Synthesis and toxicity profile in 293 human embryonic kidney cells of the β D-glucuronide derivatives of ortho-, meta- and para-cresol

Article abstract of DOI:10.1016/j.carres.2020.108225

The formation of β-glucuronides is a major route by which mammals detoxify and remove breakdown products, such as L-tyrosine, as well as many xenobiotics, from their systems. In humans, dietary L-tyrosine is broken down largely by the action of the anaero

Full text of DOI:10.1016/j.carres.2020.108225

Carbohydrate Research 499 (2021) 108225
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and toxicity profile in 293 human embryonic kidney cells of the β
D-glucuronide derivatives of ortho-, meta- and para-cresol
James A. Londona, Emily C.S. Wanga, Igor L. Barsukova, Edwin A. Yatesa,
,
*
Andrew V. Stachulskib
a Department of Biochemistry and Systems Biology, Crown Street, University of Liverpool, Liverpool, L69 7ZB, United Kingdom
b Robert Robinson Laboratories, Department of Chemistry, Crown Street, University of Liverpool, Liverpool, L69 7ZD, United Kingdom
A R T I C L E I N F O
A B S T R A C T
Keywords:
The formation of β-glucuronides is a major route by which mammals detoxify and remove breakdown products,
such as L-tyrosine, as well as many xenobiotics, from their systems. In humans, dietary L-tyrosine is broken down
largely by the action of the anaerobic gut bacterium C. difficile to p-cresol, providing a competitive advantage in
the gut microbiota. Ortho- (o-) and meta- (m-), cresols, also present in the environment, may share a common
degradative pathway. Relatively little work has been done on cresyl glucuronides. Here, a direct synthesis of o-,
m-, and p-cresyl β-D-glucuronides from methyl 1,2,3,4 tetra-O-acetyl-β-D-glucuronate and the respective cresol
employing trimethylsilyltriflate as promoter is presented. The protected intermediates were hydrolysed using
aqueous sodium carbonate to yield the cresyl β-glucuronides. The toxicities of the o-, m- and p-cresyl β–D-glu-
curonides were compared. All three were less toxic to HEK293 cells than their respective cresol precursors:
toxicity followed the order o < m < p for Na+ salts and o < p < m for Ca2+ salts. The m-cresyl-glucuronide Ca2+
salt and p-cresyl-glucuronide Na+ salt reduced colony formation by 11% and 9% (v. 30% reduction from the
aglycone) respectively, whereas o-cresyl-glucuronide (both Na+ and Ca2+ salts), mildly stimulated HEK293 cell
growth.
β-D-glucuronides
o-, m-, and p-cresol toxicity
Anaerobic gram-negative gut bacteria
Human embryonic kidney cells
L-tyrosine
C. difficile
1. Introduction
Eschericia coli, Klebsiella oxytoca and Bacteroides iotathetamicron, on ac-
count of its bacteriostatic properties and ability to affect the bacterial
The formation of glucuronides, β-linked derivatives of D-glucuronic
acid, is one important route in mammals by which the toxic breakdown
products of some naturally occurring substances, such as the amino acid
L-tyrosine, and a range of pharmaceutical drugs (xenobiotics) can be
detoxified and eliminated from the body [1]. Their formation involves
the reaction of uridine 5′-diphosphoglucuronic acid (UDP-GA) with one
of several functional groups, including amines, hydroxyl or carboxyl-
ates, to form glucuronide derivatives with greater aqueous solubility.
Although, in general, glucuronides act in a detoxifying role, one open
question is the extent to which some residual reactivity and toxicity
remains [2] .
metabolites produced. There is mounting evidence that the interplay
between the bacterial species in the gut, which constitute the gut
microbiota, mediated largely by the effect of metabolites from species
influencing each other, is important not only in regulating a healthy
microbiota, but also has wider health implications [3]. A change in the
profile of the bacterial population can lead to chronic or acute illness, in
which C. difficile often plays a significant role. Serious infections
involving C. difficile, particularly following antibiotic treatment and
increasingly in hospital settings, have become a health issue of global
significance [4]. It has been observed that Gram-negative bacteria
appear more susceptible to p-cresol than Gram-positive species and
tolerance to it varies between strains of particular species [5]. Four
p-cresol producing species have been identified from Coriobacteriaceae
and Clostridium clusters XI and VIVa [6].
Mammalian dietary L-tyrosine metabolic breakdown gives rise to
para-cresol (p-cresol) by the action of anaerobic bacteria, principally the
Gram-positive bacterium Clostridioides difficile (C.difficile; formerly
Clostridium difficile) in the gut. Para-cresol is thought to bestow a
competitive advantage on C. difficile over other gut bacteria, such as
Para-cresol is a precursor of both p-cresyl β-D-glucuronide and p-
cresol sulfate, which represent the two major routes for its removal in
* Corresponding author.
E-mail addresses: stachuls@liverpool.ac.uk, stachuls@liv.ac.uk (A.V. Stachulski).
https://doi.org/10.1016/j.carres.2020.108225
Received 11 November 2020; Received in revised form 14 December 2020; Accepted 15 December 2020
Available online 17 December 2020
0008-6215/© 2020 Elsevier Ltd. All rights reserved.

J.A. London et al.
Carbohydrate Research 499 (2021) 108225
mammals, although proportions of the two derivatives vary between
species. In humans, the p-cresol sulfate route predominates while, in
mice, p-cresyl β-D-glucuronide is more significant. The p-cresol level in
humans relates principally to the protein content of the diet and can be
moderated through control of protein intake. These two metabolites are
also associated with distinct consequences. Para-cresol sulfate promotes
insulin resistance, whereas p-cresyl β-D-glucuronide is unable to do so.
The former also plays important roles in kidney disease [7,8] and is a
virulence factor [9], inhibiting phagocyte function and decreasing
leukocyte adhesion to cytokine-stimulated endothelial cells [10]. In
chronic kidney disease, p-cresyl β-D-glucuronide increases relative to
p-cresol sulfate and the level of the former is correlated with the severity
of disease and with mortality [11,12].
2. Results and discussion
2.1. Synthesis of cresyl-glucuronides
Para-, m- and o-cresols (2, 3, and 4) were glucuronidated, using
1,2,3,4-tetra-O-acetyl, 6-methyl β-D-glucopyranosiduronate (1), in
dichloromethane using trimethylsilyl trifluoromethanesulfonate at
room temperature, under a nitrogen environment (Scheme 1). The
protected cresyl-glucuronide intermediates (5, 6, and 7), obtained in
46–69% yields, each exhibited complete selectivity for the β-anomer, as
determined by proton-proton coupling analysis (3JH1-H2 = 7.4 Hz for o-,
m- and p-derivatives) following column purification to homogeneity by
TLC. Removal of the acetyl and methyl protecting groups by dissolution
in methanol and treatment with aqueous sodium carbonate at 0 ◦C,
increasing to room temperature, gave the final cresyl-glucuronides
respectively (8, 9, and 10) in moderate yields (27–40%) as their so-
dium form, Fig. 1, with high resolution one dimensional proton and
carbon as well as two dimensional homonuclear proton and hetero-
nuclear proton-carbon NMR spectra measured.
In addition to the generation of p-cresol in the gut by bacteria, there
is the possibility of exposure to other cresol derivatives. These can be
naturally occurring, such as o-cresol from wood, or from man-made
sources such as pesticides and insecticides, tobacco smoke, as well as
numerous everyday products including creosote from coal or wood tar, a
variety of synthetic resins and, in the case of m-cresol, photographic
developer. To the best of our knowledge, it has not been established
whether the same detoxification routes are employed for the o- and m-
forms as for p-cresol, i.e. via sulfation or formation of β-D-glucuronides,
but it would be reasonable to consider this highly likely. With this in
mind, we broadened the scope of the study to include the isomeric o- and
m-cresols, and conducted parallel syntheses, characterisation and
toxicity testing of their β-D-glucuronide derivatives in HEK293 cells.
Relatively little work has been conducted on p-cresyl β-D-glucuro-
nide, although the synthesis of related compounds has often employed
the Schmidt trichloroacetimidate reaction [13], as reviewed [1], cata-
lysed by Lewis acids, to produce β-linked glucuronides [14–16].
Here, we present a direct synthetic route to the o-, m- and p-cresyl
β-D-glucuronides starting from the readily available methyl 1,2,3,4
tetra-O-acetyl ester of β-D-glucuronic acid (1) and o-, m- and p-cresol,
employing trimethylsilyltriflate (TMSOTf) as promoter. There is an
obvious saving if the anomeric tetraester (1) can be used as glycosyl
donor for the compounds of interest. This compound is available in just
one step from glucuronolactone, as opposed to two steps for the bro-
mosugar and three for the Schmidt imidate; moreover, the heavy metal
catalysts required for the classical Konigs-Knorr reaction are avoided.
The direct condensation of phenols with perester 1 was introduced by
Helferich in the 1940s, as reviewed in Ref. [17], but the original con-
ditions were harsh. Similar conditions have been used to obtain the
Variation of the cation form from sodium to calcium resulted in
distinct conformations of the glucuronide ring, deduced from peak
shifting and J-coupling variation in one dimensional proton and two
dimensional 1H,13C-HSQC NMR experiments. These different salt forms
were also tested subsequently to determine variations in their cell
toxicity, or effects on colony formation, using HEK293 cell assays.
2.2. HEK293 embryonic kidney cell toxicity
Cell seeding and growth was consistent across the plates and no
significant difference was observed, leading to the generation of com-
parable plates (Fig. 2, Supplementary Fig. 4 and Supplementary Tables 1
and 2). Increasing tyrosine concentration, particularly above 20
reduced HEK293 cell colony formation.
μM,
Para-, m- and o-cresols each reduced HEK293 colony formation
compared to the control wells, the greatest effect being seen at 50
and 100 M (Fig. 2B–D). Ortho-cresol treatment caused the greatest
reduction; 37% at 100
μM
μ
μ
M, in colony formation (Fig. 2D) compared to
26% with p-cresol; (Fig. 2B) and 28% with m-cresol (Fig. 2C) at the same
concentration. Increasing concentration was generally inversely corre-
lated with colony formation for all cresols, however, interestingly, at 20
μ
M, colony formation increased, similar to the control levels. Aside from
glycoside conjugates of other sugars with phenols [18], although α/β
this increase, the cresol treated cells followed a similar trend compared
to tyrosine, in reducing colony formation, suggesting cytotoxic effects or
inhibition of HEK293 cell growth.
mixtures may then result. Thiem et al. reported a similar glucur-
onidation of a steroidal phenol: they obtained complete β-selectivity and
a good yield, using BF3 as catalyst [19]. More recently, Wei et al. studied
this reaction in detail in the 4-methylumbelliferone series [20], again
using BF3, but adding various amines to buffer the reaction medium, and
utilising a number of glycosyl donors. In the examples studying glu-
curonidation, yields of up to 51% were obtained; N, N’-dimethylami-
nopyridine was the optimum base and high β-selectivity was obtained.
We now report that, for glucuronidation of the o-, m- and p-cresols, to
yield the glucuronide series (8)–(10), trimethylsilyl tri-
fluoromethanesulfonate (TMSOTf) at 0–20 ◦C is an excellent catalyst
and no buffering is required. In all three cases, the β-glycoside was
essentially the sole anomeric product, allowing the fully-protected
products to be re-crystallised and characterized. The final products
were obtained following simultaneous removal of the acetyl and methyl
esters under mild conditions using sodium carbonate [21]. Good yields
(up to 69%) were obtained in the glucuronidation step and β-selectivity
was complete according to NMR. In order to assess their toxicity of the
glucuronide products (8)–(10) and the free cresols towards cells relevant
to toxin clearance, HEK293 human embryonic kidney cells were selected
for testing.
The effect of p-cresyl glucuronide on colony formation appears
similar to p-cresol, although the reduction in absorbance was not as
great at 50 μM for the sodium form of the glucuronide (9% reduction) as
for the aglycone (30% reduction). Compared to the sodium form of the
glucuronide and the cresol, the calcium form showed little reduction at
any concentrations apart from 20 μM (Fig. 2B).
The m-cresyl-glucuronide sodium salt eradicates the reduction in
HEK293 colony formation seen for m-cresol, with a slight reduction
evident at 100 μM (Fig. 2C). The calcium form of the glucuronide ap-
pears to mitigate the effects of m-cresol whilst not eradicating it, as was
the case with the sodium variant. The strongest effects of the calcium
form of the glucuronide were seen at 20 μM and 100 μM with an 11%
decrease in colony formation. The effect of reversing reduction in colony
formation was also observed for both the sodium and calcium salts of o-
cresyl-glucuronide, no reductions being seen at 50 μM and 100 μM for
either form. Since the 20
μ
M, 50
μ
M and 100
μM concentrations of the
calcium form of the glucuronide and 100
μ
M, and potentially higher,
concentrations of the sodium form of the glucuronide generate consid-
erably higher crystal violet stain absorption than their respective con-
trols, it is possible that both the o-cresyl-glucuronide salts increase
HEK293 cell growth at higher concentrations.
2

J.A. London et al.
Carbohydrate Research 499 (2021) 108225
Scheme 1. Reaction scheme for the production of cresyl-β-D-glucopyranosiduronate, sodium salts (B) via the 1-cresyl-2,3,4-tri-O-acetyl, 6-methyl-β-D-glucopyr-
anuronate intermediate (A).
tissue and cell types [23]. Glucuronidation of p-, m- and o-cresols
generated the tyrosine metabolism intermediate p-cresyl-glucuronide,
alongside its two naturally occurring cresol stereoisomers, in a manner
selective for the β-anomer, improving on the stereoselectivity achieved
by an earlier synthesis [24]. Variation of the cation present generated
conformational changes to the glucuronide residue evinced by indica-
tive J-coupling values and peak shifting evident by NMR. The
cresyl-glucuronide salts were all shown to have reduced toxicity, or
reduction in colony formation, comparative to their precursor cresols
when tested on HEK293 cells; toxicity following the order; o < m < p for
Na+ salts and o < p < m for Ca2+ salts. Both o-cresyl-glucuronide sodium
and calcium salts exhibited some stimulatory effects on HEK293 cells.
4. Experimental section
4.1. General experimental methods
Commercially acquired starting materials and reagents were used as
received, except for the Amberlite IR-120 beads which were converted
Fig. 1. One dimensional 13C NMR spectra for: A. o-cresyl-β-D-glucopyr-
anosiduronate (10, R1 = CH3, R2 = H, R3 = H), B. m-cresyl-β-D-glucopyr-
to their H+ form prior to use. TLC was performed on silica-coated
aluminium plates with UV and permanganate stain used for detection,
while column chromatography was performed on VWR silica gel, 40-
anosiduronate (9, R1
= H, R2 = CH3, R3 = H) and C. p-cresyl-β-D-
glucopyranosiduronate (8, R1 = H, R2 = H, R3 = CH3), with peaks labelled
63
μ
m mesh. 1H and 13C NMR spectra were collected on a 400 MHz
(structures top right).
Bruker Avance spectrometer (100 MHz for 13C spectra) fitted with a
multinuclear BBFO probe alongside 1H, 13C, 1H,1H–COSY, 1H,1H-
TOCSY, 1H,13C-HSQC, and 1H,13C-HMBC on a 600 MHz Bruker Avance
II + spectrometer (150 MHz for 13C spectra) fitted with a TCI CryoProbe
in DMSO‑d6 and d6-Acetone for the ester intermediates and D2O for the
final cresyl glucuronide products respectively, referenced to DSS in all
cases. Low- and high-resolution mass spectra were obtained by direct
injection of sample solutions into a Micromass LCT mass spectrometer
operated in the electrospray mode, by positive ion (Micromass LCT
Waters Micromass UK Ltd, Manchester, UK). Microanalyses were per-
formed by Mrs. Jean Ellis in the Department of Chemistry, University of
Liverpool.
3. Conclusion
The cresols have been shown to reduce HEK293 cell colony forma-
tion with cytotoxic effects particularly prevalent at 50 μM and 100 μM,
where o-cresol has the strongest effect on colony formation. Glucuronide
derivatisation of these cresols, as both sodium and calcium salts, miti-
gates some of the cytotoxic effects of cresol treatment. These HEK293
cell cytotoxicity tests provide initial results employing a robust kidney-
derived cell line as a result of treatment with L-tyrosine, its breakdown
product p-cresol and structural isomers m- and o-cresol, as well as the
sodium and calcium adducts of their glucuronide derivatives. The cell
line chosen enabled investigation of the effects that these compounds
have on kidney cells relevant to the elevation of p-cresol-glucuronide
levels observed in chronic kidney disease. However, further cytotoxicity
testing could be performed using other cell lines, such as HeLa and HT-
29 cells [22], and alternative cytotoxicity assays, such as the MTT assay,
may provide a wider view of the toxicity of these compounds in other
4.2. Synthesis of cresol-glucuronides
Methyl 1-p-Cresyl-2,3,4-tri-O-acetyl-β-D-glucopyranuronate (5)
[24]: Methyl 1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronate (1) (1.20 g,
3.19 mmol) and p-cresol (2) (0.33 mL) were dissolved in dichloro-
methane (12.5 mL) and stirred over 4 Å molecular sieves under N2 for 2
3

J.A. London et al.
Carbohydrate Research 499 (2021) 108225
Fig. 2. A. A representative example of a HEK293 plate following treatment with p-cresol and crystal violet staining. B-D. HEK293 Cytotoxicity profiles with
increasing tyrosine, cresol, or cresyl-glucopyranosiduronate concentration (n = 1). Error bars indicate (no-treatment, 0
the plates via standard deviation (n = 10, SD = 0.21).
μM) control well cell growth variation across
h. Trimethylsilyl trifluoromethanesulfonate (0.55 mL, 3 mmol) was
added in a single portion and stirring was continued for 3 h with
monitoring via TLC, using a 3:1 ethyl acetate:hexane solvent mixture.
The resulting solution was diluted with 50 mL diethyl ether and washed
with saturated aqueous sodium bicarbonate, water and brine then dried
over anhydrous sodium sulfate. Filtration, then evaporation yielded a
semi-crystalline residue, which was purified via chromatography on
silica using a gradient of 30–50% ethyl acetate in hexane. Appropriate
fractions were pooled and dried to give compound (5) (0.935 g, 69%) as
a further 2 h H2O (1 mL) was added giving a clear solution. After 3 h,
Amberlite IR-120 (H+) resin was added with stirring until a pH of 6.0
was obtained. The resin was filtered, then the filtrate was evaporated to
dryness followed by azeotroping with EtOH (3 × 5 mL) to provide a solid
which was recrystallised from ethanol containing a few drops of H2O to
give the title compound (8) (0.113 g, 53%) as a white solid containing
0.2 mol equiv. sodium acetate. Further crystallisation from methanol
afforded pure (8) (0.062 g, 40%), as a slightly hygroscopic white solid;
20
[
α]
D-56◦ (c 0.5, MeOH). Found: C, 46.9; H, 5.05; C13H15O7Na.1.5H2O
a white solid. An analytical sample was obtained via recrystallisation
requires C, 46.85; H, 5.4%; δ (600 MHz, D2O) 2.29 (3 H, s, ArCH3),
H
20
from 1:5 ethyl acetate:hexane; m. p. 132–133 ◦C; [
α
]
ꢀ 20◦ (c 1,
3.54–3.63 (3 H, m, 2-H + 3-H + 4-H), 3.85 (1 H, d, J = 9.4 Hz, 5-H), 5.05
(1 H, d, J = 7.4 Hz, 1-H), 7.04 (2 H, d, J = 8.6 Hz, ArH) and 7.21 (2 H, d,
J = 8.4 Hz, ArH); δ (do., 150 MHz) 19.6, 71.8, 72.8, 75.4, 76.2, 100.6,
116.8, 130.2, 133.C3, 154.5 and 175.4, shown in Fig. 1C and Supple-
mentary Figs. 1, 6 and 7. The compound did not give a satisfactory mass
spectrum but showed a single peak on LCMS: m/z 306 for the Na salt.
Alternatively, the free glucuronic acid may be isolated by adding
Amberlite IR-120 (H+) to give a pH of 2.5, followed by filtration and
evaporation as above. Final trituration with ether affords the glucuronic
acid as a white solid.
MeOH). Found: C, 56.3; H, 5.6; m/z, 442.1698. C20H24O1D0 requires C,
56.6; H, 5.7%; C20H24O10. NH4+ requires m/z, 442.1708; δH (400 MHz,
DMSO‑d6) 2.03, 2.04 and 2.05 (9 H, 3s, CH3CO), 2.27 (3 H, s, ArCH3),
3.66 (3 H, s, CH3O), 4.69 (1 H, d, J = 9.6 Hz, 5-H), 5.03–5.10 (2 H, m,
2-H + 4-H), 5.47 (1 H, t, J = 9.6 Hz, 3-H), 5.59 (1 H, d, J = 8.0 Hz, 1-H),
6.89 and 7.14 (4 H, 2d, ArH); δ (do., 100 MHz) 20.6, 20.7, 20.8, 53.0,
C
69.5, 71.0, 71.4, 71.6, 97.9, 116.8, 130.5, 132.5, 154.7, 167.6, 169.5,
169.8 and 170.0; m/z (CI, NH3) 442 (MNH+4 , base peak); NMR shown in
Supplementary Fig. 5.
p-Cresyl-β-D-glucopyranosiduronate, sodium salt (8) [25]:
Methyl 1-p-cresyl-2,3,4-tri-O-acetyl-β-D-glucopyranuronate (5) (0.212 g
(0.5 mmol) was suspended in methanol (7 mL) and stirred at 0 ◦C while a
solution of sodium carbonate (0.14 g) in water (5 mL) was added over 1
min. The mixture was allowed to regain ambient temperature, then after
Methyl 1-m-Cresyl-2,3,4-tri-O-acetyl-β-D-glucopyranuronate (6)
[26]: This was prepared as for (5) above, using methyl 1,2,3,4-tetra-O-a-
cetyl β-D-glucopyranuronate (1) (1.13 g, 3.00 mmol) and m-cresol (3)
(0.30 mL). Following chromatography there was obtained compound
(6) (0.581 g, 46%) as a white solid: m. p. 110–112 ◦C from
4

J.A. London et al.
Carbohydrate Research 499 (2021) 108225
20
EtOAc-hexane; [
α]
ꢀ 16◦ (c 1, MeOH). Found: C, 56.6; H, 5.6; m/z,
ArH).
D
447.1263. C20H24O10 requires C, 56.6; H, 5.7%; C20H24O10. Na+ re-
quires m/z 447.1260; δH (600 MHz, DMSO‑d6) 2.01, 2.03 and 2.04 (9 H,
3s, CH3CO), 2.30 (3 H, s, ArCH3), 3.66 (3 H, s, CH3O), 4.68 (1 H, d, J =
9.9 Hz, 5-H), 5.04–5.13 (2 H, m, 2-H and 4-H), 5.47 (1 H, t, J = 9.7 Hz,
3-H), 5.63 (1H, d, J = 7.9 Hz, 1-H), 6.80–6.87 (2 H, m, ArH), 6.93 (1 H,
d, J = 7.6 Hz, ArH) and 7.24 (1 H, t, J = 7.8 Hz, ArH); δC (do., 150 MHz)
20.5, 20.6, 20.7, 21.3, 53.1, 69.4, 71.0, 71.3, 71.4, 97.3, 113.6, 117.4,
124.3, 129.9, 140.0, 156.5, 167.6, 169.7, 170.0 and 170.2, shown in
Supplementary Figs. 8 and 9.
4.3. HEK293 cytotoxicity testing
Cresol and cresyl-glucuronide cytotoxicity were determined using a
crystal violet cell viability assay on human embryonic kidney 293
(HEK293) cells [27]. The crystal violet assay is a fast and versatile in
vitro cell survival assay, used to determine the effect of a compound on
the ability of cells to proliferate and divide, staining being directly
proportional to cell biomass. HEK293 cells are one of the most common
cell lines used in a variety of biological experiments, including during
biopharmaceutical production and testing, due to their high reproduc-
ibility and their ease of growth and maintenance, making them a pop-
ular choice for initial cytotoxicity tests.
m-Cresyl-β-D-glucopyranosiduronate, sodium salt (9): This was
prepared as for 1-p-cresyl-β-D-glucopyranosiduronic acid, sodium salt
(8), above. From methyl 1-m-cresyl-2,3,4-tri-O-acetyl β-D- glucopyr-
anuronate (6) (0.100 g, 0.24 mmol) the product (9) was obtained as a
20
white solid (0.027 g, 27%): [
α]
D-32◦ (c 1, MeOH). Found (ES + ve
HEK293 cells were obtained from the American Type Culture
Collection (ATCC) and maintained in Dulbecco’s Modified Eagle’s Me-
dium (DMEM) supplemented with 10% fetal bovine serum (FBS), L-
glutamine and 100 mg/mL of streptomycin/penicillin in a humidified
atmosphere containing 16% O2, 5% CO2, 79% N2 at 37 ◦C. Cells were
seeded at 10,000 cells/mL, in 2 mL media, on 6-well plates. Following
24 h incubation at 37 ◦C, the media were aspirated and replaced with
mode): m/z, 307.0793. C13H16O7Na requires m/z 307.0789; δ (600
H
MHz, D2O) 2.32 (3 H, s, ArCH3), 3.56–3.64 (3 H, m, 2-H + 3-H + 4-H),
3.86 (1 H, d, J = 9.3 Hz, 5-H), 5.10 (1 H, d, J = 7.4 Hz, 1-H), 6.95 (1 H, d,
J = 8.2 Hz, ArH), 6.97–7.01 (2 H, overlapped s + d, ArH) and 7.28 (1 H,
t, J = 8.1 Hz, ArH); δ (do., 150 MHz) 20.4, 71.7, 72.8, 75.4, 76.2, 100.1,
C
113.6, 117.2, 124.0, 129.7, 140.6, 156.7 and 175.4, shown in Fig. 1B
and Supplementary Figs. 2, 10 and 11.
that containing either 0 μM, 5 μM, 10 μM, 20 μM, 50 μM or 100 μM cresol
or cresyl-glucuronide derivative. Cells were grown at 37 ◦C until the
control well reached confluence (7d). The media was were aspirated and
cells left on a rocker for 15 min in 1 mL crystal violet solution, followed
by removal of excess dye, and left to air dry. The crystal violet stain was
re-solubilised by 1 h incubation with 1 mL methanol and absorption
measured at 595 nm on a Flexstation 3 Multi-Mode Microplate Reader
(Molecular Devices).
Methyl 1-o-Cresyl-2,3,4-tri-O-acetyl-β-D-glucopyranuronate (7):
This was prepared as for (5) above, using methyl 1,2,3,4-tetra-O-acetyl-
β-D-glucopyranuronate (1) (1.13 g, 3.00 mmol) and o-cresol (4) (0.30
mL). Following chromatography, compound (7) was obtained as a white
20
◦
solid (0.600 g, 47%): m. p. 132–133.5 C; [
α]
ꢀ 28◦ (c 1, MeOH).
Found: C, 56.7; H, 5.65; m/z, 447.1264. C20H24O1D0. requires C, 56.6; H,
5.7%; C20H24O10. Na+ requires m/z 447.1260; δ (600 MHz, d6-acetone)
2.03, 2.04 and 2.06 (9 H, 3s, CH3CO), 2.10 (3 HH, s, ArCH3), 3.67 (3 H, s,
CH3O), 4.65 (1 H, d, J = 9.9 Hz, 5-H), 5.10 (1 H, t, J = 9.7 Hz, 4-H), 5.17
(1 H, t, J = 8.9 Hz, 2-H), 5.48 (1 H, t, J = 9.7 Hz, 3-H), 5.51 (1 H, d, J =
Funding
7.9 Hz, 1-H), 7.00–7.08 (2 H, m, ArH) and 7.16–7.24 (2 H, m, ArH); δ
C
The authors gratefully acknowledge support (to JAL) from the
Liverpool-Newcastle-Durham BBSRC Departmental Training Pro-
gramme: grant code BB/M011186/1.
(do., 150 MHz) 15.3, 20.0, 20.1, 20.2, 52.8, 68.9, 70.5, 70.8, 70.9, 97.4,
114.7, 123.1, 126.8, 127.2, 131.0, 154.2, 167.5, 169.8, 170.1 and 170.2,
shown in Supplementary Figs. 12 and 13.
o-Cresyl-β-D-glucopyranosiduronate, sodium salt (10): This was
prepared as for p-cresyl-β-D-glucopyranosiduronate, sodium salt (8),
above. From methyl 1-o-cresyl-2,3,4-tri-O-acetyl-β-D-glucopyranuronate
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
(7) (0.150 g (0.35 mmol) the product (10) was obtained as a white solid
20
(0.053 g, 35%): [
α
]
D-40◦ (c 0.5, MeOH). Found (ES –ve mode): m/z,
283.0825. C13H15O7 requires m/z 283.0823; δH (600 MHz, D2O) 2.27 (3
H, s, ArCH3), 3.58–3.68 (3 H, m, 2-H + 3-H + 4-H), 3.87 (1 H, d, J = 9.5
Hz, 5-H), 5.09 (1 H, d, J = 7.4 Hz, 1-H), 7.07 (1 H, t, J = 7.4 Hz, ArH),
7.15 (1 H, d, J = 8.1 Hz, ArH), 7.24 (1 H, t, J = 7.8 Hz, ArH) and 7.28 (1
H, d, J = 7.6 Hz, ArH); δC (do., 150 MHz) 15.4, 71.8, 72.8, 75.5, 76.2,
100.6, 115.6, 123.3, 127.2, 128.3, 131.1, 154.8 and 175.5 shown in
Fig. 1A and Supplementary Figs. 3, 14 and 15.
Acknowledgements
JAL would like to thank Dr. Rudi Grosman for developing and
allowing use of scripts applied to the presentation of high resolution
NMR spectra. ECSW would like to thank Dr. Michael Batie for support
and guidance during the completion of embryonic kidney cell toxicity
testing.
p-cresyl-β-D-glucopyranosiduronate, calcium salt was formed by
passing p-cresyl-β-D- glucopyranosiduronate, sodium salt across a Dowex
Marathon cation exchange resin, pre-loaded with Ca2+ cations. Found:
δH (600 MHz, D2O) 2.29 (3 H, s, ArCH3), 3.55–3.64 (3 H, m, 2-H + 3-H
+4-H), 3.85 (1 H, d, J = 9.4 Hz, 5-H), 5.06 (1 H, d, J = 7.5 Hz, 1-H), and
7.05 and 7.21 (4 H, 2d, both J = 8.5 Hz, ArH).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.carres.2020.108225.
m-cresyl-β-D-glucopyranosiduronate, calcium salt was formed
using the same protocol as for p-cresyl-β- D-glucopyranosiduronate, cal-
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overlapped s + d, ArH) and 7.28 (1 H, t, J = 8.1 Hz, ArH).
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