Hydrogen Bonding Promoted Tautomerism between Azo and

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Hydrogen Bonding Promoted Tautomerism Between Azo and Hydrazone Forms in Calcon with Multi-Stimuli Responsiveness and Biocompatibility Dong Zheng, Yuming Gu, Xiang Li, Lizhu Zhang, Wei Zhao, and Jing Ma J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00985 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 19, 2019

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Journal of Chemical Information and Modeling

Hydrogen Bonding Promoted Tautomerism Between Azo and Hydrazone Forms in Calcon with Multi-Stimuli Responsiveness and Biocompatibility 1,2

1,2

1,3

3

4

1,2,3*

Dong Zheng, Yuming Gu, Xiang Li, Lizhu Zhang, Wei Zhao and Jing Ma 1

Key Laboratory of Mesoscopic Chemistry of Ministry of Education, School of Chemistry and

Chemical Engineering, Nanjing University, Nanjing, 210023 (P. R. China) 2

Jiangsu Key Laboratory of Advanced Organic Materials, School of Chemistry and Chemical

Engineering Nanjing University, Nanjing 210023 (P. R. China) 3

Nanxin pharm Co., Ltd. Nanjing 210023 (P. R. China)

4

Department of Clinical Laboratory, Obstetrics and Gynecology Hospital Affiliated to Nanjing

Medical University, Nanjing, 210011 (P. R. China)

Abstract: Realization of multi-stimuli responsiveness in one molecule remains a challenge due to the difficulty in understanding and control of comprehensive interplay between the external stimuli and the subtle conformation changes. The coexistence of dynamic bonding interactions, hydroxyl group and the azo chromophore in calcon causes the multi-stimuli responsiveness to external stimuli including temperature, pH-variation and light-irradiation. Density functional theory (DFT), time-dependent DFT (TDDFT), and various molecular dynamics (MD) simulations are employed to systematically investigate the azo-hydrazone tautomerism and E-Z isomerization. The inter/intramolecular hydrogen bonding interactions promote the azohydrazone tautomerism at different pH conditions. The strong n→π* absorption in the visible light region gives an advantage of calcon without the harm to living cells from UV light. The facial tautomerism renders the calcon temperature-sensitivity, which could be triggered at body temperature (311K) with distinct color change from red to blue. It is also found that in pH= 6.8 both azo and hydrazone isomers have no cytotoxicity on the human lung cells (A549 and H1299) and hepatic epithelial cell of rat (FL83B). The visible-light absorption, pH and temperature sensitiveness and biocompatibility render calcon potential candidates for biomedical applications.

1

ACS Paragon Plus Environment

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Introduction The stimuli-responsive materials have provided new perspectives in many applications of 1-4

smart sensors, optoelectronic devices, drug delivery, and therapeutic diagnosis.

Smart materials

could convert external stimulus (light, pH, temperature, chemical, electric or magnetic field) into observable changes of states, such as color variations, mechanical movements and electric 5

conductivity changes. It is desirable to select different functional motifs from the toolbox to assemble the novel multi-stimuli responsive systems. Among them, explorations of multi-stimuli responsiveness in just one system are of great importance, since single-responsive materials may not achieve the desired goals in many complicated environments including microenvironments in normal or diseased tissues with the presence of various biological and physical triggers (e.g., 4

pH, light, reduction, enzyme, reactive oxygen species, and so on). The facile stimuli-induced variation of the coordination modes of transition metals or the charge transfer between the metal and ligands were used to build multi-responsive organometallic complexes, e.g., the Platinum ( Ⅱ ) with tetradentate chelating framework, which could realize sensitive phosphorescence switching in response to the change of temperature, pressure and UV irradiation.

2

The dynamic

noncovalent interactions and host-guest interactions between macrocycle and rod in [n]rotaxane 6

make it widely used in fabricating multi-stimuli responsive supramolecular systems. The polymer also belongs to a kind of promising candidates to build multi-stimuli responsive materials by accommodating the multi-stimuli responsive units in its long backbone or sidechains.

[4]

For

example, the dual stimuli-responsive system was fabricated by grafting both pH-responsive dimethylaminoethyl methacrylate and light-responsive 2-methyl-4-phenylazo acrylate(MPA7

azo). Although azobenzene derivatives with azo chromophore are widely used as the photoswitches between the trans (E) and cis (Z) isomers, most of them have absorption peak in the ultra violet (UV) light region at the trans (or E) configurations and their isomerizations to the

cis (Z) ones could be just triggered by UV light, which is harmful to biological systems.8 The design 2


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of visible or near-infrared light triggered light-responsive systems are hence desirable to extend the application of multi-stimuli responsive materials to biomedical and photopharmaceuticals. Calcon, also called Eriochrome Blue Black B, (1-(2-hydroxy-1-naphthylazo)-2-naphthol-4sulphonic acid, is one of such multi-stimuli responsive species with strong visible light absorption. The multi-dentate ligands (O, N atom and the sulfate group) were used to coordinate with various metal ions, hence making calcon being used in wastewater treatments.

9, 10

The calcon could also

function as biological probe through π-π and electrostatic interactions of biomolecules (protein, 11

DNA and RNA) with naphthalene ring and the sulfonate group, respectively. In fact, it will be demonstrated in the present work that the coexistence of the hydroxyl group and the azo chromophore leads to the temperature, pH, and light-responsiveness. Under different pH conditions, three protons in the calcon molecule can be successively deprotonated. The hydrogen atoms in the sulfonic acid (H1), hydroxyl groups, H2 (the hydroxyl group lies in the meta-position of the sulfonic group) and H3 (the other hydroxyl group) are presented in Figure 1a. The H1 of the sulfonic acid is easily deprotonated, in corresponding to the first acid dissociation constant of pKa1 = 1.05. The two protons in two hydroxyl groups could be successively deprotonated by 12

addition of the alkaline species (pKa2 = 7.21; pKa3 = 13.43 ). The H3 will be deprotonated before H2, due to the stronger acidity of the hydroxyl group in the naphthalene ring without sulfonate group. DFT calculations with polarizable continuum model (PCM) at the level of B3LYP/6-31G(d) have been carried out to investigate the acid-base, azo-hydrazone, and aggregate equilibria of 13

calcon.

However, the influence of intermolecular and intramolecular hydrogen bonding

interactions on isomerizations of calcon in aqueous solution was neglected in the previous DFT calculations. In this article, density functional theory (DFT), time-dependent density functional theory (TDDFT) and molecular dynamics (MD) simulations are employed to systematically investigate the azo-hydrazone tautomerism and E-Z isomerization with and without the presence of water solvent molecules. It will be shown that the intermolecular and intramolecular hydrogen bonding interactions promote the azo-hydrazone tautomerism between the aromatic azo structure and 3



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the quinoid hydrazone structure in different pH conditions (Figure 1b). The UV-vis spectroscopy will reveal that both the temperature and the pH could trigger the azo-hydrazone tautomerism, in good agreement with theoretical calculations. The enhanced n→π* transition in quinoid hydrazone form gives rise to the red shift of absorption peak in visible light region, allowing the biological applications of calcon under the harmless visible light. It is also found that in pH= 6.8 both azo and hydrazone isomers have no cytotoxicity on the human lung cells (A549 and H1299) and hepatic epithelial cell of rat (FL83B). The atomic level information of the interplay between the external stimuli and the subtle conformation change might be useful in rational design of the multi-stimuli responsive materials.

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(a)

(b) pH Azo

DE DE azo-hydrazone azo-hydrazone

Hydrazone

12

1.6 kcal/mol

40 kcal/mol

-H1-H2

-H1-H2+HN2 Azo-to-hydrazone transition state

Azo (Z )

4.0 -H1

500

Azo(E)

293

kcal/mol 303

lower barrier for tautomerism with increasing pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-H1-HN1

323 Temperature (K)

313

heating triggered tautomerism

600

Azo Hydrazone Figure 1.（a）The intramolecular hydrogen transfer induced the dynamic change between aromatic structure (azo form) and quinone structure ( hydrazone form) in calcon. (b) The pH, light, and temperature-sensitiveness of the calcon.

2 Materials and methods 2.1 Density functional theory calculations of electronic structure properties. The ground states of calcon with different protonated degrees (including azo and hydrazone tautomers) were optimized by using the density functional theory (DFT) method with B3LYP hybrid functional and 5



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14

6-311+G(d, p) basis set level using Gaussian 09 program package. Polarizable continuum model (PCM) was employed to consider the long-ranged solvent effect of aqueous solutions. The shortranged solvent effects, especially the intermolecular HB between calcon and water molecules are involved in cluster models, which were sampled from the molecular dynamics simulations. The vibrational frequencies of each conformation were computed at the optimized geometries to judge the local minima or transition states on the potential energy surfaces. The activation energy (Ea) of the Z-to-E isomerization was estimated by scanning the C-N=N-C torsion angle along the potential energy curve.

2.2 Molecular dynamics simulations of aqueous solutions

2.2.1 Ab Initio Molecular dynamics simulations for azo-hydrazone tautomerism. Due to the small activation barrier（less than 4 kcal/mol at B3LYP/6-311+G(d,p) level）of the azo-hydrazone tautomerism, ab initio molecular dynamics (AIMD) simulation is able to model the azo-hydrazone tautomerism pathways. The cubic box with periodic boundary condition (PBC) was set to 13Å×13Å×13Å with two calcon molecules surrounded by 20 water molecules, 3

3

corresponding to the density of 1 g/cm and the calcon concentration of about 1.5×10 mol/L. The AIMD simulations were performed in the DMol3 module of Materials studio.

15

The

generalized gradient approximation (GGA) in Perdew-Burke-Ernzerhof (PBE) with Grimme methods for DFT-D correction and gaussian double zeta plus polarization function (DNP) basis 16-18

set were employed to perform the AIMD simulations.

The (1×1×1) k-point was used for the

brillouin zone sampling. The canonical NVT thermodynamic ensemble and Generalized Gaussian Moment were used for temperature control at 313 K. The 1.6 ps AIMD simulations were conducted with a time step of 0.4 fs, since azo-hydrazone tautomerism finishes within 1ps. 2.2.2 Molecular dynamic simulations to investigate the molecular aggregation.

Going

forward to the more concentrated aqueous solution of calcon, we resorted to the economic force-field based molecular dynamics (MD) simulations, within the framework of the polymer 6


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consistent force field (PCFF).

19-22

Starting from DFT optimized structures, two models were set to

represent the dilute and concentrated calcon solutions, respectively. The 40Å×40Å×40Å cubic box contains one (or ten) calcon molecules and 1000 water molecules, corresponding to the calcon concentration of 2.6 (or 26.0) mol/L. The MD simulations of calcon aqueous solutions were carried out in the canonical NVT ensemble at 298 K by using the Andersen thermostat. The cut-off of vdW interaction is set to be 15.5 Å. The electrostatic interaction was evaluated by the Ewald summation. Motion equations were integrated by using the velocity verlet algorithm with a timestep of 1 fs. The 3 ns MD simulations indicate the molecular aggregation of calcon molecules in the condensed solution of about 26.0 mol/L. Detailed statistic analysis was conducted to show the favorable π-π and T-shaped stacking modes of calcon molecules. 2.2.3 Reactive Molecular Dynamics simulations for Z-to-E isomerization in different pH conditions. Traditional force-field based molecular dynamics simulations could not be used to study the

Z-to-E isomerization of azobenzene derivatives through the N=N bond rotation or the C-N=N bond angle inversion. In order to simulate the dynamic Z-to-E conversion of calcon in different pH conditions, reactive molecular dynamics (RMD) simulations were performed with some 23-25

modifications on the C-N=N-C torsion potential and C-N=N inversion potential.

The RMD

simulations have been used in our group to investigate the isomerization processes of azobenzene derivatives in various complex environments, such as the self-assembling monolayers on the Au(111) surface, the collective switching behaviour of thiolate-protected Au25 nanoparticles, and the shuttling movements of azobenzene contained [2]rotaxane upon light26 24 23, 25

irradiation.

The torsion potential, Etorsion, of the N=N bond and the inversion function, Ebend,

of the C-N=N angle in the simulations are given as follows 𝐸"#$%&#' (∅) = 𝐾∅ (1 + cos (𝑛∅ − ∅5 )) 𝐸67'8 (𝜃) = 𝐾: (𝜃 − 𝜃5 );

(1) (2)

Where the Kφ is set as the lowest vertical excitation energy, which came from the TDDFT calculations or experimental UV-vis absorption spectra. We used a canonical NVT ensemble at 7



Page 8 of 34

298 K with a Nose thermostat and the velocity verlet algorithm with a time step of 1 fs. All the systems (one calcon molecule with different protonation states, solvated with 1000 water molecules) are in the presence of periodic boundary condition (PBC) model with the size of 31Å×31Å×31Å. Twenty independent simulations were performed starting from the Z form of different protonation state of calcon. The details of the modified parameters were presented in Table S1. 2.3 The pH and temperature triggered tautomerism detected by UV-vis spectra experiments and calculations. Due to the unique visible light absorption property of calcon, the UV-vis spectroscopy was employed to monitor the dynamic process of the pH and temperature triggered azo-hydrazone tautomerism and the Z-to-E isomerization. The calcon was purchased from J&K scientific and used without additional purification. Solutions of calcon were prepared by dissolving the weighed amount of the target compound in the required amount of the water solvent. UV-vis spectra of calcon have been recorded in their aqueous solutions with the 5

concentration of 4.0 ×10− M. The acidity and alkaline of the test solutions were adjusted by -1

-2

-3

-4

-5

adding different concentrations of the HCl and NaOH solutions (10 , 10 , 10 , 10 , 10 mol/L), respectively. All pH measurements were detected with a pB-10 digital pH meter (Sartorius, Germany) at room temperature. The UV-vis absorption spectra were detected from UV-2600 (SHIMADZU, Japan) using a quartz glass cell with a path length of 10 mm at different temperatures, respectively. The temperature sensitiveness of calcon was tested in the range from 293 K to 313 K by using a heating stage (TCC-100) attached to the chamber and then maintained using a calibrated temperature controller connected to the stage. The spectra of the solution were measured every 0.5 hour with an UV-vis spectrometer at different temperatures. Electronic absorption spectra of all the possible tautomeric forms, which were sampled from MD trajectories, were modeled by using time-dependent density functional theory (TDDFT) calculations of the lowest electronic excitation energies for aqueous solutions of calcon under different pH values. In addition, the frontier molecular orbital analysis of azo and hydrazone tautomeric forms were also presented to assign the possible transition modes in the UV-vis 8


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spectra. 2.4 High resolution mass spectrometry for calcon in alkaline conditions. The azonium ionic form of calcon is not very stable in strong alkaline conditions (pH=11.3) with the color loss from blue to pale yellow after one hour, suggesting the disappearance of the azo chromophore in some degree. In order to investigate the reason behind the color loss, the HPLC-MS experiment was performed in the ltq-orbitrap XL (USA, Thermofisher) to study the possible species of calcon in strong alkaline conditions. 2.5 Cell viability assays of calcon solution in different pH values The cytotoxicity of different calcon tautomers was determined by the CKK-8 assay in human lung cancer cells (A549 and H1299) and hepatic epithelial cells of rat (FL83B). Cells growing in log 4

phase were seeded in a 96-well cell-culture plate at 1×10 /well. Under different pH and temperature conditions, the species of calcon (in red color: pH=6.8, T=293 K; blue: pH=6.8, T=298 K and yellow: pH=11.3, T=298 K) at different concentrations (0.0125, 0.025, 0.05, 0.1, 0.2 ug/ml) were added to the wells of the treatment group. The wells containing medium without cells were used as blank controls. The cells were then incubated for 72 h at 37 ℃ under 5% CO2. Thereafter, CKK-8 was added to each well for additional 4h. The assay plate was allowed to stand at room temperature for 10 min. An enzyme-linked immune sorbent assay (ELISA) reader was used to measure the absorbance value of each well with background substraction at 450 nm. The following formula was used to calculate the viability of the cell growth. Cell viability (%) = (average of absorbance value of treatment group / average of A value of control) × 100

(3)

9



Page 10 of 34

3 Results and Discussion

3.1 Facial azo-hydrazone tautomerisms under different pH conditions Due to the asymmetric substitution in two naphthalene rings around the -N=N- azo bridge, the two nitrogen atoms bonded to naphthalene rings without and with sulfonic group are labelled 27

as N1 and N2, respectively. We use the same nomenclature rule as that reported in literature:

“full” means the neutral species with the presence of all three protons; the “–Hx (x=1,2,3)” or a combination of them (e.g. -H1-H2, -H1-H3) denotes the different protonation states by removing the certain x-th protons. The hydrazone form with azonium ion can be denoted as “full+HNy（y=1, 2）” or “-Hx+HNy (x=1, 2, 3; y=1, 2)” in different protonation states, where HN1 (or HN2) is the protonation at certain N1(or N2) atom. DFT optimization of all the possible tautomers in different protonation states, 3H: full, full+HN1, full+HN2; 2H: –H1, -H1+HN1, -H1+HN2; 1H: -H1-H2, -H1-H2+HN2, -H1-H3, -H1H3+HN1; 0H: -H1-H2-H3, are shown in Figure 2. There are totally eleven tautomers in all pH conditions, the background in blue color denotes the hydrazone tautomers with the pronation at azo group. The detailed geometry parameters of intramolecular hydrogen bond, relative energy, Boltzmann distribution at 298 K, and dipole moment were presented in Figure S1. The energy difference between azo and hydrazone form of the calcon is less than 2 kcal/mol with the existence of the intramolecular hydrogen bonding interactions (Figure S1). In the 3H and 2H protonation states, the azo tautomers are preferred relative to the hydrazone form. However, in the alkaline conditions (1H), the azo and the hydrazone tautomers may coexist in solution with comparable populations (Figure S1). The hydrazone tautomers are even more favorable than the azo forms with lower energy than the unprotonated azo unit. The O-H…N and O…H-N intramolecular hydrogen bond (HB) stabilize the azo and hydrazone tautomeric structures, respectively. Thus, the calculated azo-hydrazone relative energies (less than 1 kcal/mol) 13

in this work are smaller than that (5.22 kcal/mol) reported at B3LYP/6-31G level with PCM model 10


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(solvent=water), which did not consider the intramolecular HB interactions in their DFT optimization. In the O-H…N type hydrogen bond, the distance between the proton in the hydroxyl group and the nitrogen is 1.66 Å, the ÐOH…N angle is about 146°. In the hydrazone form, in which the proton is transferred from hydroxyl to the azo nitrogen atom, the O…H distance is about 1.64 Å, and the ÐNH…O angle is 140°. The intramolecular hydrogen bonding interactions were visualized by the NCI map, as shown in Figure 2 and Figure S2. The attractive part (green) dominates in the O-H…N and N-H…O intramolecular hydrogen bond in both azo and hydrazone forms.

Figure 2. The optimized geometry of different protonation states of calcon at B3LYP/6311+G(d,p) level with PCM model (solvent=water). The selected NCI map are also presented in the coloured isosurface plot, the green colour denotes a weak H-bond and the red color stands for the steric effect.

DFT calculations also revealed that the azo-hydrazone tautomerism occurred through intramolecular hydrogen transfer, switching between the aromatic and quinoid structures. The π11



Page 12 of 34

conjugation picture of aromatic/quinoid feature of the naphthalene rings and the azo bridge is Q

azo

reflected by two kinds of bond length alternation (BLA) parameters, d and d , respectively. The detailed definition of the parameters is presented in Table S2. The smaller value of BLA parameter azo

means the larger extent of electron delocalization. The values of d

in the azo form about (0.10

Å) were larger than that (0.07 Å) in the hydrazone form, which reflect the more significant single Q

and double bond alternation in the azo form. The large value of d reflects the characteristic Q

aromatic structure in the azo form, in contrast with the smaller d in the quinoid structure in hydrazone form. It will be shown in the subsection 3.3 that the existence of quinoid strcture in hydrazone form makes the maximum wavelength absorption peaks of UV-vis spectra red-shifted. The pH conditions not only change the relative stabilities of azo and hydrazone form, but also have significant influence on the activation energy of both tautomerism and the Z-to-E isomerization of calcon (Figure 3). To trace the tautomerism reaction path, the difference between O-H and N-H distance, Dd = dO-H - dN-H, was defined as the azo-hydrazone reaction coordinates (RCs). The values of Dd are the opposite to each in the two tautomers: azo form corresponds to Dd 0. In the transitions state (TS), the Dd is close to zero. In addition, for the Z-to-E isomerization, the torsion angle, ψ, is an indicator of Z (ψ=11°) and E (ψ=180°) isomers. The two-dimensional energy profile along the proton transfer pathway and the Z-E isomerization pathway as a function of reaction coordinate Dd and the torsion angle (ψ) are illustrated in Figure 3. As mentioned before, in the acidic or weak alkaline condition (3H, 2H), the azo isomers were more stable than the hydrazone isomers (3H: 1.6 kcal/mol; 2H: 1.0 kcal/mol). But in the 1H condition the relative energy order of the two tautomeric forms was reversed. The activation energy barrier for the azo-hydrazone tautomerism decreased as the pH value increases. The detailed energy profile of azo-hydrazone tautomerism was presented in Figure S3. The calculated energy barriers of the azo-hydrazone tautomerism were smaller than 4 kcal/mol, which implied the tautomerism easily takes place at the room temperature. This gives rise to the temperature responsive property of calcon, in accompany with the color change from red to blue, as addressed in subsection 3.3. 12


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Azo-to-hydrazone dO…H

dO-H dN…H

dO…H

dN…H

dN-H

Dd > 0

Dd = dO…H - dN…H ≈ 0

Dd < 0

TSA-H

Azo Ψ=180°

0.0 3.9

E

Hydrazone

1.6

3H TSE-Z Ψ=10°

44.0

Z

20.3 19.0

27.8 1.0

pH increasing

0.0 3.7

Ψ (degree)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2H 46.9 20.0

27.6 1.0 3.1

kcal/mol 40

0.0

37.3

1H

36.5

20 0

16.0

11.0

Reaction coordinate, Dd (Å) Figure 3. The potential energy profile of the azo-hydrazone tautomerism and E-Z isomerization 13



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in different pH conditions.

The Z-E isomerization process of calcon is much more difficult than the azo-hydrazone tautomerism. The activation barriers of E-to-Z isomerization in 3H and 2H conditions are as high as 44.0 and 46.9 kcal/mol, respectively. In the alkaline condition (1H), the Ea barrier decreased sharply to 36.5 kcal/mol. It can be conceived that the intramolecular hydrogen bonding (O-H…N) interaction between the hydroxyl group and azo –N=N- bridge hindered the E-to-Z isomerization. In order to test the influence of intramolecular hydrogen bond on the E-to-Z isomerization barrier, the azobenzene (AB) without and with one/two ortho-substituted OH groups were studied as model systems and their isomerization energies are shown in Figure 4 and Figure S4. The activation energy of E-to-Z isomerization decreased as the reduced number of the ortho-hydroxyl group and the loss of intramolecular HB at azo group. Going back to calcon isomerization in the alkaline condition (1H), due to the deprotonation of the hydroxyl group, the intramolecular O-H…N hydrogen bond was disappeared and the activation of E-to-Z isomerization decreased to 37.3 and 36.5 kcal/mol. The influence of pH values and the temperature on the azo-hydrazone tautomerism and E-to-Z isomerization will be further demonstrated by the UV-vis spectra in subsection 3.3.

14


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1.72 Å

1.74 Å

1.74 Å

Figure 4. The relationship between the activation energies of isomerization and the number of the ortho-substituted hydroxyl group in azo chromophore.

3.2 Molecular dynamics simulations of the azo-hydrazone and E-Z isomerizations in various pH conditions. In order to investigate the influence of intermolecular HB interaction between calcon and solvent molecules in aqueous solution on tautomerism, the AIMD simulations were performed to follow the dynamic change between the azo and hydrazone forms. Both the DFT-optimized azo and hydrazone forms are set as the initial structures for two independent AIMD simulations. The RC of Dd is defined in the same way as that in subsection 3.1. The dotted line near zero (Dd=0) highlights the crossing point of tautomerism through TS region. Both the azo and hydrazone initial structures could proceed tautomerism by passing through TS (the dotted line) and convert to the other tautomer within 1 ps, as shown in Figure 5. One of the proton in hydroxyl group is transferred to the nitrogen in azo chromophore at 581 fs (Figure 5a). In Figure 5b the hydrazone form turned to the azo form at 739 fs, and after a short period, converted back to the hydrazone 15



again at 1018 fs. The hydrazone form tends to be more favored in the solution phase. This is in good agreement with the nearly barrierless activation barrier of tautomerism. During intramolecular hydrogen transfer from the hydroxyl to nitrogen atom in the azo group, the Na cation and the solvent molecules might assist the tautomerism, as shown in the Figure 5c and Figure S5. It is evident that a five-numbered ring intermolecular HB network is formed between the water molecules and calcon and the HBs are maintained in the whole reaction process.

RC, Dd (Å)

(a)

(c) 581 fs

azo

dO-H-dN-H 0

hydrazone

739 fs dO-H-dN-H < 0

RC, Dd (Å)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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739 fs

1018 fs

Time (fs) Figure 5. The evolution of the reaction coordinate Dd (dO-H - dN-H) of azo-hydrazone in the AIMD simulations. Azo form (a) and hydrazone form (b) are set as the initial structures in two independent AIMD runs, respectively. The intermolecular hydrogen bonding network in the selected snapshots is shown in (c).

16


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With the increasing concentration of calcon, molecular aggregation was also observed in our MD simulation of aqueous solution (Figure 6). The calcon molecules tend to aggregate in the 28

solution phase, similar to that found in MD simulations of the sunset yellow aqueous solution.

Molecular self-assembly into a chromic stack was observed at about 300 ps in solution. Along the MD trajectory, the clustering of calcon molecules into dimer, trimer and tetramer appeared. Specifically, the tetramer was formed by a stepwise addition of the monomers. The formation of an octamer was found in the end of the 3 ns simulation. The self-assembly process is easily monitored by showing the radical distribution function, RDF, between the centroids of solute molecules (D). As shown in Figure 6a, RDF has two peaks in positions of 4.2 and 7.6 Å, respectively. The population of the D near 4.2 Å indicates the formation the of π-π stacking array of the calcon molecules during the MD simulation. At about 243 ps, the π-stacking trimer is found in the headto-tail (antiparallel) stacking style. The intermolecular distances are 4.2 and 3.9 Å, respectively, 28

which are slightly larger than that (3.6 Å) the stacking distance of sunset yellow. The shoulder peak near 7.6 Å is assigned to the T-shaped architecture of the two calcon molecules. Both π-π and T-shaped stacking styles are involved in the octamer array of calcon (Figure S6). DFT calculation at B3LYP/6-311+G(d,p) level reveals that the parallel (shoulder-by-shoulder) packing style is 11 kcal/mol more stable than the head-to-tail stacking, as shown in Figure S7. The favorable π-π stacking is also due to the significant polarization effect from the large dipole moment of the hydrazone form (Figure S2).

17



Aggregation in calcon solutions π-stacking

4.2 7.6

D

4.2 Å

243 ps

(a)

3.9 Å

g(r)

T-shaped

365 ps

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 34

7.3 Å Distance between centroids of calcon (Å)

(b)

243 ps

0 ps

3000 ps 365 ps

3000 ps

365 ps

Figure 6．(a) The radical distribution function, g(r), for the centroids of calcon in aqueous solution. The π-stacking and T-shaped stacking styles are sampled from in the trajectories of MD simulations. (b) snapshots showing the formation of the dimer, trimers and tetramer aggregates from an initial dispersion of the isolated ten calcon molecules in aqueous solution. (the bar represents the orientation of the calcon, and the green end represents the naphthalene ring with the sulfonic acid).

18


Page 19 of 34

Intermolecular hydrogen bond (a)

3.0 2.7

rH…Ow

rO…Hw

g(r)

3.7

g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Intermolecular rH…Ow (Å)

Intermolecular rO…Hw (Å)

(b)

300 ps

700 ps 3.0 Å 2.1 Å

3.0 Å

1.9 Å

Figure 7. (a) The radical distribution function, g(r), for the rO…Hw between oxygen atom (O) and the hydrogen in the hydroxyl group of calcon and hydrogen in water (Hw), and for rH…Ow between AZO oxygen atom (Ow) in water solvent. (b) typical snapshots for showing the intermolecular hydrogen bond between the calcon and the water molecules.

In calcon aqueous solutions, the intermolecular HB can be formed either between the nitrogen atom in the azo group and the solvent proton, or between the –OH group of the azo dye and the solvent oxygen atom. The RDFs of both types intermolecular HB are presented in the Figure 7a. Looking at the distance between oxygen atom (O) in calcon and hydrogen atom in water (Hw), the RDF has two successive peaks at 2.7 and 3.7 Å , corresponding to the radius of first and second solvation shell, respectively. In the RDF of the distance between hydrogen atom (H) in calcon and oxygen atom in water (Ow), a sharp peak locates at 3.0 Å. The free rotation of the hydroxyl group along the C-O bond induces the oscillation of the g(r). As shown in Figure 7b, the solvent molecule clusters, taken from the MD trajectories (within 3 Å from the centroids of the calcon molecule), indicate the existence of intermolecular HB between the calcon and water 19



Page 20 of 34

solvents. The azo-hydrazone tautomerism has little effect on the solvent distribution, as shown from similar RDFs of rO…Hw and rH…Ow in the hydrazone form (Figure S8) to that in azo form. The adjacent water molecules surrounded the calcon molecule could form the intermolecular hydrogen networks in both azo and hydrazone form. Specifically, hydroxyl substituted azobenzenes, commonly known as azophenols, are particularly attracting chromophores, since they are endowed with fast thermal isomerization 29

rates under ambient conditions. Such fast thermal isomerization is related to the azo-hydrazone tautomerism, the hydrazone tautomer has low barrier in rotation of the N-N bond. Reactive molecular dynamic (RMD) simulations were also performed to investigate the Z-

to-E isomerization of calcon at different protonation states in aqueous solutions (Figure 8). The torsional profile of calcon in the different pronation states shows the correlation of the torsion angle and the intramolecular HB distance of N…H (black dot) or O…N (blue dot, in the 0H condition). Here, the variation of the N…H distance reflects the intramolecular proton transfer reaction between azo (the center of each sphere) and hydrazine (the edge) forms. In accompany with azo-hydrazone tautomerism reaction, the torsion angle oscillated near the E isomer (denoted as blue sector in Figure 8a). In Z-to-E isomerization process, the initial states of calcon are in the Z configuration (denoted in the red sector in Figure 8b-d), the final states of the calcon populated in broad range around y = ±150°. Different from dynamic process of Z-to-E isomerization in the 2H and 0H conditions, the torsion angle stayed in the range between 30° and 100° in the 1H condition，indicating the difficulty in the isomerization into the E configuration. During the Z-to-E isomerization, the dynamic intermolecular and intramolecular hydrogen network exists around the aqueous solvated calcon molecule, hindering the isomerization to some extent. This is in consistence with the higher Z-to-E isomerization barrier with the presence of intramolecular HB with azo bridge than that without HB, as shown in subsection 3.1.

20


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(a) dO…H dN…H

Azo

hydrazone

(b)

Azo (E)

Azo (Z)

(c)

(d)

dN…O

Figure 8. (a) Torsional profile of azo-hydrazone tautomerism. In the last column, the radius denotes the dN-H/dO-H, with the magnitude of radius of 3 and the center of 0 (azo form) without protonation at azo bridge. The Z-to-E isomerization in different protonation states is shown in (b) 2H; (c) 1H; (d) 0H, where the radius denotes the intramolecular HB, dN-H, with the magnitude of radius of 5. In the case of 0H (d), the radius denotes the dN-O, due to the missing of intramolecular HB.

21



Page 22 of 34

3.3 The azo-hydrazone tautomerism detected by UV-vis spectra Calcon is featured by its visible light absorption ability, and UV-vis spectroscopy technique is hence applicable to detect the azo-hydrazone tautomerism at different temperatures. The azo form of calcon in aqueous solution (pH=6.3) has the absorption peak at 512 nm, with the color of dark red. With the increase of temperature from 298 K to 311 K, the UV-vis spectra of the solution are detected every half an hour at each temperature. As shown in Figure 9a, the azohydrazone reaction of calcon could be triggered at 311 K, which is close to our body temperature, with the evident color change from red to blue. The observed heating facilitated azo-hydrazone reaction in UV-vis spectroscopy technique supports our DFT calculations, from which the small activation energy barrier of tautomerism is obtained. The broad maximum absorption peak redshifts to 595 nm within 8 hours. An isosbestic point at 540 nm is observed, which is referred to the azo-hydrazone equilibrium. The azo-hydrazone tautomerism reaction is also observed at temperatures of 313 K, 303 K and 298 K (Figure S9). As expected, such a color change process during azo-hydrazone tautomerism corresponds to the electron structure transition from aromatic azo structure to the quinoid structure. It was demonstrated in previous subsections that the ortho-substituted hydroxyl group induced the dynamic change between the aromatic structure and the quinoid structure. It is also clearly shown in Figure 9b that the hydrazone form with typical quinoid structure property will enhance the n→π* transition by the smaller HOMO-LUMO gap and larger degree of electron delocalization than those in azo form. The maximum absorption peak is hence red shifted by the change from azo form to hydrazone. In addition, the azo forms involving with H2, such as -H1-H2 and corresponding hydrazone form -H1-H2+HN2 have larger dipole moment than those H3involved species like -H1-H3 and -H1-H3+HN1.

22


Page 23 of 34

(a) pH =6.3

T =311K within 8 hours Azo→Hydrazone

Absorbance

red-shift

Wavelength (nm)

(b) Hydrazone red-shift

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Azo

23



Page 24 of 34

Figure 9. (a) The azo-hydrazone tautomerism observed by the change in UV-vis spectra at 311 K, and (b) the correlation between the maximum absorption wavelength (λmax) and the dipole moment (μ) of azo and hydrazone forms of calcon with different protonation states.

The increase of pH could also induce the tautomerism reaction within seconds. The absorption spectrum in alkaline condition (pH=11.3) has two overlapped bands at 602 nm and 633 nm, respectively (Figure 10), also corresponding to the hydrazone form. Such a color change property could be used to fabricate calcon-based temperature sensors and pH indicators. It is interesting to theoretically model the pH-dependence of UV-vis spectra of calcon aqueous solution with pH ranging from 0.2 to 13.1. As shown in Figure 10a, the calcon has maximum absorption peaks at 512 nm and 636 nm in acidic and basic media, respectively. When the pH value is increased from 1.0 to 6.1, the maximum absorption band is located at λmax=512 nm. At pH=7.1, the λmax shows a broad band in the range of 500~650 nm, which means the coexistence of the azo and hydrazone form. When pH is increased to 9.2 and 10.8, the absorption band is further red-shifted to the 636 nm with an shoulder peak at 603 nm. The two overlapped absorption bands in alkaline conditions may correspond to the azo-hydrazone tautomeric equilibrium. The relatively weak band is corresponding to the azo form, and the band in the longer wavelength refers to the hydrazone form. It should be mentioned that the selection of different DFT functionals（B3LYP，CAM-B3LYP, ωB97X-D, M06-2X and LC-ωPBE）has some influences in predictions of the lowest electronic excitation energies of the azo and hydrazone tautomers, respectively (Table S3). The estimated λmax values of azo and hydrazone forms by using CAM-B3LYP (azo: 469 nm; hydrazone: 517 nm), ωB97X-D (azo: 466 nm; hydrazone: 511 nm) and M06-2X (azo: 463 nm; hydrazone: 515 nm) functionals were close to each other. The LC-ωPBE gave different λmax values (azo: 440 nm; hydrazone: 483 nm). For all the calculation results obtained from the selected functionals, the λmax was assigned to the HOMO→LUMO transition. The calcon aqueous solution is a mixture of different protonation states with different 24


Page 25 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


populations. The distribution coefficients (δ) of different pronation states (3H, 2H, 1H and 0H) of calcon in aqueous solution was calculated from formula (4)-(7). δ(3H) =

[𝐻A ]C (4) [𝐻A ]C + 𝐾DE [𝐻A ]; + 𝐾DE 𝐾D; [𝐻A ]E + 𝐾DE 𝐾D; 𝐾DC

δ(2H) =

𝐾DE [𝐻A ]; (5) [𝐻A ]C + 𝐾DE [𝐻A ]; + 𝐾DE 𝐾D; [𝐻A ]E + 𝐾DE 𝐾D; 𝐾DC

𝐾DE 𝐾D; [𝐻A ]E (6) [𝐻A ]C + 𝐾DE [𝐻A ]; + 𝐾DE 𝐾D; [𝐻A ]E + 𝐾DE 𝐾D; 𝐾DC 𝐾DE 𝐾D; 𝐾DC δ(0H) = A C (7) [𝐻 ] + 𝐾DE [𝐻A ]; + 𝐾DE 𝐾D; [𝐻A ]E + 𝐾DE 𝐾D; 𝐾DC δ(1H) =

+

Where the [H] is the concentration of the hydrogen ion in calcon aqueous solution, the Ka1, Ka2 and Ka3 are the acid dissociation constants of calcon. As shown in Figure 10b, it can be seen that 2H and 1H are the dominant species in the pH range from 2~6 and 8~12, respectively. However, many different protonation states coexisted in the pH range from 0~2, 6~8 and 12~14. In each protonation state (3H, 2H, 1H, and 0H), the simulated UV-vis spectra (column) were the ensemble sum of the calculated excitation energies of the isolated azo and hydrazone tautomers and clusters (including tautomers and solvents) sampled from the trajectories of the MD simulation every 200 ps (Figure 10c). It should be mentioned that the 0H only has the azo form. While other protonation states (3H, 2H and 1H) have both azo and hydrazone forms. The enhanced n→π* transition in quinoid hydrazone form gives rise to the red shift of absorption peak compared with the aromatic azo form (Figure S11, S12, S13), giving the promise in the biological applications under the harmless visible light.

25



Page 26 of 34

Figure 10. (a) Experimental UV-vis spectra (3D waterfall color mapping) of calcon in aqueous -5

solution (concentration 4×10 M) at pH values range from 0.2 to 13.1 and the simulated UV-vis spectra (columns) of calcon at different pH conditions based on the conformation ensemble of the cluster model. (b) Distribution coefficient δ of 3H, 2H, 1H and 0H species at different pH 26


Page 27 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


values. (c) The molecular clusters are sampled in the first solvation shell along the 1 ns MD trajectory. In order to simulate the UV-vis spectrum at certain experimental pH values, the populations of various possible protonation states of calcon (Figure S10）were taken as the weights to assemble the TDDFT excitation energies (at B3LYP/6-311+G(d,p) level) into the UV-vis spectra. In other words, the simulated UV-vis spectra (column) at experimental pH values were the δweighted sum of the calculated UV-vis spectra at each protonation states. Taken pH=1.0 as an example, the dominant protonation states in calcon aqueous solution are 3H (53%) and 2H (47%), the populations of 1H and 0H are negligible. Hence, the simulated UV-vis spectra at pH=1.0 is the ensemble of the 3H (with the population of 53%) and the 2H (with 47%). The short-ranged solvent effect is involved in the cluster models in the first solvent shell, and the long-ranged solvent effects from distant water molecules are considered in PCM model in each TDDFT calculation of cluster model. It can be seen that such a combined discrete/continuum strategy for TDDFT calculations of conformation ensemble well reproduced experimental UV-vis spectra of calcon at different pH values. The light driven E-to-Z were also investigated by irradiation the solution with UV light (365 nm), the intramolecular hydrogen bonding interactions prohibit the UV-light driven E-to-Z isomerization (Figure S14), which is in agreement with theoretical results presented in subsection 3.1 and 3.2. The hydrazone form of calcon is not stable in pH=6.3 (Figure 11a), the color loss takes place -5

-1

with the rate constant of about 2×10 s . As shown in Figure 11b, however, in strong alkaline condition (pH=11.3), the rate constant of color loss is much faster (40 times) than that in neutral condition, as shown in Figure 11c-d and Figure S15. The high-resolution mass spectrometry (MS) was thus employed to investigate the reason behind the color loss. Two fragments were found from the MS spectra, shown in Figure 11e and Figure S16. Both the C-N and N-N bond are easily broken in alkaline conditions, suggesting the damage of the azo chromophore. The calculated UV-vis spectra based on the fragments are in agreement with the spectrum in pH =12.6 (Figure 27



S17).

(a)

(b)

pH =6.3 T=311 K

(c)

Color loss

pH =11.3 T=298 K

Color loss

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 34

(d)

2×10-5 s-1

8×10-4 s-1

(e) Fragment A

Fragment B

143.0496

m/z=143.0502

m/z=238.0180 238.0172

Figure 11. The color loss in neutral pH condition (a) and in strong alkaline condition (b). The evolution of the absorbance at 633 nm in neutral (c) and alkaline condition (d). (e) The representation of the two fragments detected in the higher resolution mass spectrometry, with the whole experimental spectrum presented in the supporting information.

28


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3.4 The cell viability of the species in calcon aqueous solution The visible light absorption feature and multi stimuli-responsive functions of calcon are very attracting in the extending the applications to biomedical fields. In this sense, the cytotoxicity of calcon species were detected by Cell Counting Kit (CCK-8) assays against A549, H1299 and FL83B cells, as shown in Figure 12. Three samples were prepared in different conditions. At pH=6.8, the red and blue species are in the azo and hydrazone forms, respectively. No cell viability inhibition was detected in the both azo and hydrazone samples, indicating that these two species were non-toxic toward normal cells. As mentioned before, in strong alkaline conditions (pH= 11.3), color loss to a pale yellow species is indeed the degraded form with the coexistence of Fragments A and B shown in Figure 11e. The cell viability of sample in strong alkaline condition with concentration of 0.2 ug/mL was 24.5% and 37.5% for the H1299 and FL83B, respectively. But in lower concentrations (0.0125 and 0.025 ug/mL), the pH=11.3 sample shows 100 % cell viability. This means the calcon may be applicable to the biosensors and biomedicals in the future.

29



pH=6.8 T=313 K hydrazone

pH=11.3

T=298 K fragment A+B

pH=6.8

T=298 K

azo

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 34

(a) A549

Concentration (μg/mL)

(b) H1299

(c) FL83B

Figure 12. The cell viability in (a) A549 cell, (b) H1299 cell and (c) FL83B cell with different concentrations of different forms of calcon (azo, hydrazone, and the fragment A and B in strong alkaline conditions).

Conclusions We have systematically investigated the azo-hydrazone tautomerism and E-Z isomerization 30


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in different pH conditions. The intermolecular and intramolecular hydrogen bonding interactions promoted the azo-hydrazone tautomerism between the aromatic azo structure and the quinoid hydrazone structure. The UV-vis spectroscopy revealed that both the temperature and the pH could trigger the azo-hydrazone tautomerism, in good agreement with theoretical calculations. The enhanced n→π* transition in quinoid hydrazone form gives rise to the red shift of absorption peak in visible light region, providing the promise to biological application under the harmless visible light. It was also found that in pH= 6.8 both azo and hydrazone isomers have no cytotoxicity on the human lung cells (A549 and H1299) and hepatic epithelial cell of rat (FL83B). Theoretical modeling is a useful tool in understanding the pH- and temperature-dependence of switching behaviors in stimuli-responsive systems.

Supporting Information Available: the details of DFT calculations, MD simulations (AIMD, RMD and conventional MD) and experimental sections (UV-vis spectra and High-resolution mass spectrometry).

ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Program of China (2017YFB0702601), the National Natural Science Foundation of China (Grant Nos. 21673111, 21873045). We are grateful to the High Performance Computing Centre of Nanjing University for providing the IBM Blade cluster system, and the support from Nanxin Pharm Co., Ltd., Nanjing.

Notes The authors declare no competing financial interest.

Author Information Corresponding author: Jing Ma: [email protected] ORCID: Jing Ma: 0000-0001-5848-9775 31



Page 32 of 34

Dong Zheng: 0000-0001-6667-7151

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TOC

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Hydrogen Bonding Promoted Tautomerism between Azo and

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