A gram scale synthesis of a multi-13C-labelled anthocyanin, [6,8,10,3′,5′-13C5cyanidin-3-glucoside, for use in oral tracer studies in humans

Article abstract of DOI:10.1039/c1cc14323a

The major dietary anthocyanin, cyanidin-3-glucoside, was prepared on a 4 g scale from three units of diethyl [2-¹³Cmalonate and one unit of [1,3-¹³C₂acetone, such that five isotope locations were distributed throughout the

Full text of DOI:10.1039/c1cc14323a

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COMMUNICATION
A gram scale synthesis of a multi-13C-labelled anthocyanin,
[6,8,10,30,50-13C5]cyanidin-3-glucoside, for use in oral tracer studies in
humansw
Qingzhi Zhang,a Nigel P. Bottingz*a and Colin Kayb
Received 18th July 2011, Accepted 25th August 2011
DOI: 10.1039/c1cc14323a
The major dietary anthocyanin, cyanidin-3-glucoside, was prepared
on a 4 g scale from three units of diethyl [2-13C]malonate and one
unit of [1,3-13C2]acetone, such that five isotope locations were
distributed throughout the molecule to provide a penta-13C5-
labelled anthocyanin, [6,8,10,30,50-13C5]cyanidin-3-glucoside
chloride, for use in human stable-isotope tracer studies.
Anthocyanins are naturally occurring polyphenols belonging
to the flavonoid family of natural products. These pigments are
Fig. 1 Anthocyanin 1 is a deep red solid. The photo shows the solid
pyrylium salts which are responsible for the rich colours from
and solutions of increasing concentration in dil. aq. HCl.
red, purple to blue displayed in various flowers, seeds, leaves,
urine, blood and faeces samples could be collected post bolus for
analysis via LC-MS/MS and isotope ratio mass spectroscopy
(IRMS).
fruits and vegetables. Epidemiological evidence suggests that
those who consume the highest proportions of anthocyanins in
the diet are at the lowest risk of cardiovascular diseases such as
hypertension and stroke.1–3 However, due to their low stability
at physiological pH and high propensity to bind with other
molecules, their absorption, distribution, metabolism and
elimination (ADME) in humans has yet to be fully established.
The complete characterisation of the ADME of anthocyanins
requires the use of multi-isotopically labelled anthocyanins in
human feeding studies to identify the metabolites of cyanidin-
3-glucoside (C3G) degradation products in vivo.
Due to the inherent instability of anthocyanins/anthocyanidins
in neutral and basic conditions, only a few syntheses have been
reported to date.8–14 Among them, the aldol condensation
between a phenolic aldehyde and an aryl ketone, pioneered by
Robert Robinson in the early 1900s, remains efficient and has
subsequently been further developed.15 We adopted this key
disconnection for the synthesis of [6,8,10,30,50-13C5]1 as it
allowed the incorporation of several 13C atoms into both the
building blocks, the phenolic aldehyde 2 and the glycosylated
aryl ketone 3, as illustrated in Scheme 1. Retrosynthetic
analysis of each of the required precursors 2 and 3 suggested
a synthetic route starting from commercially available diethyl
[2-13C]malonate and [1,3-13C2]acetone respectively.
Isotopically labelled anthocyanins have been prepared
previously in plant cell culture or catalysed by enzymes using
3
14C and H labels,4–6 however these radioactive isotopes are
not suitable for use in modern human feeding trials. Regio-
selective 13C-labelling studies have also been reported in plants
from [4-13C]enrichment.7 C3G 1 is the most commonly occurring
anthocyanin, found in 90% of all fruits (Fig. 1). Herein, we
report a unique synthesis with five 13C-isotopes located in both
the aryl moieties of C3G. The resultant [6,8,10,30,50-13C5]-
cyanidin-3-glucosyl chloride [[13C5]C3G] is targeted for in vivo
pharmacokinetic and metabolism studies in humans, and thus
several grams of C3G 1 were required such that aerosol (CO2),
a University of St Andrews, EaStChem School of Chemistry and
Centre for Biomolecular Sciences, North Haugh, St Andrews, Fife,
KY16 9ST, UK. E-mail: qz@st-andrews.ac.uk;
Fax: +1 (0)334 463808; Tel: +1 (0)334 463803
b Faculty of Medicine and Health Sciences, Department of Nutrition,
Norwich Medical School, University of East Anglia, Norwich,
UK NR4-7TJ. E-mail: Colin.Kay@uea.ac.uk
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc14323a
Scheme 1 Robinson’s final step in anthocyanin synthesis. Reagents
and conditions: (a) anhydrous HCl, EtOAc, ꢀ20 to 4 1C; (b) MeOH,
H2O, KOH, then 2 N HCl, 40–50%.
z N. P. Botting died 4th June 2011.
c
10596 Chem. Commun., 2011, 47, 10596–10598
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Scheme 2 Reagents and conditions: (a) EtONa, EtOH, 135–138 1C,
23–27%; (b) 60% aq. KOH, 130 1C, 96%; (c) oxalyl chloride, DMF,
acetonitrile, ꢀ17 1C to RT; (d) H2O, 70 1C, 72–81%; (e) (CH3CO)2O
(2 eq.), DMAP (0.2 eq.), EtOAc, reflux, 39–40%.
The synthesis of 2 exploited a route previously described for
the synthesis of [2,4,6-14C3]phloroglucinol (Scheme 2).16 This
approach incorporates three 13C atoms in a regiospecific manner.
The conversion of diethyl [2-13C]malonate to diethyl
2,4,6-trihydroxy-[1,3,5-13C3]isophthalate 4 proved to be the
most problematic step in the synthesis of 2. The process was
optimized with freshly dried, natural abundance substrate,
on a scale of up to 8 g of diethyl malonate to give 4 in a yield
of 40–42%, with a recovery of 20% of starting material.
However, reaction with diethyl [2-13C]malonate purchased to
order, and carefully pre-dried, gave a yield of only 23–27% of
[1,3,5-13C3]4 under the same conditions. Due to the need to
prepare multiple grams of 1, this first step was carried out on
B30–40 g of diethyl [2-13C]malonate, giving an average yield
of 25% of [1,3,5-13C3]4. The starting material (ca. 22%) was
recovered at a yield of only 10–12% for the second cycle. The
hydrolysis of 4 and in situ decarboxylation to 5 was more
efficient and was carried out on B6 g batches of [1,3,5-13C3]4.
A Vilsmeier–Haack reaction was used for formylation to
generate 6. A modification17 using oxalyl chloride in place of
POCl3 improved the conversion and led to greater recovery of
6 (72% at 6 g scale of 5 and 79–81% at 3 g of 5). The reaction
of the Vilsmeier iminium cation with 5 was extremely viscous
and due to the difficulty of stirring reactions on a larger scale
(410 g) led to di-formylation side products. Thus reactions
were kept between 3–6 grams of 5 to ensure more uniform
mixing.
Scheme 3 Regents and conditions: (a) HC(OEt)3 (2 eq.), BF3ꢁOEt 40 1C;
(b) iPrNEt2 (6 eq.), ꢀ78 1C; quant.; (c) EtOH, aqueous HCl, 80–100%;
(d) CH3COCH2COCH3 (2.53 eq.), tBuOK (1.86 eq.), tBuOH, then aq.
HCl, 47–53%; (e) Br2 (1.2 eq.), DCM, 70–76%; (f) aq. NaOH, CuSO4ꢁ
5H2O (catalytic), 88%; (g) (CH3CO)2O, DMAP (catalytic), EtOAc,
75%. Overall yield of step (e–g) 50%.
t
Treatment of 8 with an excess of acetylacetone and BuOK in
refluxing tert-butanol gave 4-hydroxy-[3,5-13C2]acetophenone
9 after acidic workup.20
Several bromination conditions were explored for the
conversion of 9 to 10, including N-methylpyrrolidin-2-one
hydrotribromide (MPHT)/H2O2,21 Br2/Selectfluor,22 and
HBr/H2O2;23 however, these gave complex mixtures. After
significant optimisation, bromination with elemental bromine
in DCM at 0 1C proved to be the most straightforward
method. A slight excess of Br2 and a relatively short reaction
time (40 min) gave the monobrominated product 10 in B76%
yield with minor amounts of unreacted starting material
(B15%) and B10% dibrominated product.
Hydrolysis of 10 using aqueous sodium hydroxide and
activated copper sulfate (as a catalyst) under reflux led
to 3,4-dihydroxy-[3,5-13C2]acetophenone 11,19 which was sub-
sequently converted into diacetate 12 with acetic anhydride in
the presence of DMAP.
Silyl enol ether 13, generated from acetophenone
[3,5-13C2]12, was treated with mCPBA, and after in situ hydro-
lysis gave a-hydroxy-3,4-diacetoxy-[3,5-13C2]acetophenone 15
as illustrated in Scheme 4.15,24
For an efficient coupling reaction, 2,4,6-trihydroxybenz-
aldehyde 6 was protected as diacetate 2.15 This reaction was
problematic as a result of competing C-aryl acylation side
products, and provided a diacetate [1,3,5-13C3]6 in a yield of
B40% after column chromatography and recrystallisation.
The construction of the ‘‘Eastern’’ aryl ring is illustrated in
Scheme 3 and utilised the method of Hobuss and co-workers,18
which was further developed in our laboratory,19 to
achieve a condensation between [1,3-13C2]acetone and triethyl
orthoformate giving 4H-[3,5-13C2]pyran-4-one 8. Accordingly,
treatment of triethyl orthoformate (2 equiv.) with BF3ꢁOEt2,
followed by the addition of [1,3-13C2]acetone (1 equiv.) and
N,N-diisopropylethylamine gave a mixture of intermediates
7a : 7b (57 : 43). Simply heating this mixture in EtOH/HCl
resulted in a cyclisation to 4H-[3,5-13C2]pyran-4-one 8, which
was an efficient reaction providing quantitative recovery of 8
without purification, or an 80% recovery after recrystallisation.
Glycosylation of 15 required avoiding the use of heavy
metals such as mercury and silver13,15,25 and therefore a BF3ꢁ
OEt2 catalysed coupling of a-hydroxyacetophenone 15, using
2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl trichloroacetimidate
was employed.26–28 This resulted in the glycosylated aromatic
ketone 3 in a modest yield of 40–50%. Finally, the condensation
and cyclisation of 2 and 3 were accomplished to generate
[13C5]C3G 1 using anhydrous HCl/EtOAc, followed immediately
by hydrolysis in MeOH and aq. KOH (Scheme 1).13,15 Puri-
fication of this rather unstable and water soluble flavylium
salt proved to be a significant challenge, and was carried out
on Amberlite XAD-7 resin, which was further purified using
cellulose microcrystalline and carbon-18 preparative column
chromatography, followed by recrystallisation from EtOH
c
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Scheme 4 Reagents and conditions: (a) TMSCl, Et3N, DMF, 70 1C;
(b) mCPBA, DCM, ꢀ20 1C to RT; (c) MeOH, yield for three steps 44%;
(d) 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl trichloroacetimidate,
BF3ꢁOEt2, 4 A MS, ꢀ20 to 10 1C; 40–50%.
and acetonitrile (0.1% HCl). This provided a satisfactory yield
of 40–50% of the product as a dark red powder.
In summary, a total of 4.30 grams of [13C5]C3G 1 was
obtained, with a purity of 499.9% (by HPLC). This carbon-13
enriched material as formulated for human consumption will
be utilised to study the ADME of anthocyanins. Ultimately any
metabolite identified will provide future targets for exploring
the mechanistic actions of anthocyanins in cellular models of
disease.
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The authors are grateful to the BBSRC for a grant
(BB/H004726) under the DRINC programme. Thanks go to
Drs C. H. Botting, C. P. A. Lawson and S. L. Shirran for
helping with the reaction purity analysis.
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Notes and references
0
´
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c
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This journal is The Royal Society of Chemistry 2011