Synthesis, separation and NMR spectral analysis of methyl apiofuranosides

Article abstract of DOI:10.1016/S0008-6215(98)00262-6

Methyl apiofuranosides were prepared by methanolysis of D-apiose. Four methyl apiofuranosides were separated by ion-exclusion and normal phase chromatography and their structures were characterized by ¹H and ¹³C NMR spectroscopy. The composition of methyl apiofuranosides was almost the same as that of d-apiose in D₂O. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(98)00262-6

Carbohydrate Research 313 (1998) 189–192
Synthesis, separation and NMR spectral analysis of
methyl apiofuranosides
Tadashi Ishii a,*, Masayuki Yanagisawa b
a Forestry and Forest Products Research Institute, P.O. Box 16, Tsukuba Norin Kenkyu Danchi-nai,
Ibaraki 305-8678, Japan
b Shimadzu Analytical and Measuring Center, 1, Kuwaharamachi, Nishinokyo, Nakagyoku, Kyoto 604, Japan
Received 25 May 1998; revised 8 September 1998; accepted 20 September 1998
Abstract
Methyl apiofuranosides were prepared by methanolysis of
D-apiose. Four methyl apiofuranosides were separated
1
by ion-exclusion and normal phase chromatography and their structures were characterized by H and 13C NMR
spectroscopy. The composition of methyl apiofuranosides was almost the same as that of
Elsevier Science Ltd. All rights reserved.
D-apiose in D2O. © 1998
Keywords: Apiose; 3-C-(Hydroxymethyl)-D-glycero-aldotetrose; Methyl apiofuranosides
1. Introduction
diastereoisometers [bis(b - D - apiofuranosyl) -
(R)-2,3:2,3 and -(S)-2,3:2,3-borate] since they
contain a chiral boron [5]. Since the chirally
induced chemical shift differences of 11B
NMR spectra are smaller than the linewidth
of the 11B signals, it is difficult to distinguish
diastereoisomers by 11B NMR spectroscopy.
The branched-chain aldopentose, D-apiose
[3-C-(hydroxymethyl)-D-glycero-aldotetrose],
plays an integral role in the biochemistry of
plants [1]. It is one of the components of
rhamnogalacturonan II (RG-II) in plant cell
walls [2]. Recent studies have revealed that
RG-II combines with boron (B) to form a
borate diol di-ester (RG-II–borate complex)
[3–7]. Glycosyl-linkage analysis indicated
that the apiosyl residues of RG-II were the
sites of borate esterification [5,7,8]. However,
determination of the borate-binding apiosyl
residues in the RG-II–B complex is compli-
cated by the fact that the RG-II–B complex
contains four apiosyl residues. The borate–
apiose (1:2) esters can exist in either of two
1
The H and 13C spectra of the RG-II–B
complex are too complicated to assign.
Snyder and Serianni [9] synthesized stable
isotope-labeled DL-apioses and characterized
the tautomeric mixture in water solution by
NMR spectroscopy. We prepared methyl
apiofuranosides as model compounds to
study the stereochemistry of the borate–
apiose complex. We now report the separa-
tion of four methyl apiofuranosides by
ion-exclusion and normal phase chromatog-
1
raphy and their characterization by H and
* Corresponding author. Tel.: +81-298-733211; fax: +81-
298-733795; e-mail: tishii@ffpri.affrc.go.jp
13C NMR spectroscopy.
0008-6215/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(98)00262-6

190
T. Ishii, M. Yanagisawa / Carbohydrate Research 313 (1998) 189–192
Fig. 1. Structures of compounds 1–4.
2. Experimental
3. Results and discussion
General methods.—1,2;3,3%-Di-O-isopropyl-
idene-3-C-(hydroxymethyl)-a-D-erythrofura-
nose was purchased from Sigma Chemicals.
Methanol–hydrogen chloride solution (5%
w/w) and silver carbonate were purchased
from Wako Pure Chemicals. 1H and 13C
NMR spectra were recorded at 30 °C with a
JEOL ALPHA 500 FT MNR (500 MHz),
using D2O as solvent.
Separation.—The treatment of D-apiose with
methanolic hydrogen chloride generated the
four methyl glycosides, which were separated by
ion-exclusion and normal phase chromatogra-
phy. The four glycosides were initially separated
into three fractions by ion-exclusion chro-
matography. Based on the evidence given be-
low, the components of Fractions 1 and 3 were
identified as methyl 3-C-(hydroxymethyl)-a- -
L
Preparation of methyl apiofuranosides.—
1,2;3,3%-Di-O-isopropylidene-3-C-(hydroxy-
methyl)-a-D-erythrofuranose (100 mg) was
hydrolyzed with 2 M CF3CO2H (10 mL) for
1 h at 100 °C. CF3CO2H was removed by
evaporation under dry air at 40 °C. Iso-
propanol (200 mL) was then added and the
residual acid was co-evaporated at 40 °C un-
der dry air.
D-Apiose (about 50 mg) was subjected to
methanolysis with 5% HCl in MeOH (2 mL)
at 100 °C for 16 h. Hydrogen chloride was
subsequently neutralized by adding silver
carbonate and the resulting silver chloride
precipitate was removed by centrifugation.
The supernatant was concentrated to a
syrup.
Separation of methyl apiofuranosides.—
Methyl apiosides were separated using a Shi-
madzu LC-6A HPLC equipped with a
refractive index detector. The initial separa-
tion was carried out with a Shim-pack SCR-
101P (lead form) (300×7.9 mm) column
equilibrated at 0.7 mL/min in H2O at 80 °C,
yielding three fractions (Fr. 1, 12.8 min; Fr.
2, 14.9 min; Fr. 3, 31.6 min). Fraction 2 was
further separated with a Asahi-pack NH2P50
column (250×4.6 mm) eluted with 97.5 (v/
v)% CH3CN at 0.5 mL/min at room temper-
ature, yielding two additional fractions (Fr.
2-1, 21.0 min; Fr. 2-2, 24.2 min).
threofuranoside (4) and methyl 3-C-(hydroxy-
methyl)-a-D-erythrofuranoside (1), respective-
ly. Fraction 2 contained a mixture of methyl
3-C-(hydroxymethyl)-b-D-erythrofuranoside
(2) and 3-C-(hydroxymethyl)-b- -threofura-
L
noside (3). These two methyl glycosides were
separated by normal phase chromatography,
yielding two fractions (2-1 and 2-2), whose
constituents were identified as methyl 3-C-(hy-
droxymethyl)-b- -threofuranoside (3) and
L
methyl 3-C-(hydroxymethyl)-b-D-erythrofura-
noside (2), respectively (Fig. 1).
1H and 13C NMR spectroscopy.—The pro-
ton chemical shifts of each methyl glycoside
were assigned by 2D HOHAHA (Table 1).
The protons of the hydroxymethyl group in
1 and 2 are equivalent, while those of 3 and
4 are nonequivalent. This may be due to
constrained rotation of the exocyclic hydro-
xymethyl group in 3 and 4 due to intra-
molecular hydrogen bonding between OH-2
and the hydroxymethyl group. Apiofurano-
side ring configuration of 1, 2, 3 and 4 was
confirmed by comparing single, vicinal
1H–1H scalar coupling constants (3J1,2) with
those reported for methyl apiosides and DL
-
3
apioses [9,10] and NOE. The values of J1,2
for 1 (4.9 Hz) and 3 (4.3 Hz) indicated that
H-1 and H-2 are cis. When H-1 and H-2 are
3
trans, J1,2 is usually close to 1 Hz, indicat-
ing that both oxygen atoms take up quasi-

T. Ishii, M. Yanagisawa / Carbohydrate Research 313 (1998) 189–192
191
Table 1
1H chemical shifts of methyl apiofuranosides in D2O
Compound
Chemical shift (ppm)a
H-1
H-2
H-3%ab
H-3%bb
H-4ab
H-4bb
OCH3
1
2
3
4
5.01 (4.9)c
4.99 (3.7)
5.20 (4.3)
4.97 (1.2)
4.02
3.95
4.13
4.08 (1.2)
3.62
3.66
3.71 (12.2)
3.70 (12.2)
3.62
3.66
3.77
3.83
3.91 (10.4)
3.91 (10.4)
3.77 (9.8)
3.9. (9.8)
4.06
4.06
4.00
4.03
3.45
3.46
3.48
3.43
a Relative to external acetone (l 2.23) in D2O.
b Protons of the hydroxymethyl group and attached to C-4 (Fig. 1) were numbered as H-3%a and H-3%b, and H-4a and H-4b,
respectively.
c Values in parenthesis are JHH observed at the indicated sites.
Table 2
13C chemical shifts of methyl apiofuranosides in D2O
Compound
Chemical shift (ppm)a
C-1
C-2
C-3
C-3%
C-4
OCH3
1
2
3
4
103.96
110.21
105.38
111.15
72.53
77.31
77.66
80.68
77.65
80.09
81.89
81.85
65.15
64.25
63.56
63.12
74.38
74.32
73.70
75.34
56.21
56.78
56.78
56.10
a Values are referenced to the carbon signal of external d4-methanol (49.7 ppm).
3
respectively. These assignments confirmed the
structure of methyl 3-C-(hydroxymethyl)-a-
and b-D-erythrofuranosides and methyl 3-C-
axial positions. Compound 4 has a J1,2 value
of 1.2 Hz, confirming its a-configuration.
3
However, J1,2 for 2 is 3.7 Hz, and Angyal et
al. [10] reported that J1,2 for methyl 3-C-(hy-
3
(hydroxymethyl)-a- and b- -threofuranosides.
L
The proportions of the methyl apiofura-
nosides were 20% 1, 53% 2, 7% 3 and 20% 4
as determined by 1H NMR spectroscopy.
These proportions are almost the same as that
of D-apiose in D2O at equilibrium, as deter-
droxymethyl)-b-D-erythrofuranoside is 3.5 Hz.
The 2D NOESY spectrum of 2 contains cross
peaks between H-2 and the protons of the
hydroxymethyl group, and H-2 and H-4b, in-
dicating that H-2, the hydroxymethyl group
and H-4b are found on the same face of the
ring for this compound and confirming its
structure to be methyl 3-C-(hydroxymethyl)-
b-D-erythrofuranoside (2). Similar NOE cross
peaks were observed between H-2 and the
protons of the hydroxymethyl group in the
spectrum of 1, indicating that H-2 and the
hydroxymethyl group are on the same side,
confirming the structure of 1 to be methyl
3-C-(hydroxymethyl)-a-D-erythrofuranoside
(1). The ring structures of 3 and 4 were confi-
rmed by 2D NOESY spectroscopy in the same
way as described above. The 13C signals were
assigned by HMQC (Table 2). The signals at l
103.96, 110.21, 105.38 and 111.15 are assigned
to the anomeric carbons of 1, 2, 3 and 4,
1
mined by H NMR spectroscopy [9].
The separation of the four methyl apiofura-
nosides described here is simple and useful for
preparing apiose derivatives.
Acknowledgements
The authors thank Drs H. Ono (National
Food Research Institute, Tsukuba, Ibaraki)
and W.S. York (Complex Carbohydrate Re-
search Center, University of Georgia, Athens,
GA) for useful comments and critical reading
of the manuscript, respectively. This research
was supported by funds from the Ministry of
Agriculture, Forestry and Fisheries (Glyco-

192
T. Ishii, M. Yanagisawa / Carbohydrate Research 313 (1998) 189–192
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