because the crystal structure of Cypridina luciferase was not described, it cannot be evaluated. On the other hand, both Cypridina and firefly are endowed with the ability to emit light. The reaction mechanisms are similar. Cypridina/firefly luciferin interacts with molecular oxygen to form a high energetic intermediate, Cypridina/firefly dioxetanone. The decomposition of Cypridina/firefly dioxetanone generates Cypridina/ firefly oxyluciferin, in a singlet excited state, which subsequently decays to the ground state with multicolor light. Moreover, under the effect of luciferase, both Cypridina and firefly oxyluciferin may exist many pro tonation states [14,22]. We guess basic amino acids but not neutral amino acids appear to work in the luciferase. For firefly biolumines cence, removal of the positive charge of Arginine (Arg)218 and Histidine (His)245 on the crystal structure of the thermostable luciferase of the Japanese firefly (2D1R) resulted in a red-shift in firefly bioluminescence [23–25], our computational results were also agree with the conclusions of experimental studies [26]. Base on these literature reviews, we think Arg, His and Lysine (Lys), may be present in the active sites of Cypridina luciferase, and play an important role in spectroscopic properties. Moreover, because only N1 atom of 3-(1-guanidino) propyl group or N4 atom of amide may join with Arg, Lys, His to form N1-H2⋯N3 or N4-H5⋯N6 hydrogen bond, Arg, Lys, His were simply added near N1 atom or N4 atom to construct six models, acylamino + NH+ 2 +His, acy + lamino + NH+ 2 +Arg, acylamino + NH2 +Lys, acylamino+His, acyla mino + Arg and acylamino + Lys (Figs. 3 and 4 ).
Table 1 Predicted emission maxima (nm) and oscillator strengths (f) for all possible emitters and acylamino-NH+ 2 /acylamino under the respective effects of His, Arg, and Lys in the gas phase, chloroform, acetone, DMSO and water at the TD wb97xd/6-31+G(d)// TD wb97xd/6-31+G(d) levels.
acylamino + NH+ 2 acylamino acylimino amide + NH+ 2 amide ion acylaminoNH+ 2 +His acylaminoNH+ 2 +Arg acylaminoNH+ 2 +Lys acylamino+His acylamino + Arg acylamino + Lys
gas phase
chloroform
acetone
DMSO
water
475 (0.0008) 352 (0.3311) 425 (0.1866) 933 (0.0004) 429 (0.1001) 358 (0.3061) 354 (0.1818) 366 (0.3017) 352 (0.3364) 355 (0.3333) 358 (0.3120)
376 (0.5764) 373 (0.6204) 444 (0.3885) 607 (0.0119) 415 (0.3526) 375 (0.5739) 377 (0.5847) 378 (0.6150) 371 (0.6229) 373 (0.6026) 374 (0.6248)
383 (0.6760) 383 (0.7195) 453 (0.4772) 549 (0.3376) 424 (0.4714) 386 (0.6870) 386 (0.6987) 385 (0.7050) 382 (0.7320) 385 (0.7064) 383 (0.7182)
385 (0.6965) 385 (0.7372) 455 (0.4963) 552 (0.3526) 428 (0.4777) 388 (0.7021) 388 (0.7196) 388 (0.7267) 383 (0.7367) 387 (0.7193) 384 (0.7358)
383 (0.7311) 386 (0.7593) 472 (0.5014) 536 (0.4824) 419 (0.5885) 390 (0.7366) 390 (0.7511) 391 (0.7587) 384 (0.7834) 392 (0.7888) 387 (0.7897)
2. Theoretical methods were relaxed during optimizations. The excited state optimizations were obtained by TD wB97XD/6− 31+G(d). Based on the optimized geome tries, the emission spectra were calculated by the same method.
All theoretical predictions were made with the Gaussian09 program package [27] suite using the wB97XD/6− 31+G(d) method [28]. This method was able to describe relatively accurate structure parameters, emission spectra for C-OxyLH2 [14] and Coelenteramide [29,30]. First, in order to consider the solvent effect on the fluorescent spectra of C-OxyLH2, the excited-state geometries of acylamino-NH+ 2 , acyla mino, acylimino, amide + NH+ 2 and amide ion were optimized by TD wB97XD/6− 31+G(d) method in vacuo, chloroform, acetone, DMSO and water using SMD model (universal salvation model based on solute electron density) [31]. Based on the optimized structure, the emission spectra were computed by the same method. Second, the compositive effects of basic amino acid and polarity of the environment surrounding acylamino + NH+ 2 and acylamino were mimicked by the simple models, acylamino + NH+ 2 +His, acyla mino + NH+ acylamino + NH+ acylamino+His, acyla 2 +Arg, 2 +Lys, mino + Arg and acylamino + Lys in vacuo, chloroform, acetone, DMSO and water using SMD model. TD wB97XD/6− 31+G(d) method were also used to optimize the excited-state structure and compute the emission spectra. At last, to simulate the different degree of covalent character of the N1-H2 and N4-H5 bonds, we constrained bond lengths of N1-H2 and N4H5, and increased it step by step. That is, for a given N1-H2 and N4-H5 distance ranging from 1.2 to 2.0 Å, all the remaining degrees of freedom
3. Results and discussion 3.1. The effect of solvent polarity and basic amino acids on the Cypridina bioluminescence To assess the effects of solvent polarity on the fluorescent spectra, the emission spectra, oscillator strengths (f) and main transition configu rations for all the possible emitters were calculated with TD wb97xd/ 6− 31+G (d) method in vacuo, chloroform, acetone, DMSO and aqueous based on the first excited-state optimized geometries. The data are listed in Table 1 and Table S1 (Supporting Information). Fitted curves by Gaussian function are shown in Fig. 5. The half peak width is set to 15 nm in the simulation. Obviously, all these emission peaks arise from S1→S0 electron transition corresponds dominated by HOMO (the highest occupied molecular orbital) →LUMO (the lowest unoccupied molecular orbital) (>90.4 %) except for acylamino and amide ion in gas phase. For acylamino in gas phase, the electronic transition is from HOMO-1 → LUMO (8.1 %) and HOMO → LUMO (80.8 %). For amide in gas phase, the electronic transition is from HOMO → LUMO (8.1 %), HOMO → LUMO + 1 (27.8 %) and HOMO → LUMO + 2 (60.0 %). For all the 3
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+ Fig. 5. The simulated emission spectra of acylamino + NH+ 2 (a), acylamino (b), acylimino (c), amide + NH2 (d) and amide ion (e) in the gas phase, chloroform, acetone, DMSO and water.
+ + Fig. 6. The simulated emission spectra of acylamino-NH+ 2 +His (a), acylamino-NH2 +Arg (b), acylamino-NH2 +Lys (c), acylamino+His (d) acylamino + Arg (e) and acylamino + Lys (f) in the gas phase, chloroform, acetone, DMSO and water.
possible emitters, the corresponding oscillator strengths increase by increasing the polarity of the solvent. This indicates that the higher the polarity of the solvent, the stronger the luminescence. For the emission spectra of acylamino and acylimino, the same trends are shown as for the oscillator strengths discussed above. For acylamino-NH+ 2, + amide + NH+ 2 in gas phase and amide + NH2 in chloroform, the oscil lator strengths of electronic transition are almost zero, which can be considered as a forbidden transition. In polar medium (acetone, DMSO and water), the emission wavelengths varied little with solvent polarity (1− 17 nm). Fig. 6 Next, taking acylamino-NH+ 2 and acylamino as an example, we dis cussed the compositive effects of solvent polarity and basic amino acids on the Cypridina bioluminescence. Their excited state geometries were
optimized with SMD model by TD wb97xd/6− 31+G(d) method. The results of N1-H2 and N4-H5 bond length are shown in Table 2. The N1-H2 + bond lengths of acylamino-NH+ 2 +His, acylamino-NH2 +Arg, acylamino+ NH2 +Lys are slightly longer (0.004− 0.030 Å) than in sole acylamino+ NH+ 2 for the same solvent. The emission maxima of acylamino-NH2 on the effect of His, Arg, and Lys should be close to each other because of the similar structures. As shown in Table 1, Table S1 (Supporting In formation) and Fig. 6, All these emission peaks arise from S1 → S0 electron transition corresponds all exclusively to the promotion of an electron from HOMO to LUMO (89.1 %). For acylamino-NH+ 2 +His, + acylamino-NH+ 2 +Arg and acylamino-NH2 +Lys, the emission maxima are shifted to the red in the same solvent, which is comparable to the computed results for sole acylamino-NH+ 2 , the exception being 4
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covalent character of N1-H2 bond decrease, the acylamino-NH+ 2 geom etry evolves toward acylamino. At R(N1-H2) = 1.8 Å, acylamino is dominant. However, the emission wavelength and oscillator strength only have a little change along this transformation, because the values of emission wavelength and oscillator strength of acylamino-NH+ 2 and acylamino are close to each other under the effect of basic amino acids. At last, the covalent characters of the N4-H5 bond in acylamino+His were discussed (see Table 4). The covalent character of the N4-H5 bond was changed by enlarging the bond length of N4-H5 step by step. When the N4-H5 bond length increases, the N6-H5 bond length decreases, the negative charge on amide ion part increases, in contrast, the positive charge on His+H5 part increases. It showed that the strength of the covalent character of N4-H5 bond decrease, the acylamino geometry evolves toward to amide ion. The emission wavelength was gradually shifted to red along this transformation, while the oscillator strength decreased (f = 0.2717 to 0.0015). The calculated results determine that the less covalent the N4-H5 bond, the larger the emission wavelength, namely, the spectroscopic properties of acylamino are modulated by the covalent character of the N4-H5 bond. With N4-H5 bond length increasing, the weight of amide ion in the complex increase, the emis sion spectra shift to red. At R(N4-H5) = 1.7 Å, amide ion is dominant, the emission wavelength is equal to our previously calculation (see Table 1). It means amide ion is a free anion, that is to say, amide ion nearly forms a free ion-couple with His+H+ 5 . The currently computational results confirm that the emission spectrum of acyamino can be modulated by the covalent character of the N4-H5 bond. Furthermore, the fact that the formation of the amide ion is associated with f values close to zero, in dicates that this species should not be taken into consideration as a possible emitter.
Table 2 The N1-H2 and N4-H5 bond lengths of the first excited state acylamino-NH+ 2/ acylamino under the respective effects of His, Arg, and Lys in the gas phase, chloroform, acetone, DMSO and water predicted by TD wB97xd/6-31+G(d) method (unit: Å). acylamino-NH+ 2 acylamino-NH+ 2 +His acylamino-NH+ 2 +Arg acylamino-NH+ 2 +Lys acylamino acylamino+His acylamino + Arg acylamino + Lys
gas phase
chloroform
acetone
DMSO
water
1.009 1.030 1.022 1.039 1.015 1.029 1.036 1.031
1.011 1.027 1.026 1.038 1.016 1.031 1.040 1.039
1.011 1.026 1.028 1.039 1.017 1.032 1.041 1.042
1.011 1.026 1.028 1.040 1.017 1.031 1.041 1.042
1.012 1.016 1.030 1.039 1.014 1.026 1.036 1.041
acylamino-NH+ 2 +His in chloroform. For acylamino+His in chloroform, the emission maximum is slightly shifted to the blue, comparable to the computed results for sole acylamino-NH+ 2 His (1 nm). Due to the strong hydrogen bond typical of water adducts, we would focus on the emission properties of the adducts with His, Arg and Lys in water. According to Table 1, the predicted emission spectrum for acylamino-NH+ 2 +His is 390 nm which is shifted to the red compared to sole acylamino-NH+ 2. When Arg and Lys were included, the maxima shifted to 390 and 391 nm, respectively. The three red-shifts are due to N1-H2⋯N3 hydrogen bonds between the N1, H2 atom of the acylamino-NH+ 2 and N3 atom of His/Arg/Lys. Moreover, when the effect of basic amino acids was considered, the oscillator strengths also increased. To explain this, the Kohn-Sham frontier orbitals of acylamino + NH+ 2 in the gas phase (a) and chloro form (b), acylamino-NH+ 2 +His in the gas phase (c) and chloroform (d) are shown in Fig. 7. As shown in Fig. 7, the electronic clouds in HOMO of acylamino + NH+ 2 in the gas phase are mainly on the (S)-2-butyl and acetamidopyrazin moieties, while those acylamino + NH+ 2 in chloro form, acylamino-NH+ 2 +His in the gas phase and chloroform are delo calized over the 3-indolyl and acetamidopyrazin moieties. For LUMO, the electronic clouds of all cases are mainly on the acetamidopyrazin moiety. In a word, the blue shift and the change of oscillator strengths can be attributed to the different electron distribution. The same trends are shown for acylamino under the compositive effects of solvent polarity and basic amino acids. The N4-H5 bond lengths of acylamino+His, acylamino + Arg, acylamino + Lys are slightly longer (0.014− 0.027 Å) than in sole acylamino for the same solvent. The emission maxima of acylamino under the effect of His, Arg, and Lys are also close to those of sole acylamino in gas phase and solvents. It is concluded that the basicity of amino acid and polarity of the environ ment should only slightly affect the structure and spectroscopic prop erties of the acylamino-NH+ 2 and acylamino in the solvents.
4. Conclusion The solvent effect, the basic of amino acids and the degree of cova lent character of hydrogen bond on the spectrochemistry of Cypridina oxyluciferin were studied by a DFT and TD-DFT method. First, the solvent effect on the fluorescent spectra for all the possible emitters was calculated in vacuo, chloroform, acetone, DMSO and aqueous. For all the possible emitters, the corresponding oscillator strengths increase by increasing the polarity of the solvent. In polar medium (acetone, DMSO and water), the emission wavelengths varied little with solvent polarity. Second, the compositive effects of solvent polarity and basic amino acids on the Cypridina bioluminescence were considered. The emission maxima of acylamino-NH+ 2 and acylamino on the effect of His, Arg, and Lys are slightly shifted to the red in the same solvent. Moreover, the + emission spectra of acylamino-NH+ 2 +His, acylamino-NH2 +Arg and acylamino-NH+ 2 +Lys caused strong blue shift of 109− 121 nm compared with that of sole acylamino-NH+ 2 in the gas phase because of the different electron distribution. Third, the covalent character of hydrogen bond was considered by acylamino-NH+ 2 +His and acylamino+His models. For acylaminoNH+ 2 +His, when the N1-H2 bond length increases, the weight of acyla mino moiety increases. However, the emission wavelength and oscil lator strength only have a little change along this transformation. For acylamino+His, when the N4-H5 bond length increases, the negative charge on amide ion part increases. The emission wavelength was gradually shifted to red along this transformation, while the oscillator strength decreased. Furthermore, because f values of the formation of the amide ion is close to zero, it indicates that this species should not be taken into consideration as a possible emitter. The calculation and discussion in the paper offer reference for the design of Cypridina oxyluciferin analogues. More importantly, they offer help to Cypridina bioluminescence for better apply to bioanalysis, bio sensors, and biological imaging and other relevant areas.
3.2. Effect of covalent character of N1-H2 and N4-H5 bonds In order to see if the basic amino acids and polarity of the environ ment surrounding of acylamino-NH+ 2 and acylamino mainly change the covalent character of the N1-H2 and N4-H5 bonds, subsequently affect the fluorescent properties of acylamino-NH+ 2 and acylamino, the cova lent characters of the N1-H2 and N4-H5 bonds were discussed. Because the emission wavelengths of acylamino-NH+ 2 +His, acylamino+ NH+ 2 +Arg, acylamino-NH2 +Lys are close to each other, the fluorescent properties of acylamino+His, acylamino + Arg, acylamino + Lys are also close to each other, acylamino-NH+ 2 +His and acylamino+His are taken as an example to discuss the effect of changing the covalent character of the N1-H2 and N4-H5 bond. As shown in Table 3, the co valent character of the N1-H2 bond was changed by enlarging the bond length of N1-H2 step by step. When the N1-H2 bond length increases, the N3-H2 bond length decreases, at the same time, the mulliken charge on the acylamino part is close to zero. In contrast, the positive charge on His+H2 part is close to +1. It is indicated that as the strength of the 5
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+ + Fig. 7. Kohn-Sham frontier orbitals for acylamino + NH+ 2 in the gas phase (a), acylamino + NH2 in chloroform (b), acylamino-NH2 +His in the gas phase (c), + acylamino-NH2 +His in chloroform (d).
Table 3 The partially optimized main geometrical parameters (Å), the mulliken charge on acylamino part, the emission spectrum (nm) and oscillator strength (f) of acylamino-NH+ 2 +His are obtained by TD wb97xd/6-31+G(d) method.
Table 4 The partially optimized main geometrical parameters (Å), the mulliken charge on amide ion part, the emission spectrum (nm) and oscillator strength (f) of acylamino+His are obtained by TD wb97xd/6-31+G(d) method.
N1-H2
N3-H2
qacylamino
wavelengths
f
N4-H5
N6-H5
qamide
1.200 1.400 1.600 1.800 2.000
1.559 1.201 1.099 1.058 1.038
0.409 0.216 0.128 0.069 0.000
357 355 354 354 353
0.3094 0.3156 0.3176 0.3182 0.3206
1.200 1.400 1.600 1.700 1.800 2.000
1.678 1.193 1.087 1.062 1.042 1.027
− − − − − −
6
0.576 0.753 0.848 0.904 0.995 0.984
wavelengths
f
370 395 416 429 519 560
0.2717 0.1880 0.1096 0.0713 0.0017 0.0015
C.-X. Liu et al.
Journal of Photochemistry & Photobiology, A: Chemistry 406 (2021) 113000
CRediT authorship contribution statement
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Chun-Xia Liu: Data curation, Writing - original draft. Qing-Bo Liu: . Kun Dong: . Shao-Jun Huang: Conceptualization, Methodology, Soft ware. Xi-Kun Yang: Supervision. Ai-Min Ren: Software, Validation. Chun-Gang Min: Writing - review & editing. Gang Liu: Visualization, Investigation. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgements This work was supported by National Natural Science Foundation of China [grant numbers 11764026, 21473071]; Science and Technology Planning Project of Yunnan Province (grant number 202001AT070080); National Key Basic Research Program (grant number 2013CB834801, 973); Special Funding to Basic Scientific Research Projects for Central Colleges. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jphotochem.2020. 113000. References [1] O. Shimomura, F.H. Johnson, T. Masugi, Cypridina bioluminescence: light-emitting oxyluciferin-luciferase complex, Science 164 (1969) 1299–1300. [2] O. Shimomura, The discovery of aequorin and green fluorescent protein, J. Microsc. 217 (2005) 3–15. [3] O. Shimomura, Bioluminescence: Chemical Principles and Methods, World Scientific Publishing Co. Pte. Ltd., Singapore, 2006. [4] J. Vieira, L. Pinto da Silva, J.C.G. Esteves da Silva, Advances in the knowledge of light emission by firefly luciferin and oxyluciferin, J. Photochem. Photobiol. B 117 (2012) 33–39. [5] A. Roda, M. Guardigli, Analytical chemiluminescence and bioluminescence: latest achievements and new horizons, Anal. Bioanal. Chem. 402 (2012) 69–76. [6] A. Roda, M. Guardigli, P. Pasini, M. Mirasoli, E. Michelini, M. Musiani, Bio- and chemiluminescence imaging in analytical chemistry, Anal. Chim. Acta 541 (2005) 25–36. [7] Y. Yamada, S.Y. Nishide, Y. Nkajima, T. Watanabe, Y. Ohmiya, K. Honma, S. Honma, Monitoring circadian time in rat plasma using a secreted Cypridina luciferase reporter, Anal. Biochem. 439 (2013) 80–87. [8] C. Wu, K. Kawasaki, Y. Ogawa, Y. Yoshida, S. Ohgiya, Y. Ohmiya, Preparation of biotinylated Cypridina luciferase and its use in bioluminescent enzyme immunoassay, Anal. Chem. 79 (2007) 1634–1638. [9] C. Wu, S. Irie, S. Yamamoto, Y. Ohmiya, A bioluminescent enzyme immunoassay for prostaglandin E(2) using Cypridina luciferase, Luminescence 24 (2009) 131–133. [10] C. Wu, K.Y. Wang, X. Guo, M. Sato, M. Ozaki, S. Shimajiri, Y. Ohmiya, Y. Sasaguri, Rapid methods of detecting the target molecule in immunohistology using a bioluminescence probe, Luminescence 28 (2013) 38–43. [11] C. Wu, K. Mino, H. Akimoto, M. Kawabata, K. Nakamura, M. Ozaki, Y. Ohmiya, In vivo far-red luminescence imaging of a biomarker based on BRET from Cypridina bioluminescence to an organic dye, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 15599–15603. [12] B.W. Ding, P. Naumov, Y.J. Liu, Mechanistic insight into marine bioluminescence: photochemistry of the chemiexcited Cypridina (sea Firefly) lumophore, J. Chem. Theory Comput. 11 (2015) 591–599. [13] L. Pinto da Silva, R.F.J. Pereira, C.M. Magalh˜ aes, J.C.G. Esteves da Silva, Mechanistic insight into Cypridina bioluminescence with a combined experimental and theoretical chemiluminescent approach, J. Phys. Chem. B 121 (2017) 7862–7871.
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