Biocatalytic synthesis of novel partial esters of a bioactive dihydroxy 4-methylcoumarin by Rhizopus oryzae lipase (ROL)

Article abstract of DOI:10.3390/molecules21111499

Highly regioselective acylation has been observed in 7,8-dihydroxy-4-methylcoumarin (DHMC) by the lipase from Rhizopus oryzae suspended in tetrahydrofuran (THF) at 45°C using six different acid anhydrides as acylating agents. The acylation occurred regioselectively at one of the two hydroxy groups of the coumarin moiety resulting in the formation of 8-acyloxy-7-hydroxy-4-methylcoumarins, which are important bioactive molecules for studying biotansformations in animals, and are otherwise very difficult to obtain by only chemical steps. Six monoacylated, monohydroxy 4-methylcoumarins have been biocatalytically synthesised and identified on the basis of their spectral data and X-ray crystal analysis.

Full text of DOI:10.3390/molecules21111499

molecules
Article
Biocatalytic Synthesis of Novel Partial Esters of
a Bioactive Dihydroxy 4-Methylcoumarin by
Rhizopus oryzae Lipase (ROL)
Vinod Kumar 1,2, Divya Mathur 3,4, Smriti Srivastava 3, Shashwat Malhotra 3,5, Neha Rana 3,
Suraj K. Singh 3, Brajendra K. Singh 3, Ashok K. Prasad 3, Anjani J. Varma 6, Christophe Len 7,
Ramesh C. Kuhad 2,6, Rajendra K. Saxena 2 and Virinder S. Parmar 3,6,8,
*
1
Centre of Innovative and Applied Bioprocessing (CIAB), SAS Nagar, Mohali 160 071, Punjab, India;
vinod_biodiesel@yahoo.co.in
Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021,
India; Kuhad85@gmail.com (R.C.K.); rksmicro@yahoo.co.in (R.K.S.)
Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India;
dmchem05@gmail.com (D.M.); smritichem10@gmail.com (S.S.); shashwatmalhotra@gmail.com (S.M.);
neharana256@gmail.com (N.R.); surajsingh565656@gmail.com (S.K.S.); chemquestbk@gmail.com (B.K.S.);
ashokenzyme@gmail.com (A.K.P.)
Department of Chemistry, Daulat Ram College, University of Delhi, Delhi 110 007, India
Department of Chemistry, Kirori Mal College, University of Delhi, Delhi 110 007, India
School of Chemical Sciences, Central University of Haryana, Mahendragarh, Haryana 123 029, India;
anjanikumar@cuh.ac.in
2
3
4
5
6
7
Sorbonne Universités, Université de Technologie de Compiègne, EA4297 Transformations Chimiques
de la Matière Renouvelable, F-60203 Compiègne CEDEX, France; christophe.len@utc.fr
Institute of Advanced Sciences, 410 Faunce Corner Mall Road, Dartmouth, MA 02747, USA
Correspondence: parmar@cuh.ac.in; Tel.: +91-965-073-4027
8
*
Academic Editor: Derek J. McPhee
Received: 31 July 2016; Accepted: 2 November 2016; Published: 9 November 2016
Abstract: Highly regioselective acylation has been observed in 7,8-dihydroxy-4-methylcoumarin
(DHMC) by the lipase from Rhizopus oryzae suspended in tetrahydrofuran (THF) at 45 ◦C
using six different acid anhydrides as acylating agents. The acylation occurred regioselectively
at one of the two hydroxy groups of the coumarin moiety resulting in the formation of
8-acyloxy-7-hydroxy-4-methylcoumarins, which are important bioactive molecules for studying
biotansformations in animals, and are otherwise very difficult to obtain by only chemical steps.
Six monoacylated, monohydroxy 4-methylcoumarins have been biocatalytically synthesised and
identified on the basis of their spectral data and X-ray crystal analysis.
Keywords: Rhizopus oryzae; lipase; 7,8-Dihydroxy-4-methylcoumarin; regioselectivity; acylation
1. Introduction
Coumarins (2H-1-benzopyran-2-ones) are versatile and a structurally-diverse class of natural
products essentially found in the family Rutaceae and Umbelifferae. Owing to their plethora of
pharmacological activities [1,2], coumarins have been a subject of extensive research over many
decades [ ]. In recent years, our laboratories have been actively engaged in studying the synthesis
3,4
and biological activities of a wide range of coumarin derivatives [5–8]. We have previously
isolated 8-acetyl-7-hydroxy-6-methoxycoumarin and 8-methoxycoumarin from Fraxinus floribunda
leaves [9], trigoforin (3,4,7-trimethylcoumarin) and trigocoumarin [3-(ethoxycarbonyl)methyl-
4-methyl-5,8-dimethoxycoumarin] from Trigonella foenumgraecum stems [10,11], and troupin
(4-methyl-6-hydroxy-7,8-dimethoxycoumarin) from Tamarix troupii [12] (Figure 1).
Molecules 2016, 21, 1499; doi:10.3390/molecules21111499
www.mdpi.com/journal/molecules

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Figure 1. Biologically-important coumarins.
Protein acetylation, largely acetyl coenzyme A (CoA)-dependent, is one of the prominent
post-translational modifications of proteins that regulate several cellular processes [13].
Acetyl CoA-independent protein acetylation is also known, but is restricted to the action of aspirin-like
drugs that would readily acetylate cyclooxygenase resulting in the inhibition of prostaglandin
synthesis and, thus, induce anti-inflammatory effects [14]. Through extensive studies, we identified
a microsomal enzyme, protein transacetylase (TAase) in mammalian cells and tissues, catalysing the
transfer of acetyl groups from 7,8-diacetoxy-4-methylcoumarin (DAMC) to certain receptor proteins,
viz., cytosolic glutathione S-transferase (GST), cytochrome P-450-linked mixed function oxidases
(MFO), Nicotinamide Adenine Dinucleotide Phosphate (NADPH) cytochrome c-reductase, protein
kinase C (PKC), nitric oxide synthase (NOS), and recombinant glutamine synthetase (rGlnA1) of
Mycobacterium tuberculosis, resulting in the modulation of their catalytic activities and associated
physiological effects [15–20]. The rat liver and human placental microsomal TAase was later identified
as calreticulin, a calcium-binding protein in the lumen of the endoplasmic reticulum and, consequently,
the acetyltransferase function of calreticulin was appropriately termed calreticulin transacetylase
(CRTAase) [21–24].
Furthermore, we had earlier demonstrated that buffalo liver TAase-mediated inhibition of GST
by DAMC was due to the acetylation of six lysine residues, viz., Lys-51, -82, -124, -181, -191, -210, and
N-terminus proline residues, as shown by Matrix-Assisted Laser Desorption Ionization–Time of Flight
(MALDI-TOF) and Liquid Chromatography–Mass Spectrometry (LC/MS/MS) studies [25]. It was
also inferred that the C-7 acetoxy group of DAMC helps the C-8 acetoxy group to orient itself most
favourably towards the oxygen heteroatom of the coumarin moiety and this phenomenon, presumably
controls the TAase-mediated transfer of acetyl groups to the functional proteins [26]. An interesting
observation was the action of TAase and DAMC on liver cytosolic GST, which resulted in the formation
of a monoacetoxy, monohydroxy 4-methylcoumarin (MAMHC), and 7,8-dihydroxy-4-methylcoumarin
(DHMC), although the former was the major metabolite (Figure 2) [27]. This substantiates the
fact that acetyl groups are preferentially transferred by TAase from a particular acetoxy group
of DAMC to the receptor protein leading to the accumulation of MAMHC. Nonetheless, the
position of mono-deacetylation in DAMC could not be ascertained. Thus, to identify and compare
the monoacetoxy, monohydroxy 4-methylcoumarin (MAMHC), it was important to synthesize
it chemically. However, its chemical synthesis is still not known, since regioselective chemical
mono-acylation of polyhydric phenols often requires a series of tedious steps leading to low yields of
the required product.
Figure 2. TAase catalyzed reaction.
Lipases provide an alternative, greener, and expedient route for the selective protection and
deprotection of amino, hydroxyl, and carboxyl groups in a wide range of carbohydrates, nucleosides,

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steroids, and various other classes of organic molecules. Enzyme-mediated preparation of partial
esters of multi-hydroxy compounds, such as polyphenols, has also been extensively explored owing
to the stereo- and regioselective preferences of proteases [28–32]. We have reported efficient and
straightforward regioselective protection/deprotection reactions on hydroxyl groups in an array of
different classes of substrates using a miscellany of lipases, viz. porcine pancreatic lipase (PPL), Candida
antarctica lipase-B (Novozyme®-435), lipase Amano PS, Pseudomonas sp. lipase, Candida rugosa lipase
(CRL), and Thermomyces lanuginosus lipase (Lipozyme® TL IM) in dry organic solvents [33–36].
As part of our ongoing research programme towards screening of various lipases/esterases for
carrying out selective protection/deprotection of biologically-active scaffolds, and also to identify the
MAMHC formed by the action of TAase, we have explored the possibility of using lipases for selective
mono-acylation of 7,8-dihydroxy-4-methylcoumarin (Figure 2).
2. Results and Discussion
In the present investigation, we have identified Rhizopus oryzae lipase (ROL), a fungal lipase that
assisted in the formation of monoacyloxy, monohydroxy 4-methylcoumarins from the biocatalytic
acylation of 7,8-dihydroxy-4-methylcoumarin (DHMC) with different acid anhydrides (Scheme 1).
It has been observed that greater selectivity and feasibility is achieved in the enzymatic deacylation
reactions, particularly in the case of polyphenols [30,31]. With only a few reports dealing with the
lipase-catalyzed regioselective acylation of phenolic hydroxy groups, such substrates seem to be
difficult targets for acylation, notably because a phenolic hydroxyl group is less nucleophilic and is
also known to adversely affect the enzymatic activity [37]. Therefore, we first conducted deacylation
studies on 7,8-diacetoxy-4-methylcoumarin (DAMC, Figure 1). The enzymatic substrate was prepared
by the well-known Pechmann condensation of pyrogallol with ethyl acetoacetate yielding DHMC,
followed by its acetylation using acetic anhydride in pyridine (Scheme 1) [38].
Scheme 1. Synthesis of 7,8-dihydroxy-4-methylcoumarin and 7,8-diacetoxy-4-methylcoumarin.
Interestingly, when we investigated the selective deacetylation of DAMC using lipases, viz.,
Lipozyme® TL IM, PPL, CRL, and ROL in the presence of n-butanol as an acyl scavenger in different
organic solvents, it did not yield any appreciable amount of any deacetylated product (Scheme 2).
Monoacetoxy, monohydroxy 4-methylcoumarin (MAMHC) was formed in very low yields with DHMC
as the major product in the case of Novozyme®-435 in THF at 50 ◦C.
Scheme 2. Enzymatic deacetylation studies on 7,8-diacetoxy-4-methylcoumarin (2a).
Consequently, we tried acylation of 7,8-dihydroxy-4-methylcoumarin (DHMC,
1) using
various acid anhydrides and were successful in obtaining the monoacyloxy, monohydroxy
4-methylcoumarins exclusively. Five different lipases, viz., Novozyme®-435, Lipozyme® TL IM,
PPL, CRL, and ROL were screened for regioselective acylation of one of the two hydroxyl groups
in 7,8-dihydroxy-4-methylcoumarin (1) using acetic anhydride in different organic solvents, i.e.,

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THF, 1,4-dioxane, diisopropyl ether (DIPE), dimethylformamide (DMF), toluene, and acetonitrile at
different temperatures (35–60 ◦C) and at 200 rpm in an incubator shaker. No appreciable conversion
was observed during acylation reaction mediated by Lipozyme® TL IM, PPL, or CRL in any of t◦he
six organic solvents. Novozyme®-435 showed limited regioselectivity towards DHMC in THF at 40 C
for acylation with the concomitant formation of MAMHC and DAMC. Additionally, the reaction
was rather slow and did not lead to complete consumption of DHMC even after two days and at
higher temperatures.
It has been established that fungal lipases have benefits over bacterial lipases due to their low
cost of extraction, thermal and pH stability, substrate specificity, and activity in organic solvents [39].
Keeping this in mind, the regioselective acylation of 7,8-dihydroxy-4-methylcoumarin (1) using acetic
anhydride (4a) was attempted using lipase from Rhizopus oryzae, available in our laboratory culture
collection, in different organic solvents, i.e., THF, 1,4-dioxane, DIPE, DMF, toluene, and acetonitrile, at
different temperatures. Rhizopus oryzae lipase (ROL) in THF at 45 ◦C showed the best result with the
formation of a new product with higher Rf value than the starting substrate and the disappearance of
the starting compound, as indicated by TLC examination in 18 h (Scheme 3). Though, some amount of
DAMC (2a) was also isolated (but only) in traces, it is certain to say that Rhizopus oryzae lipase (ROL)
displayed better regioselectivity, as well as better catalytic activity.
Scheme 3. Rhizopus oryzae lipase-mediated regioselective acylation of DHMC (
formation of 8-acyloxy-7-hydroxy-4-methylcoumarins 3a–3f.
1) yielding exclusive
To explore the enzyme selectivity and reaction rate for different acyl groups, different acid
anhydrides, viz., propanoic (4b), butanoic (4c), pentanoic (4d), hexanoic (4e), and benzoic anhydride
(
4f) were evaluated towards the regioselective acylation reaction for selective protection of hydroxyl
◦
groups in 7,8-dihydroxy-4-methylcoumarin (
1) using ROL in THF at 45 C (Scheme 3, Table 1).
On completion of the acylation reaction, as indicated by the disappearance of the starting compound
on TLC, the enzyme was filtered off and the crude products thus obtained were purified using
column chromatography. The mono-acylated coumarin derivatives 3a–3f were obtained in 30%–82%
isolated yields.
Table 1. Rhizopus oryzae lipase (ROL◦)-catalyzed regioselective formation of 8-acyloxy-7-hydroxy-4-
methylcoumarins 3a–3f in THF at 45 C.
Isolated Yield of 8-Acyloxy-7-hydroxy-
4-methylcoumarin (3a–3f) a (%)
7,8-Diacyloxy-4-
methylcoumarin (2a–2f)
Entry
Acid Anhydride (4a–4f)
Reaction Time (h)
2a/Traces b
2b/<10% b
2c/<10% b
2d/<20% b
2e/<20% b
2f/59.0%
1
2
3
4
5
6
Acetic anhydride (4a)
Propanoic anhydride (4b)
Butanoic anhydride (4c)
Pentanoic anhydride (4d)
Hexanoic anhydride (4e)
Benzoic anhydride (4f)
3a/82.0
3b/76.5
3c/71.0
3d/62.0
3e/60.0
3f/30.5
18
20
24
24
24
36
a
All of these reactions did not yield any product when performed in the absence of the lipase; b Products not isolated, results
according to the TLC analysis.

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It was observed that by increasing the acyl chain lengths, the reaction time increases. It was also
noticed that the percentage yield of mono-acylated product 3a 3f decreased with the increase in the
acyl chain length (Figure 3). Further, it was contemplated that the increase in the acyl chain length
and reaction time also led to the increase in the formation of diacylated coumarin derivative 2a 2f
–
–
.
We found acetic anhydride (4a) as the best acylating agent, among others, in terms of reaction time,
yield, and percentage conversion to the desired regioselectively-acylated products (Table 1, Figure 3).
Figure 3. Conversion of 7,8-dihydroxy-4-methylcoumarin (1) to 8-acyloxy-7-hydroxy-4-methylcoumarins
3a–3f using Rhizopus oryzae lipase (ROL).
Some amounts of the diacyloxycoumarins 2b–2e were also observed, but were not isolated due
to low yields. In the case of benzoic anhydride (4f), the reaction was too slow to be of any particular
application with the formation of the mono-benzoyl coumarin derivative 3f in 30.5% yield after 36 h,
together with the formation of the dibenzoyl coumarin derivative 2f in 59% isolated yield (Table 1).
Thus, it can be inferred that aromatic acid anhydride did not show good selectivity, primarily due
to steric hindrance. Along the same lines, alkyl groups having longer chain lengths also lead to
poor selectivity.
The site of acylation was confirmed by single X-ray crystal analysis of compound 3a synthesized by
biocatalytic acetylation of 7,8-dihydroxy-4-methylcoumarin with acetic anhydride and ROL (Table S1,
Supplementary Materials). The structure of 3a was found to be 8-acetoxy-7-hydroxy-4-methylcoumarin
which established the fact that the enzymatic acylation occurred regioselectively at the C-8 hydroxyl
group instead of the C-7 hydroxyl group in the presence of the ROL (Figure 4). It was previously
reported by us that, in buffalo liver, TAase mediated inhibition of GST by DAMC, the C-7 acetoxy group
helps the C-8 acetoxy group to orient itself towards the oxygen heteroatom of the coumarin leading to
the formation of MAMHC [16,26]. Selective formation of 8-acyloxy-7-hydroxy-4-methylcoumarin by
ROL mediated acylation corroborates this observation.
This unique selectivity exhibited by the Rhizopus oryzae lipase (ROL) in discriminating the
esterification of one out of two phenolic hydroxyl groups of similar reactivity is not feasible to
achieve by chemical methodologies. All attempts in our laboratories during the last three decades by
any biocatalytic or chemical sequence of reactions have been futile in obtaining the partial esters of
di- or trihydric phenolic compounds which we needed for synthesizing the bioactive analogues of
naturally-occurring flavonoids, coumarins, xanthones, or isoflavonoids having interesting biological
activities in cancer, antioxidant, and antiviral research. Thus, the results presented in this study can
find wide applications in various fields of organic chemistry and medicinal chemistry.

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Figure 4. ORTEP diagram showing crystallographic atom numbering and solid-state conformation
for 8-acetoxy-7-hydroxy-4-methylcoumarin (3a; CCDC-1508122). Crystallographic data have been
deposited with Cambridge Crystallographic Data.
3. Experimental Section
3.1. Reagents and Solvents
All reagents used were of commercial grade. Solvents were dried and purified using standard
techniques. Rhizopus oryzae, obtained from our laboratory culture collection was maintained on potato
dextrose agar (PDA). The purified lipase gives a single band on SDS PAGE with a mol. wt. of
30
±
1 kDa. 1H-NMR (400 MHz) and 13C-NMR (100.5 MHz) were recorded in DMSO. Chemical
shifts are reported in parts per million (ppm). The chemical shift values are on the
δ
scale and the
coupling constants (J) are in Hz. Mass spectra were recorded on a KC ESI 455-TOF mass spectrometer
(Micromass, Manchester, UK). The IR spectra were recorded on a Perkin-Elmer model 2000 FT-IR
spectrometer (Perkin Elmer Co., Massachusetts, MA, USA) by making a KBr disc for solid samples
and thin film for oils. X-ray diffraction data was collected on an Oxford XCalibur CCD diffractometer
(Oxford Diffraction, Abingdon, Oxfordshire, UK) using graphite-monochromated Cu K
α radiation
(λ
= 0.7107 Å) at a temperature 298 K. The structure was solved by direct methods using SIR-92
(Semi-Invariants Representation) [40] and refined by the full-matrix method (SHELXL-2016/4) [41].
The reactions were monitored by thin layer chromatography (TLC) on aluminium plates coated
with silica-gel 60F254 (Merck KGaA, Darmstadt, Germany). UV radiation and iodine were used as the
visualizing agents. Column chromatography was performed on silica gel (100–200 mesh, Merck KGaA).
3.2. Lipase Production Conditions
Production of lipase was carried out in 2 L Erlenmeyer flasks containing 400 mL of optimized
production medium (% w/v: soybean oil (emulsified with 0.2% gum acacia), 1.5; cane molasses, 1.0;
maize starch, 1.0; soybean meal, 2.0; CaCl2
·
2H2O, 0.10; KH2PO4, 0.50, pH 9.0) inoculated with
8
×
107 spores and incubated at 30 ◦C, 200 rpm for 96 h. The culture broth was subjected to filtration
and centrifugation to remove the biomass, followed by a four-fold concentration by ultrafiltration
using 10 kDa cellulose acetate membranes (Millipore Merck, Darmstadt, Germany). This partially
purified and concentrated lipase was precipitated using the protocol of Shu et al. [42] and lyophilized.
This preparation had a lipase activity of 3.6 U/mg and was used as such (in the native form) in the
present study.
3.3. Determination of Lipase Activity
The lipase activity of the native lipase was measured by the procedure described by Winkler
and Stuckmann [43]. One unit (U) of enzyme activity was defined as the amount of enzyme, which
liberates 1 µmol of p-nitrophenol from p-nitrophenyl acetate per minute under assay conditions.

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3.4. Synthesis
Regioselective Rhizopus oryzae Lipase (ROL)-Mediated Acylation Reaction on 7,8-Dihydroxy-4-
methylcoumarin (1).
To a dried round bottom flask, 7,8-dihydroxy-4-methylcoumarin (
by the addition of dry THF (25 mL). To the solution, acid anhydride 4a
1
–
) (2 mmol) was added, followed
4f (2 mmol) was slowly added
with continuous stirring. Further, lipase from Rhizopus oryzae (500 mg) was carefully added into the
stirring reaction mixture. The reaction mixture was allowed to shake in an incubator shaker at 45 ◦C
and was regularly monitored on TLC using 1:9 methanol:chloroform as solvent system. On completion
of the reaction, the enzyme was filtered off and washed twice with methanol (2
was evaporated in vacuo and the crude 8-acyloxy-7-hydroxy-4-methylcoumarins 3a
were purified using column chromatography with chloroform/methanol as the eluent.
×
10 mL). The solvent
–3f thus obtained
◦
8-Acetoxy-7-hydroxy-4-methylcoumarin (3a): White solid; 82.0% yield; m.p. 234–235 C. IR (film)
vmax (cm−1): 3103, 1774, 1692, 1605, 1442, 1370, 1279, 1222, 1171. 1H-NMR (400 MHz, DMSO-d6)
δ
: 10.78 (s, 1H, OH), 7.50 (d, J = 8.79 Hz, 1H, ArH), 6.93 (d, J = 8.79 Hz, 1H, ArH), 6.17 (s, 1H, H-3), 2.37
(s, 3H, C-4 CH3), 2.34 (s, 3H, COCH3). 13C-NMR (100.5 MHz, DMSO-d6)
δ: 168.25, 159.43, 154.01, 152.90,
146.85, 125.05, 123.05, 112.97, 112.64, 110.53, 20.31, 18.27. HRMS (ESI): m/z calcd. for C12H10O5 + Na+
[M + Na]+: 257.0420. Found 257.0421.
7-Hydroxy-8-propanoyloxy-4-methylcoumarin (3b): White solid; 76.5% yield; m.p. 190–191 ◦C. IR (film):
1
ν
(cm−1); 3140, 1765, 1693, 1601, 1453, 1393, 1360, 1281, 1196, 1177. H-NMR (400 MHz, DMSO-d6)
max
δ
: 10.73 (s, 1H, OH), 7.47 (d, J = 8.68 Hz, 1H, ArH), 6.91 (d, J = 8.68 Hz, 1H, ArH), 6.16 (s, 1H, H-3), 2.64
(q, J = 7.36 Hz, 2H, COCH2CH3), 2.39 (s, 3H, C-4 CH3), 1.14 (t, J = 7.32 Hz, 3H, COCH2CH3). 13C-NMR
(100.5 MHz, DMSO-d6) δ: 171.58, 159.41, 153.89, 152.92, 146.77, 125.02, 122.88, 112.92, 112.43, 110.41,
26.47, 18.21, 9.00. HRMS (ESI): m/z calcd. for C13H12O5 + Na+ [M + Na]+: 271.0577. Found 271.0574.
8-Butanoyloxy-7-hydroxy-4-methylcoumarin (3c): Cream-coloured solid; 71.0% yield; m.p. 145–146 ◦C.
IR (film):
ν
(cm−1), 3306, 2971, 2937, 2879, 1761, 1715, 1609, 1580, 1327, 1271, 1189, 1243, 1171, 1139.
max
1H-NMR (400 MHz, DMSO-d6)
δ: 10.77 (s, 1H, OH), 7.50 (d, J = 8.80 Hz, 1H, ArH), 6.93 (d, J = 8.80 Hz,
1H, ArH), 6.18 (s, 1H, H-3), 2.62 (t, J = 7.32 Hz, 2H, COCH2CH2CH3), 2.39 (s, 3H, C-4 CH3), 1.66–1.76
(m, 2H, COCH2CH2CH3), 1.01 (t, J = 7.32 Hz, 3H, COCH2CH2CH3). 13C-NMR (100.5 MHz, DMSO-d6)
δ
: 170.63, 159.27, 153.84, 152.91, 146.76, 125.06, 122.86, 112.86, 112.50, 110.41, 39.08, 18.19, 18.10, 13.32.
HRMS (ESI): m/z calcd. for C14H14O5 + Na+ [M + Na]+: 285.0733. Found 285.0730.
◦
7-Hydroxy-8-pentanoyloxy-4-methylcoumarin (3d): Light gray solid; 62.0% yield; m.p. 143–145 C.
1
IR (film):
ν
max (cm−1), 3139, 2937.29, 1764, 1709, 1602, 1515, 1447, 1366, 1273, 1212, 1134, 1093. H-NMR
(400 MHz, DMSO-d6)
δ: 10.77 (s, 1H, OH), 7.48 (d, J = 8.70 Hz, 1H, ArH), 6.91 (d, J = 8.70 Hz,
1H, ArH), 6.16 (s, 1H, H-3), 2.63 (t, J = 7.30 Hz, 2H, COCH2CH2CH2CH3), 2.36 (s, 3H, C-4 CH3),
2.15–2.19 (m, 2H, COCH2CH2CH2CH3), 1.43–1.45 (m, 2H, COCH2CH2CH2CH3), 0.91 (t, J = 7.33
Hz, 3H, COCH2CH2CH2CH3). 13C-NMR (100.5 MHz, DMSO-d6)
δ: 174.56, 170.77, 159.28, 153.84,
152.93, 125.03, 122.87, 112.88, 110.43, 33.38, 32.79, 26.64, 21.48, 18.20, 13.64. HRMS (ESI): m/z calcd.
for C15H16O5 + Na+ [M + Na]+: 299.0890. Found 299.0889.
8-Hexanoyloxy-7-hydroxy-4-methylcoumarin (3e): Light yellow solid; 60.0% yield; m.p. 139–141 ◦C. IR
(film)
ν
(cm−1): 3166, 2932, 1777, 1698, 1602, 1515, 1440, 1369, 1172, 1128. 1H-NMR (400 MHz,
max
DMSO-d6)
δ: 10.62 (s, 1H, OH), 7.36 (d, J = 9.16 Hz, 1H, ArH), 6.80 (d, J = 9.16 Hz, 1H, ArH), 6.04
(s, 1H, H-3), 2.50 (t, J = 7.32 Hz, 2H, COCH2CH2CH2CH2CH3), 2.25 (s, 3H, C-4 CH3), 1.52–1.59 (m,
2H, COCH2CH2CH2CH2CH3), 1.20–1.28 (m, 4H, COCH2CH2CH2CH2CH3), 0.75 (t, J = 6.88 Hz, 3H,
COCH2CH2CH2CH2CH3). 13C-NMR (100.5 MHz, DMSO-d6)
δ: 170.75, 159.25, 153.81, 152.90, 146.78,
125.01, 122.84, 112.85, 112.50, 110.43, 33.02, 30.47, 24.24, 21.83, 18.19, 13.80. HRMS (ESI): m/z calcd.
for C16H18O5 + H+ [M + H]+: 291.1227. Found 291.1214.

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8-Benzoyloxy-7-hydroxy-4-methylcoumarin (3f): Cream-coloured solid; 30.5% yield; m.p. 201–203 ◦C.
IR (film):
ν
(cm−1) 3308, 2927, 1719, 1735, 1608, 1586, 1380, 1258, 1234. 1H-NMR (400 MHz,
max
DMSO-d6) δ: 10.96 (s, 1H, OH), 8.16 (d, J = 7.32 Hz, 1H, ArH), 7.92 (d, J = 7.32 Hz, 1H, ArH), 7.76
(m, 1H, ArH), 7.46–7.65 (m, 3H, ArH), 6.99 (d, J = 8.80 Hz, 1H, ArH), 6.18 (s, 1H, H-3), 2.40 (s, 3H,
C-4 CH3). 13C-NMR (100.5 MHz, DMSO-d6)
δ: 167.34, 163.58, 159.30, 153.95, 153.08, 146.87, 134.35,
132.87, 130.05, 129.27, 129.10, 128.57, 128.20, 123.16, 113.02, 112.60, 110.46, 18.24. HRMS (ESI): m/z
calcd. for C17H12O5 + Na+ [M + Na]+: 319.0577. Found 319.0571.
4. Conclusions
In conclusion, we have chemo-enzymatically synthesized a series of novel, hitherto unknown,
8-acyloxy-7-hydroxy-4-methylcoumarins in a regioselective fashion using the Rhizopus oryzae lipase
(ROL) in THF at 45 ◦C from 7,8-dihydroxy-4-methylcoumarin (DHMC), which, if not impossible, are
very tedious and cumbersome to synthesize via chemical routes. It was observed that with an increase
in the size of the alkyl group of the acylating agent, the reaction time increased with a decrease in the
isolated yield of the desired product. The acylation selectively occurred at the C-8 hydroxyl group,
which was established using single X-ray crystal analysis.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/11/1499/s1. Crystallographic data for 8-acetoxy-7-hydroxy-4-methylcoumarin (3a; CCDC-1508122) have
been deposited with the Cambridge Crystallographic Data, Table S1: Single crystal X-ray diffraction data of
compound 3a.
Acknowledgments: The authors thank the financial support provided by the University of Delhi to
Virinder S. Parmar, Ashok K. Prasad, Brajendra K. Singh, Ramesh C. Kuhad and Rajendra K. Saxena under
the DU-DST Purse Grant Scheme. Shashwat Malhotra and Divya Mathur thank the Council of Scientific and
Industrial Research (CSIR, New Delhi, India) and the Science & Engineering Research Board (SERB, New Delhi,
India; project’s reference no. PDF/2015/000604) for the grant of Research Awards.
Author Contributions: All the thirteen authors contributed towards either the synthesis of coumarins, lipase
production and characterization, optimization of biocatalytic reactions, analysis of synthesized coumarin
derivatives using 1H-NMR, 13C-NMR and X-ray diffraction studies, or preparing and editing of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest and each author contributed significantly in the
planning, analysing and execution of work, and in preparing the manuscript.
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Sample Availability: Samples of the compounds 1, 2a and 3a–3f are available with the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).