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Siddiqui et al. Chem Synth 2023;3:25 https://dx.doi.org/10.20517/cs.2023.02 Page 3 of 50

acetate (EtOAc), filtered to separate biomass, extracted thrice with DCM/EtOAc, and evaporated using a
rotary evaporator.

The crude materials were initially fractionated by column chromatography by using hexanes-ethyl acetate
or hexanes-acetone solvent systems. The fractions were finally purified by reverse phase or/and normal
phase HPLC, followed by thin layer chromatography (TLC). The structures of purified derivatives were
established by using 1D-, and 2D-NMR (Nuclear Magnetic Resonance), HREI-MS (High-Resolution
Electron Ionization Mass Spectrometry), HRESI-MS (High-Resolution Electrospray Ionization Mass
Spectrometry), HRFAB-MS (High-Resolution Fast Atom Bombardment Mass Spectrometry), IR (Infrared),
and UV (Ultraviolet–visible) spectral data, and single-crystal X-ray diffraction analyses. Fully purified
compounds were evaluated for different biological activities.

In certain cases, solid phase fermentation was also employed to increase the yields and diversity of
metabolites.

Spectroscopy is the investigation and measurement of spectral data produced by the interaction of samples
with electromagnetic radiation. NMR spectroscopy plays a major role in the structure determination of
organic molecules, and other biological macromolecules. Chemical shifts are accurately measured by NMR
parameters as sensitive probes of molecular structures. HSQC spectroscopy determines the correlations
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1
13
between two different types of nuclei ( H with C or H with N), which are separated by one bond. HMBC
spectroscopy correlates H and C nuclei through two, three, or sometimes four bonds. COSY (¹H-¹H
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1
Correlation) Spectroscopy shows the correlation between hydrogens that are coupled to each other in the
¹H-NMR spectrum. NOESY is frequently used to determine the spatial structure of organic molecules. The
CD (Circular dichroism) is the difference in absorption of left and right circularly polarized light. Only
chiral molecules display CD, and enantiomers have CD of equal magnitude but opposite sign.
KEY OBJECTIVES
The main objectives of our work on biotransformation studies were as follows: 1: To synthesize libraries of
new and novel analogues of natural, synthetic, and semisynthetic compounds through eco-friendly and
cost-effective biotransformation techniques with the aim of improving their pharmacodynamic profiles; 2:
To produce potentially interesting regio-, stereo-, enantio-selective compounds without the use of toxic
chemicals and harsh conditions; 3: To evaluate the resultant transformed products for different biological
activities, e.g., enzyme inhibition, anti-inflammatory, anti-cancer, anti-bacterial, etc.

BIOTRANSFORMATION OF MONOTERPENES
Biotransformation of (-)-α-(1), and β-pinene (6)
α-(1), and β-pinene (6) are well-known monoterpenes having a range of pharmacological activities, such as
anti-microbial, anti-inflammatory, anti-oxidant, anti-malarial, and anti-leishmanial. They are the major
constituents of many aromatic plants. Biotransformation of (-)-α-pinene (1) with the fungal culture of
Botrytis cinerea, afforded three new hydroxylated metabolites, 3-hydroxy-(-)-β-pinene (2) (16.5%), 9-
hydroxy-(-)-α-pinene (3) (13.5%), and 4-hydroxy-(-)-α-pinene-6-one (4) (20%), along with a known
[21]
metabolite verbenone (5) (28%) [Figure 1].

Similarly, biotransformation of (-)-β-pinene (6), a structural isomer of (-)-α-pinene, with the fungal culture
of Botrytis cinerea yielded four new hydroxylated metabolites, (-)-6α-hydroxy-β-pinene (7) (22%), (-)-4β,5β-
dihydroxy-β-pinene (8) (10%), (-)-2β,3β-dihydroxypinane (9) (12%), and (-)-4β-hydroxy-β-pinene-6-one (
[22]
10) (9%) [Figure 2].