Terahertz spectroscopy of enantiomeric and racemic pyroglutamic acid

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 225 (2020) 117509

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Terahertz spectroscopy of enantiomeric and racemic pyroglutamic acid Zhipeng Wu a, b, Zhongjie Zhu b, c, Chao Cheng a, b, Jianbing Zhang b, c, Yan Gong d, Mingzhu Xu d, Shaoping Li a, *, Hongwei Zhao b, c, ** a

School of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, China Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China Zhangjiang Laboratory, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China d School of Mechanical and Electrical Engineering, Key Laboratory of Modern Agricultural Engineering, Tarim University, Alar 843300, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 June 2019 Received in revised form 23 August 2019 Accepted 2 September 2019 Available online 6 September 2019

The low-frequency vibrational properties of D-, L- and DL-pyroglutamic acid (PGA) have been investigated with the terahertz time-domain spectroscopy (THz-TDS) from 0.5 to 4.5 THz. The enantiomers (Dand L-PGA) present similar absorption spectra, while the spectrum of racemate (DL-PGA) is obviously different. The temperature-dependent THz spectra of different PGA were recorded in the range of 293 e83 K. The spectral changes during the cooling process suggest that D- and L-PGA undergo a structural phase transition, and no phase change of DL-PGA was found. The results indicate that THz spectroscopy is highly sensitive to the crystal structure of molecules. The density functional theory (DFT) calculations based on the crystal structures were performed to simulate the sample’s THz spectra. It was demonstrated that the characteristic resonant absorption peaks of the enantiomeric and racemic PGA in the low-frequency THz region originate from the different vibrations, which corresponding to the specific structures and intermolecular interactions. The conformational diversity and fluctuation may help to understand the properties of PGA in biochemistry and functional material. © 2019 Elsevier B.V. All rights reserved.

Keywords: Pyroglutamic acid Terahertz time-domain spectroscopy Density functional theory Intermolecular interactions Temperature effect Chirality

1. Introduction Pyroglutamic acid (PGA) is the naturally occurring cyclized internal amide of glutamic acid, which is also regarded as a forgotten cyclic amino acid [1,2]. It was found as an N-terminal modification in many neuronal peptides and playing important functions in nervous system. As a glutamate precursor, PGA is used as a helpful tool for glutamatergic research [3]. PGA has chirality, and the molecule has a rigid five-membered skeleton ring and two different reactive carbonyl groups (Fig. 1). L-PGA is an important active pharmaceutical ingredient used in brain-enhancing effect and other clinical medicines [4]. PGA is a highly functionalized molecule and has extensive applications in asymmetric synthesis. It is also a potential nonlinear optical material [5]. Recently, Panda et al. [6] found that the single crystals of enantiomerically pure D- and LPGA are capable of self-actuation through a rapid release latent

* Corresponding author. ** Correspondence to: H. Zhao, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China. E-mail addresses: [email protected] (S. Li), [email protected] (H. Zhao). https://doi.org/10.1016/j.saa.2019.117509 1386-1425/© 2019 Elsevier B.V. All rights reserved.

strain during a structural phase transition at heated condition without deterioration. The super-elasticity is associated with the low-dimensional hydrogen bond networks. However, the racemic PGA is mechanically inactive. The self-actuation materials are structure functionality that has great potential applications in biomimetic and artificial smart materials. These exotic properties are closely related to the molecular structure, especially the intermolecular forces. The relationship between the type and strength of the molecular interactions and the structure-function is complex and specific. Vibrational spectroscopy provides fundamental and helpful information about molecular structures and interactions. Wu et al. [7] studied the thermal stability of L-PGA with Raman spectroscopy and found that L-PGA exists in three enantiotropic polymorphic forms, each with its own temperature range of thermodynamic stability. It occurs phase transition a to a0 at 147 K when cooling, and a to b at 351 K when heating. The structural differences of the polymorphic forms are associated with specific molecular packing or assembled arrangements, and in particular the interchain hydrogen bonds. Recently, Takoua et al. [8] combined theoretical calculation and FTIR to analyze the main characteristic vibrations of

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2.3. THz time-domain spectroscopy (THz-TDS)

Fig. 1. The molecule structures of D-, L- and DL-PGA.

the pyrrolidinium ring and functional groups of L-PGA from 400 to 4000 cm1. Although the intra and intermolecular interactions of PGA have been extensively studied, the vibrational properties in low frequency band which closely related to the conformation and molecular weak interactions are still unclear. In recent years, thanks to the rapid development of ultrafast laser technology, terahertz time-domain spectroscopy (THz-TDS) has become a novel and powerful method to study the far-infrared vibrational characteristics of molecules duo to the high signal-tonoise ratio [9e12]. Ruggiero et al. [13] used THz-TDS successfully obtained the elasticity of right-handed all-cis and left-handed alltrans poly-L-proline polypeptides whose residue structure is very similar to PGA. They found that the vibrations such as rotations and torsions of the pyrrolidinium ring in THz range are a key in complex spring-like elongation and contraction of the helices. They also demonstrated that THz-TDS can effectively identify a and b polymorph L-glutamic acid [14]. THz-TDS has great potential in the study of different molecular crystals. It can be used to differentiate monocrystal and polycrystal compounds such as sucrose and other €ll et al. studied the optical saccharides [15e18]. In addition, J. Kro properties of sucrose single-crystals and found that the THz absorption spectrum exhibits angular dependence [18]. Studies have been shown that the THz spectroscopy is sensitive to different amino acids and the THz fingerprints can be used in biochemistry detection and pharmaceutical analysis. Studies also suggest that the THz spectroscopy has the capability to distinguish the enantiomeric and racemic compounds [19e21]. In this study, the low-frequency vibrational spectra of enantiomeric and racemic PGA were investigated with THz-TDS in the frequency range of 0.5 to 4.5 THz at variable temperatures. The DFT calculations based on the different PGA crystalline structures were performed for better understanding the THz characteristic absorptions. Considering THz spectroscopy is sensitive to molecular crystal structure [14,22] and polymorphism of PGA, the powder Xray diffraction (PXRD) was used to examine the structure of samples. 2. Experiment 2.1. Materials D-, L- and DL-PGA (all samples >98%) used in this study were obtained from J&K(China). Cyclic olefin copolymer (COC) powder (particle size 50e100 mm) was purchased from the experimental plant of the Shanghai Institute of Nuclear Research (SINR). COC can be highly suitable for THz spectroscopy applications due to its negligible dispersion of refractive index and negligible absorption in the THz region [23]. All the samples were in solid powder and used without further purification. 2.2. Sample preparation The samples were diluted by COC at a 5% w/w and gently grind into fine particles with agate and then compressed these samples into pellets (1.5 mm thickness and 13 mm diameter) with a hydraulic press using 2 MPa pressure.

The spectrum in the frequency range from 0.5 to 4.5 THz was measured with TAS7400TS THz-TDS system (Advantest Corporation, Japan). The wavelength is 1550 nm, the pulse width is 50 fs and the frequency resolution is 1.9 GHz. The sample was placed into THz path system and purged by drying air to maintain the relative humidity of <1%. Taking the pure COC as reference, each spectrum was average of three measurements. The number of scans under room temperature was 1024, and it was 512 in the temperature effect experiment in order to obtain the real-time dynamic data at variable temperatures. 2.4. Variable temperature controller The sample was placed into a liquid nitrogen flowed cryostat cell which equipped with cyclic olefin copolymer windows (Variable temperature cell holder, Specac Ltd. U.K.). A series of THz spectra of PGA were recorded for the temperature range of 293e83 K. 2.5. Powder X-ray diffraction (PXRD) The PXRD measurements were performed on the Bruker D8 Advance (Cu tube with 1.5406 Å, 40 kV voltage, 40 mA filament emission). The data were collected with a scan ranging from 10 to 90 (2q). The scanning rate was 0.2 s1 and step value was 0.02 . 3. Quantum chemical calculation The PXRD measurements demonstrated that all the samples we used in the experiment are powder crystal. D- and L-PGA present similar diffraction patterns, the DL-PGA is different (see Supplementary materials, Figs. S1eS3). The measured PXRD patterns of both the enantiomeric and racemic PGA are consistent with the previous data published and the parameters are available in the crystal database Cambridge Crystallographic Data Centre (CCDC) [6]. The space groups and the lattice constants of D-, L-PGA and DLPGA are listed in Table 1. These crystal cell parameters were used for the calculations. The geometry optimization and energy calculations were performed based on the solid-state DFT using the Cambridge Sequential Total Energy Package (CASTEP) [24] program which is a part of Materials Studio package from Accelrys. The calculations were performed on the crystalline state within the generalized gradient approximation (GGA) at Perdew-Burke-Ernzerhof (PBE) correlation function [25], and using norm-conserving pseudopotential. The quality of the energy calculations was ultra-fine. The plane-wave cutoff energy was 830 eV. The maximum forces between atoms were all <0.01 eV/Å. The grids for the fast Fourier transform of D-, L-, DL-PGA were 90  128  114, 90  128  114 and 36  40  40 respectively. 4. Results and discussion 4.1. THz absorption spectra of PGA Fig. 2 shows the THz absorption spectra of the enantiomeric and racemic PGA in the range of 0.5 to 4.5 THz at room temperature. Dand L-PGA show similar features and present five peaks at 0.99, 1.25, 2.11, 2.67 and 3.78 THz, all the peaks are not sharp. DL-PGA has two strong peaks at 1.55, 2.18 THz and other four peaks at 3.07, 3.57, 3.94 and 4.31 THz respectively. Combined with the PXRD experiments, this situation observed by the THz spectroscopy can be explained by the different crystal structures of the samples (Table 1). Both D- and L-PGA are orthorhombic with space group P212121 (Z ¼ 12), but the DL-PGA is monoclinic with space group

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Table 1 Structure parameters of enantiomeric and racemic PGA [6]. D-PGA

a (Å) b (Å) c (Å) a (deg) b (deg) g (deg) Space group Z V (Å3) Electronic states Total energy (eV/atom)

L-PGA

DL-PGA

a (300 K)

a0 (130 K)

a (300 K)

a0 (130 K)

270 K

100 K

9.012 13.456 14.663 90 90 90 P212121 12 1778.3 211 5.0  106

8.201 14.347 14.657

9.024 13.453 14.672 90 90 90 P212121 12 1781.1 211 5.0  106

8.186 14.331 14.658

7.956 8.744 9.098 90 115.482 90 P21/c 4 571.5 322 5.0  106

7.882 8.786 8.997

1724.8

Fig. 2. THz absorption spectra of the D-, L-, and DL-PGA at room temperature.

P21/c (Z ¼ 4). Therefore, the stereoisomeric structures and crystal packing arrangements of the enantiomers and the racemate are different. These phenomena were also observed in tartaric acids, amino acids and some chiral medicines [19,21,26]. The different THz absorption fingerprints can be used as characteristics to identify and distinguish the enantiomers and their racemic compounds.

1719.8

115.426

562.67

modes to shift to higher energies and vibrational frequencies [27,28]. But based on differences in intermolecular hydrogen bonds strength, different degree of frequency shifts may occur [29]. D- and L-PGA spectral changes are consistent in the temperature effect experiments, and the Df of the absorption peaks increase with the frequency, however, it is different for DL-PGA. This can also be attributed to their different crystal structures. Furthermore, new absorption peaks of D- and L-PGA were observed in the 3 to 4 THz region when the temperature dropped to 203 K. And with further cooling to 143 K, new peaks at 3.16, 3.56 and 3.82 THz become obvious (in the solid line box). No such phenomenon was observed for DL-PGA. Panda et al. [6] studied the crystal structure of PGA at low temperature with XRD and found both chiral forms occur phase transition a to a0 at 147 K, but the racemic crystal is completely inert, see Table 1. The unit cell parameters of a are larger than that of a0 in terms of volume and bond length. The crystal structure of a0 is more compact. The decrease in the distance between N and O atoms in the neighboring molecules lead to an enhancement of intermolecular interactions, especially the hydrogen bonds. Therefore, the significant spectral changes of enantiomers in the THz region may be related to the phase transition. In addition, some THz absorption peaks observed at room temperature are generally broad, and the broadening of the peaks usually leads to band overlap, making the distribution of peaks ambiguity. As the temperature decreased, the broad absorption bands resolve into narrower peaks due to the anharmonicity of the vibrational potentials, which promoted the absorption peaks clearly displayed [15,30,31]. Therefore, the new absorption peaks appeared at the low temperatures are the result of the joint action of phase transition and anharmonicity effect. These observations indicate that THz spectra of PGA are sensitive to the variation of temperature, which is helpful to further understand the molecular structure.

4.2. Temperature effect on PGA Considering the thermal stability and polymorphism of PGA, the temperature effects of different PGA were investigated. The THz absorption spectra of D-, L- and DL-PGA in the temperature range from 293 to 83 K are shown in Figs. 3 and 4. Because of the similarity of D- and L-PGA, the spectra of D-PGA along with the variable temperatures were shown in Fig. S4. Thirty spectra were measured during the temperature change, see Figs. 3(a) and 4(a). And for the convenience of observation, eight spectra were taken at intervals of 30 K, Figs. 3(b) and 4(b). As can be seen from the figures, the absorption peaks become distinct and sharp at the low temperature. Meanwhile, the absorption peaks present blue shift as the temperature decreases, and the degrees of frequency shift (Df) are different, Figs. 3(c) and 4(c). Such a shift is usually ascribed to thermal expansion. Cooling of solid sample results in a contraction of the crystalline unit cell dimension and causes the vibrational

4.3. Quantum chemical calculation To further understand the experimental THz spectra of the different PGA, the quantum chemical calculations were performed. The calculations were based on the crystal cells which involve the intermolecular interactions and the lattice vibrations. The experimental and calculated THz spectra of the different PGA are shown in Fig. 5 (and Fig. S5). Because the theoretical calculations in all processes have a default temperature of 0 K, the crystal cell parameters available at low temperature were adopted (Table 1). Considering that D- and L-PGA have structural phase transitions at 147 K, the crystal parameters of a0 phase were used. It can be seen from the Fig. 5(a), the calculated spectrum has a rich absorption peaks distribution in the 3e4 THz region, which is consistent with the spectrum observed in the temperature effect experiment, this

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Fig. 3. (a) Evolution of the THz spectra of L-PGA from 293 to 83 K. (The color bar represents the magnitude of the absorption coefficient). (b)The temperature effect on the absorption spectra of L-PGA, (c) Df of different absorption peaks. * The temperature range of the new peak at 3.34 THz is 203e83 K.

further suggests the occurrence of phase transition of L-PGA at low temperature. Table 2 shows the absorption peaks obtained from the experimental measurements and theoretical calculations, and the tentative assignment of the vibrational property of different PGA. There is no imaginary frequency and no scaling factors are used to the calculated frequency values. The interaction of molecules in the lattice and several typical vibrational modes of L- and DL-PGA were demonstrated in Figs. 6 and 7 respectively. PGA molecule link with the neighboring molecules and form a three-dimensional structure via O-H$$$O and NH$$$O hydrogen bonds among the amide carbonyl, carboxyl group and amide -NH groups. Therefore, the structure is stabilized by NH$$$O and O-H$$$O hydrogen bonds. However, the interactions of hydrogen bonds between the enantiomers and the racemate are exist certain differences. As for L-PGA, the molecule is linked by a network of five hydrogen bonds to neighboring molecules, while DL-PGA has four hydrogen bonds [6,8,32]. The theoretical analysis shows that the resonant absorption peaks of PGA in the THz range are originate from the different vibrational motions of molecules. At the same time, vibrations such as torsion and translation of partial atoms and local groups in the PGA molecule were also

observed. These low frequencies vibrational modes are diverse and closely related to the conformational diversity of PGA. According to the calculated results, many of the vibrations of the PGA molecules belong to the collective modes, but there are some differences in the specific details. For example, the vibrations of LPGA at 0.94 and 1.04 THz are collective, however, the former is more reflected in the rotation of the C3-C4-C5, while the latter is shown as the translation of the entire PGA molecule. Besides, the behaviors of all the molecules in a unit cell is not the same, each has its own characteristics. For instance, the vibration of L-PGA at 1.99 THz is part of the rotation of the pyrrolidinium ring and the twisting of carboxyl group, not all atoms involved in a unit cell (see Fig. 6(c)). Although the vibrational modes of L-PGA at 3.43 THz and DL-PGA at 3.44 THz are both belong to the twisting of carboxyl group (Figs. 6, 7(c)), the vibration of L-PGA is arising mainly from the C¼O of carboxyl group, while DL-PGA is arising from the whole carboxyl group. The calculated THz spectra of D- and L-PGA show some similarities, but there are certain differences, such as the position of peaks and their corresponding vibrational modes. This is mainly because of the deviations in their crystal cell parameters, including the hydrogen bonds length and volume of the unit cells

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Fig. 4. (a) Evolution of the THz spectra of DL-PGA from 293 to 83 K. (The color bar represents the magnitude of absorption coefficient). (b)The temperature effect on the absorption spectra of DL-PGA, (c) Df of different absorption peaks.

Fig. 5. The experimental and calculated spectra of L-PGA (a) and DL-PGA (b).

[6]. In addition, the presence of multiple molecules in a unit cell of the enantiomers also leads to the complex and intricate interactions. Compared with D- and L-PGA, the theoretically calculated spectrum of DL-PGA is relatively simple and agrees well with

the experimental measurement. This maybe because the enantiomers have twelve molecules in a unit cell, but the racemate has four molecules, and the intermolecular interactions of the enantiomers are more complicated than that of the racemate.

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Table 2 Comparison of the absorption peaks between the experimental and calculated spectra and the vibrational modes of different PGA. PGA

Exp. (THz)

Cal. (THz)a

Main vibrational modes assignment

1.07

Partlyb v (PGA)

1.71 1.81 2.04 2.36 2.59 2.70 2.85 2.97 3.08 3.13 3.20 3.42 3.80 3.87 4.01 4.13 4.36 0.94 1.04 1.35 1.39 1.52 1.74 1.97 1.99 2.20 2.37 2.78 2.91 3.12 3.35 3.43 3.67 3.75 3.94 4.15 4.26 1.94 2.34 2.92 3.44 3.49 3.96 4.06 4.28

Partly v (PGA), mainly from h (COO) Collective, v (PGA) Partly v (PGA), mainly from v (C4-C5) Partly v (PGA), mainly from v (C4-C5) Collective, v (PGA) Collective, v (PGA) Partly v (PGA) Partly v (C3-C4) Partly v (PGA), mainly from h (COO) Partly v (C3-C4) Partly v (PGA), mainly from v (C4-C5) Partly v (C3-C4-C5) Partly v (PGA), mainly from v (ring) Partly v (ring) Partly v (PGA) Collective, v (PGA) Partly v (PGA) Collective, mainly from v (C3-C4-C5) Collective, t (PGA) Collective, t (PGA) Collective, mainly from v (C3-C4) þv (COO) Collective, v (PGA) Partly h (COO) Partly v (ring), mainly from v (C3) Partly v (PGA), mainly from v (ring) þ h (COO) Collective, mainly from v (C2-C3-C4) þ h (COO) Collective, v (C3-C4) Collective, mainly from v (C2-C3-C4) Partly v (C3-C4) Partly v (PGA) Collective, mainly from v (C3-C4) Partly h (COO), mainly from v (C¼O) of carboxyl group Partly v (C3-C4) Partly v (C3-C4) þ r (COO) Partly v (C3-C4) þ h (COO) Partly v (PGA) Partly v (PGA) Collective, v (PGA) Collective, t (PGA) Collective, mainly from v (C4-C5) Collective, h (COO) Collective, v (PGA) v (C3-C4) Collective, mainly from r (C3-C4) þh (COO) v (ring)

293 K 83 K D-PGA

0.99 1.25

1.05 1.40 1.83

2.11

2.25

2.67

2.87

3.18 3.54 3.83

3.78

4.22

0.95

1.05

1.24

1.39

L-PGA

1.79 2.05 2.07

2.25

2.67

2.87 3.16

3.56

DL-PGA

3.07

3.82 4.13 4.21 1.70 2.23 2.68 3.41

3.57

3.91

3.94

4.14

3.84 1.55 2.18

r, rocking; v, rotation; t, translation; h, twisting. a The calculations based on the unit cell parameters of D-, L-PGA at 130 K and DL- PGA at 100 K. b Partly refers to a part of molecules in a unit cell.

Different vibrational modes respond differently to temperature changes. The study shows that the translational vibrations are more in the low frequencies. The temperature-dependent absorption peak frequency shift (Df) is small. The rocking and rotation of partly atoms more appear in the higher frequencies, and its Df tends to be large. For example, the vibrational mode of L-PGA at 1.04 THz is translation, its Df is 0.1 THz at the temperature effect experiment. The vibrational mode at 4.26 THz is rotation, with Df 0.37 THz, obviously larger than that of the former. The phenomenon was also found in DL-PGA. The vibrational mode of 2.34 THz is translation and the 3.96 THz is rotation, their Df are 0.13 and 0.34 THz respectively. The results show that the absorption peak’s frequency shift in temperature change process depends on its vibrational modes. The study indicates that the collective vibrations have distributions widely in the 0.5 to 4.5 THz region. Besides, specific vibrational behaviors of the different resonant absorption peaks are due to the molecular structure and intermolecular interactions. The

result also illustrates the conformational diversity and fluctuation of PGA. It should be noted that the calculated spectra have some deviations from the observed experimental spectra. The main reasons are as follows. One is the samples factor. There are some differences between the samples used in the experiments and the theoretical calculations. The parameters of the samples used in the calculations are cited from the reference [6]. The purity and pretreatment of the samples used are different. For example, the DLPGA in this reference was recrystallized by slow evaporation from methanol. Generally, the recrystallization can effectively improve the quality of the crystal. Another is the temperature factor. The default temperature of the theoretical calculation is 0 K, while the lowest temperature in our experiments is 83 K. From the Table 1, it can be seen that the crystal parameters such as the cell volume and the bond length of PGA show some changes at different temperatures. Meanwhile, the experiments are often influenced by the environments such as humidity and pressure.

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Fig. 6. (a) The crystal structure of L-PGA, (b) the hydrogen bonds (the blue dash lines) of L-PGA molecules, (c) the calculated vibrational modes of L-PGA at 1.99, 2.78 and 3.43 THz. Blue arrows represent the rotation, orange arrow represents the twisting.

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Fig. 7. (a) The crystal structure of DL-PGA, (b) the hydrogen bonds (the blue dash lines) of DL-PGA molecules, (c) the calculated vibrational modes of DL-PGA at 1.94, 2.34 and 3.44 THz. Blue arrows represent the rotation, bright yellow arrows represent translation, orange arrow represents the twisting.

5. Conclusion

Acknowledgements

The low-frequency vibrational spectra of D-, L- and DL-PGA at the range from 0.5 to 4.5 THz were investigated with THz-TDS system. It was observed that the absorption spectra of enantiomeric and racemic compounds are significantly different. The absorption peaks of DL-PGA are clear and sharper than that of D- and L-PGA. The characteristic absorption spectra can be used as fingerprints to detect and identify isomers in the THz region. In the variety temperature experiment, the appearance of new absorption peaks of enantiomers at low temperature suggests the occurrence of structural phase transition. The differences in THz spectra between the enantiomeric and racemic PGA are mainly due to their different crystal structures and intermolecular interactions. The DFT calculations based on the different PGA crystal cells were used to illustrate the experimental observations. Combined with theoretical calculations, the degree of Df is temperature-dependent and related to the molecular vibrational behavior. The THz characteristic resonant absorption peaks of PGA contain different vibrational modes which closely related to the conformation and intermolecular interactions. The study may helpful to further understand the structural properties of PGA in biological activity and functional material applications.

This work was supported by the Main Direction Program of Knowledge Innovation and Open Project Program of Key Laboratory of Interfacial Physics and Technology, Chinese Academy of Sciences. The National Defense Science and Technology Innovation Special Zone.

Declaration of competing interest The authors declare that they have no conflict of interest.

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