Theoretical Study on UV Spectrum and Active Site of Penicillin

doi:10.21203/rs.3.rs-1539159/v1

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Theoretical Study on UV Spectrum and Active Site of Penicillin

https://doi.org/10.21203/rs.3.rs-1539159/v1

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The research of frontier orbit and active site of penicillin molecule has theoretical and practical significance to better understand the physical and chemical properties of penicillin molecule, to synthesize penicillin molecule and to develop its new pharmacological properties. In this paper, based on density functional theory, the spatial structure of penicillin molecules was firstly constructed by using Gaussian View 6.0 software, and the configuration optimization and frequency calculation of penicillin molecules were performed by using Gaussian09 software at the B3LYP/ 6-311g (D, P) basis set level. On the basis of structural optimization, based on time-dependent density functional theory, 25 excited states of penicillin molecules in aqueous solution were calculated, and then the frontier orbitals and active sites of penicillin molecules were analyzed by Multiwfn3.6 software, Fukui function method and frontier orbital theory method. The UV spectrum was plotted with Origin2017 software. The results show that the highest occupied molecular orbital (HOMO) and the lowest unoccpied molecular orbital (LUMO) of penicillin molecule are the 88th and 89th electron orbital, respectively. The maximum absorption peak of penicillin molecule UV spectrum is at 220.89nm, and its absorption mole coefficient is 6960.18L/mol/cm. It is mainly formed by the transition from the ground state S0 of the electronic state to the excited state S2, S4, S9, S11 and S12 of the electronic state, and the transition from S0 to the excited state S4 is the biggest contributor to the UV spectrum. The nucleophile site of penicillin molecule is C35 and the potentiophile site is S25.

Penicillin

Density functional theory

Frontier orbital

Active site

UV spectrum

Penicillin (Penicillin G, PNC) was discovered accidentally by Professor Fleming (Tompsett 1996). In 1938, Florey et al. successfully isolated and obtained high purity penicillin (Chain et al. 1940). The antibacterial ability of penicillin far exceeded that of the mainstream sulfa antibiotics at that time, and it was widely used in antibacterial medicine, making it the most widely used broad-spectrum antibiotic up to now (Kumar et al. 2021; Ternák et al. 2021). Crowfoot et al were the first to determine the spatial structure of the penicillin molecule (Crowfoot et al. 1949). Hodgkin et al. solved the problem of penicillin instability by proposing that the structure of penicillin was composed of b-lactam ring and tetrahydrothiazole ring double ring, and obtained the exact structure of penicillin (Kademani et al. 2005; Söylemez et al. 2019). Penicillin belongs to the b-lactam class of antibiotics, it is composed of b-lactam ring, tetrahydrothiazole ring and side chain. Its organic chemical name is: (2S, 5R, 6R)-3,3-dimethyl-6-(2-acetyl amino benzene)-7-4-sulfur impurity oxygen generation-1-nitrogen mixed double loop [3.2.0] heptane-2-formic acid. It has 41 atoms, its molecular formula is C₁₆H₁₈N₂O₄S, and its molecular weight is 334.4.

Penicillin molecules are extremely unstable and break down easily in acidic and alkaline conditions, but the final product has the same structure. The b-lactam ring of penicillin molecule is the main part of the antibacterial. When attacked by bacteria, penicillin molecule has its property; when attacked by other substances, it is easy to decompose and lose its activity (Tompsett 1996; Chakraborty et al. 2021; Li et al. 2016). Penicillin can be hydrolyzed like most antibiotics (Yan et al. 2020; Zhang et al. 2019; Feng et al. 2017). In dilute acid solution (pH4.0) and at room temperature, lone pair electrons on carbonyl oxygen atoms in the side chain of penicillic acid, a metabolite of penicillin, attack b-lactam ring as a nucleophile to form intermediates, and then rearrange to form penicillic diacid. Penicillic diacid can further form penicillamine and penicillaldehyde. Under alkaline conditions and under the action of certain enzymes (such as b-lactamase), the alkaline group or nucleophilic group of the enzyme attacks the b-lactam ring to produce penicillic acid. When heated, penicillic acid tends to lose carbon dioxide molecules to form penicillic thiazolic acid, which can be further decomposed into penicillamine and penicillaldehyde in the presence of mercury chloride. Penicillin is a kind of weak acid substances, there are at least five, namely F, G, X, K, V, their structural backbone has penicillamic acid. The most commonly used penicillin in medicine is the sodium salt of penicillin G (common name penicillin). Penicillin produces penicillic amino acid after acidic hydrolysis in the human body. b-lactam ring is the main antibacterial part of penicillins, mainly destroying the cell wall of bacteria. Because human cell membranes have no cell walls, penicillin has little impact on people. However, penicillin is also highly toxic and sensitizing (Li et al. 2010; Guo et al. 2008), which can cause symptoms such as anaphylactic shock in severe cases, and the incidence of allergy is as high as 5%-10%. In recent years, penicillin has been abused in medical and animal feed, which poses a certain threat to people's health. Therefore, a simple and convenient detection method for penicillin residue is urgently needed. Li et al. studied the binding mechanism of penicillin and b-casein by fluorescence spectroscopy, and obtained the binding constant of penicillin and b-casein (Li et al. 2013). Ma et al. made a quantitative analysis of penicillin content in milk, and the results showed that it was feasible to analyze penicillin content in milk by near infrared spectrum (Ma et al. 2012). Rosa et al.studied the selective flucloxacillin hydroxylation by cytochrome P450 BM3 variants (Rosa et al. 2018). Many researchers have studied the determination of penicillin residues by chromatographic and spectral methods (Horii et al. 2020; Guo et al. 1994). Some other scholars explored the spatial structure of penicillin from the perspective of b-lactam antibiotic drug design (Qian et al. 2020; Levy et al. 2019).

Previous studies on penicillin mainly focused on the measurement of penicillin content and the detection of penicillin residue in food, but the frontier orbits, excited state properties, UV spectrum and active sites of penicillin molecules have not been reported. In this paper, the frontier orbit, UV spectrum, active site and excited state properties of penicillin molecule were analyzed based on quantum chemistry method, which has certain guiding significance for understanding the physical and chemical properties of penicillin molecule, designing and synthesizing new penicillin drugs and developing new pharmacological uses of penicillin.

Gauss View 6.0 software was used to construct the geometric structure of penicillin molecule, then, based on the B3LYP method of density functional theory(DFT), Gaussian09 software (Frisch et al. 2013, 2016) was used to optimize the configuration and calculate the frequency of penicillin molecules at the 6-311g (d, p) basis set level. The optimization results were convergent and without virtual frequency. This shows that the optimized structure is reasonable. On the basis of optimization, based on time-dependent density functional (TD-DFT), 25 excited states of penicillin molecules in aqueous solution were calculated, and then the wave function analysis software Multiwfn3.7 (Lu et al. 2012; Lu 2020) was used to calculate the UV excited properties of penicillin molecules and the prediction of the active site, and then the UV spectrum of penicillin were drawn by the software Origin2017.

The geometric structure of the penicillin molecule

Figure 1 shows the optimized spatial structure of penicillin molecules. It can be seen from the figure that the tension of the four-atom β-lactam ring and the five-atom tetrahydrothiazole ring is very high, resulting in the torsion of the structure. Thus, the penicillin molecule is not a simple planar structure, but a complex three-dimensional structure.

The results of configuration optimization and frequency calculation of penicillin molecules are shown in Table 1. According to the table, the maximum force change is 0.00004 a.u., the root mean square (RMS) of force change is 0.00001 a.u., the maximum displacement is 0.000573 a.u., and the root mean square value of displacement is 0.000150 a.u.. All of them meet the convergence standard and have no virtual frequency. This indicates that the molecular structure of penicillin is optimized to a stable structure with a local minimum energy value, and the structural optimization is reasonable. The energy of the optimized penicillin molecule is -1429.44Hartree.

Table 1

Optimization results of penicillin molecular geometry configuration
Maximum force change	RMS of force change	Maximum displacement	RMS of displacement
0.000004a.u.	0.00001a.u.	0.000573a.u.	0.000150a.u.
convergence	convergence	convergence	convergence

The properties of excited states

The highest occupied molecular orbital (HOMO) and the lowest unoccpied molecular orbital (LUMO) of penicillin are the 88th and 89th electron orbital, respectively. The excited state properties of penicillin are shown in Table 2. It can be seen from Table 2that the contribution of the electron transition to the excited state is not only related to the HOMO and LUMO, but also related to the orbital adjacent to the HOMO and LUMO. These are the frontier orbits of penicillin molecules (Parr et al. 1984).

Table 2

Properties of excited states of penicillin molecules
Excited state	wavelength (nm)	Energy(eV)	Oscillator strength (a.u.)	Orbital transitions	Contribution weight (%) (> 10%)
2	235.09	5.28	0.018	88→92	24.37%
				88→90	19.42%
				85→92	12.43%
				85→89	11.47%
4	226.88	5.47	0.101	87→90	49.34%
4	226.88	5.47	0.101	83→90	12.97%
9	214.33	5.79	0.034	88→93	29.14%
9	214.33	5.79	0.034	88→90	26.77%
11	209.08	5.93	0.028	85→89	21.03%
				87→92	18.49%
				82→89	14.69%
12	208.17	5.96	0.028	87→90	19.41%
				86→91	16.77%
				87→92	16.38%

3.3 Ultraviolet spectrum

On the basis of configuration optimization of penicillin molecule, 25 excited states of penicillin molecule in aqueous solution were calculated by using B3LYP method and Gaussian09 software based on TD-DFT. The threshold value of vibrator intensity is set as 0.01a.u., and the excited state with vibrator intensity lower than 0.01a.u. is not considered. Five excited states with vibrator intensity greater than 0.01a.u. of penicillin molecule can be obtained. The UV spectrum was drawn by Origin2017 software, as shown in Fig. 2, where S0 represents the ground state in UV excitation, and Sx represents the x state in UV excitation. The ultraviolet spectrum of penicillin molecule is mainly formed by the electron transition from the ground state S0 to the excited states S2, S4, S9, S11 and S12. The theoretical calculation results of UV spectrum agree well with experiment (Qian 2002; Sun et al. 2012; Faith et al. 1977; Martínez et al. 2009).

As can be seen from Fig. 2, the maximum absorption peak of penicillin molecule is located at 220.89nm, and its molar absorption coefficient is 6960.18L/mol/cm. The contribution of the main excited state transition of penicillin molecule to the maximum absorption peak of ultraviolet spectrum is shown in Table 3.

Table 3

Contribution of different transitions to UV spectrum
transition	Molar absorption coefficient(L/mol/cm)	Contribution (%)
S0→S2	360.77	5.18%
S0→S4	3575.26	51.37%
S0→S9	1146.89	16.48%
S0→S11	595.83	8.56%
S0→S12	543.30	7.81%

Active site

Fukui function method

Three states of penicillin molecule were calculated with Gaussian09 respectively: uncharged state, a positive charge state and a negative charge state, that is, N, N + 1 and N-1 states. The isosurface of the molecule is set to 0.003a.u., and the Fukui function (Fukui 1970) in the software Multiwfn3.7 is used for calculation. The isosurface of the value of the Fukui function is obtained as shown in Fig. 3 and Fig. 4 respectively, where the green area of the isosurface is the positive part, and the blue area of the isosurface is the negative part. In general, the Fukui function value of the active site of molecular chemical reaction is large, that is, the higher the electron cloud density on an atom or group, the more likely that atom or group will be the active site of the molecular chemical reaction (Fu et al. 2014; Cao et al. 2015). On the f ⁺ isosurface, the density of electron cloud is the highest around C35 atom, that is, C35 atom may be the nucleophilic site of this molecule. On the f ⁻ isosurface, the electron cloud density around the atom S25 is the highest, that is, the atom S25 may be the electrophilic site of the molecule.

3.4.2 Frontier orbital theory

Hirshfeld method was used to calculate the contribution of each atom to HOMO and LUMO, and only the top 10 contributing atoms were listed, as shown in Table 4. It can be seen from Table 4 that the atom S25 makes the largest contribution to HOMO, with a contribution rate of 55.46%. It is vulnerable to electrophilic reaction due to electrophilic reagent attack, that is, it may be the electrophilic site of penicillin. Atom C35 has the largest contribution to LUMO, with a contribution rate of 22.05%. It is vulnerable to attack by nucleophile and nucleophile reaction occurs, that is, it is most likely to be the nucleophile site of penicillin. The active sites predicted by frontier orbit are consistent with those predicted by Fukui function.

Table 4

Contribution of each atom to the frontier orbital
Atomic number	Contribution to HOMO	Atomic number	Contribution to LUMO
25S	55.46%	35C	22.05%
20C	3.93%	25S	19.57%
16N	3.80%	36O	15.11%
6C	3.04%	26C	6.69%
18C	2.99%	37O	6.43%
22O	2.94%	23C	6.28%
3C	2.86%	24N	3.65%
41O	2.67%	20C	3.47%
26C	2.57%	40H	2.28%
31C	2.17%	19C	2.23%

Penicillin is a broad-spectrum antibiotic. However, the overuse of penicillin will lead to the abuse of antibiotics, which will have negative effects on animals and humans. Therefore, the study of the structure, properties and active sites of penicillin will provide a theoretical reference for the design and synthesis of new penicillin drugs and the understanding of the physical metabolism of penicillin.

In this paper, firstly, Gauss View6.0 software was used to optimize the spatial geometric structure of penicillin molecule. Based on DFT, Gaussian09 software was used to optimize the structure and calculate the frequency of molecular configuration at the level of B3LYP/ 6-311g (D, P) base group. The optimization results were all convergent and had no virtual frequency. It shows that the optimized structure is reasonable. On the basis of optimization, the threshold value of vibrator strength was set as 0.01. Fifteen excited states of penicillin molecule were calculated based on TD-DFT in aqueous solution, and the vibrator strength of five excited states was greater than the threshold value. Original 2017 software was used to draw the ultraviolet spectrum of penicillin molecules. According to the analysis, the maximum absorption peak of UV spectrum is located at 220.89nm, and the molar absorption coefficient is 6960.18L/mol/cm, which is mainly formed by the transition from the ground state S0 to the second, fourth, ninth, 11th and 12th excited states. Among them, the transition from S0 to S4 contributed the most to the maximum absorption peak of the UV spectrum, which was as high as 51.37%. Finally, based on Multiwfn3.7 software, Fukui function and frontier orbital theory were used to calculate and analyze the active site of penicillin molecule. The results showed that the nucleophilic site of penicillin molecule was C35 and the electrophilic site was S25. The results of active site calculation are consistent with those of penicillin metabolism (Nielsen et al. 1995).

Acknowledgements: This work is supported by the National Natural Science Foundation of China (11164004), the Guizhou Provincial Photonic Science and Technology Innovation team (Qianke Joint talents team [2015]4017) and the First-class Physics Promotion Programme of Guizhou University.

Conflict of interest: The authors declare that they have no conflict of interest.

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No competing interests reported.

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Theoretical Study on UV Spectrum and Active Site of Penicillin

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Abstract

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Introduction

Computational Method

Results And Analysis

The geometric structure of the penicillin molecule

Active site

Discussions and Conclusion

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References

Additional Declarations

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