Influence of a 4′-substituent on the efficiency of flavonol-based fluorescent indicators of β-glycosidase activity

Article abstract of DOI:10.1039/d0ob01505a

This article presents novel fluorescent probes, based on the excited-state intramolecular proton transfer (ESIPT) phenomenon and flavonols, sensitive to the action of specific glycosidases. 4′-Substituted flavonols were synthesized, using various approaches, and glycosylated withd-glucose,N-acetyl-d-glucosamine andd-glucuronic acid. Evaluation of the β-glycosidase activities was performed in neutral and acidic pH. In all the cases examined, an acidic environment accelerated enzymatic hydrolysis. It was demonstrated that the 4′-chloroflavonyl glycosides of all sugars tested, both in neutral and acidic pH, are the ones most sensitive to the presence of hydrolase. In turn, 4′-dimethylaminoflavonyl glucoside is not sensitive to glucosidase action at all. Generally, the rate of enzymatic hydrolysis increases as the electron-withdrawing nature of the 4′-substituent increases. An exception is the trifluoromethyl group which, in spite of having the most favourable Hammett constant, does not contribute enough to increase the rate of hydrolysis of its glucoside. The presented experimental results are supported by the electrostatic potential (ESP) analysis and related to the mechanisms of glycoside bond enzymatic hydrolysis.

Full text of DOI:10.1039/d0ob01505a

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DOI: 10.1039/D0OB01505A.
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DOI: 10.1039/D0OB01505A
ARTICLE
Influence of the 4’-substituent on the efficiency of flavonol-based
fluorescent indicators of β-glycosidase activity
Milena Reszka,a Illia E. Serdiuk,b Karol Kozakiewicz,a Andrzej Nowacki,a Henryk Myszka,a
Received 00th January 20xx,
Accepted 00th January 20xx
Piotr Bojarski,b Beata Libereka,*
DOI: 10.1039/x0xx00000x
This article presents novel fluorescent probes, based on the excited-state intramolecular proton transfer (ESIPT)
phenomenon and flavonols, sensitive to the action of specific glycosidases. 4’-Substituted flavonols were synthesized,
using various approaches, and glycosylated with D-glucose, N-acetyl-D-glucosamine and D-glucuronic acid. Evaluation of
the β-glycosidases activities were tested in neutral and acidic pH. In the all cases examined, an acidic environment
accelerated enzymatic hydrolysis. It was demonstrated that the 4’-chloroflavonyl glycosides of all sugars tested, both in
neutral and acidic pH, are the ones most sensitive to the presence of hydrolase. In turn, 4’-dimethylaminoflavonyl
glucoside is not sensitive to glucosidase action at all. Generally, the rate of enzymatic hydrolysis increases as the electron-
withdrawing nature of the 4’-substituent increases. An exception is the trifluoromethyl group which, in spite of having the
most favourable Hammett constant, does not contribute enough to increase the rate of hydrolysis of its glucoside.
Presented experimental results are supported by the electrostatic potential (ESP) analysis and referred to the mechanisms
of glycoside bond enzymatic hydrolysis.
medicine and technology, because their action leads to release of
biologically active molecules from the respective glycosides. For
example, the activity of β-glucosidase was correlated with
Introduction
Glycosidases represent a large group of enzymes commonly found
in all living organisms ‒ bacteria and fungi,1-4 plants,5-7 mammals
and other animals8-11 ‒ where they play crucial roles in these
organisms’ functions. The main role of glycosidases is to catalyse
the hydrolysis of glycosidic linkages. In this way, oligosaccharides,
glycoconjugates, and other glycosylated molecules are degraded.
Inappropriate activity of glycosidases usually causes serious
dysfunctions in a cell and/or a whole organism. Many of the
lysosomal storage diseases (LSDs) in humans result from deficiency
of glycosidases.12,13 Such a deficiency disables degradation of the
large molecules containing sugars, thereby these are accumulated
within the cell and eventually killing it. Among the LSDs caused by
insufficient or any activity of glycosidases are the following: Tay-
Sachs and Sandhoff,14 Krabbe,15 Gaucher,16 Fabry,17 and Pompe
diseases18 as well as some from mucopolysaccharidoses (MPSs).13
Monitoring the respective glycosidase’s activity is crucial for quick
and proper diagnosis as well as for efficient therapy in these
diseases.19-22
concentrations of isoflavone released from soybean extracts
containing isoflavone glucosides. Such isoflavone extracts inhibit
angiogenesis caused by colon carcinoma cells in mice.23 Activity of
β-glucosidase was measured to prove that its increase results from
a disease process caused by Fusarium oxysporum f. sp. lupini.24
Control of β-glucosidase activity is extremely important during the
production of biofuels from lignocellulosic biomass.25,26 Activities of
the β-glucosidases of lactic acid bacteria make a contribution to the
quality of fermented food.27 Evaluation of β-glucosidase activity in
soil can be used as an indicator of its quality.28 Activity of α- and β-
glucosidases was correlated with the phytoplankton bloom29 and
with organic matter abundance in the environment.30 Activities of
different glycosidases are involved in the release of aroma to
wine31,32 and in the ripening of fruits.33 Activities of β-
glucuronidases of microorganisms in the large intestine were
determined to control carcinogenesis.34 In turn, the urine level of N-
acetyl-β-D-glucosaminidase is used as an indicator of proximal
tubular cell necrosis.35
Besides diagnostics of lysosomal diseases, glycosidases have
been investigated and employed in different fields of science,
Since glycosidases affect processes essential for various fields of
life and science, the approaches enabling determination of their
activity are of high interest. Among the variety of physicochemical
methods applied for the determination of enzyme activity,
fluorescence spectroscopy offers high sensitivity, reproducibility
and, under certain conditions, the possibility of nondestructive
analysis.
a.Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland
b.Faculty of Mathematics, Physics and Informatics, University of Gdańsk,
Wita Stwosza 63, 80-308 Gdańsk, Poland
*E-mail: beata.liberek@ug.edu.pl
Electronic Supplementary Information (ESI) available: NMR spectra of the compounds
investigated; fluorescence spectra measured at pH 5.2; results of the ESP analysis. See
DOI: 10.1039/x0xx00000x
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ARTICLE
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In a search of new alternative indicators of β-glycosidase activity, The separation of 2’-hydroxychalcone should be reVlaietwivAerltiycleqOunilcinke.
DOI: 10.1039/D0OB01505A
we focused our attention on flavonol, a naturally occurring and Otherwise, it tends to cyclize with formation of a flavanone. This
nontoxic fluorophore. Its especially attractive fluorescent features took place in the case of 2’-hydroxy-4-methoxychalcone (3b) when it
are due to the excited state intramolecular proton transfer (ESIPT), was classically synthesized and kept in an acidic solution for three
which enables desirable red-shifted emission with a large Stokes days in a refrigerator. As a consequence the solid obtained was a
shift and low susceptibility to the self-reabsorption effect.36,37 The mixture of chalcone 3b and flavanone 3b’ (Scheme 1) in the ratio of
use of flavonol instead of common non-ESIPT fluorophores avoids 2:1, as established from the 1H NMR spectrum. The basic difference
problems connected with concentration restrictions, and together in the spectra of these two isomers lays in the location of α and β
with its low toxicity, it provides more possibilities for biochemical proton (Fig. S1) signals. In the case of chalcone (3b) these protons
and medical analyses. These attractive spectral features of flavonol are attached to the double bond and are highly deshielded (δ 7.91
and its derivatives have been successfully used for investigations of for Hβ and 7.55 for Hα). In the case of flavanone (3b’) these protons
their interactions with β-cyclodextrin,38 proteins,39,40 duplex and are attached to the tetrahydropyran ring, which shifts their signals
tetraplex DNAs41 as well as for human carboxylesterase detection42 in the 1H NMR spectrum towards much higher fields (δ 5.43 for Hβ,
and monitoring of penetratin (a cell penetrating peptide) 3.10 for Hα and 2,86 for Hα’). There are reports in the literature,
interactions with lipid membranes.43
which indicate that an acidic environment favours 2’-
hydroxychalcone‒flavanone isomerization.48-50 It was also
demonstrated that this transformation can be photoinduced.51
2’-Hydroxy-4-methoxychalcone (3b) was solely synthesized with
microwave assistance followed by neutralization with 5% aqueous
HCl and separation on the same day. Probably, the short contact of
3b with an acidic environment avoids the formation of 3b’ since the
rate constant of chalcone cyclization to flavanone is small, ca. 10-5
min-1.48 Importantly, it was not necessary to separate 2’-
hydroxychalcone (3b) from flavanone (3b’) in order to synthesize the
flavonol 4b. In the conditions of the AFO reaction, both 3b and 3b’
undergo transformation to 4b.
In our initial studies, we introduced 4’-fluoroflavonyl -D-
glucoside to interaction with β-glucosidase.44 We demonstrated
that the active β-glucosidase catalyses hydrolysis of 4’-
fluoroflavonyl -D-glucoside, which results in release of 4’-
fluoroflavonol. Electronically excited species of 4’-fluoroflavonol
undergo ESIPT and thus the course of enzymatic hydrolysis in 4’-
fluoroflavonyl -D-glucoside was monitored by the rise of red-
shifted fluorescence at around 530 nm.
In this paper we enlarge the scope of glucosidases, whose
activity can be monitored by the derivatives of flavonol indicators
and demonstrate how the substituent at the 4’ position in flavonol
influences the sensitivity of the flavonyl β-D-glucosides to β-
glucosidase. With this aim, respective glucosides of 4’-substituted
flavonols were synthesized and subjected to enzymatic hydrolysis at
the physiological pH (7.4) and at pH 5.2, which was monitored by
fluorescence measurements. Electron-withdrawing (F, Cl, CF3) and
electron-donating (OMe, NMe2) groups were selected for these
studies. Additionally, the glucoside of the unsubstituted flavonol
was used. For an adequate interpretation of the experimental
findings, the kinetics of each probe’s enzymatic hydrolysis was
investigated at both pH 7.4 and 5.2. This enables a discussion of the
influence of the 4’-flavonol substituent on the rate of enzymatic
hydrolysis in terms of its mechanism. The most distinctive
fluorophores, selected during the investigation of β-D-glucosides,
were used further for verification of the N-acetyl-β-D-glucos-
aminidase’s and β-D-glucuronidase’s activities. Kinetic studies on
the hydrolysis catalysed by these enzymes at pH 7.4 and 5.2 are also
presented.
O
O
O
OH
i
O
ii
+
R
OH
R
OH
O
3b-f
H3O
2b-f
4b-f
) R = OMe
) R = NMe2
) R = F
1
R
+
HO
b
c
d
e
(
(
(
(
O
(i) 1. KOH, MeOH; 2. HClaq
(ii) 1. KOH, H2O2; 2. HClaq
) R = Cl
f
( ) R = CF3
O
3b'
OMe
Scheme 1 Synthesis of 2’-hydroxychalcones (3b-f) followed by AFO reaction
leading to flavonols (4b-f).
Taking into account that both the synthesis of 2’-hydroxy-
chalcones (3b-f) and the synthesis of flavonols (4b-f) are conducted in
methanolic alkaline solutions, we obtained 4e and 4f in a one-pot
reaction without isolation of the respective chalcones (3e and 3f).
Such an approach simplified the procedure, reduced the reaction
time, and provided the flavonols 4e and 4f with reasonable yields
relative to 1 (28% and 18%, respectively). However, in the one-pot
procedure, the precipitated flavonols (4e and 4f) were contaminated
with the respective 4-substituted benzoic acids. The latter are
products from oxidation of the 4-substituted aldehydes (2e and 2f),
which were not separated after the 2’-hydroxychalcones (3e and 3f)
synthesis. Extraction with alkaline aqueous solution allows us to
separate acids from flavonols. It has to be mentioned that the
chalcone’s synthesis was always carried out with the use of a 1:1
molar ratio of 1 and 2a-f. Despite the prolongation of the reaction
time, trace amounts of substrates were always present in the post-
reaction mixture.
Results and discussion
Synthesis
Flavonols (4b-f) were synthesized by the Algar-Flynn-Oyamada
(AFO) reaction.45,46 This involves the oxidative cyclization of 2’-
hydroxychalcones (3b-f) with hydrogen peroxide in alkaline alcohol
solution (Scheme 1).47 2’-Hydroxychalcones (3b-f) were prepared via
Claisen-Schmidt condensation of 2-hydroxyacetophenone (1) and
the respective 4-substituted benzaldehyde (2b-f). Typically, isolation
of 2’-hydroxychalcone requires neutralization of the alkaline
solution with an acid and separation of the precipitated product.
2 | J. Name., 2012, 00, 1-3
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Glucosylations of 4a-f were carried out with commercially H-bonding ability. In a majority of cases this increase is several–fold
View Article Online
DOI: 10.1039/D0OB01505A
available bromide 5 in a two-phase system (CH2Cl2/H2O) in the (3.5–7.0). The exception is 4’-dimethylaminoflavonol (4c); its
presence of K2CO3 and tetrabutylammonium bromide (TBAB) as a quantum yield rises 58-fold in the presence of BSA, which is due to
phase-transfer catalyst (Scheme 2). These reaction conditions the fact that the fluorescence of 4c in the absence of BSA is
require the use of an excess of bromide (5) over flavonol (4a-f
)
relatively small (0.4%) in comparison with other probes (4a,b,d-f).
because the former willingly hydrolyzes and additionally eliminates
HBr to form the corresponding glycal. Therefore, by-products
always accompany this reaction. Two molar excess bromide (5)
allowed per-O-acetylated flavonyl β-D-glucosides (8a-f
obtained in good yields; these gave 11a-f when deprotected.
Peracetylated flavonyl β-D-glucosaminosides (9a,b,e and β-D-
) to be
)
glucuronides (10a,b,e) were obtained in an analogous way with the
use of the respective glycosyl donors (6 and 7), which were
synthesized according to commonly used procedures. Methanolysis
of 9a,b,e led to 12a,b,e, whereas hydrolysis of 10a,b,e gave 13a,b,e
.
Figure 1 Fluorescence spectra of 4’-fluoroflavonol (4d) in the presence of
various amounts of BSA.
R
O
R
R2
R2
OR3
Table 1. Spectral parameters of fluorophores 4a-f measured in the absence
O
O
and presence of BSA.
O
O
i
O
OAc
+
φ [%]
φ
/φ
fl-BSA fl
OH
Comp.
4a
cBSA [M]
λab [nm]
342
344
354
358
402
408
342
344
346
346
342
348
λfl [nm]
413; 516
412; 535
438; 540
436; 538
R3O
AcO
fl
X
O
R1
R1
0
1.8
10.0
5.2
4a-f
R1 = OAc, R2 = CH2OAc, R3 = Ac
R1 = OH, R2 = CH2OH, R3 = H
R1 = NHAc, R2 = CH2OAc, R3 = Ac
R1 = OH, R2 = CH2OH, R3 = H
R1 = OAc, R2 = COOMe, R3 = Ac
R1 = OH, R2 = COOH, R3 = H
5.5
3.5
58
X = Br, R1 = OAc, R2 = CH2OAc
X = Cl, R1 = NHAc, R2 = CH2OAc
X = Br, R1 = OAc, R2 = COOMe
8a-f
5
6
7
7.5 · 105
a
b
c
d
e
f
R = H
ii
11a-f
R = OMe
R = NMe2
R = F
R = Cl
R =CF3
9a,b,e
0
ii
12a,b,e
10a,b,e
13a,b,e
4b
4c
4d
4e
4f
7.5 · 105
18.4
0.4
iii
0
550; –
(i) K2CO3 / TBAB / CH2Cl2 / H2O; (ii) MeONa / MeOH; (iii) LiOH / MeOH
7.5 · 105
511; 553
413; 512
411; 538
413; 524
413; 541
410; 512
413; 543
23.2
1.9
Scheme 2 Synthesis of 4’-substituted flavonyl β-D-glucosides (11a-f),
β-D-glucosaminosides (12a,b,e) and β-D-glucuronides (13a,b,e).
0
5.6
7.0
6.0
7.5 · 105
10.7
1.9
Fluorescent measurements
0
Selection of measurements conditions
7.5 · 105
13.6
1.1
An aqueous environment is necessary to evaluate glycosidase
activity. However, intensity of the proton transfer fluorescence of
flavonols is significantly diminished in water in comparison with
polar aprotic solvents.52,53 This feature of flavonols and of ESIPT
fluorophores in general is caused by the formation of inter-
molecular hydrogen bonding between flavonols in the ground state
and water molecules. This inhibits formation of the intra-molecular
hydrogen bonding in the ground state of flavonol, which is a
prerequisite for ESIPT occurrence.40 To solve this problem, the
concentration of water in the local environment of a fluorophore
can be decreased by its complexation with a protein, which is
accompanied by a substantial increase in the ESIPT fluorescence. In
this regard the most promising proteins are serum albumins, which
have been successfully used by us previously instead of organic
solvent.54 Therefore, the most efficient concentration of BSA (7.5 ·
10-5 M) was established (Fig. 1) and used herein to measure spectral
parameters of fluorophores 4a-f (Table 1).
0
7.5 · 105
6.6
cBSA – concentration of BSA; λab – wavelength of the absorption maximum;
λfl – wavelength of the N* and T* fluorescence maxima, respectively;
φ
– quantum yield of fluorescence, measured relative to quinine bisulfate in 0.05
fl
M H2SO4; φ
/φ – ratio of quantum yields of fluorescence measured in
fl-BSA
fl
a presence and absence of BSA.
Enzymatic hydrolysis with β-glucosidase
To investigate the effect of the 4’-substituent in flavonol derivatives
on the sensitivity of flavonyl β-D-glucosides to β-glucosidase,
interactions of glucosides 11a-f with β-glucosidase were
investigated. The action of this enzyme was expected to cause
hydrolysis of the glucosidic bond in 11a-f and thus release of
fluorophores 4a-f that readily form complexes with the BSA present
in solution. After light excitation, the latter complexes produced N*
species which further underwent ESIPT and transformation to the
T* species emitting in the 530-550 nm range and in this way
indicating the course of enzymatic cleavage (Scheme 3).
Thus, the mixture of a respective probe (11a-f, 5 · 10-5 M) and β-
glucosidase (5 · 10-3 units per l) in phosphate buffer saline (PBS, pH
= 7.4) and in the presence of 7.5 · 10-5 M BSA was incubated at 37
oC and fluorescence spectra were measured at specified time
The results presented in Table 1 show that complexation of
flavonols (4a-f
) with BSA results in strong increase of T*
fluorescence, with a slight bathochromic shift of its maximum,
whereas weak N* fluorescence in the range 411–511 nm is
generally not affected by addition of BSA. These indicate a change
of the molecular environment of 4a-f to one with lower polarity and
This journal is © The Royal Society of Chemistry 20xx
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intervals. At the zero point of hydrolysis, the fluorescence of glycosidases’ action, such as in the stomach, tumourVieewxtArraticcleeOllunllianer
DOI: 10.1039/D0OB01505A
glucosides 11a and 11d-f is very weak, whereas glucosides 11b and environment, and some soils. The results of investigations at pH 5.2
11c exhibit relatively strong fluorescence (Fig. 2). This finding were similar to those performed at pH 7.4 (Fig. S2). Thus, the rise of
correlates well with the fluorescence properties of respective the T* fluorescence, centered at around 540 nm, is characteristic of
flavone derivatives lacking a hydroxyl group at position 3; only the probes 11a, 11b and 11d-f. However, the ESIPT fluorescence
flavones bearing electron-releasing 4-methoxy and 4- reaches its maximum within 2-12 h at pH 5.2 whereas it was 4-20 h
dimethylamino groups exhibit relatively strong fluorescent at pH 7.4. Again, no changes in the fluorescence spectra of 11c
properties.55 The emission of flavones without 4’-substituents or of solution were observed during 24 h, evidencing that this compound
those bearing electron-withdrawing groups is very weak due to the is also relatively stable towards hydrolysis under such acidic
n-π* character of their S1 state. By such a comparison one can reaction conditions.
conclude that the glucose attached to the flavonols breaks the
conjugation between oxygen at position
3 and the rest of
chromophore fragment, probably by changing the orientation of
the non-bonding oxygen orbitals to the chromophore ring. For this
reason, glucosides 11a-f exhibit fluorescent features close to
flavones but not flavonols.
R
11a
11c
11e
11b
11d
11f
O
R
OH
OH
O
O
O
O
O
-Glucosidase
O
OH
OH
OH
+
H2O / BSA
OH
HO
HO
OH
OH
D-glucose
11a-f
4a-f
Excitation
a
( ) R = H
b
(
) R = OMe
*
*
R
R
Fluorescence
c
( ) R = NMe2
d
(
) R = F
O
+
O
O
ESIPT
e
( ) R = Cl
f
( ) R = CF3
O
OH
OH
T*
N*
Scheme 3 The assumed mode of action of flavonol-based β-glucosidase
indicators.
According to the plots in Fig. 2, as the enzymatic hydrolysis
proceeds, the rise of red-shifted T* fluorescence centered around
540 nm is observed for the probes 11a, 11b and 11d-f. Although
glucoside 11b exhibits relatively strong blue fluorescence at the zero
point, the course of its enzymatic hydrolysis is also followed by an
increase in the T* fluorescence. Simultaneously the blue band shifts
from 440 to 430 nm and its intensity decreases twice. The reason
for such changes in the blue region is that the fluorescence of
glucoside 11b is gradually replaced by the one of N* species of 4b.
Exceptionally, under the same conditions, no changes in the
fluorescence spectra of 11c solution were observed during 22 h
evidencing the relatively high stability of this compound towards
enzymatic hydrolysis. The same fluorescence spectra at the zero
point of hydrolysis and after 22 hours with maximum at about 500
nm correspond to the emission of electronically excited glucoside
11c. The ESIPT fluorescence of the remaining probes reaches its
maximum in 4-20 h, depending on the probe, indicating completion
of hydrolysis. The time needed for hydrolysis of the tested probes
decreases in the order 11c >> 11b > 11f > 11d > 11a > 11e.
Figure 2. Changes in the fluorescence spectra during enzymatic hydrolysis of
probes 11a-f at pH 7.4. Insets: plots of the relative intensity of the T*
fluorescence (IiT*/ ImaxT*) versus time.
Kinetic studies on enzymatic hydrolysis with β-glucosidase
To find out which of the studied fluorescence indicators of β-
glucosidase activity is the most efficient, kinetic parameters were
evaluated and compared for probes 11a,b,d-f. Based on the
assumption that intensity of the T* fluorescence corresponds to the
concentration of released flavonol (4a,b,d-f), the plots of ln co/ct
versus time were analysed (Fig. 3), where co is the initial
concentration of the respective 11a,b,d-f in relative units (equal to 1)
and ct is the concentration of the respective 11a,b,d-f at time ti. The
latter value was calculated from equation 1:
T*
i
T*
I
ct  co 
(1)
I
max
T*
where I
is the intensity of the T* fluorescence band at ti and I
i
T*
is the intensity of the maximal T* fluorescence measured
max
To check how an acidic environment affects the action of
probes 11a-f, fluorescence measurements analogous to those
during enzymatic hydrolysis for respective probe (each hydrolysis
T*
T*
reaches its plateau). Thus, the I / I
ratio corresponds to the
max
i
described above were also performed in the presence of PBS at pH
5.2. Acidic conditions are typical for some environments of
concentration of 4a,b,d-f released in relative units at time ti,.
4 | J. Name., 2012, 00, 1-3
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was concluded from the experiments with β-glucosidase, the best
View Article Online
DOI: 10.1039/D0OB01505A
and the worst among those investigated aglycone fluorophores
were the 4’-chloroflavonol (4e) and 4’-methoxyflavonol (4b),
respectively. So, they were used for these experiments.
Additionally, the unsubstituted flavonol (4a) was tested. The
experiments were performed under analogous conditions as for the
enzymatic hydrolysis of 11a-f. Thus, the mixture of the respective
probe (12a,b,e or 13a,b,e, 5 · 10-5 M) and the respective enzyme (1.1 ·
103 units per l of N-acetylglucosaminidase, 2.1 · 106 units per l of
glucuronidase at pH 7.4 and 2.1 · 105 units per l of glucuronidase at
pH 5.2) in phosphate buffer saline (PBS, pH 7.4 and 5.2,
respectively) and in the presence of 7.5 · 10-5 M BSA was incubated
pH 7.4
pH 5.2
Figure 3 The plots of ln co/ct vs time for enzymatic hydrolysis of 11a,b,d-f
at pH 7.4 and 5.2.
As one can notice, the dependence of ln co/ct on time for
enzymatic hydrolysis of 11a,b,d-f, at both 7.4 and 5.2 pH, is linear
with R2 = 0.99 in all cases (Fig. 3). This means that the enzymatic
cleavage follows first-order or pseudo-first-order kinetics and can
be described by equation 2
o
at 37 C and fluorescence spectra were measured at specified time
intervals.
co
According to our results, both N-acetyl-β-D-glucosaminosides
(12a,b,e) and β-D-glucuronides (13a,b,e) are sensitive to the presence
of the respective glycosidases at pH 7.4 (Figs. 4 and 5) as well as at
pH 5.2 (Figs S3 and S4). As in the case of β-glucosides, the most
sensitive to enzymatic hydrolysis are 4’-chloroflavonyl glycosides
with the constants k of 0.33 h-1 (pH 7.4) and 1.83 h-1 (pH 5.2) for
12e, and 0.42 h-1 (pH 7.4) and 4.05 h-1 (pH 5.2) for 13e (Table 3). 4’-
Methoxyflavonyl glycosides are again the least sensitive to
enzymatic hydrolysis with the measured rate constants k of 0.20 h-1
(pH 7.4) and 0.88 h-1 (pH 5.2) for 12b and 0.11 h-1 (pH 7.4) and 2.04
h-1 (pH 5.2) for 13e. The results presented demonstrate that both
enzymatic hydrolysis reactions are running faster in the acidic
environment, which is particularly seen in the case of β-D-
glucuronides (13a,b,e).
ln  k ti
(2)
ct
where k is a respective rate constant. This rate constant does not
change in the course of hydrolysis, which indicates that neither
11a,b,d-f nor 4a,b,d-f inhibit or activate β-glucosidase at the conditions
applied. Thus, all tested glucosides (11a,b,d-f) can be considered to be
fluorescence probes although the most efficient one seems to be
the glucoside 11e bearing the chloro substituent at the 4’ carbon
atom of flavonol. For this probe the rate constant of the enzymatic
hydrolysis is the highest and amounts to 0.63 h-1 at pH 7.4 and 4.51
h-1 at pH 5.2 (Table 2). In turn, the lowest rate constant was
measured for glucoside 11b with the 4’-methoxy substituent; it
amounts 0.12 h-1 at pH 7.4 and 0.23 h-1 at pH 5.2. It should be
emphasized that the mentioned substituents substantially differ in
their electronic properties related to the flavonol ring; the chloro
atom is weakly electron withdrawing whereas the methoxy group is
strongly electron donating. The calculated rate constants confirm
that the enzymatic hydrolyses studied run faster in the acidic
environment than in physiological pH. This statement is also
supported by the half-reaction times that are in the range of 1.1 h
(11e) ‒ 6.24 h (11b) in pH 7.4, and in the range of 0.15 h (11e) ‒ 4.62
h (11b) in pH 5.2 (Table 2).
12a
12b
12e
13a
13b
13e
Table 2 Kinetic parameters of the enzymatic hydrolysis
of glucosides 11a,b,d-f (c BSA 7.5 · 10-5)a
pH 7.4
pH 5.2
Probe
k (h-1)
τ1/2 (h)
1.9
6.24
2.7
1.1
3.9
k (h-1)
0.81
0.23
2.86
4.51
0.96
τ1/2 (h)
0.86
4.62
0.24
0.15
0.73
11a
11b
11d
11e
11f
0.38
0.11
0.29
0.63
0.18
a k – rate constant of enzymatic cleavage;
τ1/2 – time of half-reaction.
Enzymatic hydrolysis with β-N-acetylglucosaminidase and β-
glucuronidase
To check if the developed approach and flavonol-based indicators
can be applied to other glycosidases, flavonyl N-acetyl-β-D-
Figure 4. Changes in the fluorescence spectra during enzymatic hydrolysis of
probes 12a,b,e and 13a,b,e at pH 7.4. Insets: plots of the relative intensity of
the T* fluorescence (IiT*/ ImaxT*) versus time.
glucosaminosides (12a,b,e) and β-D-glucuronides (13a,b,e) were
synthesized and treated with β-N-acetylglucosaminidase and β-
glucuronidase in water solution at pH 7.4 and 5.2, respectively. As
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Journal Name
(Figure 6C). In this way an oxazoline intermediate isVfieowrmArteicdle, Othnleinne
DOI: 10.1039/D0OB01505A
subsequently cleaved by the deprotonated water molecule.
enzyme
enzyme
O
O
H
O
O
HO
HO
OR
O
O
+
ROH
A
OH
H
O
12a,b,e
13a,b,e
H
H
O
Figure 5. The plots of ln co/ct vs time for enzymatic hydrolysis of 12a,b,e and
13a,b,e at pH 7.4.
O
O
O
enzyme
enzyme
It should be added that substituent effects for 12a,b,e and 13a,b,e
(Table 3) are relatively small compared to substituent effects
measured for 11a,b,d-f (Table 2). Replacing of the OMe substituent
with the Cl substituent in glucosides (11b→11e) increases the rate
constant 5.7 times at pH 7.4 and 19.6 times at pH 5.2. Analogous
replacing in glucosaminosides (12b→12e) causes the rate constant
to increase 1.6 times at pH 7.4 and 2.1 times at pH 5.2 whereas in
glucuronides (13b→13e) the rate constant increases 3.8 times at pH
7.4 and 2.0 times at pH 5.2.
enzyme
enzyme
O
enzyme
O
H
O
H
H
O
O
O
O
HO
HO
HO
H
OR
OH
O
O
O
B
+
ROH
O
O
O
O
O
O
enzyme
enzyme
enzyme
enzyme
enzyme
enzyme
Table 3 Kinetic parameters of the enzymatic hydrolysis
O
H
O
O
H
H
O
O
O
O
of N-acetyl-β-D-glucosaminosides (12a,b,e
)
a
HO
HO
HO
H
and β-D-glucuronides (13a,b,e
)
OR
C
OH
O
O
O
+
ROH
C
pH 7.4
Probe
pH 5.2
O
k (h-1)
0.27
0.20
0.33
0.15
0.11
0.42
τ1/2 (h)
2.59
3.54
2.09
4.54
6.52
1.66
k (h-1)
τ1/2 (h)
0.55
0.79
0.38
0.18
0.34
0.17
O
HN
HN
O
O
HN
C
C
12a
12b
12e
13a
13b
13e
1.26
0.88
1.83
3.88
2.04
4.05
CH3
O
O
O
O
O
CH3
CH3
enzyme
enzyme
enzyme
Figure 6. Mechanism of inverting glycosidase (A), retaining glycosidase (B)
and β-N-acetylglucosaminidase (C) actions.
Being a single SN2 reaction or combination of two SN2 reactions,
enzymatic hydrolysis of the probes 11a-f, 12a,b,e and 13a,b,e depends
on the kind of leaving group, particularly when other conditions are
more or less the same. Generally, a good leaving group has to be
stable after diffusion from the reactant. In the case of nucleophilic
substitution (SN) the leaving group is a base, which can leave in the
anionic form (base) or in the neutral form (protonated base).
Importantly, the basicity of a series of bases and their protonated
forms are the same since they form the acid-base conjugated pairs.
Considering the basicity of 4a-f in either anionic or neutral form,
one has to take into account the effect of the 4’-substituent, which
differentiates these leaving groups. Except for the hydrogen (4a),
the electron donating groups, i.e. -OCH3 (4b) and -N(CH3)2 (4c) as
well as the electron withdrawing groups, i.e., -F (4d), -Cl (4e), -CF3
(4f) are among these substituents. Undoubtedly, if a more electron
withdrawing group is attached to the 4’ carbon atom of flavonol
(para position relative to the chromen-4-one ring), then the
negative charge of the flavonol anion is delocalized better, and thus
the leaving group is more stable. Therefore, according to Hammett
constants for a para substituted benzene ring (listed in the
brackets),59 the leaving ability order in the case of presented study
should look like this: CF3 (0.54) > Cl (0.227) > F (0.062) > H (0.00) >
OCH3 (-0.268) > N(CH3)2 (-0.83). With the exception of the CF3
group, the effect of other substituents on the rate of enzymatic
hydrolysis is rather compatible with the Hammett constants. For all
of the tested enzymes, the most sensitive probe is 4’-chloroflavonyl
glycoside (11e, 12e, 13e) whereas 4’-dimethylaminoflavonyl
a k – rate constant of enzymatic cleavage;
τ1/2 – time of half-reaction.
The context of enzymatic hydrolysis mechanism
To rationalize the observed series of reactivity of the studied probes
towards enzymatic hydrolysis, a discussion of the mechanism of
glycosidases action is provided. In terms of the
stereochemical outcome of glycosidic bond cleavage, glycosidases
are classified as either inverting or retaining enzymes.56,57 In the
case of inverting glycosidases enzymatic hydrolysis occurs via a
single-step SN2 mechanism in which the anomeric leaving group is
directly displaced by a water molecule (Figure 6A).58 Due to enzyme
participation, the attacking water molecule is deprotonated at the
same moment, which makes it a stronger nucleophile, whereas the
leaving group is protonated, which makes it easier to leave. In the
case of retaining glycosidases enzymatic hydrolysis is a combination
of two SN2 reactions (Figure 6B). In the first, carboxylate from the
enzyme substitutes the anomeric alkoxy group protonated by
enzyme to facilitate its leaving. In the second, water, deprotonated
at the same moment by enzyme, displaces the carboxylate ion.
Both SN2 reactions occur with the inversion of the anomeric carbon
configuration, therefore its final configuration is retained. β-N-
Acetylglucosaminidase catalyses hydrolysis via the retaining
mechanism, in which the carbonyl oxygen from the 2-acetamido
group of the sugar substrate is the first attacking nucleophile
6 | J. Name., 2012, 00, 1-3
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glucoside (11c) is not hydrolysed at all. Since the situation with 4’- on spectrometer Q-TOF UPLC/MS Agilent 6550 as VmiewonArotiicsleoOtonpliniec
DOI: 10.1039/D0OB01505A
substituted flavonol is much more complicated than with 4- masses. Thin-layer chromatography was conducted on aluminium
substituted benzene, we verified the charge distribution in the plates coated with E. Merck Kieselgel 60 F254 (0.2 mmol) using the
anionic forms of 4a-f with the electrostatistic potential (ESP) analysis following eluent systems: (v/v) A, 1:1 CHCl3 : n-hexane; B, 99:1
at the B3LYP/6-311++G** level of theory (Table S1). We assume CHCl3:isopropanol; C, 3:1 toluene : AcOEt; D, 5:1 toluene : AcOEt; E,
that the smaller the negative charge on the O3 oxygen atom in the 6:1 toluene : AcOEt; F, 4:1 toluene : AcOEt; G, 1:2 toluene : AcOEt;
anionic forms of 4a-4f, the better the anion’s negative charge is G, 1:5 toluene : AcOEt; K, 3:1 CHCl3 : MeOH; L, 1:3 CHCl3 : MeOH; M,
delocalized and the more stable is a leaving group. The order of 1:3 CHCl3 : MeOH + 5% Et3N. Detection was performed by heating
anions according to the increasing value of the negative charge on at ca. 250 oC in the stream of the hot air. Flash column
the O3 oxygen atom looks as follows: 4f (-0.5634) < 4e (-0.5838) < 4d chromatography was conducted on puriFlash 450 Interchim
(-0.5878) < 4b (-0.5948) < 4a (-0.6036) < 4c (-0.6116) and resembles instrument with MN-Kiesgel 60 (<0.8 mmol) using one of the above-
the order of the substituent Hammett’s constants. Again, the ESP mentioned eluent systems. Electronic absorption and emission
analysis indicated that 4’-trifluoromethylflavonol (4f) should be the were measured using Perkin Elmer Lambda 18 UV-Vis
best leaving group whereas our results show that the next in the spectrophotometer and Varian/Cary Eclipse spectrofluorimeter,
series 4’chloroflavonol (4e) is the fastest released from sugar. The equipped with solid and liquid sample-carriers.
worst leaving group according to the ESP analysis is 2’-Hydroxy-4-methoxychalcone and 4-methoxyflavanone (3b+3b’)
4’dimethylaminoflavonol (4c). This finding is consistent with results
2’-Hydroxyacetophenone (1) (0.6 mL, 5 mmol) and 4-
regarding the course of enzymatic hydrolysis as well as with the methoxybenzaldehyde (2b) (0.6 mL, 5 mmol) were added to the
order of the substituent Hammett’s constants. The observed suspension of KOH (1.4 g, 25 mmol) in methanol (15 mL) and stirred
discrepancy regarding 4f may indicate that in the case of enzymatic at rt. Progress of the reaction was monitored with TLC (solvent A).
hydrolysis kinetics, in addition to the leaving group stability, other After 48 h pH of the mixture was adjusted to 3 using 5% HCl.
factors also have to be taken into account e.g. matching the Addition of HCl caused formation of the precipitate, which was left
substrate (flavonyl glycoside) to the enzyme (glycosidase). One in supernatant for 3 days. After that time precipitate was filtered,
should also consider the possibility that since the enzymatic washed with mixture of methanol and water (1:1) and dried. Crude
hydrolysis requires the leaving group to be protonated, the product was purified by flash column chromatography (solvent B) to
strongest withdrawing group (CF3) lowers the basicity of the give about 2:1 mixture of 2’-hydroxy-4-methoxychalcone (3b) and 4-
flavonol oxygen atom enough that the protonation is more difficult, methoxyflavanone (3b’) as a yellow solid (0.702 g, 50%); 3b: Rf 0.37
1
which slows the reaction compared to the weaker withdrawing Cl (solvent A); H NMR (CDCl3): δ 12.93 (s, 1 H, OH), 7.92 (dd, 1 H, J
group.
7.93 Hz, 1.53 Hz, H6’), 7.91 (d, 1 H, J 15.26 Hz, Hβ), 7.63 (d, 2 H, J
Finally, it is important to recall our kinetic studies (Tables 2 and 8.54 Hz, H2, H6), 7.55 (d, 1 H, J 15.26 Hz, Hα), 7.49 (bt, 1 H, J 8.85
3), which indicate that substituent effects for N-acetyl-β-D-glucos- Hz, 7.02 Hz, H4’), 7.03 (d, 1 H, J 8.54 Hz, H3’), 6.94 (m, 3 H, H3, H5,
1
aminosides (12a,b,e) and β-D-glucuronides (13a,b,e) are relatively H5’), 3.87 (s, 3 H, OCH3); 3b’: Rf 0.26 (solvent A); H NMR (CDCl3): δ
small compared to substituent effects measured for β-D-glucosides 7.93 (dd, 1 H, J 7.93 Hz, 1.22 Hz, H6’), 7.50 (dt, 1 H, H4’), 7.41 (d, 2
(11a,b,d-f). This may suggest that in the case of β-N-acetylglucos- H, J 8.85 Hz, H2, H6), 7.03 (m, 2H, H3’, H5’), 6.95 (d, 2 H, J 8.85, H3,
aminidase and β-glucuronidase action the leaving group ability of H5), 5.43 (dd, 1 H, J 13.43 Hz, 2.45 Hz Hβ), 3.84 (s, 3 H, OCH3), 3.10
the flavonol is less important than in the case of β-glucosidase (dd, 1 H, J 16.79 Hz, 13.12 Hz, Hα), 2.86 (dd, 1 H, J 16.79 Hz, 2.75 Hz,
action.
Hα’).
2’-Hydroxy-4-methoxychalcone (3b)
2’-Hydroxyacetophenone (1) (0.6 mL,
Experimental
5
mmol) and 4-
General remarks:
methoxybenzaldehyde (2b) (0.6 mL, 5 mmol) were added to the
suspension of KOH (1.4 g, 25 mmol) in methanol (15 mL). Such the
mixture was divided in half and transferred into two microwave
reactor tubes. Every tube was heated in four 10 min cycles (reactor
power 100 W, tmax 80 °C). Then, content of the tubes was
transferred into beaker and pH of the mixture was adjusted to 3
with 5% HCl. Obtained precipitate was filtered off, washed with the
1:1 (v/v) mixture of methanol and water and dried. In this way 2’-
hydroxy-4-methoxychalcone (3b) was obtained (0.952 g, yellow
solid, 75%).
Solvents and chemical reagents, including 2’-hydroxyacetophenone
(1) 4-substituted benzaldehydes (2b-f), 2,3,4,6-tetra-O-acetyl-α-D-
glucopyranosyl bromide (5) and flavonol (4a), were purchased and
used without further purification. For enzymatic hydrolysis
commercially available (Sigma-Aldrich) BSA, β-glucosidase (from
almonds), β-N-acetylglucosaminidase (from Canavalia ensiformis)
and β-glucuronidase (from bovine liver) were used. The IR spectra
were recorded in KBr disks on spectrophotometer Bruker IFS 66.
1
The H and 13C NMR spectra were recorded on a Varian Merkury
500 MHz instrument (500.13/125.76 MHz), using standard
experimental conditions in CDCl3, DMSO-d6 or D2O with internal
Me4Si. Positive-ion mode MALDI-TOF mass spectra were obtained
using a Bruker Biflex III spectrometer with 2,5-dihydroxybenzoic
acid matrixes. High-resolution mass spectra (HRMS) were marked
4’-Methoxyflavonol (4b)
From 3b+3b’
The mixture of 2’-hydroxy-4-methoxychalcone (3b) and 4-
methoxyflavanone (3b’) (0.682 g, 2.69 mmol) was dissolved in 80%
aqueous methanol solution (54 mL) and NaOH (0.538 g, 13.45
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mmol) was added. Then, 30% H2O2 (1.37 mL, 13.45 mmol) was mixture (0.683 g) was dissolved in CHCl3 (50 mL),ViwewasAhrtiecdle Ownliitnhe
DOI: 10.1039/D0OB01505A
added dropwise to the mixture, which was stirred. Progress of the saturated aqueous Na2CO3 solution (3 x 20 mL), next with water (20
reaction was monitored with TLC (solvent A). Importantly, the mL) and dried over MgSO4. Dried extract was evaporated to give 4’-
product formation was accompanied by the disappearance of both trifluoromethylflavonol (4f) as a creamy solid (0.279 g, 18%); Rf 0.56
the spot corresponding to 3b and the spot corresponding to 3b’. (solvent A); 1H NMR (500 MHz, DMSO-d6): δ 10.07 (bs, 1 H, OH),
After 1 h some precipitate was formed and reaction was completed. 8.45 (d, 2 H, J 8.17 Hz, H2’, H6’), 8.14 (dd, 1 H, J 7.75 Hz, 1.72 Hz,
The mixture was acidified with 5% HCl to pH 3. Obtained precipitate H5), 7.95 (d, 2 H, J 8.60 Hz, H3’, H5’), 7.85 (ddd, 1 H, J 7.75 Hz, 8.17
was filtered off, washed with the 1:1 (v/v) mixture of methanol and Hz, 1.72 Hz H7), 7.80 (d, 1 H, J 8.17 Hz, H8), 7.50 (t, 1 H, J 7.74 Hz,
water and dried. In this way 4’-methoxyflavonol (4b) was obtained 7.31 Hz, H6); 13C NMR (125 MHz, DMSO-d6): δ 173.2 (C4), 154.6,
(0.386 g, creamy solid, 54%); Rf 0,15 (solvent A); 1H NMR (500 MHz, 143.3, 140.1, 135.3 (C2, C3, C9, C4’), 134.1 (C7), 128.2 (C2’, C6’),
DMSO-d6): δ 9.44 (s, 1 H, OH), 8.22 (d, 2 H, J 9.16 Hz, H2’, H6’), 8.12 125.4 (q, 3JC,F 3.81 Hz, C3’, C5’), 124.9 (C5), 124.7 (C6), 123.2, 121.3
(d, 1 H, J 7.02 Hz, H5), 7.80 (t, 1 H, J 8.54 Hz, 7.02 Hz, H7), 7.77 (d, 1 (C10, C1’), 118.5 (C8); MALDITOF-MS: m/z 307.2 (M+H)+.
H, J 8.24 Hz, H8), 7.47 (t, 1 H, J 7.02 Hz, 6.71 Hz, H6) 7.15 (d, 2 H, J Flavonyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosides (8a-f
)
8.85 Hz, H3’, H5’), 3.86 (s, 3 H, OCH3). These data are in accordance General procedure:
with the literature.60
From 3b
Water (5-10 mL) was added to the mixture of the respective
flavonol (4a-f) (1 mmol), K2CO3 (276.4 mg, 2 mmol) and TBAB (322,4
This synthesis was carried out analogously to that above mg, 1 mmol). Such the mixture was stirred and 2,3,4,6-tetra-O-
described. The difference is that 2’-hydroxy-4-methoxychalcone (3b) acetyl-α-D-glucopyranosyl bromide (5) (2 mmol), dissolved in DCM
(0.947 g, 3.74 mmol) was used as a substrate. Additionally, final (5-10 mL), was added in four portion during 2 h. With every portion
product was washed solely with water (instead of the 1:1 (v/v) of 5, the additional portion of K2CO3 (138.2 mg, 1 mmol) was added.
mixture of methanol and water). In this way 4’-methoxyflavonol The mixture was stirred at rt and monitored by TLC (eluent C).
(4b) was obtained (0.635 g, creamy solid, 63%).
4’-Dimethylaminoflavonol (4c)
Synthesized according to procedure previously reported.54
4’-Fluoroflavonol (4d)
Synthesized according to procedure previously reported.44
4’-Chloroflavonol (4e)
When the reaction was ended the mixture was diluted with DCM
(90 mL) and extracted with water (5 x 20 mL). The organic layer was
dried over MgSO4, filtered and concentrated in vacuum. The residue
was purified using column flash chromatography (eluent D or E).
Flavonyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (8a)
Synthesized according to procedures previously reported.54
2’-Hydroxyacetophenone (1) (0.6 mL,
5
mmol) and 4- 4’-Methoxyflavonyl
2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside
chlorobenzaldehyde (2e) (0.703 g, 5 mmol) were added to the (8b)
suspension of KOH (1.4 g, 25 mmol) in methanol (15 mL) and stirred
Reaction of 4’-methoxyflavonol 4b (169.1 mg; 0.63 mmol) with
at rt. Progress of the reaction was monitored with TLC (solvent A). bromide 5 gave 8b (350 mg, 93%): Rf 0.45 (eluent F); 1H NMR
After 24 h 30% H2O2 (2.5 mL, 25 mmol) was added dropwise to the (CDCl3): δ 8.16 (dd, 1 H, J 7.93 Hz, 1.22 Hz, H5), 8.00 (d, 2 H, J 8.85
mixture. After 1 h some precipitate was formed and reaction was Hz, H2’, H6’), 7,61 (td, 1 H, J 8.54 Hz, 1.53 Hz, H7), 7.46 (d, 1 H, J
complete. The mixture was acidified with 5% HCl to pH 3. Obtained 8.54 Hz, H8), 7.34 (t, 1 H, J 8.85 Hz, 7.93 Hz, H6), 6.94 (d, 2 H, J 9.16
precipitate was filtered off and dissolved in DCM (40 mL). DCM Hz, H3’, H5’), 5.66 (d, 1 H, J 7.63 Hz, H1”), 5.22 (t, 1 H, J 9.46 Hz,
solution was washed with saturated aqueous Na2CO3 (3 x 20 mL) 9.16 Hz, H3”), 5.16 (dd, 1 H, J 9.76 Hz, 7.63 Hz, H2”), 5.02 (t, 1 H, J
and with water (2 x 20 mL). DCM solution was dried with MgSO4. 9.77 Hz, 9.16 Hz, H4”), 3.93 (dd, 1 H, J 12.21 Hz, 4.27 Hz, H6a”), 3.87
Dried extract was evaporated to give 4’-chloroflavonol (4e) as (dd, 1 H, J 12.21 Hz, 2.44 Hz, H6b”), 3.83 (s, 3 H, OCH3), 3.55 (ddd, 1
yellow needle-like crystals (0.379 g, 28%); Rf 0,56 (solvent A); 1H H, J 10.07 Hz, 4.27 Hz, 2.44 Hz, H5”), 2.05, 1.95, 1.93, 1.80 (4 x s, 12
NMR (500 MHz, DMSO-d6): δ 9.85 (s, 1 H, OH), 8.27 (d, 2 H, J 8.54 H, 4 x OCOCH3); 13C NMR (125 MHz, CDCl3): δ 173.8 (C4), 170.0,
Hz, H2’, H6’), 8.13 (d, 1 H, J 7.63 Hz, H5), 7.83 (t, 1 H, J 7.32 Hz, 7.02 169.9 (4 x OCOCH3), 161.6 (C4’), 157.3, 155.2 (C2, C9), 135.7 (C3),
Hz, H7), 7.78 (d, 1 H, J 8.54 Hz, H8), 7.66 (d, 2 H, J 8.54 Hz, H3’, H5’), 133.5 (C7), 130.9 (C2’, C6’), 128.2 (C1’), 125.6 (C5), 124.8 (C6), 123.0
7.49 (t, 1 H, J 7.32 Hz, H6). These data are in accordance with the (C10), 118. (C8), 113.6 (C3’, C5’), 98.8 (C1”), 71.6 (C5”), 69.8 (C2”),
literature.60
4’-Trifluoromethylflavonol (4f)
2’-Hydroxyacetophenone (1) (0.6 mL,
69.2 (C4”), 67.9 (C3”), 61.5 (C6”), 55.4 (OCH3), 20.9, 20.7, 20.6, 20.5
(4 x OCOCH3).
mmol) and 4- 4’-N,N-Dimethylaminoflavonyl 2,3,4,6-tetra-O-acetyl-β-D-gluco-
5
trifluorometylbenzaldehyde (2f) (0.87 g, 5 mmol) were added to the pyranoside (8c)
suspension of KOH (1.4 g, 25 mmol) in methanol (15 mL). Such the
Synthesized according to procedures previously reported.54
mixture was stirred at rt and monitored with TLC (solvent A). After 4’-Fluoroflavonyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (8d)
24 h 30% H2O2 (2.5 mL, 25 mmol) was added and stirring was
continued next 1 h. Then the reaction mixture was acidified with 5% 4’-Chloroflavonyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (8e)
HCl to pH 3, which caused formation of the precipitation, which was
Synthesized according to procedures previously reported.54
Synthesized according to procedures previously reported.44
filtered off, dried and identified (NMR) as a 1:1 mixture of 4’- 4’-Trifluoromethylflavonyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyrano-
trifluoromethylflavonol (4f) and 4-trifluoromethylbenzoic acid. This side (8f)
8 | J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
Reaction of 4’-trifluoromethylflavonol 4f (200 mg; 0.65 mmol) 1 H, J 8.24, Hz, H8), 7.45 (t, 1 H, J 8.24 Hz, 7.45 Hz, H6V),ie7w.3Ar3tic(lbe ,O1nliHne,
1
DOI: 10.1039/D0OB01505A
with bromide 5 gave 8f (157.1 mg, 38%): Rf 0.44 (eluent F); H NMR NH), 6.99 (d, 2 H, J 8.85 Hz, H3’, H5’), 5.31 (d, 1 H, J 8.85 Hz, H1”),
(CDCl3): δ 8.25 (dd, 1 H, J 7.93, 1.53, H5), 8.20 (d, 2 H, J 8.24, H2’, 5.16 (t, 1 H, J 9.77 Hz, 9.46 Hz, H4”), 5.11 (t, 1 H, J 10.07 Hz, 9.46 Hz,
H6’), 7.78 (d, 2 H, J 8.54, H3’, H5’), 7.74 (ddd, 1 H, J 8.24, 7.32, 1.83, H3”), 4.41 (q, 1 H, J 8.85 Hz, 7.63 Hz, H2”), 4.04 (dd, 1 H, J 12.21 Hz,
H7), 7.58 (d, 1 H, J 8.24, H8), 7.45 (t, 1 H, J 7.93, 7.32, H6), 5.75 (d, 1 4.58 Hz, H6a”), 3.95 (dd, 1 H, J 12.21 Hz, 2.44 Hz, H6b”), 3.90 (s, 3 H,
H, J 7.93, 1H”), 5.29 (t, 1 H, J 9.46, H3”), 5.19 (dd, 1 H, J 9.77, 7.93 OCH3), 3.57 (ddd, 1 H, J 9.77 Hz, 4.58 Hz, 2.75 Hz, H5”), 2.06, 2.05,
H2”), 5.07 (t, 1 H, J 9.46, 9.77, H4”), 4.02 (dd, 1 H, J 12.21, 3.97 2.00, 1.93 (4 x s, 12 H, 4 x COCH3); 13C NMR (125 MHz, CDCl3): δ
H6a”), 3.91 (dd, 1 H, J 12.21, 2.44, H6b”), 3.62 (dt, 1 H, J 10.07, 3.97, 174.4 (C4), 171.4, 170.8, 170.4, 169.3 (4 x COCH3), 162.0 (C4’),
2.75, H5”), 2.14, 2.02, 2.00, 1.85 (4 x s, 12 H, 4 x OCOCH3); 13C NMR 157.8 (C2), 155.3 (C9), 137.0 (C3), 133.9 (C7), 131.1 (C2’, C6’), 125.6
(CDCl3): δ 174.0 (C4), 170.3, 170.0, 169.9, 169.4 (4 x COCH3), 155.5, (C5), 125.1 (C6), 123.8 (C1’), 122.4 (C10), 118.1 (C8), 113.7 (C3’,
155.3 (C2, C9), 136.8 (C3), 134.1 (C7), 134.1 (C4’), 129.5 (C2’, C6’), C5’), 101.0 (C1”), 73.9 (C3”), 72.3 (C5”), 68.3 (C4”), 61.8 (C6”), 55.4
3
125.8 (C5), 125.2 (C6), 125.1 (q, JC,F 3.63, C3’, C5’), 124.0 (C10), (OCH3), 54.4 (C2”), 23.4 (NHCOCH3), 20.8, 20.6, 20.5 (3 x OCOCH3);
118.2 (C8), 98.9 (C1”), 72.6 (C3”), 71.8 (C5”), 71.5 (C2”) 68.1 (C4”), HRMS (ESI): m/z calcd for C30H31NO12 597.1846, found 597.1841
61.1 (C6”), 20.8, 20.6, 20.5, 20.3 (4 x OCOCH3); MALDITOF-MS: m/z (M)+, 598.1915 (M+H)+, 620.1729 (M+Na)+.
637 (M+H)+; 659 (M+Na)+; 675 (M+K)+.
Flavonyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyrano- copyranoside (9e)
4’-Chloroflavonyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glu-
sides (9a,b,e
General procedure:
)
Reaction of flavonol 4e (215 mg; 0.9 mmol) with chloride 6 gave
9e (430 mg, 80%): Rf 0.42 (eluent G); H NMR (CDCl3): δ 8.23 (dd, 1
1
Water (5-10 mL) was added to the mixture of the respective H, J 7.93 Hz, 1.53 Hz, H5), 8.11 (d, 2 H, J 8.85 Hz, H2’, H6’), 7.75
flavonol (4a,b,e) (1 mmol), K2CO3 (276.4 mg, 2 mmol) and TBAB (ddd, 1 H, J 8.54 Hz, 7.78 Hz, 1.52 Hz, H7), 7.58 (d, 1 H, J 8.54 Hz,
(322,4 mg, 1 mmol). Such the mixture was stirred and 2-acetamido- H8), 7.47 (t, J 7.93 Hz, H6) 7.46 (d, 2 H, J 8.54 Hz, H3’, H5’), 7.11 (d, 1
3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride (6) (2 H, J 7.32 Hz, NH), 5.31 (d, 1 H, J 8.85 Hz, H1”), 5.16 (t, 1 H, J 9.76,
mmol), dissolved in DCM (5-10 mL), was added in four portion 9.46, H4”), 5.10 (t, 1 H, J 10.07 Hz, 9.46 Hz, H3”), 4.39 (q, 1 H, J 9.16
during 2 h. With every portion of 6, the additional portion of K2CO3 Hz, 8.54 Hz, H2”), 4.02 (dd, 1 H, J 12.20, 4.28, H6a”), 3.96 (dd, 1 H, J
(138.2 mg, 1 mmol) was added. The mixture was stirred at rt and 12.21 Hz, 2.75 Hz, H6b”), 3.55 (ddd, 1 H, J 9.46 Hz, 4.27 Hz, 2.75 Hz,
monitored by TLC (solvent G). When the reaction was ended the H5”), 2.06, 2.05, 2.00, 1.96 (4 x s, 12 H, 4 x COCH3); 13C NMR (125
mixture was diluted with DCM (90 mL) and extracted with water (5 MHz, CDCl3): δ 174.5 (C4), 171.3, 170.8, 170.4, 169.2 (4 x COCH3),
x 20 mL). The organic layer was dried over MgSO4, filtered and 156.6 (C2), 155.4 (C9), 137.6. 137.4 (C3, C4’), 134.3 (C7), 130.6
concentrated in vacuum. The residue was purified using column (C2’,C6’), 128.5 (C3’, C5’), 125.6 (C5), 125.4 (C6), 123.8 (C1’), 118.2
flash chromatography (eluent H).
(C8), 100.9 (C1”), 73.7 (C3”), 72.3 (C5”), 68.0 (C4”), 61.5 (C6”), 54.3
Flavonyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyrano- (C2”), 23.4 (NHCOCH3), 20.7, 20.6, 20.5 (3 x OCOCH3); HRMS (ESI):
side (9a)
m/z calcd for C29H28NO11Cl 601.1351, found 601.1351 (M)+,
Reaction of flavonol 4a (300 mg; 1.26 mmol) with chloride 6 602.1423 (M+H)+, 624.1242 (M+Na)+.
gave 9a (168 mg, 24%): Rf 0.4 (eluent G); 1H NMR (500 MHz, CDCl3):
δ 8.24 (d, 1 H, J 7.93 Hz, H5), 8.14 (d, 2 H, J 7.02 Hz, H2’, H6’), 7.75
(t, 1 H, J 8.24 Hz, 7.32 Hz, H7), 7.60 (d, 1 H, J 8.24 Hz, H8), 7.49 (m, 4
H, H6, H3’, H4’, H5’), 7.23 (b, 1 H, NH), 5.31 (d, 1 H, J 8.54 Hz, H1”),
5.15 (t, 1 H, J 9.46 Hz, H4”), 5.10 (t, 1 H, J 9.77 Hz, H3”), 4.39 (q, 1 H,
J 8.24 Hz, 7.63 Hz, 7.32 Hz, H2”), 4.01 (dd, 1 H, J 12.21 Hz, 4.58 Hz,
H6a”), 3.95 (dd, 1 H, J 12.21 Hz, 2.44 Hz, H6b”), 3.57 (m, 1 H, H5”),
2.05, 2.00, 1.94 (3 x s, 12 H, 4 x COCH3); 13C NMR (125 MHz, CDCl3):
δ 174.7 (C4), 171.3, 170.8, 170.4, 169.3 (4 x COCH3), 157.9 (C2),
155.5 (C9), 137.6 (C3), 134.2 (C7), 134.2 (C4’), 129.2 (C2’, C6’),
128.9 (C3’, C5’), 125.7 (C5), 125.2 (C6), 123.8 (C1’), 118.3 (C8), 100.9
(C1”), 73.9 (C3”), 72.3 (C5”), 68.2 (C4”), 61.8 (C6”), 54.4 (C2”), 23.4
(NHCOCH3), 20.8, 20.61, 20.59 (3 x OCOCH3); HRMS (ESI): m/z calcd
for C29H29NO11 567.1741, found 567.1739 (M)+, 568.1804 (M+H)+,
590.1635 (M+Na)+.
Methyl (flavonyl 2,3,4-tri-O-acetyl--D-glucopyranosid)uronates
(10a,b,e
)
General procedure:
Water (8 mL) was added to the mixture of the respective flavonol
(4a-f) (1 mmol), K2CO3 (276.4 mg, 2 mmol) and TBAB (322,4 mg, 1
mmol). Such the mixture was stirred and methyl 2,3,4-tri-O-acetyl-
α-D-glucopyranosyluronate bromide (7) (2 mmol), dissolved in DCM
(8 mL), was added in four portions during 2 h. With every portion of
7, the additional portion of K2CO3 (138.2 mg, 1 mmol) was added.
The mixture was stirred at rt and monitored by TLC (eluent F).
When the reaction was ended the mixture was diluted with DCM
(90 mL) and extracted with water (5 x 20 mL). The organic layer was
dried over MgSO4, filtered and concentrated in vacuum. The residue
was purified using column flash chromatography (eluent D or E).
Methyl (flavonyl 2,3,4-tri-O-acetyl--D-glucopyranosid)uronate
(10a)
4’-Methoxyflavonyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranoside (9b)
Reaction of flavonol 4b (300 mg; 1.11 mmol) with chloride 6
gave 9b (204 mg, 33%): Rf 0.42 (eluent G); 1H NMR (500 MHz,
CDCl3): δ 8.22 (dd, 1 H, J 7.93 Hz, 1.53 Hz, H5), 8.17 (d, 2 H, J 8.85
Hz, H2’, H6’), 7.72 (ddd, 1 H, J 8.24 Hz, 7.32 Hz, 1.83 Hz, H7), 7.57 (d,
Reaction of flavonol 4a (236.2 mg; 0.99 mmol) with bromide 7
gave 10a (410 mg, 75%): Rf 0.45 (eluent F); 1H NMR (500 MHz,
CDCl3): δ 8.24 (dd, 1 H, J 7.93 Hz, 1.22 Hz, H5), 8.08 (2 x d, 2 H, J
7.32, H2’, H6’), 7.70 (ddd, 1 H, J 8.55 Hz, 7.02 Hz, 1.83 Hz, H7), 7.54
(m, 4 H, H8, H3’, H4’, H5’, H4’), 7.42 (t, 1 H, J 7.93 Hz, 7.02 Hz, H6),
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J. Name., 2013, 00, 1-3 | 9
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Organic & Biomolecular Chemistry
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ARTICLE
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5.80 (d, 1 H, J 7.93 Hz, H1”), 5.35 (t, 1 H, J 9.46 Hz, H3”), 5.20 (dd, 1 washed by MeOH (2 mL). The solution was evaporated to dryness
View Article Online
DOI: 10.1039/D0OB01505A
H, J 9.46 Hz, 7.93 Hz, H2”), 5.19 (t, 1 H, J 9.77 Hz, H4”), 3.98 (d, 1 H, and diethyl ether (2 mL) was added to cause product precipitation.
J 9.77, H5”), 3.58 (s, 3 H, COOCH3), 2.09, 2.02, 2.01 (3 x OCOCH3); Flavonyl β-D-glucopyranoside (11a)
13C NMR (125 MHz, CDCl3): δ 173.9 (C4), 169.9, 169.8, 169.5 (3 x
Synthesized according to procedures previously reported.54
OCOCH3), 166.8 (COOCH3), 157.5 (C2), 155.3 (C9), 136.2 (C3), 133.7 4’-Methoxyflavonyl β-D-glucopyranoside (11b)
(C7), 130.9, 130.5 (C1’, C4’), 129.2 (C2’, C6’), 128.3 (C3’, C5’), 125.7
O-Deacetylation of 8b (300 mg; 0.5 mmol) gave 11b (82.6 mg,
(C5), 125.00 (C6), 124.11 (C10), 118.1 (C8), 98.7 (C1”), 72.4 (C5”), 38%): Rf 0.8 (eluent K); 1H NMR (500 MHz, DMSO-d6): δ 8.20 (d, 2 H,
72.1 (C3”), 71.4 (C2”), 69.4 (C4”), 52.6 (COOCH3), 20.8, 20.6, 20.5 (3 J 9.16 Hz, H2’, H6’ ), 8.11 (dd, 1 H, J 7.93 Hz, 1.53 Hz, H5), 7.83
x OCOCH3); HRMS (ESI): m/z calcd for C28H26O12 554.1424, found (ddd, 1 H, J 7.02 Hz, 1.53 Hz, H7), 7.76 (d, 1 H, d, J 7.93 Hz, H8 ), 7.51
554.1419 (M)+, 555.1493 (M+H)+, 577.1312 (M+Na)+.
Methyl (4’-methoxyflavonyl 2,3,4-tri-O-acetyl--D-glucopyrano-
sid)uronate (10b)
(t 1 H, J 7.93 Hz, 7.02 Hz, H6), 7.11 (d, 2 H, J 8.85 Hz, H3’, H5’), 5.58
(d, 1 H, J 7.63 Hz, H1”), 5.42 (d, 1 H, J 4.58 Hz, 2”-OH), 5.08 (d, 1 H, J
4.88 Hz, 3”-OH), 4.96 (d, 1 H, J 5.19 Hz, 4”-OH), 4.26 (t, 1 H, J 5.80
Hz, 5.49 Hz, 6”-OH), 3.87 (s, 3 H, OCH3), 3.56 (dd, 1 H, J 11.90 Hz,
5.80 Hz, H6a”), 3.35 (dd, 1 H, J 11.60 Hz, 5.80 Hz, H6b”), 3.24 (m, 1 H,
H3”), 3.21 (m, 1 H, J 7.63 Hz, 4.58 Hz, H2”), 3.09 (m, 2 H, H4”, H5”);
13C NMR (125 MHz, DMSO-d6): δ 173.9 (C4), 161.6 (C4’), 156.1 (C2),
155.1 (C9), 136.2 (C3), 134.5 (C7), 131.3 (C2’, C6’), 125.5, 125.4 (C5,
C6), 123.8 (C10), 118.8 (C8), 114.2 (C3’, C5’), 101.2 (C1”), 77.9 (C5”),
76.9 (C3”) ,74.7 (C2”), 70.3 (C4”), 61.3 (C6”), 55.9 (OCH3); HRMS
(ESI): m/z calcd for C22H22O9 430.1258, found 430.1259 (M)+,
431.1332 (M+H)+, 453.1153 (M+Na)+.
4’-N,N-Dimethylaminoflavonyl β-D-glucopyranoside (11c)
Synthesized according to procedures previously reported.54
4’-Fluoroflavonyl β-D-glucopyranoside (11d)
Synthesized according to procedures previously reported.44
4’-Chloroflavonyl β-D-glucopyranoside (11e)
Reaction of flavonol 4b (81 mg; 0.3 mmol) with bromide 7 gave
1
10b (104 mg, 59%): Rf 0.49 (eluent F); H NMR (500 MHz, CDCl3): δ
8.22 (dd, 1 H, J 7.93 Hz, 1.53 Hz, H5), 8.11 (d, 2 H, J 9.16 Hz, H2’,
H6’), 7.68 (ddd, 1 H, J 8.85 Hz, 7.32 Hz, 1.83 Hz, H7), 7.52 (d, 1 H, J
8.85 Hz, H8), 7.41 (t, 1 H, J 8.85 Hz, 7.93 Hz, H6), 7.03 (d, 2 H, J 9.16
Hz, H3’, H5’), 5.83 (d, 1 H, J 7.93 Hz, H1”), 5.36 (t, 1 H, J 9.77 Hz,
9.46 Hz, H3”), 5.24 (dd, 1 H, J 9.46 Hz, 7.93 Hz, H2”), 5.21 (t, 1 H, J
9.77 Hz, 9.46 Hz, H4”), 3.98 (d, 1 H, J 10.07 Hz, H5”), 3.91 (s, 3 H,
COOCH3), 3.58 (s, 3 H, OCH3), 2.11, 2.03, 2.01 (3 x OCOCH3); 13C
NMR (125 MHz, CDCl3): δ 173.7 (C4), 169.9, 169.6, 169.3 (3 x
OCOCH3), 166.7 (COOCH3)), 161.7 (C4’), 157.4 (C2, C9), 135.6 (C3),
133.5 (C7), 131.0 (C2’, C6’), 125.7 (C5), 124.8 (C6), 122.8 (C10),
118.0 (C8), 113.7 (C3’, C5’), 98.7 (C1”), 72.4 (C5”), 72.1 (C3”), 71.4
(C2”), 69.5 (C4”), 55.4 (OCH3), 52.5 (COOCH3), 20.8, 20.6, 20.5 (3 x
OCOCH3); HRMS (ESI): m/z calcd for C29H28O13 584.1530, found
584.1523 (M)+, 585.1594 (M+H)+, 607.1415 (M+Na)+.
Synthesized according to procedures previously reported.54
4’-Trifluoromethylflavonyl β-D-glucopyranoside (11f)
O-Deacetylation of 8f (100.3 mg; 0.21 mmol) gave 9f (63 mg,
85%): Rf 0.83 (eluent K); H NMR (500 MHz, DMSO-d6): δ 8.39 (d, 2
Methyl (4’-chloroflavonyl 2,3,4-tri-O-acetyl--D-glucopyrano-
sid)uronate (10e)
1
H, J 8.24 Hz, H2’, H6’), 8.14 (dd, 1 H, J 7.93 Hz, 1.53 Hz, H5), 7.93 (d,
2 H, J 8.24 Hz, H3’, H5’), 7.87 (ddd, 1 H, J 8.54 Hz, 7.02 Hz, 1.53 Hz,
H7), 7.87 (d, 1 H, J 7.93 Hz, H8), 7.55 (t, 1 H, J 8.24 Hz, 7.02 Hz, H6),
5.59 (d, 1 H, J 7.93 Hz, H1”), 5.38 (b, 1 H, 2”-OH), 5.08 (b, 1 H, 4”-
OH), 4.98 (b, 1 H, 3”-OH), 4.33 (b, 1 H, 6”-OH), 3.57 (d, 1 H, J 10.68
Hz, H6a”), 3.36 (m, 1 H, H6b”), 3.25 (m, 1 H, H3”), 3.16 (m, 1 H, H2”),
3.11 (m, 2 H, H4”, H5”); 13C NMR (125 MHz, DMSO-d6): δ 174.2 (C4),
155.3 (C2), 154.7 (C9), 137.4, 135.2 (C3, C4’), 134.9 (C7), 130.4 (C2’,
Reaction of flavonol 4e (301.4 mg; 1.11 mmol) with bromide 7
gave 10e (250.9 mg, 38%): Rf 0.56 (eluent F); 1H NMR (CDCl3): δ 8.23
(d, 1 H, J 7.93 Hz, 1.53 Hz, H5), 8.06 (d, 2 H, J 8.85 Hz, H2’, H6’), 7.71
(ddd, 1 H, J 8.54 Hz, 7.02 Hz, 1.53 Hz, H7), 7.53 (d, 1 H, J 8.24 Hz,
H8), 7.51 (d, 2 H, J 8.85 Hz, H3’, H5’), 7.43 (t, 1 H, J 7.94 Hz, 7.33 Hz,
H6), 5.78 (d, 1 H, J 7.93 Hz, H1”), 5.36 (t, 1 H, J 9.46 Hz, H3”), 5.21
(dd, 1 H, J 9.15 Hz, 7.94 Hz, H2”), 5.17 (t, 1 H, J 9.77 Hz, 9.46 Hz,
H4”), 3.97 (d, 1 H, J 10.08 Hz, H5”), 3.59 (3H, s, COOCH3), 2.13, 2.04,
2.01 (3 x OCOCH3); 13C NMR (125 MHz, CDCl3): δ 173.8 (C4), 169.9,
169.8, 169.5 (3 x OCOCH3), 166.7 (COOCH3), 156.4 (C2), 155.2 (C9),
137.1, 136.3 (C3, C4’), 133.9 (C7), 130.6 (C2’, C6’), 128.9 (C1’), 128.6
(C3’, C5’), 125.7 (C5), 125.1 (C6), 124.1 (C10), 118.1 (C8), 98.8 (C1”),
72.4 (C5”), 71.8 (C3”), 71.3 (C2”), 69.4 (C4”), 52.7 (COOCH3), 20.8,
20.6, 20.5 (3 x OCOCH3); HRMS (ESI): m/z calcd for C28H25O12Cl
588.1035, found 588.1040 (M)+, 589.1113 (M+H)+, 611.0930
(M+Na)+.
3
C6’), 125.8 (C6), 125.6 (C5), 125.5 (q, JC,F 3.63 Hz, C3’, C5’), 123.3
(C10), 119.0 (C8), 101.2 (C1”), 78.0 (C5”), 76.9 (C3”), 74.5 (C2”), 70.2
(C4”), 61.3 (C6”); HRMS (ESI): m/z calcd for C22H19O8F3 468.1032,
found 468.1028 (M)+, 469.1102 (M+H)+, 491.0917 (M+Na)+.
Flavonyl 2-acetamido-2-deoxy-β-D-glucopyranoside (12a)
O-Deacetylation of 9a (120.2 mg; 0.22 mmol) gave 12a (97 mg,
1
90%): Rf 0.82 (eluent L); H NMR (500 MHz, DMSO-d6): δ 8.25 (d, 2
H, J 7.93 Hz, H2’, H6’), 8.11 (d, 1 H, J 7.93 Hz, H5), 7.98 (d, 1 H, J 9.16
Hz, NH), 7.84 (t, 1 H, J 7.93 Hz, 7.32 Hz, H7), 7.76 (d, 1 H, J 8.54, H8),
7.55 (m, 3 H, H3’, H4’, H5’), 7.51 (t, 1 H, t, J 7.63 Hz, 7.32 Hz, H6),
5.74 (d, 1 H, J 8.54 Hz, H1”), 5.02 (d, 1 H, J 4.88 Hz, 4”-OH), 5.00 (d,
1 H, J 5.80 Hz, 3”-OH), 4.25 (t, 1 H, J 5.80 Hz, 5.49 Hz, 6”-OH), 3.72
(q, 1 H, J 9.46, 8.85 Hz, H2”), 3.55 (dd, 1 H, J 11.29 Hz, 5.49 Hz,
H6a”), 3.39 (m, 1 H, H3”), 3.31 (m, 1 H, H6b”), 3.08 (m, 2 H, H4”,
H5”), 1.84 (s, 3 H, NHCOCH3); 13C NMR (125 MHz, DMSO-d6): δ
174.0 (C4), 169.8 (NHCOCH3), 155.1, 155.3 (C2, C9), 136.9, 135.9
Flavonyl β-D-glucopyranosides (11a-f) and 2-acetamido-2-deoxy-β-
D-glucopyranosides (12a,b,e
)
General procedure:
Respective acetate (8a-f or 9a,b,e) (1 mmol) was dissolved in MeOH
(10 mL) and 0.1 N MeONa in MeOH (2.32 mL, 0.232 mmol) was
added. The mixture was stirred for 1 h at rt. The reaction was
followed by TLC using eluent K or L. The resulting precipitate was
filtered off and filtrate was neutralized using Dowex (H+) and
10 | J. Name., 2012, 00, 1-3
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(C3, C4’), 134.6 (C7), 131.4 (C2’, C6’), 129.9 (C1’), 128,8 (C3’, C5’), 7.86 (ddd, 1 H, J 8.54 Hz, 7.02 Hz, 1.53 Hz, H7), 7.77 V(idew, 1ArHtic,leJO8n.l5in4e
DOI: 10.1039/D0OB01505A
125.6, 125.4 (C5, C6), 123.8 (C10), 118.9 (C8), 99.3 (C1”), 78.0 (C5”), Hz, H8), 7.54 (m, 4 H, H6, H3’, H4’,H5’), 5.60 (d, 1 H, J 7.63 Hz, H1”),
74.6 (C3”), 70.9 (C4”), 61.2 (C6”), 56.3 (C2”), 23.6 (NHCOCH3); HRMS 3.23 (t, 1 H, J 8.54 Hz, H3”), 3.19 (d, 1 H, J 9.46 Hz, H5”), 3.16 (dd, 1
(ESI): m/z calcd for C23H23NO8 441.1424, found 441.1421 (M)+, H, J 10.07 Hz, 7.93 Hz, H2”), 3.15 (t, 1 H, J 9.16 Hz, H4”); 13C NMR
442.1494 (M+H)+, 464.1314 (M+Na)+.
(125 MHz, DMSO-d6): δ 174.2 (C4), 171.8 (COOH), 156.2 (C2), 155.2
4’-Methoxyflavonyl
(12b)
2-acetamido-2-deoxy-β-D-glucopyranoside (C9), 137.0 (C3), 134.7 (C7), 131.1 (C4’), 129.8 (C2’, C6’), 128.7 (C3’,
C5’), 125.7, 125.5 (C5, C6), 123.8 (C10), 118.9 (C8), 100.9 (C1”), 77.1
O-Deacetylation of 9b (141.1 mg; 0.24 mmol) led to 12b (72 mg, (C3”), 74.6, 74.5 (C4”, C5”), 72.6 (C2”); HRMS (ESI): m/z calcd for
1
89%): Rf 0.82 (eluent L); H NMR (500 MHz, DMSO-d6): δ 8.27 (d, 2 C21H18O9 414.0951, found 414.0955 (M)+, 415.1029 (M+H)+,
H, J 8.85 Hz, H2’, H6’), 8.08 (d, 1 H, J 7.93 Hz, 1.53 Hz, H5), 8.01 (d, 1 437.0844 (M+Na)+.
H, J 8.85 Hz, NH), 7.82 (ddd, 1 H, J 8.24 Hz, 7.02 Hz, 1.53 Hz, H7),
7.75 (d, 1 H, J 8.24 Hz, H8), 7.49 (t, 1 H, J 7.93 Hz, 7.63 Hz, H6), 7.08
(d, 2 H, J 9.16 Hz, H3’, H5’), 5.75 (d, 1 H, J 8.54 Hz, H1”), 5.03 (d, 1 H,
J 4.27 Hz, 4”-OH), 5.02 (d, 1 H, J 5.49 Hz, 3”-OH), 4.27 (t, 1 H, J 5.49
Hz, 6”-OH), 3.87 (s, 3 H, OCH3), 3.75 (q, 1 H, J 8.85 Hz, 10.07 Hz,
H2”), 3.54 (dd, 1 H, J 11.90 Hz, 5.19 Hz, H6a”), 3.35 (m, 1 H, H3”),
3.31 (m, 1 H, H6b”), 3.08 (m, 2 H, H4”, H5”), 1.85 (s, 3 H, NHCOCH3);
); 13C NMR (125 MHz, DMSO-d6): δ 173.8 (C4), 169.8 (NHCOCH3),
161.57 (C4’) 155.4 (C2), 155.0 (C9), 136.1 (C3), 134.3 (C7), 131.4
(C2’, C6’), 125.4, 125.3 (C5, C6), 123.7, 123.3 (C10, C1’), 118.7 (C8),
114.2 (C3’, C5’), 99.2 (C1”), 77.9 (C5”), 74.7 (C3”), 70.9 (C4”), 61.2
(C6”), 56.3 (C2”), 55.8 (OCH3), 23.6 (NHCOCH3); HRMS (ESI): m/z
calcd for C24H25NO9 471.1529, found 471.1521 (M)+, 472.1594
(M+H)+, 494.1411 (M+Na)+.
4’-Methoxyflavonyl -D-glucopyranosiduronic acid (13b)
O-Deacetylation of 10b (60 mg; 0.1 mmol) yielded 13b (46 mg,
1
91%): Rf 0.65 (eluent M); H NMR (500 MHz, D2O): δ 7.73 (d, 2 H, J
8.85 Hz, H2’, H6’), 7.71 (d, 1 H, J 8.55 Hz, H5), 7.58 (t, 1 H, t, J 7.63
Hz, H7), 7.28 (d, 1 H, J 8.24 Hz, H8), 7.26 (t, 1 H, 7.63 Hz, H6), 6.65
(d, 2 H, J 8.85 Hz, H3’, H5’), 4.91 (d, 1 H, J 7.63 Hz, H1”), 3.66 (s, 3 H,
OCH3), 3.49 (t, 1 H, J 8.55 Hz, 8.24 Hz, H2”), 3.45 (m, 3 H, H3”, H4”,
H5”); 13C NMR (125 MHz, D2O): δ 175.8 (COOH), 174.9 (C4), 161.1
(C4’), 157.9 (C2), 154.5 (C9), 135.8 (C3), 134.5 (C7), 130.9 (C2’, C6’),
125.2 (C6), 124.4 (C5), 122.2, 121.7 (C10, C1’), 118.2 (C8), 113.6
(C3’, C5’), 102.0 (C1”), 77.3, 75.6, 71.6 (C3”, C4’, C5”), 73.5 (C2”);
HRMS (ESI): m/z calcd for C22H20O10 444.1056, found 444.1053 (M)+,
445.1126 (M+H)+, 467.0941 (M+Na)+.
4’-Chloroflavonyl -D-glucopyranosiduronic acid (13e)
4’-Chloroflavonyl 2-acetamido-2-deoxy-β-D-glucopyranoside (12e)
O-Deacetylation of 9e (350 mg; 0.58 mmol) gave 12e (281.5 mg,
94%): Rf 0.85 (eluent L); H NMR (500 MHz, DMSO-d6): δ: 8.28 (d, 2
O-Deacetylation of 10e (186.1 mg; 0.32 mmol) gave 13e (156.1
mg, 90%): Rf 0.67 (eluent M); H NMR (500 MHz, D2O): δ 7.85 (d, 1
1
1
H, J 8.54 Hz, H5), 7.82 (d, 2 H, J 8.54 Hz, H2’, H6’), 7.67 (t, 1 H, J 7.93
Hz, 7.32 Hz, H7), 7.43 (d, 1 H, J 8.54 Hz, H8), 7.34 (t, 1 H, J 7.93 Hz,
7.32 Hz, H6), 7.29 (d, 2 H, J 8.24 Hz, H3’, H5’), 4.99 (d, 1 H, J 7.63 Hz,
H1”), 3.45 (m, 4 H, H2”, H3”, H4”, H5”); 13C NMR (125 MHz, D2O): δ
176.2 (COOH), 174.9 (C4), 157.5 (C2), 154.9 (C9), 137.0, 136.5 (C3,
C4’), 134.8 (C7), 130.5 (C2’, C6’), 128.5 (C3’, C5’), 127.9 (C1’), 125.6
(C6), 124.7 (C5), 122.5 (C10), 118.3 (C8), 101.9 (C1”), 77.1, 75.5,
73.4, 71.6 (C2”, C3”, C4”, C5”): HRMS (ESI): m/z calcd for C21H17O9Cl
448.0561, found 448.0552 (M)+, 449.0627 (M+H)+, 471.0442
(M+Na)+.
H, J 8.85 Hz, H2’, H6’), 8.10 (d, 1 H, J 7.93 Hz, 1.22 Hz, H5), 8.00 (d, 1
H, J 9.16 Hz, NH), 7.84 (ddd, 1 H, J 8.54 Hz, 7.32 Hz, 1.53 Hz, H7),
7.77 (d, 1 H, J 8.24 Hz, H8), 7.60 (d, 2 H, J 8.54 Hz, H3’, H5’), 7.51 (t,
1 H, J 7.63 Hz, 7.32 Hz, H6), 5.72 (d, 1 H, J 8.24 Hz, H1”), 5.05 (d, 1 H,
J 3.97 Hz, 4”-OH), 5.02 (d, 1 H, J 5.19 Hz, 3”-OH), 4.30 (t, 1 H, J 5.49
Hz, 6”-OH), 3.71 (q, 1 H, J 9.77 Hz, 9.16 Hz, H2”), 3.54 (dd, 1 H, J
11.60 Hz, 4.88 Hz, H6a”), 3.38 (m, 1 H, H3”), 3.32 (m, 1 H, H6b”),
3.08 (m, 2 H, H4”, H5”), 1.84 (s, 3 H, NHCOCH3); 13C NMR (125 MHz,
DMSO-d6): δ 173.9 (C4), 169.8 (NHCOCH3), 155.1, 154.3 (C2, C9),
136.9, 135.9 (C3, C4’), 134.6 (C7), 131.4 (C2’, C6’), 129.9 (C1’), 128.8
(C3’, C5’), 125.6, 125.4 (C5, C6), 123.8 (C10), 118.9 (C8), 99.3 (C1”),
78.0 (C5”), 74.6 (C3”), 70.9 (C4”), 61.2 (C6”), 56.2 (C2”), 23.6
(NHCOCH3); HRMS (ESI): m/z calcd for C23H22NO8Cl 475.1034, found
475.1019 (M)+, 476.1102 (M+H)+, 498.0917 (M+Na)+.
Conclusion
In this work we discussed different approaches to the synthesis of
4’-substituted flavonols that were then transformed into 4’-
substituted flavonyl glycosides. We demonstrated that, with the
exception of the 4’-dimethylamino derivative, such glycosides are
sensitive to the presence of respective glycosidases in both neutral
and acidic conditions. The latter conditions definitely accelerate
enzymatic hydrolysis. Due to the ESIPT phenomenon, the
glycosidases action, associated with fluorophore release from the
probe, can be monitored by the increasing red-shifted fluorescence
near 535 nm. Flavonyl glycosides with the electron withdrawing 4’-
chloro substituent are the most sensitive to the enzyme’s action for
each sugar, glycosidase and conditions applied. In turn, flavonyl
glycosides with the electron donating 4’-methoxy substituent are
the least sensitive to the glycosidase presence. Flavonyl glucoside
with the most electron donating 4’-dimethylamino substituent is
not sensitive to glucosidase action at all. With the exception of 4’-
Flavonyl -D-glucopyranosiduronic acids (13a,b,e
)
General procedure:
Respective acetate (10a,b,e) (1 mmol) was dissolved in MeOH (10
mL) and 1 M LiOH in H2O (1 mL, 1 mmol) was added. The mixture
was stirred for 28 h at rt. The reaction was followed by TLC using
eluent M. The resulting precipitate was filtered off and filtrate was
neutralized using Dowex (H+) and washed by MeOH (2 mL). The
solution was evaporated to dryness and diethyl ether (2 mL) was
added to cause product precipitation.
Flavonyl -D-glucopyranosiduronic acid (13a)
O-Deacetylation of 10a (300 mg; 0.54 mmol) gave 13a (206.1
1
mg, 92%): Rf 0.68 (eluent M); H NMR (500 MHz, DMSO-d6): δ 8.26
(2 x d, 2 H, J 7.93 Hz, H2’, H6’), 8.12 (dd, 1 H, J 7.93 Hz, 1.53 Hz, H5),
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J. Name., 2013, 00, 1-3 | 11
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Organic & Biomolecular Chemistry
Page 12 of 12
ARTICLE
Journal Name
28. D. E. Stott, S. S. Andrews, M. A. Liebig, B. J. Wienhold, D. L.
trifluoromethylflavonyl glucoside, which has theoretically the most
favourable (the most electron withdrawing) substituent, the results
are consistent with the Hammett constants and ESP analysis as well
as with the mechanisms of glycosidic bond enzymatic hydrolysis.
The 4’-chloroflavonyl glycosides and procedures presented
constitute sensitive, reproductive and applicative method for
monitoring of β-D-glucosidase, N-acetyl-β-D-glucosaminidase and β-
D-glucuronidase activities.
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Conflicts of interest
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Acknowledgements
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