Unexpected enzyme-catalyzed regioselective acylation of flavonoid aglycones and rapid product screening

Article abstract of DOI:10.1039/c2ob06784f

Unprecedented regioselective acylation of flavonoid aglycones was achieved using Candida antarctica lipase B (CALB). The rapid screening of product formation was performed by the use of the high resolution phenol-type OH ¹H NMR spectral region recorded after the addition of picric acid.

Full text of DOI:10.1039/c2ob06784f

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COMMUNICATION
Unexpected enzyme-catalyzed regioselective acylation of flavonoid aglycones
and rapid product screening†
Eleni Kyriakou,a Alexandra Primikyri,a Pantelis Charisiadis,a Maria Katsoura,b Ioannis P. Gerothanassis,a
Haralambos Stamatisb and Andreas G. Tzakos*a
Received 23rd October 2011, Accepted 14th December 2011
DOI: 10.1039/c2ob06784f
In contrast to the current perception that CALB is incapable to
acylate flavonoid aglycones, herein we describe for the first time
the enzymatic acylation of the phenolic group of flavonoid agly-
cones (quercetin and naringenin) by transesterification with vinyl
acetate or vinyl butyrate in organic solvents.
Unprecedented regioselective acylation of flavonoid aglycones
was achieved using Candida antarctica lipase B (CALB). The
rapid screening of product formation was performed by the
use of the high resolution phenol-type OH H NMR spectral
region recorded after the addition of picric acid.
1
Factors which affect the biocatalytic process such as the acyl
donor and organic solvent were determined. Product formation
Natural products and especially plant-derived polyphenols cover
a very interesting chemical space of biological relevance due to
their vast chemical diversity, and fine-tuning for optimal inter-
actions with biological macromolecules through evolutionary
selection.1 Indeed, they present numerous anti-inflammatory,
anti-angiogenic and anti-carcinogenic effects in cell culture and
in animal models2,3 with more than 50% of the current effective
drugs in the cancer chemotherapy area originating from natural
products.4,5 Although phenol-type OH groups constitute the
most important and frequently encountered chemotypes confer-
ring bioactivity, recent studies revealed that protection of these
functional groups by O-methylation or O-acetylation resulted in
analogues with higher intestinal absorption, resistance to hepatic
metabolism and better anticancer activity in respect to the orig-
inal compounds.6,7 Thus, such protection is highly desirable but
not in a form of generic transformation of the original molecule
but rather in a regioselective way.8,9 To achieve this and to avoid
the tedious protection/deprotection steps required in a classical
chemical synthesis method, due to the numerous reactive
hydroxyl groups of polyphenols, we hypothesized that biocataly-
sis could be a powerful tool. Candida antarctica lipase B
(CALB) is an excellent biocatalyst for asymmetric organic chem-
istry with high enantioselectivity and regioselectivity in a broad
range of substrates.10–12 CALB, although it has been reported to
acylate glycosylated flavonoids,13–17 has been determined to
have low activity for a range of tertiary alcohols and polyphenol
aglycones15,18–20 and no detectable activity in flavonoids
aglycones.14,15,21–23
1
was rapidly monitored through H NMR by harnessing the great
shielding sensitivity of the phenol-type OH absorptions.24,25
Spectral resolution enhancement, for a factor of over 500, was
achieved after the addition of picric acid to the reaction products.
The resonance absorption of the OH(5) protons of flavonoid
aglycones could be used as an internal sensor to monitor the
transformation efficacy and regioselectivity. This is a novel and
rapid method for screening product formation in enzyme-
mediated polyphenol aglycones transformations without the use
of tedious and time consuming isolation techniques.
Our initial flexible docking calculations26–28 indicated that the
flavonoid quercetin could be favourably accommodated for trans-
formation in the substrate binding site of CALB and adopt a pre-
ferable orientation with its 3′ hydroxyl group facing the catalytic
active site (Fig. 1). To probe the in silico observations (Fig. 1),
the flavonoid quercetin, which presents numerous biological
activities, was chosen as the model system for such catalysis
(Scheme 1). Indeed, product formation was observed (45%
yield) only in the presence of the enzyme (ESI, Fig. S1†), and
optimum reaction conditions (solvent, acyl donor) were deter-
mined (ESI, Fig. S2†). Reaction products (tR = 14.30 min) can
aSection of Organic Chemistry and Biochemistry, Department of
Chemistry, University of Ioannina, Ioannina GR-45110, Greece.
E-mail: atzakos@cc.uoi.gr; Tel: (+30)2651-00-8387
bLaboratory of Biotechnology, Department of Biological Applications
and Technologies, University of Ioannina, Greece
†Electronic supplementary information (ESI) available: Experimental
details as also HPLC, MS and NMR data. See DOI: 10.1039/
c2ob06784f
Fig. 1 Three dimensional model of quercetin bound to the active site
of lipase B as derived from flexible docking calculations. Quercetin is
shown as sticks and CALB is shown (A) as surface and (B) as ribbons.
In (B) the 3′-OH of quercetin is illustrated as a yellow sphere and the
catalytic amino acids as sticks.
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CO(4). In the region from 9 to 11 ppm, where the OH(3′),
OH(4′), OH(3) and OH(7) protons of flavonoids were expected,
very broad resonance absorptions were observed with a compo-
site linewidth (Δν1/2 ≈ 874 Hz). Similarly, in the spectral region
from 6 to 8 ppm (Fig. 2B), where the aromatic resonances of
flavonoids are expected to absorb, very broad peaks were
observed. Therefore, the low quality and complexity of the
recorded spectrum prohibited a detailed spectral assignment of
the reaction products. Interestingly, not only resonances of the
exchangeable –OH protons but also the aromatic region suffered
from severe line broadening, possibly due to proton exchange
and/or aggregation phenomena (Fig. 2B).
In order to surmount such problems we used a novel method-
ology which can enhance spectra quality. Specifically, the reac-
tion products were titrated with picric acid. Picric acid (PA) has a
very low pKa value (0.42), in comparison to the low acidity of
quercetin (pKa ≈ 7.70), that could fine-tune the proton chemical
exchange and disrupt hydrogen bonding networks of polyphe-
nols. In addition, since picric acid has been employed in numer-
ous interactions such as electrostatic, hydrogen bonding29 and
π–π stacking interactions30 it can intercalate with aggregates
formed between stacked aromatic rings, thus disrupting potential
forms of aggregation (ESI, Fig. S3†).
Indeed, after addition of picric acid (25.30 nmol) the recorded
1H NMR spectrum was significantly improved not only in the
–OH region (Fig. 2C) but also in the aromatic region (Fig. 2D).
Specifically, the broad peak at 12.48 ppm with a composite
asymmetric linewidth (Δν1/2 ≈ 43.6 Hz), after the addition of
picric acid, was resolved into three discrete and very sharp reson-
ances at 12.37 ppm (Δν1/2 ≈ 1.4 Hz), 12.43 ppm (Δν1/2 ≈ 1.5
Hz) and 12.50 ppm (Δν1/2 ≈ 1.6 Hz). The resonance at
12.50 ppm should be attributed to the OH(5) of unreacted quer-
cetin. The other two resonances are due to the OH(5) protons of
two monoacylated products. In the spectral region of 9 to
11 ppm, nine sharp resonance absorptions appeared, with Δν1/2
Scheme 1 Flavonoids are comprised of two benzene rings (A and B)
linked through a heterocyclic pyran or pyrone ring (C) in the middle.
The OH(5) proton is implicated in the formation of an intramolecular
hydrogen bond that is depicted with a red line. Acylation of quercetin
can, in principle, take place in positions 3, 5, 7, 3′ and 4′. CALB (Novo-
zyme 435) was found to regioselectively acylate quercetin in ring B
leading to two products, Q1 and Q2. Q2 was formed with 3 times higher
yield. The reaction was performed with quercetin (3 μmol), vinyl acetate
(1 mmol) and 60 mg ml−1 of catalyst in solvent (200 μl) at 60° C.
be readily separated from unreacted quercetin (tR = 12.50 min)
through preparative HPLC. Although acylation of quercetin can
take place in five different positions (5, 7, 3, 3′ and 4′), ESI-MS
analysis confirmed that only mono-acylation took place (ESI,
Fig. S7†). In order to unambiguously determine the derived acy-
lation products, 1H NMR experiments were performed.
1
Fig. 2A illustrates selected regions of the H NMR spectrum
in DMSO-d6 of the reaction product(s). A strongly deshielded
asymmetric broad resonance was observed at 12.5 ppm. This res-
onance can be assigned to the OH(5) protons of flavonoids,
located in the benzene ring A (Scheme 1), participating in strong
intramolecular hydrogen bonding with the carbonyl group
≈ 1.6
0.4 Hz, after the addition of picric acid, which
previously appeared as extremely broad composite resonances
(Δν1/2 ≈ 874 Hz). Therefore, with the addition of picric acid a
great reduction in the linewidth by a factor of over 560 was
achieved. This enhanced spectral refinement was also evident in
the aromatic spectral region. For instance, the broad resonance
absorption at 6.12 ppm (Δν1/2 ≈ 6.2 Hz) resulted in three high
resolution resonances at 6.18 ppm, 6.19 ppm and 6.21 ppm
(Fig. 2D).
This high resolution spectrum indicated the presence of more
than a single species in the reaction products. In order to quanti-
tate the different chemical species in the reaction products, NMR
diffusion experiments (DOSY) were performed.31 The DOSY
spectra clearly suggested the presence of two reaction products
(data not shown).
Fig. 2 500 MHz 1D 1H NMR spectrum of the OH and aromatic
regions of the enzymatically obtained monoacylated derivatives of quer-
cetin (1.35 μmol) in 500 μl DMSO-d6. The –OH and aromatic spectral
regions are illustrated in (A), (B) before and in (C), (D) after the addition
of 25.30 nmol of picric acid (2.5 μl from a 10.18 mM stock solution),
respectively. The molar ratio of picric acid and acetyl quercetin deriva-
tives in the reaction was 1 : 53. Picric acid (PA) resulted in a spectral res-
olution enhancement by a factor of over 560. In (A) the inset shows a
zoom-in (×8) on the spectral region from 9 to 11 ppm. In (C) the absorp-
tions of the OH(5) protons for Q (unreacted quercetin), Q1 and Q2 (the
retrieved derivatives) are indicated with arrows and could be used as
internal sensor for monitoring the reaction products.
Application of 2D 1H–13C HMBC and 1H–13C HSQC (Fig. 3)
of the same solution as in Fig. 2(C) resulted in a significant
number of cross peaks of the –OH groups. Several long range
connectivities of 2–4J(1H,13C) couplings were observed which
allowed the complete assignment of the carbon skeleton of the
monoacylated derivatives of quercetin in Scheme 1 (see the ESI,
Tables S2–S3 and Fig. S11–S14 for the complete assignment of
the two products†). More specifically, acylation took place at the
4′ (OH(4′)) or the 3′ (OH(3′)) position of quercetin (located in
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1
Fig. 4 OH region of the 500 MHz 1D H NMR spectrum recorded of
the crude reaction products of the enzymatically obtained monoacylated
derivative of narigenin (2.30 μmol) in DMSO-d6. Assignments of the
resonances of the unreacted naringenin (N) and the monoacylated
product (N1) are denoted.
Fig. 3 Selected regions of the 500 MHz 1H–13C HSQC (A) and
1H–13C HMBC (B)–(D) spectra of the solution used in Fig. 2(C). The
cross peaks corresponding to the 3′-and the 4′-monoacylated derivatives
of quercetin are colored in red and blue, respectively. The cross peaks of
the proton H3′′ of the acetyl group to carbons C(2′′) [2J(1H,13C)] and
C3′ [4J(1H,13C)] for the 3′-derivative and the cross peaks of the proton
H3′′ of the acetyl group to carbons C(2′′) [2J(1H,13C)] and C4′ [4J
(1H,13C)] for the 4′-derivative are illustrated as arrows in the relevant
chemical substructure parts in (B). The cross peaks of the proton OH(4′)
to carbons C(5′) [3J(1H,13C)] and C3′ [3J(1H,13C)] and C4′ [2J(1H,13C)]
for the 3′-derivative and the cross peaks of the proton OH(3′) to carbons
C(2′) [3J(1H,13C)], C3′ [2J(1H,13C)] and C4′ [2J(1H,13C)] for the 4′-
derivative are illustrated as arrows in the relevant chemical substructure
parts in (C), (D).
assign a single reaction product (N1, Fig. 4) having a yield of
71%. The same approach was also applied in the case of the
enzymatic transformation of the glycosylated flavonoid hypero-
side (3-O-galactoside of quercetin) using vinyl acetate. Again
after addition of picric acid (the molar ratio of picric acid and
acetylated derivative was 1 : 44) in the crude reaction products a
high resolution 1D 1H NMR spectrum was recorded (ESI,
Fig. S16†). By simply using the OH(5) absorption, that was now
located twelve bonds apart from the acylation site (sugar
moiety), a single reaction product was determined with a 97%
yield.
The determined efficiency of CALB to transform, in a regio-
selective way, flavonoid aglycones to their relevant esters could
open a new avenue towards the production of more potent poly-
phenol derivatives. To this direction will also contribute our
NMR methodology, which can rapidly screen product formation
and achieve enhanced spectral resolution after the addition of
picric acid. Transformation efficacy and regioselectivity could be
readily monitored after directly recording the 1D 1H NMR
spectra of the crude reaction products without any prior isolation
or fractionation. The absorption of the OH(5) proton of flavo-
noids aglycones which was used here as an internal sensor of the
enzyme-mediated transformation competence could assist in the
future in situ investigation of enzyme kinetics of such
transformations.
the B ring, Scheme 1). The two acylated products did not
present the same yield, but the 3′-O-acetyl derivative was formed
with more than 3 times higher yield. We estimated that
1.012 μmol of 3′- and 0.337 μmol of the 4′-acylated product
were present in the reaction products on the basis of the defined
amount of picric acid which was added to the solution. This
observed preference of the 3′ position of quercetin for CALB-
mediated transformation to the relevant ester is in accordance
with our docking calculations (Fig. 1).
From this work it became evident that the absorptions of the
deshielded OH(5) protons of flavonoids (Fig. 2C) are not only
deficient of resonance overlapping problems, but also could be
used as an internal sensor for the rapid and direct monitoring of
product formation in enzyme-mediated flavonoid aglycones
transformations. Interestingly, this absorption, located in ring A,
presents a great shielding sensitivity to substituent effects of at
least up to eleven bonds apart (reaction took place in ring B). To
further validate the usefulness of this approach, the enzyme-
mediated naringenin aglycone (N, Fig. 4) transformation was
investigated using the same optimum conditions as used for
quercetin. The absence in naringenin of a double bond at pos-
ition C2–C3 (Fig. 4) lead to increased flexibility and thus could
result in higher yield, whereas the presence of a single phenolic
group in the B ring (4′-position) could suggest the formation of
single product. Indeed, as shown in Fig. 4, without any prior iso-
lation of the reaction products, by just monitoring the OH(5)
absorptions in the crude reaction mixture, we could clearly
Acknowledgements
All the MS, HPLC and NMR data were recorded in the MS,
HPLC and NMR centers of the University of Ioannina. This
work was supported by a grant from the Esthir Gkani Foundation
(Ioannina, Greece). Kostoula Ioanna is acknowledged for helpful
discussions.
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