Chemical synthesis of 5’-β-glycoconjugates of vitamin B6

Article abstract of DOI:10.1016/j.carres.2020.107940

Various 5’-β-saccharides of pyridoxine, namely the mannoside, galactoside, arabinoside, maltoside, cellobioside and glucuronide, were synthesized chemically according to KOENIGS-KNORR conditions using α4,3-O-isopropylidene pyridoxine and the respective acetobromo glycosyl donors with AgOTf (3.0 eq.) and NIS (3.0 eq.) as promoters at 0 °C. Furthermore, 5’-β-[¹³C₆]-labeled pyridoxine glucoside (PNG) was prepared starting from [¹³C₆]-glucose and pyridoxine. Additionally, two strategies were examined for the synthesis of 5’-β-pyridoxal glucoside (PLG).

Full text of DOI:10.1016/j.carres.2020.107940

Journal Pre-proof
Chemical synthesis of 5’-β-glycoconjugates of vitamin B
6
Thomas Bachmann, Christian Schnurr, Laura Zainer, Michael Rychlik
PII:
S0008-6215(19)30722-0
DOI:
https://doi.org/10.1016/j.carres.2020.107940
CAR 107940
Reference:
To appear in: Carbohydrate Research
Received Date: 10 December 2019
Revised Date: 3 February 2020
Accepted Date: 4 February 2020
Please cite this article as: T. Bachmann, C. Schnurr, L. Zainer, M. Rychlik, Chemical synthesis of
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’-β-glycoconjugates of vitamin B , Carbohydrate Research (2020), doi: https://doi.org/10.1016/
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Chemical synthesis of 5’-β-glycoconjugates of vitamin B6
1
,*
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1
Thomas Bachmann , Christian Schnurr , Laura Zainer and Michael Rychlik
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6
Chair of Analytical Food Chemistry, Technical University of Munich,
Maximus-von-Imhof-Forum 2, 85354 Freising, Germany; thomas.bachmann@tum.de;
michael.rychlik@tum.de; laura-zainer@online.de; christian.schnurr@tum.de
*correspondence: thomas.bachmann@tum.de
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0
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Abstract: Various 5’-β-saccharides of pyridoxine, namely the mannoside, galactoside,
arabinoside, maltoside, cellobioside and glucuronide, were synthesized chemically
according to KOENIGS-KNORR conditions using α4,3-O-isopropylidene pyridoxine and the
respective acetobromo glycosyl donors with AgOTf (3.0 eq.) and NIS (3.0 eq.) as promoters
1
1
1
1
13
at 0 °C. Furthermore, 5’-β-[ C ]-labeled pyridoxine glucoside (PNG) was prepared starting
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13
from [ C ]-glucose and pyridoxine. Additionally, two strategies were examined for the
6
synthesis of 5’-β-pyridoxal glycoside (PLG).
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5
Keywords: Glycosylation, Chemical Synthesis, Labeled, Glycoconjugates, Pyridoxine,
1
Pyridoxal, Vitamin B , PNG, PLG.
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1. Introduction
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The formation of glycoconjugates belongs to the most important mechanisms of
post-translational modification and takes place in various species and cell types.[1-9] Hence,
it is highly relevant to a number of medical fields, e.g. topics regarding the nervous[10, 11] or
immune system[12-14], alcoholism[15], inflammation[16], apoptosis[17] and cancer[12, 18,
19]. Fascinatingly, merely ten nucleotides suffice as building blocks in the manufacturing of
the mammalian glycome in its plethora of substrates.[2, 20]
Although glycoconjugates find purpose in a manifold of different aspects of biological
processes, the knowledge about the full spectrum of their impact still resides in its infancy. In
plants, glycosylation fulfills not only a function of storage, but shows a significant influence
on the signaling processes in cells, where the complexity of the information transfer results
from the structural variety of glycans.[21-29]
When glycosylations are carried out in plants that contribute to the human diet,
glycoconjugates become a subject of food-related consumers’ health.[30-33] Wheat, infected
with fungi of the genus Fusarium, was found to detoxify the mycotoxin deoxynivalenol
(DON) by converting it into the less toxic deoxynivalenol-3-glucoside (D3G).[34, 35] The

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assessment of the mycotoxin contamination in food, therefore, must include the modified
mycotoxin D3G, since cleavage of the glycosidic bond during digestions releases the free
toxin DON.[31-33, 36-41] What is crucial for the analytics of toxic substances also applies to
beneficial secondary metabolites. Micronutrients like vitamins occur in glycosylated forms,
as well, which affects the bioavailability in the human body.[30] The group of vitamin B6
constitutes an interesting example hereby, since it consists of six in vivo interconvertible[42,
43] vitamers, namely pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM) and their
respective phosphorylated compounds pyridoxine 5'-phosphate (PNP), pyridoxal
5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP), and an additional glucosylated
derivative, pyridoxine-5'-β-D-glucoside (5'-β-PNG, Figure 1), which can be found as a major
part of the B content (5 - 70 %) in plants.[44-49]
6
OH
N
O
HO
HO
O
OH
OH
OH
Pyridoxine-5'- -glucoside (PNG)
Figure 1. Plants store vitamin B as pyridoxine-5'-β-
4
4
3
4
D
-glucoside.
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47
48
49
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51
52
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56
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PNG was first identified in 1977 after its isolation from rice bran and later prepared by
chemical synthesis according to
K
OENIGS-KNORR
conditions implementing
-glucopyranosyl bromide and
α4,3-O-isopropylidene pyridoxine, 2,3,4,6-tetra-O-acetyl-α-
D
silver carbonate.[50] Other strategies regarding the chemical preparation of this molecule are
scarce and numerous methods rely on biotechnological techniques. Here, the two main paths
being followed comprise either the assistance of isolated enzymes[47, 51, 52] or the
utilization of seeds.[53-58] Regarding the latter, alfalfa seeds present a convenient way to
produce unlabeled PNG with yields ranging from 35 to 60 % and also allow the synthesis of
2
3
[ H]- and [ H]-β-PNG.[57, 59-62]
B in its active form (PLP) functions as a cofactor in a variety of processes in the human
6
metabolism.[63] Consequently, the relation between the amount of modified vitamin in food
and the total B intake includes important information regarding the impact on the nutrition.
6
Studies with deuterated PNG suggested, that the bioavailability is substantially higher in
humans (50 - 58 % relative to PN)[64, 65] than in rats (20 - 30 %).[57, 66]
Although PNG remains in the spotlight of research because of its abundant
occurrence[49], other glycoconjugated derivatives of PN were also found in nature. More
than 50 % of the glycosylated compounds in rice bran were found to be composed of

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5'-O-(β-cellobiosyl) pyridoxine, 4'-O-(β-D-glucosyl)-5'-O-(β-cellobiosyl) pyridoxine and
5'-O-(β-glucotriosyl) pyridoxine.[67] Additionally, 38 % of the total PN-content in rice bran
were assigned to 5'-O-[6-O-((+)-5-hydroxy-dioxindole-3-acetyl)-β-cellobiosyl] pyridoxine
after enzymatic and chemical hydrolysis procedures.[68] In wheat bran, popped pea and
soybeans, another compound, namely B X, exists in small amounts. Although the structure
6
could not be derived from the isolate, treatment of the latter with alkaline solution and
β-glycosidase released vitamin B , which indicates a glycosylated derivate.[69, 70] Further
6
studies revealed, that podded pea Pisum sativum L[71, 72] produces
5'-O-[6-O-(3-hydroxy-3-methyl-4-carboxybutanoyl)-β-D-glucopyranosyl)]-pyridoxine (HM
GPNG) and 5‘-O-(6-O-malonyl-β- -glucopyranosyl)-pyridoxine (MalonylPNG) during
D
germination. Furthermore, the potential occurrence of PN-oligosaccharides in potatoes was
proposed, but not further specified.[73]
Identification of new PN-saccharides proceeded not only through the process of
isolation, but was also accomplished via biotechnological approaches. Experiments with an
isolated marine exo-α-glucosidase (EC 3.2.1.20) from the anaspidean mollusk Aplysia
fasciata showed transglycosylating properties and were successfully utilized in the
preparation of 4’-/5’-α-PNG and the respective isomaltosides.[74] Suzuki et al. found a
glucuronic acid-like derivative while incubating
a
mixture of uridine
5’-diphosphoglucuronyltranserfase from rabbit liver, uridine phosphoglucuronic acid and
pyridoxine.[75] The three galactosyl conjugates 5'-O-(β-
4'-O-(β- -galactopyranosyl)-pyridoxine
4'-O-(β-O-galactopyranosyl-(1→4)-β-
D
-galactopyranosyl)-pyridoxine,
and
D
D
-galactopyranosyl)-pyridoxine were reportedly
produced in a culture of Sporobolomyces singularis growing on a medium containing 5 %
lactose, 0.75 % yeast extract and 2 % pyridoxine.[76, 77] Moreover, two fructose derivatives,
5'-O-(β-
D
-fructofuranosyl)-pyridoxine
-fructofuranosyl-(2→l)-β-
and
5'-O-[β-
D
D
-fructofuranosyl]-pyridoxine, were detected in a culture
medium of Aspergillus niger and A. sydowi containing sucrose and pyridoxine.[78] While
examining a series of glycosidases for their synthetic potential regarding pyridoxine as a
substrate, 4’-/5’-O-(2-acetamido-2-deoxy-β-
4’-/5’-O-(β- -galactopyranosyl) pyridoxine and 4’-/5’-O-(α-
were prepared using 4-nitrophenyl-2-acetamido2-deoxy-β-
4-nitrophenyl-β- -galactopyranoside and mannose as sugar donors.[79]
D
-glucopyranosyl) pyridoxine, 5’-α- and
-mannopyranosyl) pyridoxine
-glucopyranoside,
D
D
D
D
Although a variety of studies were dedicated to the isolation and identification of a
manifold of glycoconjugated derivatives of PN, indications about their related content in

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9
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food, in particular when PN-glucoside is excluded, are sparsely sown. Therefore, with raising
awareness of the relevance of glycosylated natural compounds in the scientific field, the
present study pursued to expand the catalogue of known PN-saccharides via chemical
synthesis and pave the way for further analytical studies revolving around their occurrence in
food.
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01
02
2. Results and discussion
2.1. Synthesis of the starting materials
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04
In order to fulfill this task, glycosyl donors and the acceptor had to be prepared as
starting materials, first, before implementing them into glycosylation reactions (Scheme 1).
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1
05
06
1
Scheme 1. General synthetic strategy for the preparation of the PN-saccharides.
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The preparation of the glycosyl donors started with the installation of protection groups.
Hereby, acetate groups were chosen because of their neighbouring group effect.[80-82] The
anomeric outcome of the acetylation reaction was directed using either pyridine (py, α-Glc)
or sodium acetate (β-Glc) (Scheme 2).[83] Since glucuronidation constitutes an important
phase II metabolic pathway for a broad spectrum of substances, the preparation of
glucuronides was enclosed in this study.[84-91] Acetylated glucuronic methyl ester was
prepared from glucuronic acid γ-lactone.[92] Installation of bromine as leaving group was
accomplished with hydrogen bromide (HBr).[93-95]
115
1
1
16
17
Scheme 2. General depiction of the preparation process for the saccharide donors involving an
acetylation followed by a bromination step.

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120
The selective protection of pyridoxine was undertaken with 2,2-dimethoxypropane and
p-toluenesulfonic acid (pTSA) in order to increase the regioselectivity towards the desired
5’-position and to allow the solubility of PN in organic solvents (1, 96 %, Scheme 3).
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1
1
22
23
Scheme 3. The protection of pyridoxine was accomplished with 2,2-dimethoxypropane and pTSA in
acetone.
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2.2. Glycosylation reactions
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132
The preparation of the glycosides followed the guidelines of KOENIGS-KNORR
conditions[96], but was performed with an increased amount of promoter AgOTf/NIS (3.0
eq.). The application of NIS, although presenting a well-established additive with regards to
the reaction of thioglycosides, usually doesn’t find utilization under KOENIGS-KNORR
conditions. Preliminary experiments revolving around the glucosylation of PN under these
conditions, however, resulted exclusively in the formation of orthoester-structures when low
amounts (0.1 - 0.3 eq.) of promoter (AgOTf, Ag CO , Ag O, Cu(OTf) , TMSOTf, BF ·OEt )
2
3
2
2
3
2
were used in combination with NIS.
133
1
1
34
35
Scheme 4. Preliminary experiments resulted exclusively in the orthoester formation, when low amounts of
promoter were added.
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141
Additionally, no conversion of the starting material was observed when NIS was missing
from the reaction, suggesting an NIS-mediated formation of the orthoester. Interestingly, an
increased amount of promoter/NIS (3.0 eq.) resulted both in a complete shift from orthoester
structure to the desired glycoconjugate and furthermore in the selective formation of the
β-epimer. Hence, the glycosylation of PN with various glycosyl donors was performed using
3.0 eq. of AgOTf/NIS in order to obtain the protected glycosides (Scheme 4).

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1
42
43
Scheme 5. The glycosylation reaction for the preparation of the PN-saccharides was undertaken
based on KOENIGS-KNORR conditions followed by a two-step deprotection procedure. The listed
yields refer to the deprotected glycosides obtained after three steps (glycosylation and deprotection).
1
1
44
45
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152
153
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165
The robustness of the acetonide group in basic milieu and stability of the acetate groups
towards acidic conditions demanded a two-step procedure for the subsequent removal of the
protection groups. Thus, the acetate groups were cleaved by application of 1 N KOH/NaOH
first, followed by reflux in 1 % HCOOH in order to deprotect the acetonide functionality.
Following this procedure, PN-saccharides containing mannose 9, galactose 10, glucuronic
acid 11, maltose 12, cellobiose 13, xylose 14 and arabinose 15 were synthesized with a
selective anomeric (β) outcome, which was verified through NMR experiments.
The deprotected glycosides were obtained after three steps (glycosylation and
deprotection) in approximately the same range of yields (10 - 25 %) except for PN-xyloside
(< 3 %) and PN-glucuronide (50 %). The low yield of the xyloside arose from a stronger
tendency to decomposition resulting from a higher instability of the molecule compared to
the other saccharides. This aspect was particularly salient in NMR measurements of the
glycoconjugate (which was purified by preparative HPLC and confirmed by ESI-MS
beforehand), where the signals of pyridoxine and xylose overshadowed those of the
glycoside. Storing the substrate at lowered temperatures (− 28 °C) did not improve this
aspect. By contrast, the preparation of the glucuronide worked well and resulted in the
highest yield among the reacted sugar donors. Overall, most losses occurred during the
glycosylation reaction rather than the deprotection steps. Preliminary experiments including
the variation of reaction conditions, next to promoter quantity and type, such as temperature
(− 78, − 30, 0 °C or rt) and time, although successfully shifting the formation of orthoester

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structures completely towards the β-epimer of the desired product, didn’t manage to improve
the reaction outcome over a certain point, qualifying the chosen reaction conditions (3.0 eq.
AgOTf/NIS; 0 °C; CH Cl ) as best variant using PN as substrate under KOENIGS-KNORR
2
2
conditions. On the other hand, there consequently lies much potential for improvement with
regards to aspects like increased reaction outcome in the variation of the glycosylation
method and exploration of further leaving groups.
The literature mainly focuses on the synthesis of the abundantly present PN-glucoside.
Only few preparations of other PN-saccharides are reported, among them mostly via
biotechnological methods. Charles and Divakar optimized reaction conditions for the
preparation of PN-glucoside (36 ± 10 %), -mannoside (23 ± 10 % of which being 4’α/5’α/5’β
= 24/34/42) and -galactoside (40 ± 10 % of which being 4’α/5’α/5’β = 47/27/26) with the aid
of β-glucosidase from sweet almonds starting from the respective saccharides, but struggled
with bad regio- and anomeric selectivity resulting in a mixture of 4’/5’-saccharides and
α/β-anomers.[52] Weignerova et al. utilized specific glycosidases and could improve the
regioselectivity. Here, 4’/5’-β-PN-galactosides were obtained in 32 % yield (related to the
sugar donor) utilizing β-galactosidase from A. oryzae and 4’/5’-α-PN-mannosides were
received in 2 % yield (also related to the sugar donor) with mannosidase from Canavalia
ensiformis (jack beans).[79] Tadera et al. isolated 53 mg of 5’-β-PN-cellobioside from 10 kg
defatted rice.[67] The yields of the chemical synthesis proposed in the study presented here
aligned with the results from the literature, but laborious separation of regio-isomers could be
omitted since 5’-selectivity was promoted through the acetonide protecting group.
Furthermore, contrary to many biotechnological strategies, upscaling constitutes a simple
and non-time-consuming operation because the starting materials are inexpensive and easily
prepared. The remaining PN-saccharides (glucuronide, maltoside, xyloside and arabinoside)
have not been reported in the literature to date.
In order to evaluate fragmentation patterns of the PN-saccharides and to affirm their
structure additionally to NMR-experiments, the deproteced PN-saccharides were measured
via ESI-MS (Figure 2). Protonated PN-maltoside (12, m/z 494, A) dissociated through loss of
a glucose moiety (m/z 314), followed by loss of another glucose fragment (m/z 170) and
water (m/z 152). Protonated PN-cellobioside (13, m/z 494, B) showed a similar pattern,
confirming the disaccharidic structure of the product. Fragmentation of protonated
PN-glucuronide (11, m/z 346, C) resulted in the loss of water (m/z 328) and loss of the
glucuronic moiety (m/z 152). Further fragmentation steps of PN (m/z 108) are detailed
elsewhere.[97] The ESI-MS spectrum of protonated PN-arabinoside (15, m/z 302, D)

2
2
00
01
showed fragments after the loss of water (m/z 284), the sugar moiety (m/z 152) and further
fragmentations (m/z 108).
2
2
2
02
03
04
Figure 2. LC-ESI(+)-MS/MS spectra of PN-maltoside (12, A, collision energy (CE) = − 20 V), PN-cellobioside
(13, B, CE = − 21 V), PN-glucuronide (11, C, CE = − 25 V) and PN-arabinoside (15, D, CE = − 23 V).
205
2.3. Synthesis of 5’-β-pyridoxal glucoside (5’-β-PLG)
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218
An additional branch of this study was directed towards the question, whether other
vitamers of the B group occur as glycosylated derivatives in nature. Since PLP plays an
6
important role as a cofactor in many enzymatic reactions in the human metabolism[63, 98],
PL was chosen as the target substrate. The vitamer shows the same solubility properties as
PN, thus being insoluble in organic liquids. For solving this problem, the strategy outlined
above involving the installation of an acetonide group was not available due to the aldehyde
group. Masking the latter with imine functionalities[99, 100] enhanced the solubility of the
molecule in dichloromethane, but did not result in the formation of product during
glycosylation reactions further down the road. Hence, the first step revolved around a
solubility study, where the hydrochloric salt (HCl) of PL was suspended in various solvents
suitable for glycosylation reactions. In accordance to unprotected PN, no dissolution of the
white solid was observed, and the thought arose, whether and to what extent the HCl-salt was
inhibiting a proper dissolution in certain solvents. Since vitamin B is based on a pyridine
6

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moiety, a stronger non-nucleophilic base was employed in order to test this hypothesis. Upon
addition of imidazole (1.1 eq.), the solid completely dissolved in dimethyl sulfoxide and
dimethylformamide (DMF), mediocrely in dichloromethane (DCM) and no dissolution was
observed in diethyl ether (Et O), tetrahydrofuran (THF) and toluene (Tol) (Figure 3).
2
Additional imidazole did not result in an improved solubility in the other solvents.
2
2
24
25
Figure 3. Solubility screening of PL·HCl in various solvents with the addition of imidazole (1.1 eq.).
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After finishing the solubility experiments, a preliminary glycosylation was undertaken
in DMSO and DMF according to KOENIGS-KNORR conditions (AgOTf/NIS 3.0 eq., 0 °C).
Hereby, no product formation was detected via ESI-MS in aliquots taken during the reaction
and after work-up. Since the synthesis of the PN-saccharides was successful in
dichloromethane, but an unsatisfying solubility of PL was observed in that solvent during the
screening, a mixture of CH Cl and DMF (v/v = 1/1) was tested in the next approach (Scheme
2
2
5).
O
OAc
AgOTf/NIS3.0 eq.
Imidazole 1.2 eq.
OAc
N
HO
O
O
OH
AcO
AcO
0
°C
AcO
AcO
O
OH
AcO
(CH
2
Cl
2
/DMF)
AcO
N
Cl
Br
H
O
16
17
OH
N
KOH/NaOH
O
HO
HO
O
OH
(
2
ACN/H O)
OH
O
15 % (after
two steps)
21
233

2
2
34
35
Scheme 6. The glycosylation of PL·HCl was undertaken according to KOENIGS-KNORR conditions
with an increased amount of promoter in a solvent mixture.
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250
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255
With this procedure, the product (17) was verified in the reaction mixture via ESI-MS (m/z
+
498.1 [M + H] ). After the work-up, the crude substance was deprotected as described for the
+
PN-saccharides to obtain the desired PLG (m/z 330.0 [M + H] ) in 15 % yield after two steps
starting from PL·HCl. Hereby, in accordance to the reaction outcome of the PN-saccharides,
most losses occurred during the glycosylation step rather than the deprotection steps. A
crucial part influencing the reactivity of this particular glucosylation lies in the solvent
mixture, that had to be applied in order to guarantee the complete dissolution of the starting
materials. Since DMSO and DMF, when used solely as solvent, didn’t lead to a conversion of
starting material, they could have an impact on the overall reactivity and yield. Furthermore,
as results from preliminary experiments stated, pyridine-derivatives with basic properties
exhibit an influence on the reactivity of the promoter. Imidazole, in the same way as PN,
could inhibit the formation of product due to interaction with the promoter. A furtherly
increased addition of promoter didn’t lead to an improvement regarding the reaction
outcome. The mild treatment with NaOH/KOH did not have an impact on the aldehyde
group, leaving it intact during the deprotection procedure.
A singlet at δ = 10.3 ppm and absence of a signal group correlating to a third CH -group in
2
the NMR-spectra affirmed the integrity of the aldehyde-group. A doublet at 4.55 ppm (J =
8.0 Hz) confirmed the selective formation of the β-anomer. The fragmentation pattern of the
product in LC-ESI(+)-MS/MS showed the loss of the sugar moiety (m/z 168) affirming the
structure as well (Figure 4).
2
2
56
57
Figure 4. LC-ESI(+)-MS/MS spectrum of PLG (21, CE = − 26 V).

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This method presents a strategy for the direct synthesis of PLG starting from the
commercially available hydrochloric salt of the vitamer while simultaneously proposing a
way to dissolve the substrate.
A further conceivable route for the preparation of PLG, beside the preparation starting
from the hydrochloric salt, involves the assignment of PNG (18) as the starting substrate
(Scheme 6).
264
2
2
65
66
Scheme 7. An alternative route for the synthesis of PLG led through an additional oxidation step
during the deprotection procedure.
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275
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277
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279
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282
283
Because the aldehyde functionality in 4’-position constitutes the sole difference between
PNG and PLG, the implementation of an oxidation step into the deprotection procedure
presents a convenient way for the preparation of the desired glycoconjugate. Hereby, the
acetonide group was removed by reflux in accordance to the procedure for PN-saccharides
described above unveiling the hydroxyl functionality (19) in 4’-position. Oxidation with
manganese oxide (MnO ) in dichloromethane led to the selective formation of the aldehyde
2
+
group (20), whose integrity was assured by ESI-MS measurements (m/z 498.1 [M + H] ).
Lastly, the acetate groups were cleaved in basic milieu to obtain PLG (21) in 35 % yield after
three steps starting from the protected PNG. Although the yield increased compared to the
synthesis starting from PL at first glance, it has to be noted, that a multistep procedure for the
preparation of PNG preceded the proposed strategy, increasing the overall working effort and
hampering the accessibility of the method. Additionally, the oxidation of PN reportedly
poses a challenging reaction, often accompanied by the transformation into an oxime and
subsequent acid hydrolysis in order to obtain the aldehyde.[101-103] Both methods - direct
glycosylation of PL and oxidation of PNG - were carried out under non-optimized reaction
conditions, and comprise the potential for higher yields. In this regard, other glycosylation
methods involving different protecting- and leaving groups or promoters should be tested for

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288
the direct glycosylation. Additionally, other oxidation reagents like potassium
permanganate[104] can be examined for the multistep method.
So far, the glycosylation of PL was only accomplished at the 3‘-position via
KOENIGS-KNORR
synthesis
utilizing
pyridoxal-monoethylacetal
hydrochloride,
acetobromoglucose and silver carbonate.[105]
1
3
289
2.4. Synthesis of [ C ]-5’-β-PNG
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293
294
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296
Stable isotope dilution analysis (SIDA) marks a unique way of quantifying substrates in
food, since, among other positive features, the loss of analyte during sample work-up can be
exactly compensated for.[106, 107] Although being a promising method, SIDA demands
isotopically labeled substrates, which are not always commercially available and thus have to
be synthesized. Since this study revolved around the search for glycoconjugated derivatives
of vitamin B , the last chapter involved the chemical synthesis of isotopically labeled
6
5’-β-PNG for its application in SIDA.
297
2
2
3
98
99
00
Scheme 8. The synthesis of isotopically labeled 5’-β-PNG (26) was accomplished according to
K
OENIGS-KNORR conditions. a) Ac O, Py, DMAP; b) HBr; c) AgOTf/NIS; d) NaOH/KOH; e) 1 %
2
HCOOH.
301
302
303
304
305
306
307
308
309
310
The synthesis followed the strategy for the PN-saccharides mentioned above. Isotopic
13
labeling was introduced through the glucose moiety starting from [ C ]-glucose (Scheme 7).
6
The preparation of the sugar donor proceeded through acetylation (quant.) using acetic
anhydride (Ac O), pyridine (Py) and 4-dimethylaminopyridine (DMAP) and subsequent
2
bromination (92 %), undertaken with hydrogen bromide (HBr). The glycosidic bond
between the sugar donor and α4,3-O-isopropylidene pyridoxine was established with
AgOTf/NIS (3.0 eq.) as promoter in dichloromethane at 0 °C. The following two-step
deprotection involved the application of NaOH/KOH and 1 % HCOOH to obtain the desired
1
3
5’-β-[ C ]-PNG in 33 % yield after the three steps including glycosylation and deprotection.
6
The structural validity was confirmed through NMR spectroscopy - a doublet of doublets at

+
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
4.26 ppm with a J = 7.7 Hz affirmed the β conformation - and ESI-MS (m/z 338.1 [M + H] ).
Cleaving the glycosidic bond with β-glycosidase from almonds verified the β-selectivity of
the reaction.
The currently favored preparation of pyridoxine glycosides involves the addition of
2
saccharides to sprouting alfalfa seeds and allows the isolation of [ H]- (47 %) and
3
[ H]-β-PNG beside unlabeled PNG (35 - 60 %).[57, 59-62, 64] Although the yield seems
comparingly higher by 10 % at first glance, the utilization of seeds involves the synthesis of
deuterated PN via a five step route[62, 104] beforehand in order to provide the starting
13
material. By utilization of commercially available [ C ]-glucose and the simple strategy of
6
acetylation/bromination in order to prepare the starting materials, the overall costs in
preparation in the presented method are reduced. Additionally, utilizing seeds for the
preparation of glycosides is rather optimized at this point and losses occur mostly during
handling and chromatography[62], whereas the chemical synthesis promises high potential
13
for optimization through variation of the reaction conditions. Furthermore, [ C ]-glucose
6
introduces a higher amount of labelling into the final molecule and hence reduces analytical
errors as a mass spectrometric overlap due to naturally occurring isotopologues is avoided.
3
3
27
28
3. Materials and Methods
3.1. General information
329
330
331
332
333
334
335
Reactions sensitive to air or moisture were carried out in dried glassware under a positive
pressure of argon using standard Schlenk techniques. Solvents were distilled and stored over
molecular sieves prior to use. Chemicals received from commercial sources (Acros,
Sigma-Aldrich, Fluka, Fisher Scientific) were used without further purification unless stated
1
3
otherwise. D-[ C ]-glucose was purchased from Sigma-Aldrich. Detailed preparations of the
6
sugar donors and glycosides can be found in the supplementary information. The optical
rotation was measured on a P3000 polarimeter (Fa. Kruess).
336
3.2. Column chromatography/TLC
337
338
339
340
341
Column chromatography was performed on silica gel 60 (Merck, 230 - 240 mesh) with
the eluent mixtures given for the corresponding procedures. Thin-layer chromatography
(TLC) was performed using silica-coated aluminium plates (silica gel 60). The substances
were detected by UV (λ = 254 nm, 366 nm) or after visualization with CAM (cerium
ammonium molybdate)/potassium permanganate (KMnO ) solution.
4
342
3.3. Nuclear Magnetic Resonance

343
344
345
346
347
348
349
NMR spectra were recorded either on a Bruker AV III system (400 MHz, Bruker,
Rheinstetten, Germany) or on a Bruker AV III system (500 MHz, Bruker, Rheinstetten,
1
13
Germany). H- and C NMR spectra were recorded at 400 or 500 MHz and at 101 or 126
1
13
MHz, respectively. H and C NMR spectroscopic chemical shifts δ are reported in parts per
million (ppm) relative to residual proton signal. All coupling constants (J) are reported in
Hertz (Hz). The following abbreviations were used to explain multiplicities: s = singlet, d =
doublet, dd = doublet of doublets, t = triplet, m = multiplet.
350
3.4. LC-MS/MS
351
352
353
354
355
LC-MS/MS was carried out on a Shimadzu Nexera X2 UHPLC system (Shimadzu,
Kyoto, Japan) with the mobile phase combinations water/acetonitrile or water/methanol. The
injection volume was 1 µL. The LC was interfaced with a triple quadrupole ion trap mass
spectrometer (LCMS-8050, Shimadzu, Kyoto, Japan). Data acquisition was performed with
LabSolutions software 5.80 (Shimadzu, Kyoto, Japan).
356
4. Conclusions
357
358
359
360
361
362
363
364
365
366
367
The motivation to understand the full extent of the involvement of glycoconjugates in
nature has become the driving force to a variety of research fields. Especially in terms of
vitamins, the influence of the respective glycosidic conjugates on the human metabolism
remains not thoroughly explored to this day. Studies revolving around these frontiers in
research show a demand for respective standards, but commercial availability is often the
decisive road block. Hence, this study focused on the chemical synthesis of various
13
5’-β-saccharides of PN and isotopically labeled 5’-β-[ C ]-PNG. Furthermore, strategies to
6
create other B glycosides (e.g. PLG) were illuminated. So far, the main focus in literature
6
lies on the isolation and preparation of PN-saccharides, but data on their content in food is
scarce. The synthesized substrates herein can find their purpose in future experiments
involving analytical surveys of natural samples.
3
68
69
Supplementary Materials: Experimental procedures and NMR-spectra of the new
3
substances are available in the supplementary information.
370
371
372
373
Author Contributions: T.B. and M.R. conceived and designed the experiments; T.B.
performed the experiments and analyzed the data; L.Z. contributed to the synthesis of PLG;
C.S. contributed to the evaluation of the NMRs; T.B. wrote the manuscript; M.R. revised the
manuscript. All authors contributed to the revision of the manuscript.

3
3
74
75
Funding: The study was supported by a grant from the Deutsche Forschungsgemeinschaft
(RY 19/17–1).
376
377
378
Acknowledgments: The authors gratefully acknowledge the support by the Chair of Food
Chemistry and Molecular Science, Technical University Munich, Germany, for the NMR
measurements
3
79
80
Conflicts of Interest: The authors declare no competing financial interests.
3
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644

Chemical synthesis of 5’-β-glycoconjugates of vitamin B6
Highlights
•
The chemical synthesis of the 5’-β-saccharides of pyridoxine, namely the mannoside,
galactoside, arabinoside, maltoside, cellobioside and glucuronide, was accomplished.
New strategies for the chemical glucosylation of pyridoxal were investigated.
•
•
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The chemical synthesis of 5’-β-[ C ]-labeled pyridoxine glucoside (PNG) was done
6
according to KOENIGS-KNORR conditions.

Declaration of interests
☒
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.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: