Towards the synthesis of glycosylated dihydrochalcone natural products using glycosyltransferase-catalysed cascade reactions

Article abstract of DOI:10.1039/c4gc00960f

Regioselective O-β-D-glucosylation of flavonoid core structures is used in plants to create diverse natural products. Their prospective application as functional food and pharmaceutical ingredients makes flavonoid glucosides interesting targets for chemical synthesis, but selective instalment of a glucosyl group requires elaborate synthetic procedures. We report glycosyltransferase-catalysed cascade reactions for single-step highly efficient O-β-D-glucosylation of two major dihydrochalcones (phloretin, davidigenin) and demonstrate their use for the preparation of phlorizin (phloretin 2′-O-β-d-glucoside) and two first-time synthesised natural products, davidioside and confusoside, obtained through selective 2′- and 4′-O-β-d-glucosylation of the dihydroxyphenyl moiety in davidigenin, respectively. Parallel biocatalytic cascades were established by coupling uridine 5′-diphosphate (UDP)-glucose dependent synthetic glucosylations catalysed by herein identified dedicated O-glycosyltransferases (OGTs) to UDP dependent conversion of sucrose by sucrose synthase (SuSy; from soybean). The SuSy reaction served not only to regenerate the UDP-glucose donor substrate for OGT (up to 9 times), but also to overcome thermodynamic restrictions on dihydrochalcone β-d-glucoside formation (up to 20% conversion and yield enhancement). Using conditions optimised for overall coupled enzyme activity, target 2′-O- or 4′-O-β-d-glucoside was obtained in ≥88% yield from reactions consisting of 5 mM dihydrochalcone acceptor, 100 mM sucrose, and 0.5 mM UDP. Davidioside and confusoside were isolated and their proposed chemical structures confirmed by NMR. OGT-SuSy cascade transformations present a green chemistry approach for efficient glucosylation in natural products synthesis. the Partner Organisations 2014.

Full text of DOI:10.1039/c4gc00960f

Green Chemistry
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Towards the synthesis of glycosylated
dihydrochalcone natural products using
glycosyltransferase-catalysed cascade reactions†
Cite this: Green Chem., 2014, 16,
4417
Alexander Gutmann,a Linda Bungaruang,a Hansjoerg Weber,b Mario Leypold,b
Rolf Breinbauerb and Bernd Nidetzky*a,c
Regioselective O-β-D-glucosylation of flavonoid core structures is used in plants to create diverse natural
products. Their prospective application as functional food and pharmaceutical ingredients makes
flavonoid glucosides interesting targets for chemical synthesis, but selective instalment of a glucosyl
group requires elaborate synthetic procedures. We report glycosyltransferase-catalysed cascade reactions
for single-step highly efficient O-β-D-glucosylation of two major dihydrochalcones (phloretin, davidi-
genin) and demonstrate their use for the preparation of phlorizin (phloretin 2’-O-β-D-glucoside) and two
first-time synthesised natural products, davidioside and confusoside, obtained through selective 2’- and
4’-O-β-D-glucosylation of the dihydroxyphenyl moiety in davidigenin, respectively. Parallel biocatalytic
cascades were established by coupling uridine 5’-diphosphate (UDP)-glucose dependent synthetic gluco-
sylations catalysed by herein identified dedicated O-glycosyltransferases (OGTs) to UDP dependent con-
version of sucrose by sucrose synthase (SuSy; from soybean). The SuSy reaction served not only to
regenerate the UDP-glucose donor substrate for OGT (up to 9 times), but also to overcome thermo-
dynamic restrictions on dihydrochalcone β-D-glucoside formation (up to 20% conversion and yield
enhancement). Using conditions optimised for overall coupled enzyme activity, target 2’-O- or 4’-O-
β-D-glucoside was obtained in ≥88% yield from reactions consisting of 5 mM dihydrochalcone acceptor,
100 mM sucrose, and 0.5 mM UDP. Davidioside and confusoside were isolated and their proposed chemi-
cal structures confirmed by NMR. OGT-SuSy cascade transformations present a green chemistry
approach for efficient glucosylation in natural products synthesis.
Received 24th May 2014,
Accepted 24th July 2014
DOI: 10.1039/c4gc00960f
www.rsc.org/greenchem
Table 1 Major dihydrochalcones and their glucosides
Introduction
Flavonoids are a large and structurally diverse group of natural
polyphenols.1,2 They are widely distributed in edible plants
and therefore constitute an important part of the human
diet.3,4 Some flavonoids are strong antioxidants and thus serve
as powerful inhibitors of lipid peroxidation.5,6 Dietary intake
of flavonoids has been correlated with reduced risk of chronic
diseases, coronary heart disease in particular.7 Dihydrochal-
cones present a major sub-class of flavonoids and are charac-
terized structurally by two phenolic rings connected through
Dihydrochalcone
R1
R2
R3
R4
Phloretin (1)
Davidigenin (2)
Phlorizin (3)
Trilobatin (4)
Davidioside (5)
Confusoside (6)
Nothofagin (7)
H
H
H
H
H
H
H
H
H
H
H
OH
H
OH
OH
H
H
OH
Glucose
H
Glucose
H
H
Glucose
H
Glucose
H
Glucose
aInstitute of Biotechnology and Biochemical Engineering, Graz University of
Technology, NAWI Graz, Petersgasse 12, 8010 Graz, Austria.
a flexible open-chain three-carbon linker (Table 1).8 Many
dihydrochalcones, such as the common phloretin (1), which is
abundantly present in the leaves and peel of apples,9,10 exhibit
a wide spectrum of interesting and pharmacologically relevant
E-mail: bernd.nidetzky@tugraz.at; Fax: (+43)-316-873-8434; Tel: (+43)-316-873-8400
bInstitute of Organic Chemistry, Graz University of Technology, NAWI Graz,
Stremayrgasse 9, 8010 Graz, Austria
cAustrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria
†Electronic supplementary information (ESI) available: Protein gel; HPLC traces:
distinction of 2 glucosides, purified 5 and 6; PcOGT-GmSuSy conversions at
various pH; NMR data. See DOI: 10.1039/C4GC00960F
bioactivities.11 Next to a general antioxidative property,12
1
shows antithrombotic13 and hepatoprotective properties,14
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potential suppression of metabolic carcinogen activation15 and strate, typically a nucleoside diphosphate (NDP)-sugar, for
effects intracellular drug accumulation.16 Davidigenin (2), the transfer of a glycosyl residue onto certain position(s) of the
4′-deoxy analogue of 1, is broadly distributed among different acceptor molecule.43 The impressive flexibility displayed by
plant families.17 As an antispasmodic compound present in or several promiscuous GTs allowed glycosylation of a wide range
derived from traditional medicines it is applied to the treat- of acceptors, but distinction between different phenolic
ment of intestinal disorders and asthma.17 2 also exhibits hydroxyls in flavonoid acceptors still presents a notable
weak antibacterial activity18 and shows antidiabetic activity.19
Glycosylation is an important biological mechanism for the
challenge.44–47
Practical enzyme catalysts for selective glucosylation of 1 or
structural and functional diversification of natural flavo- 2 are to be established. Up to now only one GT from apple
noids.20,21 Generally, the attachment of sugar residue(s) to a (Malus × domestica) and its ortholog from pear (Pyrus commu-
flavonoid core increases compound bioavailability due to nis) (99% sequence identity) were reported to selectively
strong water solubility enhancement.22,23 Furthermore, physio- produce analytical amounts of phlorizin (3) by glucosylation of
logical and pharmacological properties are often beneficially 1.48,49 Other recently reported glucosylations of 1 by plant GTs
altered as consequence of glycosylation.24,25 Taste properties yielded mixtures of monoglucosides.49,50 An even more pro-
(e.g. sweetness, bitterness) and colour can also be modified by miscuous bacterial GT glucosylated 1 at 2′-, 4′- and 4-OH, pro-
glycosylation.26,27 Interestingly, compared to other flavonoid ducing a complex mixture of glucosidic compounds including
sub-classes that show broadly diverging and often complex two di- and a triglucoside.51 Therefore, this emphasises that
glycosylation patterns, glycosylated dihydrochalcones occur selectivity-based GT enzyme selection is of key importance for
primarily as mono-β-D-glucopyranosides.8 Sugar attachment to successful development of a biocatalytic synthesis process.
dihydrochalcones often results from a phenolic O-glycosidic Furthermore, dedicated reaction engineering is necessary to
bond, although aromatic C-glucosylation is also found in solve the problem of cost-effective supply of the NDP-sugar
certain compounds.8,28 Phloretin is naturally present mainly substrate.52
as the 2′-O-β-D-glucoside, commonly referred to as phlorizin
Herein we present identification of GT biocatalysts for
(3).29 Due to inhibition of cellular glucose transport 3 was con- selective transformation of 1 into 3, and of 2 into 5 or 6. A paral-
sidered for type II diabetes treatment.29 The corresponding lel GT cascade reaction (Scheme 1) was established to enable
4′-O-β-D-glucoside, which is called trilobatin (4), is less dihydrochalcone glucosylation from sucrose (8) as a highly
common.30 Davidioside (5) is the 2′-O-β-D-glucoside of 2, while expedient and inexpensive glucosyl donor substrate. The
the 4′-O-β-D-glucoside is called confusoside (6). Compounds 5 overall bi-enzymatic transformations were carried out in the
and 6 were previously isolated from plant extracts and charac- presence of catalytic amounts of uridine 5′-diphosphate (UDP)
terized structurally.31,32 However, except for a low yielding total (9) to generate and constantly recycle in situ the UDP-glucose
synthesis of 6, preparation of glucosides of 2 was not (10) utilised for glucoside synthesis. Applying reaction con-
reported.33
ditions optimised for enzyme activity and designed to over-
Due to their various prospective applications as fine chemi- come thermodynamic limitations, target O-β-D-glucoside was
cals, pharmaceutical ingredients and food additives, dihydro- obtained as single transfer product in ≥88% yield and could
chalcone glucosides 3–6 are interesting targets for large-scale be isolated readily. We propose Scheme 1 to be a generally
preparation.29,34,35 However, their isolation from natural applicable green chemistry approach for efficient glucosylation
material is complicated by occurrence in complex multi-com- in natural products synthesis.
ponent mixtures and at relatively low abundance.30–32,36 Selec-
tive glucosylation of 1 and 2 would provide convenient access
to 3–6 through bottom-up synthesis. Process chemistry for this
purpose is not well developed, though, and elaborate pro-
cedures involving a substantial amount of protecting group
chemistry are required for precise instalment of the glucosyl
group.37 Alternative biotransformations using cultured plant
cells were successfully applied for direct glucosylation of a
variety of phenolic compounds including several flavo-
noids.38,39 However, presumably due to a mixture of native
plant glucosyltransferases, control of regioselectivity was
difficult and typically mixtures of various glucosides were
obtained. Isolated enzymes in contrast have earned green
credentials for being able to cope with issues of reactivity and
selectivity in glycosylation reactions.40,41 Glycosyltransferases
(GTs) are widely applied for glycosylation of flavonoids,
Scheme 1 GT-catalysed cascade reaction for synthesis of natural
product glucosides. Coupling of a “synthetic” GT with SuSy in one pot
enables cost-effective in situ formation of glucosyl donor substrate 10
whereas use of glycoside hydrolases, glycosynthases and trans-
glycosidases is so far limited by a narrow substrate range and
from 8 and catalytic amounts of 9. End-product inhibition by 9 is
typically low yields.26,41,42 GTs utilise an activated donor sub- obviated.
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lysis of samples taken at different times revealed gradual con-
sumption of 2 with concomitant appearance of a major and a
minor signal eluting at positions consistent with glucosidic
products (Fig. 1c). According to HPLC peak areas around 80%
Results and discussion
Identification of GT enzymes catalysing regioselective
glucosylation of phloretin (1) and davidigenin (2)
A GT from pear (Pyrus communis; PcOGT) was recently reported of 2 were converted to the major glycoside and less than 2% of
to be highly selective for transferring the glucosyl moiety of the by-product were formed when equilibrium was obtained.
UDP-glucose (10) to the 2′-OH of 1, producing 3.49,53 However, NMR data recorded from the reaction mixture gave preliminary
evidences were only from thin layer chromatography49 or from evidence that the 2′-OH of 2 had been glycosylated, thereby
initial-rate experiments carried out at low conversion (≤25%) indicating that regioselectivity was maintained upon exchange
of both donor and acceptor substrate.53 Furthermore, the con- of acceptor substrate. Specific activities of PcOGT related to
centrations of 1 used (300 and 100 µM, respectively) were too glucosyl acceptor substrate were determined from the time
low to be of synthetic relevance. A number of studies show courses of consumption of 1 and 2, and 1 (1.76 U mg−1
that GT regioselectivity may change substantially depending protein) was an about 500-fold better substrate than 2.
on degree of conversion or on initial substrate concen-
In search of a differently regioselective GT catalysing gluco-
tration.46,53 Therefore, this necessitated rigorous evaluation of syl transfer to the 4′-OH of 1 and 2, we tested a recently
PcOGT as catalyst for synthesis of 3 at elevated substrate described variant of a C-glycosyltransferase from rice (Oryza
concentration.
sativa; OsCGT) that had its Ile121 replaced by Asp.53 Highly
Using recombinant PcOGT (0.1 U mL−1) purified from an purified mutant enzyme was used in all experiments (ESI,†
Escherichia coli overexpression culture (ESI,† Fig. S1), trans- Fig. S1). Wild-type OsCGT was shown to catalyse glucosyl trans-
formation of 10 and 1 (5 mM each, pH 6.5) into phloretin fer from 10 to the aromatic 3′-C of 1, thus producing the C-β-D-
glucoside(s) was examined in dependence of reaction progress glucoside nothofagin (7).53,54 The particular residue substi-
until attainment of apparent equilibrium (∼70% conversion of tution in OsCGT causes change in reaction selectivity such that
1). Product analysis was done using a reversed-phase HPLC O-glucosylations of the 2′-OH and 4′-OH occur next to the
method in which the monoglucosidic regio-isomers 3 and 4 native C-glucosylation of 1.53 Using 1 at different concen-
were baseline separated (Fig. 1a). Only 3 and no 4 was detected trations between 2.5 µM and 2.5 mM, it was found that the
at all times (Fig. 1b). Moreover, close balance between 1 con- enzymatic conversion resulted invariably in a mixture of gluco-
sumed and 3 formed was consistent with enzymatic reaction sidic products that contained 3 or 7 as the main constituent
exclusively at the acceptor’s 2′-OH.
whereas 4 was present only as a by-product (≤15% of total). It
PcOGT was then examined for glucosylation of 2 from 10 was not possible to enhance the relative abundance of 4 by
(each 5.0 mM, pH 7.5). UV absorbance traces from HPLC ana- running the reaction at different pH in the range 6.2–10.5.
Ability of the OsCGT variant to synthesise substantial amounts
of 4 is nevertheless remarkable, and the question of whether
isolation of 4 from the enzymatically prepared mixture of
phloretin glucosides would still be practical was left for consider-
ation in the future. Notably, though, the OsCGT variant was com-
plementarily regioselective to PcOGT in the glucosylation of 2, as
shown in Fig. 1d. The main PcOGT glucosylation product, likely
5, was completely absent from the reaction sample obtained
with the mutated OsCGT. However, a single new product peak
appeared. Its area increased proportionally to the decreasing
peak area of the 2. Interestingly, the retention time was very
similar to that of the trace product formed from 2 by PcOGT,
however HPLC analysis of a mixture of both conversions clearly
reveals them to be distinct (ESI,† Fig. S2). Based on preliminary
NMR data of the reaction mixture it was assumed, and will be
confirmed later, that 6 was formed in the reaction of the GT
mutant. Specific activity of the OsCGT variant for glucosylation
of 2 was determined as 22 3 mU mg−1
.
In summary, therefore, GT catalysts for selective glucosyla-
tion of 1 and 2 have been identified, and they were used
further for synthesis of 3, 5 and 6.
Fig. 1 Reversed-phase C-18 HPLC-analysis of dihydrochalcone (gluco- Thermodynamic analysis of glucosylation of phloretin (1)
sides) (X = unbound compounds including 9 and 10). (a) Separation of
Glucosylation of 1 or 2 from 10 is an equilibrium-controlled
glucosides of 1 (3, 4, 7) and 2 (5, 6) is achieved. (b) Conversion of 1 by
PcOGT. (c) Conversion of 2 by PcOGT (? = unknown by-product).
process.55 Thermodynamic restrictions on the synthesis of
(d) Conversion of 2 by OsCGT I121D.
dihydrochalcone β-D-glucosides therefore required clarification.
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inexpensive donor substrate. Only catalytic amounts of 9 as
compared to stoichiometric amounts of 10 are required in the
process. By using 8 in suitable excess over the acceptor sub-
strate, thermodynamic restrictions on acceptor glucosylation
are overcome effectively (see later). Equilibrium of the SuSy
reaction (Keq ≥ 0.5) favours formation of 10 in a wide pH range
(pH ≤ 7.5).52 Finally, problems of pronounced end-product
inhibition by 9, which have severely restricted direct synthetic
use of several flavonoid glycosyltransferases in the past, are
brought under control.56,57 Continuous removal of 9 due to
formation of 10 decreases the steady-state concentration of 9
in the reaction to a value even smaller than the one estab-
lished from the low (catalytic) amount that was initially added.
To optimise conditions for synthesis of 3, the coupled reac-
tion of SuSy and PcOGT was studied at different pH values in
the range 5.7–8.5. Recombinant SuSy from soybean (Glycine
max; GmSuSy) purified from an E. coli expression culture was
used (ESI,† Fig. S1). The applied concentrations of 1, 8 and 9
were 5.0 mM, 100 mM and 0.5 mM, respectively. Despite
addition of 20% DMSO as co-solvent, poor aqueous solubility
of 1 restricted use of concentrations higher than about 5 mM.
The time course of 3-formation was measured at each pH
(ESI,† Fig. S3). The data was used for calculation of initial pro-
duction rates of 3 (r3), final yields at reaction equilibrium
(24 h) and space-time yields (STY) for a target product concen-
tration of 4.0 mM (Fig. 3). The optimum pH range for high r3
was between pH 6.2 and 7.0 (Fig. 3a). Except for pH 5.7 and
8.5 where r3 was small and reaction equilibrium was therefore
not attained within the 24 h timespan of the experiment, the
final concentrations of 3 were around 4.4 mM, equivalent to a
yield of ∼90% based on conversion of 1 (Fig. 3b). With increas-
ing pH a marginal improvement of equilibrium concentrations
of 3 from 4.36 (pH 6.2) to 4.46 (pH 7.8) was observed.
However, STY for formation of 4 mM 3 (Fig. 3a) confirmed that
pH dependency of initial rates is predominantly determining
efficiency of conversions. A clear optimum of STY was found
Fig. 2 Phlorizin (3) synthesis (grey, 5 mM 1 and 10) and degradation
(black, 5 mM 3 and 9) by PcOGT (pH 6.5) level out at the same equili-
brium concentrations. (a) 1 (triangles), 3 (circles); (b) 10 (triangles),
9 (circles).
Fig.
2 displays results of time-course analyses for syn-
thesis and degradation of 3, catalysed by PcOGT at pH 6.5 and
30 °C. All compounds present in the reaction were quantified
(1, 3, 9, 10). Close balance for the proposed overall conversion,
1 + 10 ↔ 3 + 9, was obtained at each time and in each direction
of reaction. Therefore, this indicated absence of enzyme-cata-
lysed or spontaneous side reactions, such as hydrolysis of 10
for example. Reactions run in forward and reverse direction
levelled out at exactly the same end concentrations of 1 and 3
within limits of experimental error (Fig. 2a), clearly suggesting
that the true thermodynamic equilibrium had been attained.
It was affirmed that addition of fresh enzyme to a reaction
mixture at (apparent) equilibrium in Fig. 2 did, as expected,
not induce further concentration changes. An equilibrium con-
stant (Keq) of 4.6 ( 0.7) was calculated from the data. Under
the conditions used, therefore, only about 70% of substrate 1
can be converted to product 3. Development of a glucosyl
transfer cascade (Scheme 1) that involved in situ generation of
the UDP-sugar substrate 10 from sucrose (8) and UDP (9) was
highly useful to overcome these thermodynamic restrictions.
Glucosyl transfer cascade for efficiency-enhanced glucosylation
of phloretin (1)
The general principle of the glucosyl transfer cascade is shown
in Scheme 1. Two parallel glucosyl transfer reactions are con-
nected in a one-pot biotransformation via their common 10/9
substrate/product pair. Conversion of 8 and 9 is catalysed by
sucrose synthase (SuSy) and yields 10 and D-fructose (11). The
SuSy reaction, which represents the invariant part of the pro-
posed cascade, is flexibly coupled to different GT-catalysed
glucosylations of target acceptors under utilisation of 10 as the
donor substrate. Benefit of performing synthetic glucosylations
through SuSy-GT cascades as compared to single GT reactions
is manifested in significant improvement of key parameters of
overall transformation efficiency. First of all, cost-effective
Fig. 3 Influence of pH on efficiency of PcOGT-GmSuSy cascade con-
versions using BisTris (circles) and TAPS (triangles) buffers (5 mM 1,
0.5 mM 9, 100 mM 8). (a) Initial rate of formation of 3 (black), STY for
formation of 4 mM 3 (grey). (b) Final equilibrium concentrations of 3
supply of 10 is accomplished using 8 as a highly expedient and after 24 h (grey: equilibrium was not reached).
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around pH 6.5 with more than 50% loss at pH 6.2 and 7.1, mation in the early reaction phase of cascade reactions bene-
respectively. Overall, compared to synthesis of 3 directly from 1 fited somewhat (∼25% enhancement of r3) from the presence
and 10 (each 5 mM), introduction of the SuSy-GT cascade rep- of external 10, probably because the in situ produced steady-
resented a substantial yield enhancement from ∼70 to ∼90%. state concentration of 10 was not sufficient for PcOGT to
The effect of “thermodynamic push” from 8 on glucoside become fully saturated with glucosyl donor substrate. Use of
product formation is noted. It is explained from the net reac- 5 mM 10 instead of 0.5 mM 9 in SuSy-GT cascade reactions
tion of the glucosyl transfer cascade, which is sucrose (8) + furthermore caused moderate gain in final conversion (∼3%)
phloretin (1) ↔ phlorizin (3) + D-fructose (11).
and a more significant improvement of STY for production of
To further examine the role of 8 in driving the glucosylation 4.0 mM of 3 by about 35%. However, in our opinion a roughly
of 1, the cascade reaction was performed at pH 6.5 under con- 30% reduced reaction time to obtain a very similar product
ditions where both 8 (100 mM) and 10 (5 mM) were present as concentration is not compensating additional costs of replac-
donor substrates for glucosylation of 1 (5 mM). Fig. 4 shows ing UDP with 10-times higher concentrations of the more
the full time course of the conversion along with the corres- expensive 10. Both SuSy-PcOGT cascade reactions clearly out-
ponding time courses from the coupled reaction without sup- performed direct glycosylation of 1 by PcOGT. By addition of
plementation of 10 (but 0.5 mM 9) and the direct glucosylation GmSuSy the final concentration of 3 was increased from
in the absence of GmSuSy. Final conversions, initial rates and 3.61 mM to 4.44 (0.5 mM 9) and 4.60 mM (5 mM 10),
space time yields are summarised in Table 2. Product for- respectively.
Synthesis of davidoside (5) and confusoside (6) via glucosyl
transfer cascade reaction
In order to compensate in some degree the 200-fold decrease
in specific activity of PcOGT caused by change of acceptor sub-
strate from 1 to 2, the pH of the cascade reaction was raised to
7.5 where PcOGT is optimally active and the specific 2 gluco-
sylation rate was therefore enhanced around 2.5-fold as com-
pared to pH 6.5. The Ile121 → Asp variant of OsCGT is also best
active at a pH of around 7.5.53 Loss in specific activity of
GmSuSy caused by the rise in pH was negligible in comparison
(∼35%). The optimum pH of conversion of 8 by GmSuSy was
recently determined to be about 6.0.52
Relevant time courses for glucosylation of 2 by PcOGT and
OsCGT variant are shown in Fig. 5 and summarised in Table 2.
The substrate concentrations used were the same as before in
the conversions of 1. The concentration of 2 (5.0 mM) was set
according to aqueous solubility in the water-DMSO solvent
Fig. 4 PcOGT catalysed formation of 3 (circles) from 5 mM 1 (triangles)
at pH 6.5: black: without GmSuSy, 5 mM 10; dark grey: GmSuSy, 5 mM
10; light grey (dashed): GmSuSy, 0.5 mM 9.
used. Coupled enzyme reactions carried out in the presence
and absence of added 10 are compared to the single enzyme
reaction. Confusoside (6) was always obtained as single gluco-
syl transfer product in conversions with OsCGT I121D
(Fig. 5b). However, in PcOGT catalysed conversions besides
davidioside (5) a second compound, distinct from 6, accumu-
lated during the first 4 h and diminished again upon
prolonged incubation (Fig. 5a). Very low concentrations
(∼0.08 mM, <2% of total products) prevented structural analy-
sis of the by-product by NMR. However, most likely the by-
product is the third potential mono O-glucoside of 2, resulting
from glucosylation at the 4-OH. Its decline in favour of for-
mation of 5 at longer incubation times is explained by reversi-
bility of the glycosylations.55 This causes accumulation of the
thermodynamically favoured product (here 5) at equilibrium
conditions. Final mixture of the conversion of 2 contained the
main product 5 in about 50-fold excess over the by-product,
Table 2 Parameters of direct/GT-cascade catalysed synthesis of 3, 5
and 6a
3b
5c
6d
GT 5 mM 10
Conversione (%)
72.2
6.9
nd
91.9
8.2
7.6
88.9
6.5
5.6
79.6
1.3
nd
91.4
1.9
0.8
88.0
1.7
0.7
88.3
1.9
1.2
95.3
2.2
1.4
91.7
1.3
1.0
rglucoside f (mM h−1
)
)
)
STYg (mM h−1
)
GT-GmSuSy 5 mM 10
GT-GmSuSy 0.5 mM 9
Conversione (%)
rglucoside f (mM h−1
STYg (mM h−1
)
Conversion e (%)
rglucoside f (mM h−1
STYg (mM h−1
)
a Data extracted from Fig. 4 and 5. b 0.1 U mL−1 PcOGT (0.1 U mL−1
GmSuSy), pH 6.5, 5 mM 1. c 0.1 U mL−1 PcOGT, (0.1 U mL−1 GmSuSy),
pH 7.5, 5 mM 2. d 0.04 U mL−1 OsCGT I121D (0.04 U mL−1 GmSuSy), clearly demonstrating that highly regiospecific glucosylation of
pH 7.5, 5 mM 2. e At equilibrium after 6 (3), 24 (5) or 20 (6) h of
2 can be achieved by PcOGT. Comparison of the different reac-
tion conditions used revealed that the initial conversion of 2
proceeded slightly faster and the overall conversion was ∼3.5%
conversion. f Rate of glucoside formation during initial 25 (3) or
60 min (5, 6). g STY for formation of 4 mM glucoside (nd: less than
4 mM product).
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Fig. 5 Glucosylation of 5 mM 2 (triangles) at pH 7.5: black: without GmSuSy, 5 mM 10; dark grey: GmSuSy, 5 mM 10; light grey (dashed): GmSuSy,
0.5 mM 9; (a) PcOGT forms 5 (squares) and an unknown compound (circles). (b) OsCGT I121D only produces 6 (diamonds).
higher in GT-GmSuSy cascade reactions that contained exter- running the reaction in an aqueous-organic two-phase
nal 10. Benefit of coupling the synthetic glucosylation to con- system59 and feeding the acceptor substrate in accordance to
version of 8 was again manifested primarily in the final reaction progress52 are all viable strategies to enhance the
product concentration, which was enhanced by 12% (5) and effective acceptor concentration in the reaction. However,
7% (6), respectively in the SuSy-GT cascade reaction as com- detailed reaction engineering for targeted “de-bottlenecking”
pared to the single GT reference reaction. Product yields in of an enzymatic production was beyond the scope of this
coupled enzyme conversions of 2 were 88% or higher. The study. Alternative isolation of single flavonoids from complex
STYs of glucosylations of 2 were 5.5 (OsCGT variant) and 8 mixtures of phenolic compounds in plant materials involves
times (PcOGT) lower than those for glucosylation of 1.
time consuming multi-step procedures and large quantities of
organic solvents. With the notable exception of highly abun-
dant 3, no yields above 5 mg g−1 dry weight were reported for
extraction of 4–6 from plant leaves.30–32,36 Due to simplifica-
tion of work-up typical yields of the herein described GT-SuSy
cascade reactions of around 2 g L−1 (∼4.5 mM) seem sufficient
to compete with extraction from natural sources. Furthermore,
natural availability is no longer a bottleneck on supply of agly-
cons 1 and 2 because they are readily obtained by high yielding
single step Friedel–Crafts acylation.60 A two-enzyme one-pot
glucosyl transfer cascade for convenient synthesis of the
corresponding dihydrochalcone β-D-glucosides (3, 5, 6) was
developed. Coupling of the synthetic enzymatic glucosyl trans-
fer to conversion of 8 and 9 catalysed by SuSy presents a gener-
ally applicable strategy to provide UDP-glucose (10) donor
substrate in a cost-effective manner and to cope with issues of
unfavourable thermodynamics and UDP (9) product inhi-
bition. It can be applied as a universal approach for green
enzymatic synthesis of flavonoid glucosides.
Product isolation and NMR spectroscopic characterization
The two products synthesised by glucosylation of 5 mM 2 were
isolated from 5 mL reaction mixtures using preparative
reversed phase HPLC. 5 and 6 were obtained as white powder
after freeze-drying and recovered in ≥80% yield. Identities of 5
and 6 were unambiguously assigned from results of a detailed
NMR spectroscopic characterization that involved besides
1H and 13C NMR, HMQC (only 5), COSY and HMBC. Results
are summarized in Table S1 and Scheme S1 (ESI†). Although
separation was achieved, concentrations of the by-product of 2
glucosylation by PcOGT were too low for NMR analysis.
Davidioside (5) and confusoside (6) were recovered from gluco-
sylation of 2 by PcOGT and OsCGT variant as single compounds
at purities >98% based on HPLC peak areas (ESI,† Fig. S4).
Conclusions
Glucosyltransferases catalysing highly regiospecific β-D-glucosyl-
transfer from UDP-glucose (10) to the 2′-OH of the dihydro-
chalcones 1 and 2, and to the 4′-OH of 2 were identified. So far
no glycosyltransferase for selective synthesis of trilobatin (4) by
Experimental
Materials
glucosylation of the 4′-OH of 1 was found, but it presumably Unless otherwise indicated, all chemicals were from Sigma-
exists in Malus trilobata which only forms 4 but not 3.30 Aldrich (Vienna, Austria) in the highest purity available.
Maximum glucoside product concentrations were mainly Phloretin (98%) was from AK Scientific (Union City, CA, US)
restricted by aqueous solubility of the comparably hydrophobic and phlorizin dihydrate (≥98%), from Carl Roth (Karlsruhe,
dihydrochalcone acceptors. Optimised use of co-solvent,58 Germany). Strep-Tactin® Sepharose® and desthiobiotin were
4422 | Green Chem., 2014, 16, 4417–4425
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Paper
from IBA (Goettingen, Germany). BCA Protein Assay Kit was
from Thermo Scientific (Waltham, MA, US).
An reversed phase HPLC-based assay for quantification of
dihydrochalcones (1, 2) and their glycosides (3–6) was used to
determine activities of PcOGT and the OsCGT variant.53 In
short, conversions of 5 mM dihydrochalcone with 0.5 mM 10
were started by GT addition and conducted under agitation
(400 rpm) and temperature control (30 °C) using a thermo-
mixer. Typically 4 aliquots of 100 µL were taken within 60 min
to determine linear initial rates. Reactions were stopped by
mixing with 100 µL acetonitrile. Precipitated protein was
removed by centrifugation (13 200 rpm, 20 min) before apply-
ing 5–10 µL of supernatant to HPLC analysis. An Agilent 1200
HPLC equipped with a Chromolith® Performance RP-18e
column (100 × 4.6 mm) was used at 35 °C, and UV-detection at
288 nm was applied. A water (A) to acetonitrile (B) gradient
(0.1% trifluoroacetic acid each) was applied for elution:
20–47.5% B (7.5 min, 1 mL min−1), 47.5–100% B (0.05 min,
1 mL min−1), 100% B (1.45 min, 1.5 mL min−1), 100–20% B
(0.05 min, 1.5 mL min−1), 20% B (2.45 min, 1.5 mL min−1).
One unit of GT activity was defined as the amount of enzyme
glucosylating 1 µmol 1 per min under following conditions:
30 °C, 5 mM 1, 0.5 mM 10 in 50 mM BisTris, pH 6.5 (PcOGT)
or 7.5 (OsCGT I121D) containing 13 mM MgCl2, 50 mM KCl,
0.13% BSA and 20% DMSO.
Synthesis of davidigenin (2)
Synthesis of 2 was done in a single step from 1,3-benzenediol
(resorcinol) and 3-(4-hydroxyphenyl)propanoic acid (phloretic
acid) using an adopted Friedel–Crafts acylation method.60
Phloretic acid (6 mmol, 990 mg) and resorcinol (6 mmol,
661 mg) were stirred in 4 mL BF3Et2O (32 mmol) at 90 °C for
120 min under argon. Complete conversion of resorcinol was
verified by TLC. The mixture was poured into 200 mL 10%
aqueous NaOAc and stirred for 2 h at room temperature. The
solution was 3 times extracted with 200 mL EtOAc. Combined
EtOAc was washed with 40 mL water and 40 mL brine before
drying over MgSO4. The solvent was evaporated under reduced
pressure and the residue was chromatographed over silica gel
column using cyclohexane–EtOAc mixtures. 2 (61% isolated
yield) was confirmed by 1H- and 13C-NMR spectroscopy
(Bruker AVANCE III 300 spectrometer).
Enzyme production
Escherichia coli BL21-Gold (DE3) expression strains for PcOGT
(UGT88F2; GenBank: FJ854496),53 OsCGT (GenBank:
FM179712) Ile121 to Asp mutant53 and GmSuSy (GenBank:
AF030231)52 were described elsewhere in detail. Enzyme
expression in lysogeny broth (LB)-medium as N-terminal Strep-
tag II fusion proteins and enzyme purification by affinity
chromatography on Strep-Tactin® Sepharose® columns was
also reported elsewhere.52 Expected molecular mass and purity
of enzymes were affirmed by SDS polyacrylamide gel electro-
phoresis (PAGE). Aliquots of final preparations were stored at
−70 °C and thawed only once prior to their use.
Equilibrium constant (Keq) of glucosylation of 1 by PcOGT
Keq of PcOGT was determined by running glucosylation of 1 in
forward (5 mM 1, 5 mM 10) and reverse direction (5 mM 3,
5 mM 9) until no further conversion was observed. Reactions
were run at 30 °C and started by addition of 100 mU mL−1
PcOGT (50 mM BisTris, pH 6.5, 13 mM MgCl2, 50 mM KCl,
0.13% BSA and 20% DMSO). Concentrations of 1 and 3 were
determined by the HPLC based GT activity assay.
The same samples (10 µL) were applied for quantification
of 9 and 10 using an anion exchange HPLC protocol. An
Agilent 1200 HPLC system was used for separation on an
Agilent ZORBAX SAX (4.6 × 250 mm) column at 30 °C. 9 and
10 were monitored by UV detection at 254 nm. Using 20 and
500 mM potassium phosphate buffer (pH 6.8) as solvent A and
B, respectively following gradient was applied at a constant
flow rate of 1.5 mL min−1: 0–100% B (7 min), 100% B (2 min),
100–0% B (0.05 min), 0% B (3.95 min).
Activity assays
Sucrose cleavage by GmSuSy was measured spectrophotometri-
cally using a discontinuous two-step enzymatic assay. Oxi-
dation of 10, formed from 8 (100 mM) and 9 (0.5 mM), to
UDP-α-D-glucuronic acid is coupled to reduction of two NAD+
molecules to NADH by human UDP-α-D-glucose 6-dehydro-
genase (hUGDH) as described elsewhere in detail.52 In short,
reactions were started by GmSuSy addition and samples of 150 µL
were stopped by heating (95 °C, 5 min). Precipitated protein
was removed by centrifugation (13 200 rpm, 20 min) and 100 µL
of the supernatant were mixed with 400 µL of measuring
Formation of 3, 5 and 6 by direct GT and GT-GmSuSy cascade
reactions
solution (2.5 mM NAD+, 0.05% TritonTM X-100 in 100 mM 5 mM 1 and 2 were converted in 50 mM BisTris at pH 6.5 and
HEPES, pH 8.0) in a Half Micro Cuvette. Absorbance at 340 nm 7.5, respectively. All reactions contained 13 mM MgCl2, 50 mM
was measured before and after incubation with 1.5 mU of KCl, 0.13% BSA and 20% DMSO. As glucose source conver-
hUGDH and concentration of 10 was calculated from the sions without GmSuSy contained 5 mM 10 while for those with
increase of absorbance (εNADH = 6220 M−1 cm−1). Typically 4 GmSuSy either 5 mM 10 and 100 mM 8 or 0.5 mM 9 and
distinct measurements were used to calculate linear initial 100 mM 8 were used. Conversions were started by addition of
rates. One unit of GmSuSy was defined as the amount of 100 mU mL−1 PcOGT (and 100 mU mL−1 GmSuSy) or 40 mU
enzyme producing 1 µmol 10 (2 µmol NADH) per minute mL−1 OsCGT I121D (and 40 mU mL−1 GmSuSy). Samples were
under following conditions: 30 °C, pH of cascade reactions incubated at 30 °C and the GT HPLC protocol was applied for
(6.5 or 7.5) 0.5 mM 9, 100 mM 8 in 50 mM BisTris, 13 mM sampling after distinct incubation times and quantification of
MgCl2, 50 mM KCl, 0.13% BSA and 20% DMSO.
1, 2, 3, 5 and 6.
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Paper
Green Chemistry
Coupled glucosylation of 5 mM 1 by PcOGT and GmSuSy
(100 mU mL−1 each) in presence of 0.5 mM 9 and 100 mM 8
9 A. Picinelli, E. Dapena and J. J. Mangas, J. Agric. Food
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Isolation and identification of 5 and 6
5 and 6 from glucosylation of 2 by PcOGT and OsCGT I121D
mutant were purified by preparative reversed phase C-18 HPLC
on an Agilent 1200 system equipped with a SphereClone 5 µm
ODS(2) (250 × 10.0 mm) column. Water was used as solvent A
and acetonitrile as solvent B (0.1% formic acid each). Separ-
ation of 5 and 6 from other compounds was achieved by step
gradients from 10% to 100% B at room temperature. 5 eluted
at 25% and 6 at 35% B. After removing acetonitrile under
reduced pressure water was removed by freeze drying. Purity
and identity were confirmed by HPLC and NMR. 1H-NMR,
13C-NMR, COSY, HMBC and HMQC (5 only) were recorded on
a Varian Unity Inova 500 MHz spectrometer.
Acknowledgements
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Financial support from the Austrian Science Fund (FWF, DK
Molecular Enzymology W901-B05) and the EU FP7 project
SuSy (Sucrose Synthase as Effective Mediator of Glycosylation)
is gratefully acknowledged. L. B. is supported from a scholar-
ship of Ramkhamhaeng University (Bangkok, Thailand).
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24 C. M. M. Gachon, M. Langlois-Meurinne and
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