Chromane derivatives of small aromatic molecules: Chemoenzymatic synthesis and growth inhibitory activity on human tumor cell line LoVo WT

Article abstract of DOI:10.1016/j.bmc.2009.06.061

Aromatic substrates tyrosol (p-hydroxyphenylethanol) and 2,6-dihydroxynaphthalene (2,6-DHN) were converted into chromane derivatives by means of chemoenzymatic reactions catalyzed by the aromatic prenyltransferase of bacterial origin NovQ, using dimethyla

Full text of DOI:10.1016/j.bmc.2009.06.061

Bioorganic & Medicinal Chemistry 17 (2009) 6003–6007
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chromane derivatives of small aromatic molecules: Chemoenzymatic
synthesis and growth inhibitory activity on human tumor cell line LoVo WT
Alberto Macone a, Eugenio Lendaro a, Alessandra Comandini a, Irene Rovardi b, Rosa Marina Matarese a,
Antonio Carraturo c, Alessandra Bonamore a,
*
a Dipartimento di Scienze Biochimiche, Sapienza Università di Roma, P.le A. Moro 5, 00185 Roma, Italy
b Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, P.le A. Moro 5, 00185 Roma, Italy
c Laboratorio di Patologia Clinica, Ospedale Santa Maria Goretti, ASL Latina, Via Canova, 04100 Latina, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Aromatic substrates tyrosol (p-hydroxyphenylethanol) and 2,6-dihydroxynaphthalene (2,6-DHN) were
converted into chromane derivatives by means of chemoenzymatic reactions catalyzed by the aromatic
prenyltransferase of bacterial origin NovQ, using dimethylallyl bromide as allylic substrate instead of the
natural isoprenyl pyrophosphate substrate. Stereoselective prenylation occurred in o-position with
respect to the phenol hydroxyl in both compounds. Prenylated derivatives were readily converted into
chromane products via a selective 6-endo-trig cyclization involving the oxygen atom from the phenol
moiety and the double bond of the prenyl substituent, a process catalyzed by FeCl3. These findings set
up the basis of a most convenient two-step, one-pot process which allows for easy recovery of the chro-
mane products in high yields. The chromane derivatives thus obtained were tested for cytotoxicity and
pro-apoptotic activity using LoVo WT cells, a line of human colon adenocarcinoma.
Received 23 March 2009
Revised 25 June 2009
Accepted 26 June 2009
Available online 3 July 2009
Keywords:
Aromatic prenyl transferase
Chromane
Growth inhibitory activity
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
ural sources. Thus, chemical or enzymatic synthesis of large
amount of prenylated aromatic products is highly desirable.
Prenylated aromatic compounds exhibit a wide spectrum of
pharmacological effects as anti-microbial activity, pro-apoptotic
activity, anti-proliferative activity against cancer cell lines, etc.1–6
Among the rich variety of natural prenylated molecules, chro-
mane and chromene derivatives, with an extra pyrano or dihydro-
pyrano ring, represent a family of compounds endowed with most
interesting properties.7 All these molecules are generally charac-
terized by low cellular toxicity and good membrane permeability,
properties that make them ideal drug template compounds. Some
of these molecules have been shown to be able to inhibit mycobac-
terial growth,8 to be promising therapeutic agents for AIDS9 and to
posses antitumoral activity.10–12 Recently, the use of chromane
derivatives as therapeutic agents in the treatment of cancer and
cell proliferative disorders has also been reported.13,14 Based on
these data, the development of novel chromane-like molecules
with potentially high biological activity for the design of new drugs
or as molecular building blocks for chemical synthesis is a compel-
ling target for pharmaceutical applications.
The advances in the research on aromatic prenylated compounds
led to the identification of a novel class of prenyl transferases (PTa-
ses) from streptomycetes, which are able to transfer dimethylallyl
or geranyl chains to phenyl- and naphthyl-rings. Among these
enzymes, the best characterized is Streptomyces sp. prenyltransfer-
ase Orf2 (recently renamed NphB), involved in the biosynthesis of
isoprenoid-polyketide antibiotics. NphB is capable of transferring
isoprenoid chains to a broad range of polycyclic aromatic mole-
cules.17,18 This enzyme has been tested for the selective geranylation
towards flaviolin, flavonoids and dihydroxynaphthalenes. The less
characterized PTases CloQ from Streptomyces roseochromogenes
and NovQ from Streptomyces spheroids, instead, are involved in the
specific prenylation of monocyclic aromatic compounds like p-
hydroxyphenylpyruvate and p-hydroxybenzaldehyde.19 Although
all these enzymes are able to work in vitro in the presence of these
aromatic molecules and isoprenyl pyrophosphate, the reaction
yields are rather low, partially due to the spontaneous dephospho-
rylation of dimethylallyl and geranyl pyrophosphates in water.
Moreover, the high cost of pyrophosphoriled substrates makes the
process impractical for the large scale synthesis of prenylated aro-
matic products.
So far, most of such active compounds have been obtained as
natural products via direct isolation mainly from higher plants.15,16
However, prenylated compounds often exist at trace levels in nat-
In the present paper we report the overexpression of the NovQ
prenyltransferase in Escherichia coli and the construction and over-
expression of a double mutant, which is able to accommodate
* Corresponding author. Tel.: +39 0649910990; fax: +39 064440062.
E-mail address: alessandra.bonamore@uniroma1.it (A. Bonamore).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.061

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A. Macone et al. / Bioorg. Med. Chem. 17 (2009) 6003–6007
larger aromatic substrates. Both the wild type protein and the mu-
tant have been tested by using the less expensive and more stable
dimethylallyl bromide as aliphatic substrate, and tyrosol and 2,6-
DHN as aromatic substrates. Moreover, in order to modulate and
improve the activity of the prenylated compounds obtained by
the enzymatic reaction, we performed an iron-catalyzed cycliza-
tion leading to chromane derivatives. The cytotoxic properties of
chromane derivatives have been evaluated against LoVo WT
cultured cells of human colon adenocarcinoma.
NovQ catalyzed prenylations. As a control, blank reactions in the
absence of enzyme have also been performed and yielded a mix-
ture of o- and m-prenylated compounds.
2.2. Chemoenzymatic synthesis
2.2.1. Chemoenzymatic synthesis of 2-(2,2-dimethyl-3,4-
dihydro-2H-chromen-6-yl)ethanol (compound 1)
Tyrosol is efficiently prenylated by wt NovQ in isopropanol/buf-
fer mixtures in the presence of prenylbromide. As demonstrated by
GC/MS analysis performed on the reaction mixture tyrosol was
converted into o-prenyl-tyrosol (1) after 12 h with a yield >80%.
Mass spectrum of compound 1 derivatized with trimethylsilyl
chloride showed a fragmentation pattern characterized by the
molecular ion m/z = 350, which accounts for the presence of a tri-
methylsilyl-group on both the aliphatic and phenol hydroxyl
groups. Similarly to what reported for metal-catalyzed chromane
and chromene formation,20 addition of catalytic amounts of FeCl3
to the reaction mixture containing prenyl derivative 1 led to bicy-
clic compound 2. The process most likely occurred via a selective
6-endo-trig cyclization involving the oxygen atom from the phenol
moiety and the double bond of the prenyl substituent. As 2 is insol-
uble in aqueous solutions, the compound was easily recovered by
centrifugation as a light brown powder and characterized by GC/
MS, 1H NMR and 13C NMR. As shown by the GC/MS chromatogram,
2 has been obtained with a 98% purity and 78% isolated yield.
The derivatization of 2 with TMS chloride led to the substitution
of only one silyl group on the aliphatic hydroxyl; the phenolic
hydroxyl is unreactive as it is part of the chromane backbone.
2. Results and discussion
2.1. Enzymes
Aromatic prenyltransferases (PTAses) have been demonstrated
to be useful as biocatalysts for the production of prenylated com-
pounds both in vivo and in vitro. In particular, Streptomyces sp. pre-
nyltransferase NphB showed broad specificity and good
stereoselectivity in the prenylation of polycyclic aromatic
molecules.17,18
In contrast, NovQ was shown specifically recognizes monocyclic
aromatic compounds as substrate.19 In order to broaden the sub-
strate specificity of NovQ a set of mutants has been designed on
the basis of a three-dimensional theoretical model obtained by
homology modeling on the X-ray structures of NphB (Fig. 1). The
topological positions R160 and Y233 appeared as the best candi-
dates for mutagenesis studies in that these residues occupy the
‘aromatic binding pocket’ of the active site, located within the b-
barrel (see Fig. 1, panel B). Thus, single and double mutants
R160A, Y233A and R160A-Y233A have been designed cloned and
overexpressed in E. coli under the control of an IPTG inducible pro-
moter as soluble proteins of 34 kDa (about 100 mg soluble protein/
l culture).
2.2.2. Chemoenzymatic synthesis of 3,3-dimethyl-2,3-dihydro-
1H-benzo[f]chrome-8-ol (compound 5) and 2,2,8,8-tetramethyl-
2,3,4,8,9,10-hexahydrochromeno[6,5-f]chromene (compound
6)
The catalytic activities of all four enzymes were tested against
the aromatic substrates tyrosol and 2,6-DHN in the presence of
dimethylallyl pyrophosphate, the natural substrates. The reaction
products were obtained in moderate isolated yield (50–70%) and
good selectivity with respect to blank reactions in the absence of
enzymes, as shown by GC/MS analysis carried out at the end of
the reaction (see Section 4). As a result, it was observed that
whereas NovQ was the most active enzyme on tyrosol, the double
mutant R160A-Y233A performed better on 2,6-DHN (data not
shown). However, similar reaction yields were observed by using
dimethylallyl bromide instead of the more expensive and less sta-
ble pyrophosphate. The experimental situation changed radically
when using alcohols (ethanol, 2-metoxyethanol, isopropanol and
isoamylic alcohol) as cosolvents to allow for better solubilization
of the alkyl bromide. In particular, 20% isopropanol/phosphate buf-
fer mixtures at pH 7.0 proved to be the best reaction medium for
NovQ R160A-Y233A was able to catalyze the prenylation of 2,6-
dihydroxynaphthalene in the presence of dimethylallyl bromide
(Scheme 1). Similarly to tyrosol, the prenylation reaction on 2,6-
DHN occured on the o-position to the phenolic hydroxyls. How-
ever, the formation of two compounds is observed, the first (3)
bearing one prenyl group on C1 and the second (4) bearing two
prenyl groups on C1 and C5. These findings were confirmed by
GC/MS analysis on TMS derivatives obtained after reaction of the
crude mixture with trimethylsilyl chloride. Also in this case the
addition of catalytic amounts of FeCl3 led to the formation of
monochromane 5 and dichromane 6 from compounds 3 and 4,
respectively. Chromane derivatives 5 and 6 were obtained in 30%
yield each as yellow solids. The mixture of the two products could
be easily recovered via centrifugation and filtration of the crude
reaction mixture.
Figure 1. Structural model of NovQ. The overall fold is shown in panel A as a ribbon diagram. A detail of the active site is shown in panel B. R160 and Y233 residues subjected
to mutations are indicated in ball-and-stick. The picture was generated with PYMOL.

A. Macone et al. / Bioorg. Med. Chem. 17 (2009) 6003–6007
6005
Scheme 1. Reagents and conditions: (a) prenyl bromide, NovQ, isopropyl alcohol/phosphate buffer pH 7.5 (20% v/v), 16 h, 25 °C; (b) prenyl bromide, NovQ R160A-Y233A,
isopropyl alcohol phosphate buffer pH 7.5 (20% v/v), 16 h, 25 °C.
2.3. Bioactivity
Compounds 2, 5 and 6, obtained as described before, were fur-
ther purified by preparative HPLC and characterized by NMR and
GC/MS spectrometry (see Section 4.6). All compounds, dissolved
in DMSO, have been tested for their in vitro antineoplastic activity
towards LoVo WT cells. All these molecules, whose internalization
into the cells has been demonstrated by uptake kinetics at 24 and
48 h, have been evaluated for their cytotoxic activity.
As shown in Figure 2, compound 2 is inactive, while 2,6-DHN
derivatives posses a moderate cytotoxicity after 24 h incubation.
The observed effect is time and concentration dependent. The
IC50 calculated after 24 or 48 h incubation time in the presence
of 5 and 6 were, respectively, 34.5 and 257.6 lM (Table 1). In order
Figure 2. Cytotoxic effect of compounds 2, 5 and 6 in LoVo WT cells. Cell viability
was determined by the MTS assay and the percentage of cell viability was
calculated as a ratio of drug-treated cells and control cells (treated with 0.2% DMSO
only). The data are presented as the mean SD of eight independent experiments.
to assess the ability of 5 and 6 to induce apoptosis, the hypodip-
loidy (i.e., loss of fragmented DNA) of LoVo WT cells has been eval-
uated by flow cytometry.21 As apparent from the results reported
in Table 1, 24 h exposure of the tested cell line to 5 and 6 has
shown that only 5 has a pro-apoptotic effect and is able to induce
a mean increase in the percentage of hyplodiploid cells. As a con-
trol, 2,6-DHN does not show cytotoxicity neither pro-apoptotic
activity leading to the conclusion that only its chromane-like func-
tionalization is able to confer the desired biological activity.
3. Conclusion
The results of the present investigation indicate that aromatic
PTAase enzymes can be conveniently used in biotransformation

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A. Macone et al. / Bioorg. Med. Chem. 17 (2009) 6003–6007
known structures performed with the DSSP program22 was down-
Table 1
IC50 values (mM) of compounds 5 and 6. IC50 values (
LoVo WT cells by MTS assay after 24 or 48 h treatment
l
mol/l) were determined in
IC50
loaded from the PDB web site. Secondary structure predictions
23
24
for NovQ were performed with the programs PROF
,
PSIPRED and
SAM-T0225
.
Compound
Incubation time (h)
The SASA of known structures was calculated with the NACCESS
program, and residues were defined as buried when the SASA val-
ues were 620 Å. Prediction of solvent accessibility for NovQ was
performed with the program PROF. The reliability of the prediction
was considered to be ‘low’ and ‘high’ for values of expected predic-
tion accuracy of 0–4 and 5–9, respectively. InsightII26 and its
Biopolymer module (Accelrys Inc.) were used to perform: (i) struc-
ture visualization and analysis; (ii) optimal structure superposi-
tions and RMSD calculations; (iii) all the steps required for model
building; (iv) main-chain dihedral angle, hydrogen bond and con-
tact calculations. Two atoms were defined as being in contact if
their distance is lower than or equal to 3.5 Å.
5
24
48
34.51
27.35
6
24
48
257.6
395.4
processes to yield chromane derivatives. In particular, two inter-
esting results are highlighted within the proposed biotransforma-
tion. First, NovQ PTAase recognizes the easily affordable
prenylbromide substrate as an alternative to the highly unstable
and expensive natural substrate, isoprenyl pyrophosphate. Second,
the o-prenyl derivative thus obtained can be easily converted into
the chromane scaffold by addition of FeCl3 within the original reac-
tion mixture. These findings set up the basis of a most convenient
two-step, one-pot process which allows for easy recovery of the
chromane products in high yield. The present process paves the
way to the development of variously functionalized derivatives of
aromatic phenols through easy and efficient modifications that
are expected to increase their bio-availability and bioactivity. Fur-
ther chemoenzymatic methods will be planned to synthesize novel
functionalized aromatic compounds starting from 2, 5 and 6, in
order to create more soluble and highly active molecules.
4.3. Prenylation assays
Catalytic activities were explored for the recombinant purified
proteins NovQ and R160A-Y233A mutant, by using tyrosol and
2,6-DHN as aromatic substrates, and dimethylallyl bromide as
cosubstrates. The reaction condition consisted of 5 mM aromatic
substrate, 10 mM dimethylallyl bromide, 10
alcohol/50 mM phosphate pH 7.5 (20% v/v), in a final volume of
500 l. After overnight incubation at room temperature, 100 l of
l of diethyl ether,
lM enzyme, isopropyl
l
l
the reaction mix were extracted twice with 200
l
dried and analyzed by GC/MS and NMR.
4. Experimental
4.4. Cyclization runs
4.1. Proteins expression and purification
Crude reaction mixtures containing 1 or 3 and 4 were treated
with FeCl3 (1:20, w/w with respect to the aromatic substrate). After
3 h the conversion of the starting substrates was complete. The
products 2 or 5 and 6 were recovered as light brown solids by cen-
Synthetic genes coding for Streptomyces spheroids prenyltrans-
ferase NovQ have been constructed by GENEART (GmbH, Germany)
with optimised E. coli codons. The gene, designed with NdeI/BamHI
restriction sites, was cloned directly within a pET22-b expression
vector (Novagen, Darmstadt, Germany) cut with the same restric-
tion enzymes. Competent E. coli BL21 (DE3) cells were transformed
with the ligation mixture and the colonies with the correct DNA in-
sert were selected through PCR screening.
trifugation and filtration. After being washed with 500 ll of ethyl
alcohol the solids were dried under vacuum (30% isolated yield).
Samples of 5 and 6 for NMR analysis were purified by means of
preparative HPLC.
Site directed mutagenesis was carried out on the pET22-b-novQ
plasmid with the Quikchange mutagenesis kit (Stratagene, La Jolla,
CA, USA) according to the manufacturer’s instructions. The mutants
obtained were sequenced and transformed in E. coli BL21 (DE3) cells.
E. coli cells were grown in Luria–Bertani medium containing
4.5. GC/MS and NMR analysis
Before GC/MS analysis the samples were derivatized with 50 ll
of silylation reagent at 25 °C for 30 min. The derivatized samples
were suspended in ethyl acetate and analyzed by GC/MS on a
Agilent 6850A gas chromatograph coupled to a 5973N quadrupole
mass selective detector (Agilent Technologies, Palo Alto, CA, USA).
Gas-chromatographic separations were carried out on a Agilent
HP-5MS fused-silica capillary column (30 m  0.25 mm i.d., film
100
lg/ml ampicillin. Wild type NovQ, R160A, Y233A and R160A-
Y233A mutants expressions were induced by adding 1 mM IPTG
to cells in exponential growth phase (OD600 = 0.6). After induction
the cells were incubated overnight at 25 °C, pelleted, resuspended
in a minimum volume of lysis buffer (50 mM Tris–HCl pH 8.0,
1.0 mM EDTA, 0.5 mM DTT, 100 mM NaCl, 10% glycerol, 1 mM
PMSF) and sonicated until the supernatant was clear. After centri-
fugation, the supernatant was dialyzed versus 20 mM phosphate
buffer pH 7.0, loaded on a DEAE–cellulose column (Whatman
International Ltd, Maidstone, England) equilibrated with the same
buffer and eluted with a NaCl linear gradient (0–0.5 M).
thickness 0.25 lm). Injection mode: splitless at a temperature of
260 °C. Column temperature program: 70 °C (1 min) then to
280 °C at a rate of 10 °C/min and held for 15 min. The carrier gas
was helium at a constant flow of 1.0 ml/min. The spectra were
obtained in electron impact mode at 70 eV ionization energy and
a mass range scan from m/z 30 to 500; ion source temperature
280 °C, ion source vacuum 10À5 Torr.
NMR experiments were taken in CDCl3 at room temperature on
a Varian Unity Inova at 400 MHz and 296 K. Chemical shifts are
expressed in d (ppm) values relative to tetramethylsilane (TMS)
as internal reference.
4.2. Model building of NovQ
Molecular model of NovQ was built as follows. Orf2, whose
crystallographic structure is available,17 was chosen as main tem-
plate, from which all conserved main-chain regions were retained.
Side-chains of conserved residues were imported from the main
structural template, those of mutated residues from a library of
side-chain conformations frequently observed in protein struc-
tures (rotamers). Assignment of secondary structure elements to
4.6. Spectral data of new compounds
4.6.1. 2-(2,2-Dimethylchroman-6-yl)ethanol
Compound 2 1H NMR spectrum (400 MHz, CDCl3): d 6.94 (1H,
dd, J = 8, 2.5 Hz; H-7), 6.92 (1H, d, J = 2.5 Hz; H-5), 6.73 (1H, d,

A. Macone et al. / Bioorg. Med. Chem. 17 (2009) 6003–6007
6007
J = 8 Hz; H-8), 3.81 (2H, t, J = 7 Hz, b-CH2), 2.77, 2.76 (2 Â 2H, 2 Â t,
tion, Madison, WI, USA) and PMS (Sigma–Aldrich Chemie GmbH)
dissolved in Dulbecco’s phosphate buffered saline (DPBS). The
absorbance of formazan (metabolite of MTS by viable cells) was
measured at 490 nm. The absorbance value was used to calculate
the surviving cell number. The IC50 value (concentration of a com-
pound that is required to inhibit 50% of cell growth) was calculated
with dose–response curves.
J = 7 Hz; H2-4,
a-CH2), 1.80 (2H, t, J = 7 Hz, H-3), 1.33 (6H, s,
2 Â Me). 13C NMR (100 MHz, CDCl3): d 153.7 (C-9), 129.8 (C-6),
128.3 (C-5), 126.1 (C-7), 120.0 (C-10), 111.6 (C-8), 73.1(C-2), 63.1
(b-CH2), 39.5 (
a
-CH2), 31.7 (C-3), 22.6 (C-4), 26.9 (2 Â Me). GC/
MS (EI, m/z): 206 (34.8%), 175 (100%), 151 (25.0%), 133 (15.1%),
91 (15.4%).
4.6.2. 3,3-Dimethyl-2,3-dihydro-1H-benzo[f]chrome-8-ol
Compound 5 1H NMR spectrum (400 MHz, CDCl3): d 7.74 (1H, d,
J = 8 Hz, H-9), 7.46 (1H, d, J = 8 Hz, H-5), 7.13 (1H, dd, J = 8 and
2.5 Hz, H-6), 7.12 (1H, d, J = 2.5 Hz, H-8), 7.00 (1H, d, J = 8, H-10),
3.00 (2H, t, J = 7 Hz, H2-3), 1.96 (2H, t, J = 7 Hz, H2-4), 1.37 (6H, s,
2 Â Me). 13C NMR (100 MHz, CDCl3): d 154.2 (C-7), 151.8 (C-10a),
130.6 (C-8a), 127.8 (C-5), 126.9 (C-4-b), 126.2 (C-6), 126.1 (C-9),
124.4 (C-8), 119.1 (C-10), 111.7 (C-4a), 75.1 (C-2), 33.1 (C-3),
26.9 (2 Â Me), 20.6 (C-4). GC/MS (EI, m/z): 228 (77.3%), 173
(100%), 144 (22.2%), 115 (27.7%), 127 (10.6%).
Acknowledgments
We gratefully acknowledge Professor Alberto Boffi, Professor
Bruno Botta, Professor Andrea Tafi and Dr. Aldo Caiazzo for helpful
discussions and valuable suggestions.
This work was supported by FIRB 2003 to A.M. and A.B.
References and notes
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Compound 6 1H NMR spectrum (400 MHz, CDCl3): d 7.64 (2H, d,
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4.7. Cytotoxicity assay
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The cell line utilized in this study is LoVo WT, an human colon
adenocarcinoma, obtained from the American Type Culture Collec-
tion (Manassas, VA). Cells were grown in D-MEM F12 supple-
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50 lg/ml streptomycin sulfate, and maintained in humidified
atmosphere with 5% CO2 at 37 °C. Cell disassociation was obtained
with 0.05% trypsin—0.02% EDTA.
Compounds 2, 5 and 6 were dissolved in dimethyl sulfoxide
(DMSO) to give a stock solution of 100 mM from which further
dilutions in culture medium were prepared. The residual solubiliz-
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cytotoxic.
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