Practical synthesis of [1-13C]- and [6-13C]-D-galactose

Article abstract of DOI:10.1016/S0040-4039(03)01098-0

The chemical synthesis of ¹³C-labeled D-galactose as useful molecular probes for studying the conformation of oligosaccharides attached to proteins was performed. The method for synthesizing the title labeled compounds was newly developed via t

Full text of DOI:10.1016/S0040-4039(03)01098-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 4903–4907
Practical synthesis of [1-13C]- and [6-13C]-
-galactose
D
Ken-ichi Sato,* Shoji Akai, Mayumi Sakuma, Masaru Kojima and Ken-ju Suzuki
Laboratory of Organic Chemistry, Faculty of Engineering, Kanagawa University, 3-27-1, Rokkakubashi, Kanagawa-ku,
Yokohama 221-8686, Japan
Received 3 March 2003; revised 23 April 2003; accepted 25 April 2003
Abstract—The chemical synthesis of 13C-labeled
oligosaccharides attached to proteins was performed. The method for synthesizing the title labeled compounds was newly
developed via the corresponding 1-ene and 5-ene compounds derived from 1,2:5,6-di-O-isoproppylidene-a- -galactofuranose by
D-galactose as useful molecular probes for studying the conformation of
D
considering the efficient introduction of the atom. All protons of galactose from H-1 to H-6 were observed by the HMQC–
HOHAHA technique using 1:1 mixtures of methyl [1-13C]- and [6-13C]-b-
D-galactopyranoside, which were prepared from the title
compounds. © 2003 Elsevier Science Ltd. All rights reserved.
Sialyl glycoconjugates are important cell surface com-
ponents active in a variety of intercellular recognition
events. Therefore, a study of the conformation and
dynamics of these sialyloligosaccharides and their
analogs should provide useful insights into how these
cell surface oligosaccharides interact with the corre-
sponding receptor molecules. However, although the
conformational properties1,2,3a–c of sialyloligosaccha-
ride analogs of low molecular weight have been
reported by many research groups, the conformation
and dynamics of sialyloligo-saccharides and their
analogs attached to glycoproteins have not yet been
fully analyzed. To address this problem, 13C-labeled
sialic acid (NeuAc) has been utilized for the conforma-
tional analysis of sialyloligosaccharides on artificial
membrane surfaces4a,b and TRNOE experiments.5
Recently, novel NMR techniques, HSQC–TOCSY–
NOESY–TOCSY, for observation of all protons of
NeuAc, H-3 to H-9, even with only a single 13C-atom at
the 3-position of NeuAc, bound to a glycoprotein are
reported.2 However, since combined multi-pulse tech-
niques generally suffer from low sensitivity, the authors
have synthesized minimal [3,9-13C]-labeled NeuAc for
convenient observation of all protons of NeuAc from
H-3 to H-9 by the HMQC–HOHAHA technique. The
authors have also synthesized [3-13C]- and [9-13C]-
labeled NeuAc, respectively, and demonstrated that
identical results are obtained by NMR for [3,9-13C]-
NeuAc and 1:1 mixtures of [3-13C]- and [9-13C]-NeuAc.6
It is generally accepted that it is rather difficult to
detect 1H information from [U–13C]-sugars, in high
sensitivity, because of 13C–13C couplings and long-range
couplings. This minimal and efficient 13C-labeling
method promised to enable us to prepare practical
amounts of important 13C-labeled component monosac-
charides, such as NeuAc, KDN, galactose, mannose,
mannosamine, and fucose for studying how the func-
tions and roles of cell surface sialyl and KDN oligosac-
charides intact with the corresponding receptor
molecules.
In this paper, a practical synthesis of [1-13C]- and
[6-13C]-
D-galactose and the labeling effect of 1:1 mix-
tures of the galactoses by the HMQC–HOHAHA tech-
nique are described. 13C is preferably introduced at the
last stage for saving the labeled compound, 13CH3I,
that was commercially available. The introduction of
13C was performed by extension of either end of pen-
toses derived from the same intermediate, 1,2:5,6-di-O-
isopropylidene-a-
D-galactofuranose with phosphonium
salt (Ph3P13CH3I).
Synthesis of [1-13C]-
D
-galactose from
D
-glucose
[1-13C]-
D
-Galactose was synthesized from D-glucose as
follows (Scheme 1). 1,2:5,6-Di-O-isopropyridene-a-D-
galctofuranose (3) was synthesized by the modified
Weygand method.7 That is, the starting compound
3-O-triflate 1 instead of 3-O-tosylate was derived from
1,2:5,6-di-O-isopropyridene-a-
D
-glucofuranose
(1)
quantitatively. Compound 1 was treated with DBU/
DMSO to give the elimination compound 2 in 98%
yield. The hydroboration–oxidation reaction of 2 gave
Keywords: carbohydrates; labeling; NMR.
* Corresponding author. Tel.: +81-45-481-5661, ext. 3853; fax: +81-
45-413-9770; e-mail: satouk01@kanagawa-u.ac.jp
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)01098-0

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K. Sato et al. / Tetrahedron Letters 44 (2003) 4903–4907
Scheme 1. Reagents and conditions: (a) DBU/DMSO; (b) BH3–THF, then NaOH aq., H2O2/THF; (c) 70% AcOH aq.; (d) NaH,
BnBr/DMF; (e) 85% AcOH aq.; (f) NaIO4/MeOH–H2O, then 1 M HCl aq.; (g) isopropenylmethylether, PPTS; (h) Ph3P13CH3I,
n-BuLi/THF, then AcOH aq.; (i) NaH, MOMCl/DMF; (j) m-CPBA/C2H4Cl2; (k) (i) 1 M KOH aq./DMSO; (ii) TEMPO,
trichloroisocyanuric acid/CH2Cl2, (iii) CSA/CH2Cl2; (l) 10% Pd–C, H2/EtOH–H2O; (m) 1 M KOH aq./DMSO; (n) BzCl/Py., then
MsCl; (o) 1 M KOH aq./MeOH.
the corresponding 3 in 93% yield. The partial hydrolysis
of 3 at rt gave 1,2-O-isopropylidene-a- -galactofura-
respectively. The required epoxide 12 was hydrolyzed
with 1 M KOH aq. in DMSO to give the 1,2-di-ol
compound in 99% yield. The primary hydroxyl group
of the product was then selectively oxidized with
D
nose (4) in 87% yield. Compound 4 was then benzylated
with NaH, BnBr in DMF to give the tri-O-benzylated
compound 5 in 94% yield. The hydrolysis of 5 at 70°C
gave 6 in 96% yield. Periodate oxidation of 6 with
NaIO4 and 1 M HCl in MeOH–H2O gave the corre-
sponding pentose derivative 7 quantitatively. The
hydroxyl group of the unstable carbonyl compound 7
was protected with MIP (methoxyisopropyl) by using
isopropenyl methyl ether and a catalytic amount of
PPTS at 0°C to give 8 in 95% yield. The Wittig reaction
of 8 with labeled phosphonium salt Ph3P13CH3I (1.0
equiv.), which was quantitatively synthesized from
13CH3I, and treating with 30% AcOH aq. gave labeled
hexose derivative 9 in 72% yield. The MIP protecting
group proved to be suitable for this carbonyl com-
pound and the reaction conditions. The product was
too unstable, even to a column of silica gel. We treated
the resulting mixture of the Wittig reaction with acid
(30% AcOH aq.) to obtain a single deprotected (MIP)
compound 9. Re-protection of 9 with an MOM group,
which could not be introduced to 7, gave 10 in 90%
yield. Peracid oxidation of 10 with m-CPBA in 1,2-
dichloroethane at 60°C gave the corresponding
epimeric epoxides 11 and 12 in 33 and 52% yields,
2,2,6,6-tetramethyl-1-piperidinyloxy,
a
free radical
(TEMPO7) and trichloroisocyanuric acid in CH2Cl2 to
give the galactose derivative in 90% yield, which, in
turn, afforded the corresponding galactofuranose
derivative 13 in 90% yield by treatment with ( )-10-
camphorsulfonic acid (CSA) in CH2Cl2 (three steps,
80% yield). Removal of the benzyl group of 13 by
catalytic reduction with 10% Pd–C, H2 in EtOH–H2O
gave [1-13C]-galactose quantitatively. It is also possible
to obtain 13 from 12 directly by treatment with BF3-
Et2O, DMSO,8 albeit in lower yield (68%). On the other
hand, conversion of the diastereomeric epoxide 11 to 12
was possible for saving the labeling compound as fol-
lows. Compound 11 was hydrolyzed to give the corre-
sponding 1,2-di-ol derivative 14, in 98% yield, by
treatment with 1 M KOH aq./DMSO at 70°C. Selective
benzoylation and mesylation of each hydroxyl group of
14 gave 15 in 88% yield. Compound 15 was then
treated with 1 M KOH aq./MeOH to give epoxide 12 in
91% yield. A direct acylation and mesylation of 11 gave
unsatisfactory results. As a result of this synthesis, the
percentage of the utilization of 13CH3I was 40%.

K. Sato et al. / Tetrahedron Letters 44 (2003) 4903–4907
4905
Synthesis of [6-13C]-
[6-13C]-
-Galactose was synthesized from
D
-galactose from
D
-glucose
NMR data for the non-labeled and 13C-labeled galac-
toses are shown in Figure 1. In order to measure the
HMQC–HOHAHA spectra of a 1:1 mixture of [1-13C]-
D
D
-glucose as
follows (Scheme 2). The same intermediate 3 used for
and [6-13C]-
labeled
ing methyl b-
O-acetyl-a-
-galactopyranosyl trichloroacetimidate.10a,b
NMR data for the HMQC–HOHAHA spectra of a 1:1
-galacto-
D
-galactose as a single isomer, both 13C-
the synthesis of [1-13C]-
D
-galactose was benzylated to
D
-galactoses were converted into its correspond-
-galactopyranoside via 2,3,4,6-tetra-
give 16 quantitatively. Compound 16 was then partially
hydrolyzed to give di-ol compound 17, which was
quantitatively converted into the corresponding alde-
hyde 18 by oxidation with NaIO4. The introduction of
13C was achieved by the Wittig reaction of 18 and
Ph3P13CH3I (1.0 equiv.) to give the labeled olefin com-
pound 19 in 74% yield. Peracid oxidation of 19 with
m-CPBA in a similar manner as mentioned above gave
epimeric epoxides 20 and 21 in 58 and 25% yields,
respectively. Hydrolysis of an epoxide 21 with 1 M
KOH aq./DMSO gave the corresponding di-ol 22 in
90% yield. Galactofuranose derivative 22 was then
D
D
mixture of methyl [1-13C]- and [6-13C]-b-
D
pyranoside are shown in Figure 2, which were prepared
from 13C-labeled galactoses for convenient measure-
ment as a single compound.
As described above, a practical synthesis of minimally
13C-labeled
D-galactose should facilitate studies on the
conformational properties and dynamic behavior of
oligosaccharides that contain galactose. This short and
efficient 13C-labeling method may also be extended to
other important component monosaccharides and the
oligosaccharides derived from these labeled monosac-
charides reacted with the corresponding receptor
molecules to determine the mechanism of interaction.
hydrogenolyzed and hydrolyzed to obtain [6-13C]-
D-
galactose. The yield was 96% (two steps). On the other
hand, the undesired epoxide 20 can be converted into
the desired 21 as follows.
Compound 20 was hydrolyzed to give the correspond-
ing 1,2-di-ol derivative 23, in 89% yield, by treatment
with 1 M KOH aq./DMSO at 70°C. Selective benzoyla-
tion and mesylation of each hydroxyl group of 23 gave
24 in 78% yield. Compound 24 was then treated with 1
M KOH aq./MeOH to give the epoxide 21 in 95%
yield. This procedure brings the percentage of utiliza-
tion of 13CH3I to 40%. Compounds 20 and 21 should
also be useful 13C-labeled precursors for synthesizing
analogs to study the functional properties of
oligosaccharides.
[1-13C]- and [6-13C]-
a-
D
-galactose were synthesized from
D
-glucofuranose derivative 1 in 17 steps (total yield,
27%) and 13 steps (total yield, 32%), respectively. Our
new work promises to be an effective method to obtain
gram-scale 13C-labeled galactoses.
Acknowledgements
This work was partially supported by a ‘High-Tech
Research Center Project’ of Ministry of Education,
Science, Sports and Culture, Japan and a Grant-in-Aid
for Scientific Research from the Ministry of Education,
Science, Sports and Culture, Japan.
1H and 13C NMR data for each 13C-labeled compound,
[1-13C]- and [6-13C]-
D
-galactose, are given in Ref. 9. H
1
Scheme 2. Reagents and conditions: (p) BnBr, NaH/DMF; (q) 70% AcOH aq.; (r) NaIO4/MeOH–H2O; (s) Ph3P13CH3I,
n-BuLi/THF; (t) m-CPBA/C2H4Cl2; (u) 1 M KOH aq./DMSO; (v) BzCl, Py., then MsCl; (w) n-Bu4NOH/THF; (x) 1 M KOH
aq./DMSO; (y) 10% Pd–C, H2/EtOH; (z) Dowex H+ form/H2O.

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K. Sato et al. / Tetrahedron Letters 44 (2003) 4903–4907
Figure 1. 1H NMR data of non-labeled and 13C-labeled
D
-galactoses (a:i=35:65).
Figure 2. HMQC-HOHAHA spectra of a 1:1 mixture of methyl [1-13C]- and [6-13C]-b-
D-galactopyranoside.
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