Phytochemistry 173 (2020) 112297

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Discovery of a stable vitamin C glycoside in crab apples (Malus sylvestris) T

a b c a b
Alistair T. Richardson , Jung Cho , Tony K. McGhie , David S. Larsen , Robert J. Schaffer ,
Richard V. Espleyb, Nigel B. Perrya,d,∗
a
Department of Chemistry, University of Otago, P. O. Box 56, Dunedin, New Zealand
b
The New Zealand Institute for Plant and Food Research Limited, Private Bag 92169, Auckland, 1142, New Zealand
c
Plant & Food Research, Private Bag 11600, Palmerston North, New Zealand
d
Plant & Food Research, Department of Chemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand

A R T I C LE I N FO A B S T R A C T

Keywords: Non-targeted LC-MS metabolomics on fruit of three wild and domesticated apple species (Malus sylvestris, M.
Malus sylvestris sieversii and M. domestica) showed that two crab apple (M. sylvestris) accessions were distinguished by high
Rosaceae concentrations of an ascorbic acid glycoside (AAG). This was partly purified, but key NMR signals were masked
Crab apple by inseparable sucrose. Reference samples of 2-O-β-D-glucopyranosyl L-ascorbic acid and 2-O-β-D-galactopyr-
Metabolomics
anosyl L-ascorbic acid were synthesised, but both coincided with the crab apple AAG on LC-MS. Peracetylation of
LC-MS
the crab apple extract allowed both purification and characterisation, and the AAG was proven to be 2-O-β-D-
Peracetylation
Vitamin C glucopyranosyl L-ascorbic acid by comparison of 1H NMR, HRMS and HPLC data with synthesised peracetylated
Ascorbic acid ascorbyl glycoside standards. The stability of the natural AA 2-β-glycoside was similar to synthetic 2-O-α-D-
Ascorbyl glycoside glucopyranosyl L-ascorbic acid, used widely in cosmetic and pharmaceutical products. This discovery in crab
2-O-β-D-Glucopyranosyl L-ascorbic acid apples (Rosaceae) is only the fourth reported occurrence of any ascorbyl glycoside from plants, the others being
from Cucurbitaceae, Solanaceae and Brassicaceae. It is hypothesised that AAGs may be more widespread in
plants than currently realised.

1. Introduction
All plants biosynthesise ascorbic acid (1, AA, vitamin C) primarily to
detoxify reactive oxygen species produced during photosynthesis
(Smirnoff, 2018). Despite this ubiquity, only two naturally occurring
derivatives of AA have been reported from plants (Fig. 1). 2-O-β-D-
Glucopyranosyl L-ascorbic acid (2) was isolated from goji berry fruit
(Lycium barbarum and L. chinense, Solanaceae), (Toyoda-Ono et al.,
2004). Ascorbic acid glycoside (AAG) 2 was also found in Arabidopsis
thaliana (Brassicaceae) plants overexpressing a UDP-dependent glyco-
syltransferase (von Saint Paul, 2010). The other AAG known in plants is
6-O-β-D-glucopyranosyl L-ascorbic acid (3), isolated from phloem
exudates of zucchini (courgettes, Cucurbita pepo, Cucurbitaceae)
(Hancock et al., 2008).
While natural AAGs are reportedly rare, synthetic 2-O-α-D-gluco-
pyranosyl L-ascorbic acid (4) (Fig. 1) is commonly used in cosmetics
and pharmaceuticals due to its superior stability over AA (Gudiminchi
et al., 2016; Han et al., 2012). The stability of 4 is a consequence of
substitution at O-2 of AA, which protects the 2,3-enediol against de-
gradation via oxidation (Mandai et al., 1992). When ingested or applied
to the skin, 4 acts as pro-vitamin C by hydrolysis with an endogenous
glucosidase to release AA (Han et al., 2012). The β-glucoside 2 has been
shown to act in a similar manner when ingested by rats, but the oxi-
dative stability of 2 has not been specifically examined (Toyada-Ono
et al., 2005). Other potential beneficial effects of AAG 2 are preventing
oxidative stress (Wang et al., 2019) and palliating colitis (Huang et al.,
2019).
α-Glucoside 4 was first prepared using a microbial transglucosidase
to catalyse reaction between maltose and AA (Suzuki et al., 1973).
Many different methods of enzymatic synthesis have since been re-
ported, and optimised to facilitate industrial-scale production of 4
(Gudiminchi et al., 2016; Han et al., 2012). Chemical syntheses of AAGs
have also been reported (Li and Shi, 2009; Ma et al., 2017; Toyoda-Ono
et al., 2004). Glycosyl bromide donors gave both 2- and 3-β-glycosides
of AA, with phase transfer glycosylations producing 2, the 2-β-ga-
lactoside and the 3-β-glucoside (Ma et al., 2017).
We now describe the discovery of an AAG in crab apple. Apples
(Malus spp., Rosaceae) are one of the world's largest fruit crops and are
known for their beneficial effects on human health. The major phyto-
chemicals present include catechin, quercetin, and chlorogenic acid, all
with antioxidant activity (Boyer and Liu, 2004; Espley and Martens,
2013). The AA content of apples is low (5–30 mg/100 g fresh weight)

∗
Corresponding author. Department of Chemistry, University of Otago, P. O. Box 56, Dunedin, New Zealand.
E-mail address: nigel.perry@plantandfood.co.nz (N.B. Perry).

https://doi.org/10.1016/j.phytochem.2020.112297
Received 10 October 2019; Received in revised form 8 February 2020; Accepted 8 February 2020
Available online 15 February 2020
0031-9422/ © 2020 Elsevier Ltd. All rights reserved.
A.T. Richardson, et al.
Fig. 1. Structures of AA (1) and the only known naturally occurring derivatives
in plants, 2-O-β-D-glucopyranosyl L-ascorbic acid (2) and 6-O-β-D-glucopyr-
anosyl L-ascorbic acid (3). Synthetic 2-O-α-D-glucopyranosyl L-ascorbic acid
(4) is commonly used in cosmetic products.
compared to crops such as kiwifruit (80–100 mg/100 g fw) (Bassi et al.,
2017; Yahia, 2018). Crab apples are wild apple species with small,
astringent fruit that are not grown commercially. Consequently re-
search into crab apples is more limited, but they may contain valuable
traits lost during domestication (Harris et al., 2002; Volk et al., 2015).
We report below LC-MS discovery of an AAG in crab apples, and show
this to be 2-O-β-D-glucopyranosyl L-ascorbic acid (2) by acetylation of
crab apple extract and isolation of the peracetylated derivative of 2, and
matching its properties with a fully synthesised sample.
2. Results and discussion
2.1. Detection of an AAG in crab apples
In order to identify secondary metabolites that may have been lost
through centuries of breeding for other traits, a non-targeted metabo-
lomics study was carried out on three wild and domesticated apple
species: Malus sylvestris (L.) Mill.; M. sieversii M. Roem.; and M. do-
mestica (Suckow) Borkh. Three accessions of each species were selected
for their previously determined genetic diversity (Kumar et al., 2014).
Extracts of four fruit from each of these nine different accessions were
analysed by LC-MS. Principal Component Analysis (PCA) was used to
interpret the results (Fig. 2). Fruits from the same accession were
grouped most tightly, then accessions were grouped according to their
gene pool, indicating that each species was characterised by a distinct
combination of metabolites. M. domestica samples were identifiable
based on their polyphenol composition, M. sieversii by a selection of
fatty acids, and M. sylvestris (crab apples) by specific dihydrochalcones
(Fig. 2).
PCA also identified an ion of m/z 337.0749 as separating two of the
M. sylvestris accessions from the other samples. This ion was prominent
in extracts of all fruits of two of the three M. sylvestris accessions, but
barely detectable or absent in all of the other samples. The observed
[M-H]- m/z was consistent with a molecular formula of C12H18O11,
which did not match any known apple components. MS2 data showed a
key daughter ion of m/z 174.0171 (C6H6O6) indicating loss of a hexose.
Using this MS information to search a public chemical structure data-
base (www.chemspider.com) suggested that the M. sylvestris metabolite
could be an AAG. A second fragment, with m/z 277.0566, was con-
sistent with the loss of –CHOH–CH2OH (the 5,6-diol) from AA, but with
2
Phytochemistry 173 (2020) 112297
retention of the sugar. This ruled out a 5- or 6-glycoside, narrowing the
list of possibilities to a 2- or 3-O-hexoside of AA. Commercial AAG 4
was found to elute later on LC-MS than the AAG in crab apples (Fig. 3).
Therefore the unknown glycoside was not 4, and isolation was required
for a definitive characterisation.
Preparative LC of a M. sylvestris fruit extract yielded a fraction
containing the AAG by UV detection, but HRMS and 1H NMR spectro-
scopy (Fig. 4) showed that this fraction was mostly sucrose. However,
there was a minor AAG ion in the HRMS and minor signals in the 1H
NMR spectrum (Fig. 4). Signals from sucrose obscured most potential
AAG signals, but a resolved doublet (4.51 ppm, J = 7.8 Hz) was typical
of an anomeric proton of a β-glycoside (Fig. 4). Other purification
methods, including HILIC, C18 and silica-gel chromatography, did not
separate the target AAG from sucrose, so an alternative approach was
required.
2.2. Synthesis of ascorbyl glycosides 2 and 2a
AAGs 2 and 2a (Fig. 5) were chosen as the first synthetic targets
because glucose and galactose are the most common hexose sub-
stituents in plants, and 3-glycosides of AA were unreported at the time
(Shah et al., 2013).
Protected AA derivative 5 (Fig. 5) was prepared by literature pro-
cedures with a 65% yield (Toyoda-Ono et al., 2004). Compound 5 re-
acted with glycosyl bromide 6 under phase transfer conditions to give
the protected glucoside 7 in 45% yield. Removal of the 5,6-iso-
propylidene group with HCl, hydrogenolysis of the 3-O-benzyl group,
and subsequent deacetylation with methoxide gave glucoside 2 with an
overall yield of 17% from AA. Galactoside 2a was synthesised using the
same procedures with an overall yield of 24%. The lower yield of glu-
coside 2 was attributed to loss of glucosyl donor via β-elimination,
which occurs more readily for glucosides than galactosides (Kleine
et al., 1985).
After we had completed these syntheses, similar approaches were
published for the preparation of 2, 2a and the 3-β-glucoside of AA (Ma
et al., 2017). Our NMR data were mainly consistent with those of Ma
et al. and Toyoda-Ono et al. (2004) but we observed the anomeric
proton of 2a as a doublet of doublets (4.5 and 3.2 Hz), rather than the
expected 8 Hz doublet. We attribute this to virtual long-range coupling,
which occurs when an isolated proton signal receives “false” coupling
information from protons strongly coupled to each other, as previously
reported for anomeric protons of other β-galactosides (Dahmen et al.,
1984; Ma et al., 2017).
We also found that the 1H NMR spectra of AAG 2 from our several
syntheses gave spectra with the 1′-H and 4-H proton signals at varying
chemical shifts (HRMS and LC-MS retention times were the same).
Spectra of 2 in acidic, neutral and basic D2O revealed that this was a pH
effect (Fig. 6). The upfield shift of both 1′-H and 4-H signals with base
can be explained by de-protonation of the acidic 3-OH, giving more
electron density shielding the adjacent protons.
The synthesised AAGs 2 and 2a eluted at about the same time as the
crab apple AAG on LC-MS (Fig. 3). We could not find any LC conditions
that clearly resolved 2 and 2a. Therefore we tried a different approach
to characterise the crab apple compound.
2.3. Isolation and characterisation via derivatisation
Acetylation was chosen to facilitate isolation and characterisation
because conversion of multiple hydroxyl groups to acetates would allow
silica-gel chromatography to be used, providing an alternative separa-
tion mode to RPLC. Derivatisation would also aid characterisation be-
cause acetylation of hydroxyl groups results in downfield shifts of the
geminal protons, generally leading to better resolution of glycoside
proton resonances (Larsen et al., 2015). Acetylation also removes acidic
protons and thus minimises pH variation in 1H NMR spectra.
The acetylation procedure was tested on synthetic AAGs 2 and 2a
A.T. Richardson, et al. Phytochemistry 173 (2020) 112297

Fig. 2. Left: PCA (Pareto scaling with 95% confidence interval) of the LC-MS analyses of extracts from four fruit of each of nine apple accessions: green
triangles = M. sylvestris, red circles = M. domestica, purple Xs = M. sieversii. Right: PCA of molecular features showing that M. domestica accessions were char-
acterised by polyphenols (red), M. sieversii by fatty acids (blue) and M. sylvestris by dihydrochalcones (green). A molecular feature tentatively identified as an ascorbyl
glycoside (top left) was also associated with two M. sylvestris accessions. (For interpretation of the references to colour in this figure legend, the reader is referred to
the Web version of this article.)

Fig. 3. LC-MS chromatograms (Instrument B, method I) of crab apple extract, commercial 2-O-α-D-glucopyranosyl L-ascorbic (4), and two AAGs produced by
chemical synthesis, 2-O-β-D-glucopyranosyl L-ascorbic (2) and 2-O-β-D-galactopyranosyl L-ascorbic (2a). The traces are extracted ion chromatograms for m/z 337 [M
– H]-. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

(Fig. 7). Pyridine catalysed acetylation of 2 with acetic anhydride did
not yield the desired hepta-acetate, but gave a product with a major
HRMS ion at m/z 595.1270. This was 60 Da less than expected, and the
1
H NMR spectrum showed only six acetyl signals, 5-H was a triplet as
opposed to the expected triple doublet, and 4-H was not seen. These
data were consistent with base-catalysed elimination of 5-OAc to give 8
(Fig. 7), a previously unreported structure. This was obtained as a single
isomer, but we could not assign it as Z/E because there were no diag-
nostic NOE interactions and no model compounds in the literature.
Acid catalysis was then tested (Fig. 7). Acetic anhydride with
perchloric acid successfully acetylated both AAGs, giving 9 and 9a in
reasonable yields (> 70%) without the need for further purification.
The NMR spectra (Fig. 8 and Table 1) showed signals arising from the
AA lactones of each glycoside consistent with the expected structures
and in line with NMR data for synthetic intermediates (Supporting In-
formation). Key HMBC correlations from 2-C to 1′-H confirmed that the
sugars were still attached at O-2 of AA. The sugar signals reflected the
different orientation of 4′-OAc in the glucoside (9) and galactoside (9a),
with the 3′-H signal a triplet for 9 (9.3 Hz) and a double doublet (10.4
and 3.5 Hz) for 9a (Fig. 8). These clear differences between 1H NMR

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A.T. Richardson, et al. Phytochemistry 173 (2020) 112297

Fig. 4. 1H NMR spectra (400 MHz, D2O) of a crab apple extract fraction (I) and sucrose (II). The circled signal was consistent with the anomeric proton of a β-AAG.

Fig. 5. Synthetic route to AAGs 2 and 2a. I. Acetone, CH3COCl, overnight, room temp. (RT), II. PhCH2Br, K2CO3, DMSO, 50 °C, 3.5 h, III. NaOH(aq), TBAB, CHCl3,
50 °C, 1 h, IV. Conc. HCl(aq), MeOH, 40 °C, 45 min, V. H2/Pd–C, MeOH, RT, 3 h, VI. MeONa, MeOH, 20 min, RT, IRA 120 (H+), overall yield 17% (2) and 24% (2a).

spectra of the peracetylated AAGs were not observed for the un-
protected AAGs 2 and 2a (Supporting Information).
The peracetylated AAGs were also compared by LC-MS (Fig. 9).
Unlike the AAGs 2 and 2a, the derivatised equivalents were retained on
a C18 reverse phase column and were readily resolved by manipulation
of the solvent gradient, providing an additional tool to identify the AAG
in crab apples.
An extract of M. sylvestris fruit was acetylated and fractionated by
silica-gel chromatography. Fractions containing the target compound
were combined and re-subjected to silica-gel chromatography to give
an AAG peracetate. 1H, 13C and 2D NMR analyses (Fig. 8, Table 1) and
LC-MS analysis (Fig. 9) showed that this was hepta-O-acetyl-2-O-β-D-
glucopyranosyl L-ascorbic acid (9).
2.4. Stability of 2-O-β-D-glucopyranosyl L-ascorbic acid (2)
The stability to oxidation of synthetic AA α-glucoside 4 is the reason
for its commercial applications (Han et al., 2012). It has been assumed
that glucoside 2 would also resist oxidation due to protection of as-
corbate O-2, but this has not been specifically examined (Toyada-Ono
et al., 2005). Acidic and basic solutions of AA (1) and AAGs 2 and 4
under air were analysed by HPLC (see Supporting Information). As

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A.T. Richardson, et al. Phytochemistry 173 (2020) 112297

Fig. 6. 1H NMR spectra of β-glucoside 2 in D2O (400 MHz) demonstrating the effect of pH on proton resonances 1′-H and 4-H. Top: one drop of 2 M NaOH added to
NMR tube; middle: nothing added; bottom: one drop of conc. HCl added to NMR tube.

expected, AA was stable with acid, but degraded rapidly with base,
consistent with acid buffering of extracts for AA analyses (Bassi et al.,
2017; Hijaz and Killiny, 2014; Yuan and Chen, 1998). The β-glycoside 2
and α-glycoside 4 were equally stable: neither degraded in acidic or
basic solutions over 12 h.
2.5. Conclusions
Our discovery of 2-O-β-D-glucopyranosyl L-ascorbic acid (2) in crab
apples is only the fourth reported occurrence of an AAG, or any AA
derivative, in plants (Hancock et al., 2008; Smirnoff, 2018; Toyoda-Ono
et al., 2004; von Saint Paul, 2010). This paucity of naturally occurring

Fig. 7. Acetylation of 2 under basic conditions gave the unsaturated glycoside 8, but acidic conditions led to the successful acetylation of both 2 and 2a. I. Ac2O,
pyridine, 0 °C, overnight, 44%. II. Ac2O, HClO4, 0 °C, 1 h, 71% (9) and 73% (9a) yield.

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A.T. Richardson, et al. Phytochemistry 173 (2020) 112297

Fig. 8. The downfield regions of the 1H NMR spectra for acetylated ascorbyl glycosides (structures in Fig. 7.). I. Peracetyl galactoside 9a, II. Acetylated and purified
ascorbyl glycoside from crab apples, III. Peracetyl glucoside 9.

Table 1
NMR data of acetylated crab apple AAG and acetylated synthetic AAGs (400 MHz, CDCl3).
Ac-Crab apple compound Peracetyl glucoside (9) Peracetyl galactoside (9a)

13 1 13 1 13 1
C# C H (HSQC) C H (HSQC) HMBC C H (HSQC) HMBC

Ascorbic acid unit

1 164.7 – 164.7 – 4 164.7 – 4
2 127.5 – 127.4 – 1′, 4 127.6 – 1′, 4
3 146.6 – 146.6 – 5 146.5 – 4, 5
4 74.6 5.40 (1H, m)a 74.6 5.39 (1H, m)a 6a, 6 b 74.6 5.41 (1H, m)a 6a, 6 b
5 66.8 5.40 (1H, m)a 66.7 5.39 (1H, m)a 6a, 6 b 66.8 5.41 (1H, m)a 6a, 6 b
6 62.1 4.35 (1H, dd, J = 11.8, 5.2 Hz) 62.1 4.35 (1H, dd, J = 11.7, 5.3 Hz) 4, 5 62.1 4.36 (1H, dd, J = 11.7, 5.2 Hz) 4, 5
4.27 (1H, m)a 4.27 (1H, m)a 4.26 (1H, dd, J = 11.7, 7.0 Hz)
Glycosyl component
1′ 97.5 5.61 (1H, d, J = 7.9 Hz) 97.5 5.61 (1H, d, J = 7.8 Hz) 3′, 5′, 2′ 98.3 5.51 (1H, d, J = 8.0 Hz) 5′
2′ 70.9 5.13 (1H, m)a 70.9 5.12 (1H, m)a 3′ 68.4 5.33 (1H, dd, J = 10.4, 8.0 Hz) 1′, 3′, 4′
3′ 72.7 5.24 (1H, t, J = 9.3 Hz) 72.6 5.24 (1H, t, J = 9.3 Hz) 2′, 4′ 70.7 5.06 (1H, dd, J = 10.4, 3.5 Hz) 2′, 4′
4′ 68.0 5.13 (1H, m)a 68.0 5.12 (1H, m)a 3′, 6′a, 6′b 67.0 5.39 (1H, m)a 3′, 6′
5′ 72.5 3.81 (1H, ddd, J = 10.2, 4.9, 2.3 Hz) 72.5 3.81 (1H, ddd, J = 10.1, 4.9, 2.3 Hz) 1′, 6′a, 6′b 71.7 4.01 (1H, td, J = 6.6, 1.0 Hz) 6′
6′ 61.6 4.27 (1H, m)a 61.6 4.27 (1H, m)a 4′ 61.2 4.13 (2H, dd, J = 6.5, 1.3 Hz) 5′
4.11 (1H, dd, J = 12.4, 2.3 Hz) 4.10 (1H, dd, J = 12.4, 2.3 Hz)
Acetate groups
Ac-CO 170.7 – 170.6 – 170.6 –
Ac-CO 170.4 – 170.4 – 170.4 –
Ac-CO 170.2 – 170.2 – 170.3 –
Ac-CO 169.9 – 169.8 – 170.1 –
Ac-CO 169.6 – 169.5 – 169.8 –
Ac-CO 169.5 – 169.5 – 169.5 –
Ac-CO 165.3 – 165.3 – 165.5 –
Ac-CH3 20.8 2.26 (3H, s) 20.8 2.26 (3H, s) 20.9 2.27 (3H, s)
Ac-CH3 20.8 2.08 (3H, s) 20.7 2.08 (3H, s) 20.8 2.16 (3H, s)
Ac-CH3 20.8 2.07 (3H, s) 20.7 2.07 (3H, s) 20.8 2.09 (3H, s)
Ac-CH3 20.8 2.07 (3H, s) 20.7 2.07 (3H, s) 20.8 2.07 (3H, s)
Ac-CH3 20.8 2.03 (6H, s)a 20.7 2.03 (6H, s)a 20.7 2.04 (3H, s)
Ac-CH3 20.6 2.03 (6H, s)a 20.6 2.03 (6H, s)a 20.6 2.03 (3H, s)
Ac-CH3 20.5 2.02 (3H, s) 20.4 2.01 (3H, s) 20.5 2.00 (3H, s)

a
Signals overlapped in 1H NMR spectrum; HSQC - 1H signals correlated to the carbon; HMBC 1H signals correlated to the carbon.

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A.T. Richardson, et al. Phytochemistry 173 (2020) 112297

Fig. 9. LC-MS (Instrument B, method II) extracted ion chromatograms for m/z 655, [M + Na]+, of acetylated AAGs 9 (red, 7.0 min) and 9a (green, 8.1 min), and
acetylated and purified crab apple AAG (blue, 7.0 min). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version
of this article.)

Table 2 3. Experimental
Apple samples in the non-targeted metabolomics study.
Accession Malus species Sample codea Fruit collection date
3.1. General materials and methods

1 domestica R6T91.92 2/Mar/2015 Laboratory-grade solvents were used for syntheses and purifica-
2 domestica R6T43.44 2/Mar/2015 tions, and HPLC or MS grade solvents were used for chromatography
3 domestica R5T53.54 9/Mar/2015
and MS. All commercial solvents and reagents were used as received.
4 sieversii R3T39.40 2/Mar/2015
5 sieversii R3T67.68 9/Mar/2015 TLCs were run on Merck silica gel F254 plates visualised under UV light
6 sieversii R5T115.116 18/Mar/2015 then with vanillin dip (2% vanillin, 1% conc. H2SO4, in EtOH). Column
7 sylvestris R5T33.34 2/Mar/2015 chromatography used silica-gel 60, 200–400 mesh, 40–63 μm. 1H, 13C
8 sylvestris R5T115.116 9/Mar/2015
and 2D NMR spectra were recorded at either 400 MHz or 500 MHz on
9 sylvestris R5T93.94 31/Mar/2015
Varian spectrometers, with samples at 25 °C in 5 mm tubes. 1H chemical
a
R = Row in orchard, T = Tree unique code. shifts are reported in ppm relative to the CHCl3 singlet at 7.26 ppm in
CDCl3, the CHD2OD quintet at 3.31 ppm in CD3OD, or the HOD singlet
AAGs in the literature seems to suggest that these metabolites are rarely at 4.79 ppm in D2O. 13C chemical shifts are reported in ppm relative to
produced by plants, despite the ubiquity of AA and the frequency of the solvent triplet at 77.16 in CDCl3, or the solvent septet at 49.00 ppm
glycosides in plant metabolism. The reported natural AAGs are from in CD3OD. HRMS were recorded on a Bruker microTOFQ instrument
four different plant families: Cucurbitaceae, Solanaceae, Brassicaceae using electrospray ionisation in positive or negative mode as indicated.
and now Rosaceae. The way that these known AAGs were discovered
may explain why they have not been detected in other plants. 6-β- 3.2. Plant materials
Glucoside 3 was found in phloem exudates, a plant tissue rarely sam-
pled for phytochemical analyses, while searching for AA itself (Hancock All apple fruit samples were collected from the Plant & Food
et al., 2008). 2-β-Glucoside 2 was found in goji fruit by a targeted Research orchard in Havelock North, New Zealand. The trees used in
search for AA derivatives (Toyoda-Ono et al., 2004). Non-targeted the metabolomics study are listed in Table 2, with details reported
metabolomics studies with LC-MS led to the discovery of AAG 2 in elsewhere (Cho, 2016). The first isolation used fruit collected from two
experimentally manipulated A. thaliana plants (von Saint Paul, 2010) M. sylvestris crab apple trees with unique location and identifier codes
and in crab apples (this work). R01T014 (Block R8) and R01T047 (Block R8) in autumn 2017. The
We hypothesise that AAGs are widespread in plants and could be second isolation with acetylation used fruit collected from a M. sylvestris
found by targeted LC-MS analyses of various plant parts, including crab apple tree with unique location and identifier code R01T024
phloem. The stability of 2-β-glucoside 2 suggests that the special pre- (Block R21) on April 18, 2018.
cautions needed for AA quantification would not be needed. However,
we have shown that 2-β-glucoside 2 and 2-β-galactoside 2a (a likely but
3.3. Chromatography instruments and methods
as yet unreported natural AA derivative) are difficult to distinguish by
LC-MS, and may require peracetylation to separate on LC-MS or to
Instrument A. The UHPLC-UV system consisted of a Dionex Ultimate
characterise by NMR spectroscopy.
3000 Rapid separation LC system (Thermo Scientific) equipped with
PDA detection and Foxy fraction collector. Instrument control and data
analysis were performed using Chromeleon Chromatography Data
System software. Method I: A Phenomenex (Luna HILIC 150 × 4.6 mm,

7
A.T. Richardson, et al.
3 μm) column was used and eluted isocratically at 1.0 ml/min over
10 min with 80:20 MeCN and H2O + 20 mM ammonium formate (pH
5), injection volume 50 μL.
Instrument B. The LC-MS system consisted of a Shimadzu LC20
Prominence LC system linked to a Shimadzu LCMS-2020 mass spec-
trometer with electrospray ionisation controlled by LabSolutions soft-
ware. Method I: A Phenomenex (Luna HILIC, 150 × 4.6 mm, 3 μm,
200 Å) column equipped with a HILIC guard cartridge was maintained
at 40 °C and eluted with a mobile phase of A: H2O + 10 mM ammo-
nium formate (pH 6.7) and B: MeCN. An injection volume of 15 μl and
flow rate of 0.7 ml/min were used. The gradient used was as follows:
0–8 min 90% B, 8–12 min 90–50% B, 12–14 min 50% B, 14–15 min
50–90% B, 15–18 min 90% B. Method II: A C18 (Vision HT,
50 × 2.1 mm, 3 μm) column equipped with C18 guard cartridge was
maintained at 40 °C and eluted with a mobile phase of A: H2O + 0.1%
formic acid and B: MeOH + 0.1% formic acid with a flow rate of
0.4 ml/min. The gradient used was as follows: 0–3 min 10–40% B,
3–8 min 40% B, 8–12 min 40–100% B, 12–14 min 100% B, 14–15 min
100–10% B, 15–18 min 10% B. Method III: Column and mobile phase
composition the same as for Instrument B, Method I. The column was
eluted isocratically at 90% B for 15 min. Single wavelength UV detec-
tion (260 nm) was incorporated prior to MS detection.
Instrument C. The LC-HRAM-MS/MS system was composed of a
Dionex Ultimate® 3000 Rapid Separation LC and a micrOTOF QII high
resolution mass spectrometer (Bruker Daltonics, Bremen, Germany)
fitted with an electrospray ion source. The micrOTOF QII parameters
were: temperature 225 °C; drying N2 flow 6 l min−1; nebulizer N2
1.5 bar, endplate offset 500 V, mass range 100–1500 Da, data were
acquired at 5 scans s−1. Negative ion electrospray was used with a
capillary voltage of 3500 V. Post-acquisition internal mass calibration
used sodium formate clusters with the sodium formate delivered by a
syringe pump at the start of each chromatographic analysis. Method I: A
Thermo Fisher Scientific (Hypersil GOLD 200 × 2.1 mm, 1.9 μm)
column was maintained at 40 °C and eluted with a mobile phase of A:
H2O + 0.2% formic acid and B: MeCN with a flow rate of 0.4 ml/min.
The gradient used was as follows: 0–0.5 min 30–55% B, 0.5–25 min
0.5–98% B, 25–45 min return to starting conditions. Method II: The LC
conditions were the same as for Method I. Fragmentation of the ion at
m/z 337.0781 was achieved at 20 eV using N2 as the collision gas. Data
were recorded using a data dependant acquisition (DDA) mode with no
exclusions.
3.4. Metabolomics study
Fruit peel of each accession (Table 2) was frozen under liquid N2
and freeze dried for transport and storage. Samples of freeze-dried
material (ca. 100 mg) were extracted with EtOH:H2O (80%) overnight
at 4 °C, centrifuged (13,000 rpm, 5 min), diluted with MeOH (1:2) and
analysed by LC-MS (Instrument C, method I). The same sample set was
then analysed using a second method (Instrument C, method II) so that
MS2 data could be collected on the major ions. Standards of known
apple components were analysed alongside apple peel extracts and
were used to identify these metabolites. Molecular features of high in-
tensity were identified with the aid of mass databases.
3.5. Attempted isolation of AAG
M. sylvestris fruit (980 g) were homogenised with dry ice using a
Hobart food chopper. A portion of the chopped fruit (664 g) was ex-
tracted with 3 l of MeOH:H2O:formic acid (50:50:1) before it was fur-
ther homogenised with an Omni GLH blender. The mixture was then
left to extract overnight at 4 °C. The extract was filtered and solid re-
sidue extracted with a further 2 l of MeOH:H2O:formic acid (50:50:1)
and left overnight at 4 °C. The second extract was filtered, MeOH re-
moved in vacuo, and freeze dried and separated by HPLC (Instrument A,
method I) to give a fraction containing AAG (5 mg including buffer).
8
Phytochemistry 173 (2020) 112297
HRMS (negative ion mode) found m/z 341.1112 (calcd. for C12H21O11,
341.1084), and 337.0803 (calcd for C12H17O11, 337.0776); 1H NMR
(400 MHz, D2O) Fig. 4; HPLC (Instrument B, method I) was used to
analyse the apple fraction, plus commercial 2-α-glucoside 4 and syn-
thetic glycosides, 2 and 2a (Fig. 3).
3.6. Isolation of peracetylated AAG
M. sylvestris fruit peel (13.4 g) was frozen under liquid N2, ground to
a powder with a pestle and mortar, and extracted by shaking overnight
with MeOH:H2O + 5% w/v mHPO4 (100 ml). The extract was filtered,
concentrated and freeze dried to give the crude material as a pale pink
powder (3.5 g). To a stirred mixture of this crude extract (2.0 g) in
acetic anhydride (40 ml) was added perchloric acid in acetic anhydride
(10 drops, in 5 ml) at 0 °C. The reaction was stirred for 2 h at 0 °C and
then allowed to warm to room temperature over an hour. Ice water was
used to quench the reaction before it was extracted with EtOAc, washed
with NaHCO3 and brine, dried over anhyd. MgSO4 and solvent removed
under reduced pressure to give the acetylated extract as a brown sludge
(4.1 g). The acetylated extract (4.1 g) was subsequently fractionated
with silica-gel chromatography (DCM:Et2O). The most promising frac-
tions were combined and solvent removed in vacuo to give a mixture of
acetylated products (150 mg). A second silica-gel column (DCM:Et2O)
was used to isolate Ac-AAG 9 (5.8 mg). 1H NMR (400 MHz, CDCl3), 13C
NMR (100 MHz, CDCl3) and 2D NMR in Table 1; HRMS (positive ion
mode) m/z 655.1457 [M + Na]+ (calcd for C26H32O18Na, 655.1481).
HPLC (Instrument B, method II) was used to analyse the acetylated
glycoside from crab apple alongside acetylated synthetic glycosides 9
and 9a (Fig. 9).
3.7. Synthesis and derivatisation
Syntheses of AAGs 2 and 2a were achieved using methods analo-
gous to those now reported by Ma et al. (2017) described in Supporting
Information.
2-O-(2′,3′,4′,6′-Hexa-O-acetyl)-β-D-glucopyranosyl 3-O-acetyl-4,5-
dehydro-L-ascorbic acid (8).
To a stirred solution of AAG 2 (40 mg, 0.12 mmol) in acetic anhy-
dride (5 ml, 52.9 mmol) was added pyridine (5 ml, 62.1 mmol) at 0 °C.
The solution was allowed to warm to room temperature and stirred
overnight before it was diluted with ice water, extracted with EtOAc,
washed with HCl (1 M), water, 5% NaHCO3, and water again before it
was dried over anhyd. MgSO4 and solvent removed under reduced
pressure to give the unsaturated derivative 8 as a colourless oil (30 mg,
0.05 mmol, 44%).
1
H NMR (400 MHz, CDCl3) δ 2.01 (3H, s, Ac-Me), 2.03 (3H, s, Ac-
Me), 2.07 (3H, s, Ac-Me), 2.07 (3H, s, Ac-Me), 2.07 (3H, s, Ac-Me),
2.31(3H, s, Ac-Me), 3.87 (1H, ddd, J = 10.0, 4.8, 2.3 Hz, 5′-H), 4.12
(1H, dd, J = 12.4, 2.3 Hz, 6′-Ha), 4.27 (1H, dd, J = 12.4, 4.8 Hz, 6′-
Hb), 4.83 (2H, d, J = 7.34 Hz, 6-H), 5.12 (2H, m, 3′-H & 4′-H), 5.24
(1H, dd, J = 9.8, 8.7 Hz, 2′-H), 5.36 (1H, t, J = 7.3 Hz, 5-H), 5.73 (1H,
d, J = 7.9 Hz, 1′-H); 13C NMR (100 MHz, CDCl3) δ 170.6 (Ac-CO),
170.6 (Ac-CO), 170.0 (Ac-CO), 169.4 (Ac-CO), 169.4 (Ac-CO), 164.6
(Ac-CO), 160.1 (C1), 142.9 (C4), 138.6 (C3), 130.1 (C2), 103.8 (C5),
97.3 (C1′), 72.4 (C5′), 72.4 (C2′), 70.6 (C3′), 67.9 (C4′), 61.4 (C6′), 57.4
(C6), 20.8 (Ac-CH3), 20.7 (2 x Ac-CH3), 20.6 (Ac-CH3), 20.6 (Ac-CH3),
20.1 (Ac-CH3); HRMS (positive ion mode) m/z 595.1270 [M + Na]+
(calcd for C24H28O16Na, 595.1271).
3,5,6,2′,3′,4′,6′-Hepta-acetyl-2-O-β-D-glucopyranosyl L-ascorbic-acid
(9).
To a stirred mixture of AAG 2 (50 mg, 0.15 mmol) in acetic anhy-
dride (5 ml, 52.9 mmol) was added perchloric acid at 0 °C. The reaction
was stirred for 2 h at 0 °C and then allowed to warm to room tem-
perature over an hour. Ice water was used to quench the reaction before
it was extracted with EtOAc, washed with NaHCO3 and brine, dried
over anhyd. MgSO4 and solvent removed under reduced pressure to
A.T. Richardson, et al.
give peracetate 9 as a colourless oil (66 mg, 0.11 mmol, 71%).
1
H NMR (400 MHz, CDCl3), 13C NMR (100 MHz, CDCl3) and 2D
NMR in Table 1; HRMS (positive ion mode) m/z 655.1470 [M + Na]+
(calcd for C26H32O18Na, 655.1481).
3,5,6,2′,3′,4′,6′-Hepta-acetyl-2-O-β-D-galactopyranosyl L-ascorbic-
acid (9a).
To a stirred mixture of AAG 2a (300 mg, 0.89 mmol) in acetic an-
hydride (15 ml, 158.7 mmol) was added perchloric acid at 0 °C. The
reaction was stirred for 2 h at 0 °C and then allowed to warm to room
temperature over an hour. Ice water was used to quench the reaction
before it was extracted with EtOAc, washed with NaHCO3 and brine,
dried over anhyd. MgSO4 and solvent removed under reduced pressure
to give peracetate 9a as a colourless oil (411 mg, 0.65 mmol, 73%).
1
H NMR (400 MHz, CDCl3), 13C NMR (100 MHz, CDCl3) and 2D
NMR in Table 1; HRMS (positive ion mode) m/z 655.502 [M + Na]+
(calcd for C26H32O18Na, 655.1481).
3.8. Stability study
Stock solutions of AA, 4 and 2 were made up in H2O (1 mg/ml).
Solutions of HCl and NaOH (0.133 M) were also prepared in H2O.
Stability studies were initiated by addition of an ascorbate derivative
solution (250 μl) to either acid or base solution (750 μl). This gave final
concentrations of 250 μg/ml for the ascorbate derivative and 0.1 M for
the acid or base. Once initiated, sub-samples were analysed by HPLC
(Instrument B, method III, Section 4.3) at 30 min intervals up to 6 h,
and 60 min intervals up to 12 h. Quantification was based on peak area
at 260 nm and achieved by comparison to standards of each of the three
ascorbate derivatives over a concentration range of 5–500 μg/ml.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgement
We thank Satish Kumar, Richard Volz and Claire Molloy for assis-
tance with plant selection and sampling, and William Laing for sam-
pling and valuable discussions (all Plant & Food Research). AR thanks
the University of Otago for a PhD scholarship and publishing bursary.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2020.112297.
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