# Determination of absolute configuration of lasdiploic acid (natural product) using TD-DFT calculations

## Abstract

Lasdiploic acid is a natural product which is extracted from an endophytic fungus. It has got medicinal importance due to its potent xanthine oxidase inhibiting activity. Over activity of this enzyme may cause increased level of uric acid in blood, which may eventually lead to gout. This was extracted and isolated by a group of researchers and its biological activity was discovered. This compound has got three stereocentres, so eight different stereoisomers are possible. Determination of absolute configuration of this natural product using conventional spectroscopic techniques is not possible. However, in recent past, Electronic Circular Dichroism (ECD) have been extensively utilized for the purpose of determination of absolute configuration of natural products. The results have been found to be so convincing and reliable that researchers from natural products are now using this technique at their choice. With revolutionary advancements in the area of quantum chemical calculations of chiroptical spectroscopy over the past decade, the Time Dependent Density Functional Theory (TD-DFT) calculation of Electronic Circular Dichroism (ECD) spectra has emerged as a very promising tool. The principle is simply based on the comparison of the calculated and experimental ECD spectra: the more closely they match, the more reliable conclusion for the absolute configuration assignment can be drawn.

**Keywords**: xanthine oxidase, lasdiploic acid, endophytic fungus, Electronic Circular Dichroism

## ABBREVIATIONS

DFT | Density Functional Theory |

TD-DFT | Time Dependent Density Functional Theory |

AC | Absolute Configuration |

ECD | Electronic Circular Dichroism |

## INTRODUCTION

## Background

## Statement of the Problem

Since NMR couldn't give satisfactory results regarding absolute configuration determination, TD-DFT calculations are performed on each of the stereoisomers of lasdiploic acid.

## Objectives of the Research

On the basis of the nature of the project following two objectives are proposed.

## Objective 1:

To learn and carry out the optimization of all the possible conformers.

To learn and compare the stability of all conformers on the basis of relative energies.

## Objective 2:

To learn and perform TD-DFT calculations.

To analyze the simulated UV-VIS spectra and compare with the literature report.

To analyze the simulated ECD spectra of Lasdiploic acid and compare with the literature report.

## Scope

TDDFT is used in variety of applications like chemistry, biology, solid state physics, and materials science^{1}. A big hand of TD-DFT calculation lie in spectroscopy, yielding response and excitations of atoms, molecules and solids. Overall, results for excitation energies tend to be fairly good but depend significantly on the system and type of excitation considered, also on the type of basis set used and level of theory applied. Computational methods can provide satisfying results regarding absolute configuration determination.

## LITERATURE REVIEW

## Information

In figure 2, solid line represents the experimental ECD spectrum while the dotted line belongs to simulated ECD spectra^{2}. It is can be seen clearly that the two spectra look like very similar, thus, the absolute configuration of Eucophylline can be determined.

## METHODOLOGY

## Concepts

## Quantum chemical study

The quantum chemical study will be carried out to understand stability of all possible stereoisomers. Schrödinger wave equation is the most important theory in quantum chemistry. Electronic excitations are performed computationally in order to determine various properties of compounds. These electronic excitations are based on Frank-Condon principle. Serious and fruitful research activities are going on this area of science.

## Methods

## Method of calculation

The optimization of stereoisomers will be carried out using Density Functional Theory (DFT) with 6-31+G(d) basis set.

TD-DFT calculations will be carried out using the CAM-B3LYP method and aug- cc- pVDZ basis set.

## RESULTS AND DISCUSSIONS

## To learn and carry out the optimization of all the possible stereoisomers.

Optimization of all possible stereoisomers has been carried out by using B3LYP method with 6-31+G(d) basis set. The optimized geometries are given below.

## To learn and compare the stability of all conformers on the basis of relative energies.

The absolute energies (Hartree) of all stereoisomers were converted to kcal/mol by multiplying a factor of 627.5095. The relative energy of all stereoisomers are given below (Table 1).

Absolute Configuration | Hartree | Kcal/mol |

3E, 8E, 7A | -734.041945 | 0.00 |

3A, 8E, 7A | -734.041464 | 0.30183207 |

3A, 8A, 7E | -734.040119 | 1.14583235 |

3E, 8E, 7E | -734.039974 | 1.23682122 |

3E, 8A, 7E | -734.039941 | 1.24121379 |

3A, 8A, 7A | -734.039941 | 1.25752904 |

3A, 8E, 7E | -734.039273 | 1.50727782 |

3E, 8A, 7A | -734.037897 | 2.54015846 |

## To learn and perform TD-DFT calculations.

TD-DFT calculations are carried out on all optimized stereoisomers.

TD-DFT calculations are carried out using the CAM-B3LYP method and aug-ccpVDZ basis set.

So far UV-VIS spectra are generated and analyzed at various excitation states (N).

N ranges from 6 to 20. Simulated UV-VIS spectra of 3E 8E 7A at various excitation levels (N) is given below (Figure 4)

** **

**Graphical Representation of UV-VIS spectra**

Figure 5 justifies the number of states (N= 6 to 10) which give the most relevant results with respect to the experimental UV-VIS spectra as it can be seen that when N= 20, the spectra is some what different.

** ****Mathematical Representation of UV-VIS spectra **

States(N) | Energy(eV) | Energy(nm) | Oscillator Strength (f) |

N _{1} | 3.9239 | 315.97 | 0.0007 |

N_{ 2} | 6.2668 | 197.84 | 0.5685 |

N_{ 3} | 6.5403 | 189.57 | 0.0007 |

N_{ 4 } | 6.9258 | 719.02 | 0.0008 |

N _{5}_{} | 7.2585 | 170.81 | 0.2319 |

N_{ 6} | 7.5081 | 165.13 | 0.0162 |

N _{7} | 7.7416 | 160.15 | 0.0043 |

N _{8} | 7.869 | 157.56 | 0.0021 |

N _{9} | 7.9022 | 156.9 | 0.0014 |

N_{10} | 7.9427 | 156.1 | 0.0195 |

## Generatating ECD spectra using TD-DFT calculations

All other ECD spectra which are also matching with the reported method are given below, however, the overlapping figure is not given which is given only for the above said stereoisomer (3R, 8S, 7R).

## Inference from simulated and experimental ECD spectra

ECD calculations were carried out on all possible conformers using CAM-B3LYP method and aug-cc-pVDZ basis set. The ECD spectra generated were analysed by using Guassview 5.09. All the ECD spectra of possible stereoisomers were compared with the literature report and it was found that all the spectra were compared with reported literature. This shows the results are reproducible and reliable. However, the stereoisomer 3R, 8S, 7R (3A, 8E, 7A) was the point of discussion. Literature says the solution form of lasdiploic acid exists as 3R, 8S, 7R (3A, 8E, 7A) on the basis of showing identical simulated and experimental ECD spectra and also it is the second most stable stereoisomer both from our studies and the literature report. From our study, the ECD spectra of 3R, 8S, 7R (3A, 8E, 7A) generated with the reported basis set also shows the same ECD spectra Figure 5. Hence, it can be concluded the results produced from our study are matching exactly with the literature report.

## Other ECD Spectra

**3A, 8A, 7E**

**3A, 8E, 7E **

** 3E, 8A, 7A **

**3E, 4E, 5A**

**3E, 8E, 7E **

**3A, 8A, 7A**

**3A, 8E, 7A **

**3E, 8A, 7E **

## CONCLUSION

Techniques involving electronic excitations like UV-VIS and ECD are among the spectroscopic methods used for the structural elucidation of molecules. UV-VIS is mainly used to characterize the molecules possessing electronic conjugation or simply the chromophores or coloured compounds by determining the characteristic wavelength ( λ max) of the molecule. Now, recently the ECD has been widely used to characterize the stereochemistry of natural products. To learn more about excitation spectroscopic techniques (simulated) I undertook one more project which is to determine the absolute configuration of natural products. The calculations for optimization and ECD were done by using Gaussian. TD-DFT is the method used for ECD calculations. The results from my study are well in accordance with the literature report.

## REFERENCES

1. X. C. Li, D. Ferreira, Y. Ding. Determination of Absolute Configuration of Natural Products: Theoretical Calculation of Electronic Circular Dichroism as a Tool. Curr. Org. Chem. **2010**, 14, 1678–1697.

2. D. M. McCann, P. J. Stephens. Determination of Absolute Configuration Using Density Functional Theory Calculations of Optical Rotation and Electronic Circular Dichroism: Chiral Alkenes. J. Org. Chem.** 2006**, 71, 6074-6098.

## ACKNOWLEDGEMENTS

* I thank Indian Academy of Sciences for selecting me under the Summer Research Fellowship Program 2019. *

** **I would like to express my sincere gratitude to my project guide **Prof. P. V. Bharatam** for the continuous support. His guidance helped me in all the time throughout my work. He always made sure that I’m comfortable here in this lab.

*Thanks to ***National Institute of Pharmaceutical Education and Research, Mohali*** for treating me as I belong to you. *

*My sincere thanks also goes to my mentor ***Mr. Aabid Wani***, who was patient enough to explain me everything from the beginning. I heartly thank*** Ms. Deepika Kathuria***, ***Mr. Gurudutt Dubey****, Mr. Pravin Wanjari **and all other Ph.D. fellows of PVB lab.

*Thanks to my institution S. V. National Institute of Technology, Surat for allowing to do my project here at NIPER. Also thanks to Dr. Ketan. C. Kuperkar for giving me faculty recommendation. *

*Also I would like to thank ***Dr. Suchandra Goswami***, ***Ms. Marudhvati Kudumula, Ms. Sumi Aisha Salim, Ms. Kavyasree S. N, Ms. Samima Khatun, Mrs. Nisha, Mr. Ravindra Gurjar*** and all other masters’ students working in PVB lab. *

*Thanks to my parents (Suresh and Uma) for always being supportive for their little girl*.

*And last but not least I’m ever grateful to the divine supreme power, Bhagwan for all the *

*things in my life.*

**Arya MP **

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