Chemoenzymic approaches to the preparation of 5-C- (hydroxymethyl)hexoses

Article abstract of DOI:10.1021/jo970232p

Synthesis of 5-C-(hydroxymethyl)hexoses, carbohydrates resistant to metabolism, is described. These compounds are obtained in the reaction of hexose 6-aldehydes with formaldehyde. 5-C-(Hydroxymethyl)-L-arabino- hexopyranoses can be efficiently obtained from D-galactosides by a two-step chemoenzymic synthesis using galactose oxidase for the preparation of required hexose 6-aldehydes. This method is an example of carbohydrate synthesis without use of protecting groups. Other 5-C-(hydroxymethyl)hexoses are prepared by a typical chemical methodology requiring specific protection of the hexose hydroxyl groups.

Full text of DOI:10.1021/jo970232p

J . Org. Chem. 1997, 62, 4471-4475
4471
Ch em oen zym ic Ap p r oa ch es to th e P r ep a r a tion of
5-C-(Hyd r oxym eth yl)h exoses
Adam W. Mazur* and George D. Hiler, II
Procter & Gamble Pharmaceuticals, Health Care Research Center, P.O. Box 8006,
Mason, Ohio 45040-8006
Received February 10, 1997X
Synthesis of 5-C-(hydroxymethyl)hexoses, carbohydrates resistant to metabolism, is described.
These compounds are obtained in the reaction of hexose 6-aldehydes with formaldehyde. 5-C-
(Hydroxymethyl)-L-arabino-hexopyranoses can be efficiently obtained from D-galactosides by a two-
step chemoenzymic synthesis using galactose oxidase for the preparation of required hexose
6-aldehydes. This method is an example of carbohydrate synthesis without use of protecting groups.
Other 5-C-(hydroxymethyl)hexoses are prepared by a typical chemical methodology requiring specific
protection of the hexose hydroxyl groups.
In tr od u ction
5-C-(Hydroxymethyl)hexoses constitute an interesting
group of sugars that resist metabolism by mammalian
enzymes and by intestinal anaerobic bacteria. The
additional hydroxymethyl group present at the hexose
C-5 appears to not only prevent degradation of the hexose
backbone but also inhibit cleavage of the glycosidic
linkages in simple glycosides and in disaccharides.1 Due
to these properties, 5-C-(hydroxymethyl)hexose deriva-
tives have been proposed as components of foods2 and
pharmaceuticals.3
This report describes a methodology used to introduce
the hydroxymethyl group at the 5-carbon in hexoses. A
general scheme for hydroxymethylation of carbohydrates
at the penultimate carbon is shown in Figure 1. The
primary alcohol group in hexose is converted first into
the aldehyde and then subjected to an aldol-type con-
densation with formaldehyde. The hydroxymethyl sugar
aldehyde, initially formed in condensation, undergoes a
spontaneous Canizzaro reduction with the excess of
formaldehyde to give the (hydroxymethyl)hexose. The
hydroxymethylation procedure was first applied to car-
bohydrate transformations by by Schaeffer4 and later
used to make a number of 2-C- and 4-C-(hydroxymeth-
yl)pentose and -hexose derivatives in the syntheses of
natural products such as D- and L-apioses5 and D-
hamamelose.6 Hydroxymethylation of C-2 in D-mannose
derivatives produced branched alditols proposed as ar-
tificial sweeteners.7 The hydroxymethylation of pentose-
1,5-dialdoses led to the preparation of 4-C-(hydroxyme-
thyl)pentose-based aminosugars8 and nucleosides.9-11
Interestingly, syntheses of 5-C-(hydroxymethyl)hexoses
have not been reported in the literature prior to our
research.
F igu r e 1. Hydroxymethylation of the R-carbon in aldehydes.
The availability of hexose 6-aldehydes is an important
factor in the synthesis of 5-C-(hydroxymethyl)hexoses. In
general, these aldehydes can be obtained by oxidation of
the hexose primary alcohol group12 or azide13 or periodate
oxidation of heptosides.14 Also, a reduction of glucuronic
acid ester to gluco-1,5-dialdohexose via organoboron
intermediates has been reported.15 We will describe two
approaches to the synthesis of 5-C-(hydroxymethyl)hex-
oses. One method is based on a traditional chemical
synthesis and requires the use of protecting groups to
selectively oxidize the hexose 6-hydroxyl to the corre-
sponding aldehyde. The second method greatly simplifies
synthesis of the title compounds. It utilizes enzyme
galactose oxidase, which allows for carrying out this
oxidation as well as subsequent hydroxymethylation on
the unprotected carbohydrate in the aqueous solution.
However, the latter method is currently limited to the
galactose-containing carbohydrates due to the enzyme
specificity for this sugar structure.
Resu lts a n d Discu ssion
For the chemical oxidations of the primary hydroxyl
group in hexoses having D-gluco, D-manno, and D-fructo
configurations, we have chosen pirydinium dichromate16
(Figure 2) and the Swern reagent17 (Figure 3). Since
these oxidations do not discriminate between the unpro-
tected primary vs secondary hydroxyl groups, the hexose
X Abstract published in Advance ACS Abstracts, J une 15, 1997.
(1) Mazur, A. W.; Mohlenlamp, M. J .; Hiler, G. D., II; Wilkins, T.
D.; Van Tassell, R. L. J . Agric. Food Chem. 1993, 41, 1925.
(2) Mazur, A. W. US 5,106,967.
(3) Mazur, A. W.; Pikul, S.; Daggy, B. US 5,591,836.
(4) Schaffer, R. J . Am. Chem. Soc. 1959, 81, 5452.
(5) Ho, P.-T. Can. J . Chem. 1979, 57, 381.
(12) Singh, S.; Nambiar, S.; Porter, R. A.; Sander, T. L.; Taylor, K.
G. J . Org. Chem. 1989, 54, 2300. Dziewiszek, K.; Zamojski, A.
Carbohydr. Res. 1986, 150, 163. Garegg, P. J .; Samuelsson, B.
Carbohydr. Res. 1978, 67, 267. Ward, D. J .; Szarek, W. A.; J ones, J .
K. N. Carbohydr. Res. 1972, 21, 305. Godman, J . L.; Horton, D.
Carbohydr. Res. 1967, 6, 229.
(6) Ho, P.-T. Tetrahedron Lett. 1978, 19, 1623.
(7) Witczak, Z. J .; Whistler, R. L. Carbohydr. Res. 1987, 169, 252.
(8) Wright, J . J .; Luce, C. H. J . Org. Chem. 1978, 43, 1968.
(9) Leland, D. L.; Kotick, M. P. Carbohydr. Res. 1974, 38, C9.
(10) Youssefyeh, R. D.; Verheyden, J . P. H.; Moffatt, J . G. J . Org.
Chem. 1979, 44, 1301.
(11) J ones, G. H.; Taniguchi, M.; Tegg, D.; Moffatt, J . G. J . Org.
Chem. 1979, 44, 1309.
(13) Clode, D. M.; Horton, D. Carbohydr. Res. 1971, 17, 365. Horton,
D.; Luetzow, A. E.; Wease, J . C. Carbohydr. Res. 1968, 8, 366.
(14) Petterson, B.; Theander, O. Acta Chem. Scand. B 1974, 21, 29.
(15) Koster, R.; Idelmann, P.; Dahlhoff, W. V. Synthesis 1982, 82,
650.
(16) Andersson, F.; Samuelsson, B. Carbohydr. Res. 1984, 129, C1.
(17) Amura, K.; Swern, D. Tetrahedron 1978, 34, 1651.
S0022-3263(97)00232-6 CCC: $14.00 © 1997 American Chemical Society

4472 J . Org. Chem., Vol. 62, No. 13, 1997
Mazur and Hiler
galactose oxidase (GOase).20 This is the only known
enzyme allowing for conversion of the hexose C-6 carbon
bearing the primary hydroxyl group into the aldehyde
functionality. Galactose oxidase has been known for over
30 years21 and used extensively in biochemical analysis.20
Yet, attempts to apply GOase-catalyzed oxidation in the
carbohydrate synthesis were in the past only partially
successful, and consequently, GOase was not used in
practical synthesis. Low levels of substrate conversion22
and simultaneous formation of galacturonic acid23-25 were
reported to be the key limitations for the synthetic
applications of the enzyme. These results were construed
as indicating that the synthetic utility of GOase might
be limited by the intrinsic catalytical properties of the
protein. In contrast, we have found that GOase allows
for efficient oxidation of galactose-containing carbohy-
drates to the corresponding 6-aldehydes if the enzyme is
properly purified.20,26 In our hands, the oxidation of
D-galactose-based carbohydrates has been almost quan-
titative (>95% conversion) and practically has not created
by products. In particular, we have not detected (by C-13
NMR) formation of galacturonic acid. Obtained alde-
hydes (1,5-dialdoses) have been identified by 13C NMR
of lyophilized samples from crude oxidation mixtures.
Purification of galactose 6-aldehydes is difficult because
they have a tendency, particularly upon drying, to form
impurities that are probably intermolecular acetal struc-
tures.27 In order to minimize these side reactions, the
crude solution of the oxidation product was used directly
for the condensation with formaldehyde (Figure 5). In
contrast with the hydroxymethylation of 2, we have not
detected the retro-Michael elimination during the con-
densation of the unprotected hexodialdo-1,5-pyranoses.
This result is not surprising since the unprotected
aldehydes do not contain a good leaving group at the
â-carbon. On the other hand, there was a possibility that
the hydroxyl group at the â-carbon might epimerize. This
phenomenon was detected in the hydroxymethylation of
pentofuranose 5-aldehydes11 as a result of a reversed
aldol cleavage of the bond between the aldehyde R- and
â-carbon atoms in the initial carbohydrate aldehyde-
formaldehyde adduct. A careful examination of a model
condensation leading to 24 (Figure 5) revealed the
presence of only 1% of the C-4 epimer. This result
indicates that the stereochemistry at C-4 is highly
conserved in this reaction compared to hydroxymethy-
lation of pentofuranose 5-aldehydes.
F igu r e 2. Chemical synthesis of 5-C-(hydroxymethyl)-L-
arabino-hexopyranose derivatives.
substrates had to be selectively protected. Consequently,
we have prepared protected hexose 6-aldehydes from 1,2:
3,4-di-O-isopropylidene-R-D-galactose (1), methyl 2,3,4-
tri-O-benzyl-R-D-glucopyranoside (5), 1,2,3,4-tetra-O-
benzyl-R-D-mannopyranoside (6), and methyl 1,3,4-tri-O-
benzyl-R-D-fructofuranoside (13). Compounds 5 and 6
were obtained in three steps from D-glucose and benzyl
R-D-mannoside, respectively, by known procedures.18 The
synthesis of 13 consisted of seven steps from sucrose by
a method analogous to one used by Szarek et al.19
Condensation of 1,2:3,4-diisopropylidene-R-D-galacto-
hexodialdo-1,5-pyranose (2) with formaldehyde (Figure
2) is accompanied by replacement of the 3,4-isopropy-
lidene with the methylene group. The exchange mech-
anism is believed to involve a retro-Michael elimination
of the acetone molecule from the aldehyde followed by a
Michael addition of formaldehyde to the resulting R,â-
unsaturated aldehyde (Figure 4). The major product of
this reaction was 5-C-(hydroxymethyl)-1,2-isopropylidene-
3,4-methylene-5-C-(hydroxymethyl)-â-L-arabino-hexopy-
ranose (3), while 1,2:3,4-di-O-isopropylidene-5-C-(hy-
droxymethyl)-â-L-arabino-hexopyranose (4) was only a
minor product with a 10:1 ratio of 3 to 4. The presence
of a 3,4-methylene group in 3 was confirmed by a
characteristic carbon-13 resonance at 94.4 ppm, while the
signals from the isopropylidene acetal carbons in 4 were
observed at 108 ppm. Also, proton NMR of 3 showed two
signals at 4.95 and 4.68 ppm, each corresponding to one
geminal proton from the methylene group. A similar
exchange of acetal groups has been noticed by others
during the reaction of 2,3-O-isopropylidene-â-D-ribo-
pentodialdofuranose,9 although the reported ratio of 2,3-
O-methylene vs 2,3-O-isopropylidene derivatives was
only 1:9. The difference in the ratios of products in these
two reactions may reflect a different degree of competi-
tion between the retro-Michael reactions of hexodial-
dopyranose and pentodioaldofuranose and their conden-
sations with formaldehyde. In our case, the complete
conversion of 2 requires about 4 h, which seems to be a
relatively slow process compared to 30 min reported for
the hydroxymethylation of 2,3-O-isopropylidene-â-D-ribo-
pentodialdofuranose.10 Clearly, the slower condensation
increases the probability of the acetal exchange.
All the transformations of protected sugars, that is
oxidation and subsequent condensation of aldehydes to
5-C-(hydroxymethyl) derivatives, can be conveniently
followed by TLC since the substrates and the products
have distinctive Rf values on the normal-phase silica
plates. In enzymic oxidation of unprotected sugars,
however, we found TLC techniques to be less reliable
(20) Mazur, A. W. Galactose Oxidase. Selected Properties and
Synthetic Applications. In Enzymes in Carbohydrate Synthesis; Bed-
narski, M. D., Simon, E. S., Eds.; ACS Symposium Series 466; 1991;
American Chemical Society, Washington, DC, pp 99-110.
(21) Copper, J . A. D.; Smith, W.; Bacila, M.; Medina, H. J . Biol.
Chem. 1967, 119, 508. Avigad, G.; Amaral, D.; Asensio, C.; Horecker,
B. L. J . Biol. Chem. 1962, 237, 2736.
(22) Root, R. L.; Durrwachter, J . R.; Wong, C.-H. J . Am. Chem. Soc.
1985, 107, 2997.
(23) Matsamura, S., et al. Chem. Lett. 1988, 1747.
(24) Kelleher, F. M.; Bhavanandan, V. P. J . Biol. Chem. 1986, 261,
11045.
In the case of unprotected D-galactose, the primary
hydroxyl group can be efficiently oxidized to the corre-
sponding aldehyde by air in the presence of enzyme
(25) Pazur, J . H.; Knull, H. R.; Chevalier, G. E. J . Carbohydr.
Nucleosides Nucleotides 1974, 4, 129.
(26) Crane, L. J .; Mazur, A. W.; Nau, D. R.; Kluesener, B. W. US
Patent 5,149,646.
(18) Dziewiszek, K.; Zamojski, A. Carbohydr. Res. 1986, 150, 163.
(19) Szarek, W. A.; Zamojski, A.; Tiwari, K. N.; Ison, E. R. Tetra-
hedron Lett. 1986, 27(33), 3827.
(27) Maradufu, A.; Perlin, A. S. Carbohydr. Res. 1974, 32, 127.

Preparation of 5-C-(Hydroxymethyl)hexoses
J . Org. Chem., Vol. 62, No. 13, 1997 4473
F igu r e 3. Chemical synthesis of 5-C-(hydroxymethyl)-D-xylo-hexose, -D-lyxo-hexose, and -D-erythro-hexulose derivatives. Key:
(a) (COCl)2, DMSO, TEA; (b) CH2O, NaOH; (c) Pd(OH)2, H2; Ac2O/pyridine for 12.
The sugar 6-aldehydes and 5-C-(hydroxymethyl)hex-
oses are easy to identify by carbon-13 NMR. Formation
of the aldehyde during oxidation of the protected hexoses
is evidenced by appearance of the carbonyl signal at
about 200 ppm with simultaneous disappearance of the
primary alcohol carbon resonance at about 62 ppm. On
the other hand, the carbonyl signal in unprotected
aldehydes emerges at about 88 ppm and no signal is
detected at 200 ppm. This clearly indicates a presence
of the hydrated aldehyde group. A transient formation
of the nonhydrated aldehyde 17 was detected by proton
NMR when the enzymic reaction was run in deterium
oxide. Conversion of the aldehyde group into 5-C-
(hydroxymethyl)hexose was confirmed by the absence of
the resonances corresponding to the carbonyl carbon and
the appearance of the signals at 57-61 ppm of two
hydroxymethyl groups formed in the reaction between
sugar 6-aldehyde and formaldehyde.
F igu r e 4. Mechanism of isopropylidene/methylene exchange
during hydroxymethylation of 2.
Finally, a word on the catalysis of hydroxymethylation
reaction. Both stages of this process (Figure 1), that is
the initial aldehyde condensation and the following
Canizzarro reaction, reqiure basic catalysts. In fact, it
is necessary to use a base in amounts well exceeding the
catalytic requirement because formic acid created in the
Canizzarro reaction neutralizes the catalyst. If an alkali
metal hydroxide serves as the catalyst in the condensa-
tion of unprotected carbohydrates, the soluble salts
present at the end of reaction are difficult to separate
from the carbohydrate product. A remedy for this situ-
ation proved to be the catalysis provided by the ion-
exchange resin IRA-400(HO-). The resin serves not only
as the catalyst but also simultaneously removes formic
acid produced in the reaction.
F igu r e 5. Chemoenzymic synthesis of 5-C-(hydroxymethyl)-
L-arabino-hexopyranose derivatives.
Su m m a r y
We have shown that 5-C-(hydroxymethyl)hexoses can
be obtained by condensation of hexose 6-aldehydes with
formaldehyde. Preparation of 5-C-(hydroxymethyl)-L-
arabino-hexopyranose mono- and oligosaccharides is
simple since one can easily obtain the required hexose
6-aldehyde by enzymic oxidation of the galactose primary
hydroxyl group. The entire synthesis in this case includ-
ing oxidation and condensation is conducted in water. It
does not involve protecting groups and can be conve-
niently applied on a kg scale. The condensation of
unprotected hexodialdo-1,5-pyranoses produces highly
pure 5-C-(hydroxymethyl)hexoses without substantial
epimerization at C-4 or retro-Michael elimination from
the carbohydrate aldehyde. In contrast, the synthesis
of 5-C-(hydroxymethyl)hexoses from carbohydrates that
do not have the “galacto” configuration at C-4 and C-5
mostly due to a low resolution of aldehydes from other
reaction components (e.g., Whatman KF5 plates). An
effective method to quantitate the formation of aldehyde
during the enzymic oxidation proved to be the 2,3,5-
triphenyltetrazolium assay28 adapted for this reaction.
Alternatively, HPLC was used to measure the disappear-
ance of the carbohydrate substrate in the oxidation and
formation of 5-C-(hydroxymethyl)hexose during the sub-
sequent condensation with formaldehyde. Unfortunately,
we have not found an HPLC method to simultaneously
detect the substrate and the product, during the oxida-
tion, using the same column.
(28) Dische Z. In Methods in Carbohydrate Chemistry; Whistler, R.
L., Wolfrom, M. L., Eds.; Academic Press: New York, 1962; Vol. 1, p
513.

4474 J . Org. Chem., Vol. 62, No. 13, 1997
Mazur and Hiler
hydroxide (700 mL) and stirred at room temperature for 18-
24 h until TLC showed completion of the reaction. After
neutralization to pH 6-7 with 44% formic acid and concentra-
tion under reduced pressure to 500 mL (removal of THF and
formaldehyde), the reaction mixture was diluted to 1200 mL
with distilled water. The product was extracted with ethyl
acetate (5 × 300 mL), washed with distilled water (2 × 200
mL), and dried with sodium sulfate (anhydrous). Evaporation
of the solvent under reduced pressure produced a viscous oil
that contained substantial amounts of formaldehyde polymers.
These impurities are usually not seen on TLC but can easily
be detected by proton NMR and can be removed by heating
and stirring of the impure product for 2 h at 120 °C under
vacuum. The final purification was accomplished by column
chromatography (silica gel, e.g., Matrex 60A) or HPLC (gives
better yield) with solvent toluene:acetone ) 4:1. Three
products were eluted in the following order: l,2:3,4-di-0-
isopropylidene-R-D-galactopyranose (1), l,2:3,4-di-0-isopropy-
lidene-5-C-(hydroxymethyl)-â-L-arabino-hexopyranose (4), and
5-C-(hydroxymethyl)-l,2-isopropylidene-3,4-methylene-â-L-ara-
bino-hexopyranose (3) in a ratio of approximately 8:8:84. The
yield of 3 was 35.0 g (59%). For 3: TLC (Analtech GF), Rf )
0.22, AcOEt:hexanes ) 45:55; 13C NMR (CDCl3, 67.8 MHz) d
94.8 (C-1), 70.8 (C-2), 72.3 (C-3), 72.2 (C-4), 76.5 (C-5), 62.1
(C-6), 61.7 (C-7), 108.6, (CH3CHCH3), 94.4 CH2, 25.9, 24.5
in the hexose molecule generally require elaborate chemi-
cal transformations.
Exp er im en ta l Section
Galactose oxidase was isolated from the fermentation
medium of Dactylium dendroides according to the method of
Tressel and Kosman29 or by our procedure described below.
Activity of enzyme was measured by a coupled horseradish
peroxidase assay,30 and protein content was determined by a
standard Bio-Rad assay. The HPLC measurements of the
substrate concentration during the GOase catalyzed oxidation
were done on a Supelco-NH2 column with acetonitrile-water
85:15 at a flow rate 1.25 mL/min. NMR spectra were mea-
sured with QE-300 (General Electric) and J EOL FX-270
spectrometers. Molecular weights of products were confirmed
by mass spectroscopy, chemical ionization, or FAB methods
using HP5985B (Hewlett-Packard) or ZAB2F (VG-Fisons)
spectrometers.
P r ep a r a tion of Ga la ctose Oxid a se. Fermentation of
Dactylium dendroides (ATCC 46032, NRRL 2903) was done
according to the literature procedure.29 The fermentation
broth (24 L, 6.5 units/mL) was filtered through a nylon bag to
remove mycelia. The supernatant containing copper sulfate
(5 mM) and histidine (10 mM) at pH 6-7 was concentrated
by ultrafiltration to about 5% of the initial volume. The
retentate was equilibrated with the buffer containing 20 mM
NaH2PO4, 10 mM histidine, and 5 mM CuSO4 at pH 5.3. The
Millipore Pellicon system equipped with a 10 000 MWCO
membrane was used for concentration and equilibration. The
final volume of the equlibrated crude enzyme concentrate was
1120 mL and the activity 136 units/mL. The enzyme (960 mL,
125 000 units) was then purified on a carboxysulfone column
21.4 mm × 35 cm (J . T. Baker Co.) equilibrated with the same
buffer (4 L). The superntant was loaded in 4 × 200 mL and
1 × 120 mL portions on the column. Each load was followed
by elution of the void volume with the fresh equlibration buffer
(200 mL). Following the last void volume, the column was
washed with the buffer containing 100 mM NaH2PO4, 10 mM
histidine, and 5 mM CuSO4 at pH 7.0 (250 mL). The final
elution was done with a buffer made of 300 mM NaH2PO4, 10
mM histidine, and 5 mM CuSO4 at pH 7.0. The enzyme was
collected in the fractions between 130 and 250 mL. The
activity of the recovered enzyme in 120 mL of eluant was 932
u/mL giving a total of 111 900 units (89% yield of chromato-
graphic purification) with a specific activity of 1435 u/mg.
1,2:3,4-Di-O-isop r op ylid en e-r-D-ga la cto-h exod ia ld o-l,5-
p yr a n ose (2). 1,2:3,4-Di-O-isopropy1idene-R-D-galactose31 (1)
(100 g, 0.38 mol) was added to a solution of pyridinium
dichromate (PDC) (72 g, 0.19 mol), acetic anhydride (124 mL,
1.31 mol), and N,N-dimethylformamide (240 mL, 3.1 mol) in
dichloromethane (850 mL). The reaction mixture was refluxed
for approximately 3-4 h until TLC showed complete conver-
sion of the carbohydrate substrate. The dark green solution
was then cooled to room temperature. Most of the chromium
salts were precipitated by diluting the reaction mixture with
toluene (6000 mL). To remove these salts thoroughly, the
toluene mixture was filtered twice: through a Celite pad (24
× 7.5 cm) and silica gel (35-70 pm) pad (24 × 7.5 cm). Silica
gel was finally washed with toluene (2000 mL) followed by
ethyl acetate (1000 mL). The slightly green solution was
evaporated at reduced pressure to a viscous oil, which after
distillation (0.06 mmHg, 125-135 °C) gave 58.0 g (58%) of the
product (2): TLC (Analtech GF) Rf ) 0.50, AcOEt:hexanes )
45:55; 13C NMR (CDCl3, 67.8 MHz) 199.72 (CdO) 109.60,
108.60 ((CH3)2C), 95.89 (C-1), 72.8 (C-5), 71.87, 71.35, 70.09
(C-2, C-3, C-4), 25.63, 25.45, 24.48, 23.90 ((CH3)2C).
1
(CH3); H NMR (CDC13, 300 MHz): d 5.22 (d, 1H, J 1,2 ) 5.1
Hz, H-1), 4.72 (s, 1H, CH2), 4.40 (s, 1H, CH2), 4.05 (d, 1H, J 3,4
) 7.3 Hz, H-4), 3.88 (dd, 1H, J 1,2 ) 5.1 Hz, H-2), 3.63 (d, 1H,
J 3,4 ) 7.3, H-3), 3.30-3.50 (m, 4H, H-6, H-7). For 4: TLC
(Analtech GF) Rf ) 0.45, CHCl3:MeOH ) 9:1; 13C NMR (CDCl3,
67.8 MHz) δ 94.5 (C-1), 70.3 (C-2), 71.8 (C-3), 72.8 (C-4), 76.5
(C-5), 62.0 (C-6), 61.4 (C-7), 108.0, 107.9 (CH3CHCH3), 26.0,
1
26.7 (CH3); H NMR (CDC13, 300 MHz) δ 5.65 (d, 1H, J 1,2
)
5.2 Hz, H-1), 4.60 (dd, 1H, J 3,4 ) 7.4 Hz, H-3), 4.24 (dd, 1H,
J 1,2 ) 5.2 Hz, J 2,3 ) 1.0 Hz, H-2), 4.09 (d, 1H, J 3,4 ) 7.4 Hz,
H-4), 3.58-3.86 (m, 4H, H-6, H-7), 2.84 (m, 1H, OH), 2.48 (m,
1H, OH), 1.56 (s, 3H, CH3), 1.47 (s, 3H, CH3), 1.36 (s, 3H, CH3),
1.35 (s, 3H, CH3). Anal. Calcd for C11H18O7: C, 50.38; H, 6.92.
Found: C, 50.65; H, 6.92
Met h yl 2,3,4-Tr i-O-b en zyl-5-C-(h yd r oxym et h yl)-r-D-
xylo-h exop yr a n osid e (9). An oxidizing mixture17 was made
by a careful addition at -70 °C of a dry solution of DMSO
(3.84 mL, 53.7 mmol) and CH2Cl2 (30 mL) into a solution of
oxalyl chloride (2.16 mL, 24.7 mmol) in dry CH2Cl2 (110 mL)
and stirring the resulting solution for 10 min. While the
temperature was maintained -70 °C, a solution of methyl
2,3,4-tri-O-benzyl-R-D-glucopyranoside (5)18 (12.0 g, 26 mmol)
in dry CH2Cl2 was added over a period of 10 min, and stirring
was continued for additional 45 min. After subsequent addi-
tion of dry Et3N (18.1 mL, 130 mmol), the reaction mixture
was allowed to warm to room temperature. The product
solution was washed with water, 1 N HCl, NaHCO3, and H2O.
After evaporation of the solvent, the crude residue was
dissolved in dioxane (225 mL) and stirred with 37% aqueous
formaldehyde (40 mL) and 1 M NaOH (40 mL) at room
temperature for 20 h. The product solution was neutralized
with 1 N HCl and evaporated to dryness. The crude product
was purified on a silica column using a solution of hexane and
ethyl acetate 45/55. Two components were collected: a minor,
faster moving starting material (5) (2 g) and a major, the
product 6 (5.75 g, 45%): TLC (Analtech GF) Rf ) 0.62, AcOEt:
hexanes ) 45:55; 13C NMR (CDCl3, 75 MHz) d 100.40 (C-1),
80.77 (C-2), 80.40 (C-3) 79.43 (C-4), 80.98 (C-5), 65.08 (C-6),
65.01 (C-7) 139.35, 138.71, 138.61, 129.19, 129.08, 128.68,
128.55, 128.32 (Ph), 74.13, 76.36, 76.75 (PhCH2) 57.12 (CH3);
1H NMR (CDC13, 300 MHz) δ 7.50-7.20 (m, 15H, Ph’s), 5.02
(d, 1H, J a,b ) 10,8 Hz, CH2 Ph), 4.94 (d, 1H, J a,b ) 10.9 Hz,
CH2 Ph), 4.85 (d, 1H, J a,b ) 10.8 Hz, CH2 Ph), 4.77 (d, 1H, J a,b
) 11.9 Hz, CH2 Ph), 4.60-4.50 (m, 3H, CH2 Ph, H-1), 4.24 (t,
1H, J 2,3 ) J 3,4 ) 9.7 Hz, H-3), 3.88 (d, 1H, J 3,4 ) 9.7 Hz, H-4),
3.80-3.60 (m, 4H, H-6, H-7), 3.51 (dd, 1H, J 1,2 ) 3.9 Hz, J 2,3
) 9.7 Hz H-2), 3.42 (S, 3H, CH3).
5-C-(Hyd r oxym eth yl)-l,2-O-isop r op ylid en e-3,4-O-m eth -
ylen e-â-L-a r a bin o-h exop yr a n ose (3). A homogeneous solu-
tion of l ,2:3,4-di-O-isopropylidene-R-D-galacto-hexodialdo-l,5-
pyranose (2) (60.0 g, 0.225 mol) in 36% formaldehyde (700 mL)
and tetrahydrofuran (1750 mL) was treated with 1 N sodium
Ben zyl 2,3,4-tr i-0-ben zyl-5-C-(h yd r oxym eth yl)-r-L-lyxo-
h exop yr a n osid e (10) was prepared from benzyl 2,3,4-tri-O-
benzyl-R-D-mannoside (6)18 (23.7 g, 44 mmol) analogously to
(9). The final product was purified on a silica column with a
solution of hexane and ethyl acetate 65/35. The yield of 10
was 9 g (36%): 13C NMR (CDCl3, 75 MHz) δ 98.45 (C-1), 75.22
(C-2), 76.83 (C-3), 76.74 (C-4), 80.51 (C-5), 65.06 (C-6), 65.02
(29) Tressel, P.S.; Kosman, D. J . Anal. Biochem. 1980, 105, 150.
(30) Amaral, D.; Kelly-Falcoz, F.; Horecker, B. L. In Methods in
Enzymology; Wood, W. A., Ed.; Academic Press: New York, 1966; Vol.
9, p 87.
(31) Schmidt, O. T. In Methods in Carbohydrate Chemistry; Whistler,
R. L., Wolfrom, M. L., Eds.; Academic Press: New York, 1963; Vol. 2,
p 324.

Preparation of 5-C-(Hydroxymethyl)hexoses
J . Org. Chem., Vol. 62, No. 13, 1997 4475
(C-7) 138.29, 138.13, 138.04, 137.02, 128.59, 128.47, 128.03,
127.75 (Ph) 70.31, 72.43, 72.90, 76.22 (PhCH2).
remaining aldehyde. About 60-85% of the galactose oxidase
activity was usually recovered after each run. A sample of
the crude oxidation mixture was lyophilized for 13C NMR
determination of the product.
Meth yl 5-C-(h yd r oxym eth yl-r-D-xylo-h exop yr a n osid e
(11) was obtained by hydrogenolysis of methyl 2,3,4-tri-O-
benzyl-5-C-(hydroxymethyl)-R-D-xylo-hexopyranoside (10) (10
mmol) in a solution of water (50 mL), methanol (50 mL), and
acetic acid (15 mL) containing a catalyst, 20% Pd(OH)2 on
charcoal (3.0 g), under normal conditions for 16 h. The
reaction mixture was filtered through Celite, the filtrate was
evaporated, and the residue was dissolved in water (50 mL),
deionized with IRA-400(OH-), and evaporated. The yield of
the product (11) was in excess of 90%: TLC (Whatman K5F)
Rf ) 0.34, MeCN:H2O ) 75:25; 13C NMR (CDCl3, 75 MHz) d
101.03 (C-1), 70.62, 71.21 (C-2,C-3) 69.24 (C-4), 80.35 (C-5),
62.47 (C-6), 61.90 (C-7), 57.0 (CH3).
5-C-(Hyd r oxym eth yl)-(r+â)-D-1yxo-h exop yr a n ose (12)
was obtained analogously to 11 from benzyl 2,3,4-tri-O-benzyl-
5-C-(hydroxymethyl)-â-D-lyxo-hexopyranoside (10). The prod-
uct 12, TLC (Whatman K5F) Rf ) 0.26, MeCN:H2O ) 4:1, was
identified as the peracetylated derivative, which was made by
acetylation of the crude product with Ac2O in pyridine. The
acetyl derivative was extracted with CH2Cl2 after a treatment
of the acetylation mixture with water and purified on HPLC
using a Whatman Particil 5 column with EtOAc:hexanes )
1:1. 5-C-(Acetoxyxymethyl)-1,2,3,4-tetraacetyl-(R+â)-D-lyxo-
hexopyranose (12): 13C NMR (CDCl3, 75 MHz) δ 87.50, 90.18
(C-1), 67.85, 66.43 (C-2), 67.47, 68.23 (C-3), 66.82, 66.03 (C-
4), 76.62, 77.08 (C-5), 64.71, 63.86 (C-6), 61.87, 62.94 (C-7).
Resonance peaks from the acetyl groups are at about 20 ppm
(CH3) and 168-170 ppm (CdO).
Met h yl 5-C-(h yd r oxym et h yl)-1,3,4-t r i-O-b en zyl-r-D-
er yth r o-h exu lofu r a n osid e (15a ) a n d m et h yl 5-C-(h y-
d r oxym eth yl)-1,3,4-tr i-O-ben zyl-â-D-er yth r o-h exu lofu r a -
n osid e (15b) were prepared analogously to 9 from methyl
1,3,4-tri-O-benzyl-(R+â)-fructofuranosides19 (13) (13.5 g, 29
mmol). The crude product containing 15a and 15b was
fractionated on a silica column using a mixture of toluene:
acetone 10:1. The yields of the R-somer 15a was 2.25 g
(15.7%): 13C NMR (CDCl3, 75 MHz) δ 137.89, 137.73, 137.45,
129.14, 128.54, 128.35, 128.16, 127.86 (phenyl rings), 107.01
(C-2), 86.94 (C-3), 85.35 (C-5), 83.52 (C-4), 73.71, 73.07, 72.85
(CH2-Ph), 67.74 (C-1), 64.54, 64.16 (C-6, C-7), 49.56 (CH3). The
yield of the â-isomer 15b was 1.75 g (12.2%): 13C NMR (CDCl3,
75 MHz) δ 137.07, 137.87, 137.23, 128.48, 128.33, 128.23,
127.95 (phenyl rings), 102.31 (C-2), 84.35 (C-3), 83.57 (C-4),
83.13 (C-5), 73.56, 73.51, 73.12 (CH2Ph), 68.60 (C-1), 65.66,
63.43 (C-7, C-6), 49.51 (CH3); TLC (Analtech GF), Rf (15a ) )
0.17, Rf (15b) ) 0.11, toluene:Me2CO ) 2:1.
Meth yl 5-C-(Hyd r oxym eth yl)-r-D-er yth r o-h exu lofu r a -
n oside (16a) an d Meth yl 5-C-(Hydr oxym eth yl)-â-D-er yth r o-
h exu lofu r a n osid e (16b). These products were obtained from
methyl 5-C-(hydroxymethyl)-1,3,4-tri-O-benzyl-R-D-erythro-
hexulofuranoside (15a ) (2.25 g, 4.5 mmol) and methyl 5-C-
(hydroxymethyl)-1,3,4-tri-O-benzyl-â-D-erythro-hexulofurano-
side (15b) (1.75 g, 2.5 mmol) by hydrogenolysis under normal
conditions. The hydrogenation was carried out in methanol
(50 mL), in the presence of 10% Pd(OH)2 on charcoal (3.0 g),
during 20 h. The reaction mixture was filtered through Celite,
the cake was washed with hot water, and the filtrate was
evaporated to dryness. The yields of the products were in
excess of 90%. Methyl 5-C-(hydroxymethyl)-R-D-erythro-hexu-
lofuranoside (16a ): 13C NMR (CDCl3, 67.8 MHz) δ 108.34 (C-
2), 86.92 (C-5), 80.24 (C-3), 77.25 (C-4), 64.99 (C-6), 61.99 (C-
7), 59.98 (C-1), 49.09 (CH3). Methyl 5-C-(hydroxymethyl)-â-
D-erythro-hexulofuranoside (16b): 13C NMR (CDCl3, 67.8 MHz)
δ 102.87 (C-2), 83.70 (C-5), 77.94 (C-3), 77.25 (C-4), 64.99 (C-
6), 61.99 (C-7), 59.98 (C-1), 49.09 (CH3).
Meth yl â-D-ga la cto-h exodialdo-1,5-pyr an oside (17): 13C
NMR (CDCl3, 75 MHz) δ 104.31 (C-1), 88.71 (C-6), 77.13 (C-
5), 73.16 (C-3), 70.97 (C-2) 68.67 (C-4), 57.73 (CH3).
Ben zyl â-D-ga la cto-h exod ia ld o-1,5-p yr a n osid e (18): 13C
NMR (CDCl3, 75 MHz) δ 136.67, 128.71, 128.44 (Ph), 101.95
(C-1), 88.30 (C-6), 76.82 (C-5), 72.71 (C-3), 71.45 (C-2), 70.59
(CH2), 68.18 (C-4).
2′,3′-Isop r op ylid en e-(D + L)-glycer yl â-D-ga la cto-h exo-
d ia ld o-1,5-p yr a n osid e (19): 13C NMR (CDCl3, 75 MHz) δ
110.59 (C(CH3)2), 103.74, 103.51 (C-1) 88.71 (C-6), 77.19 (C-
5), 74.77 (C-2′), 73.04 (C-3), 71.03, 70.45 (C-2, C-1′′, 68.61 (C-
4), 65.90 (C-3′), 26.05, 24.67 (CH3).
4-O-(â-D-ga la cto-Hexod ia ld o-1,5-p yr a n osyl)-(1f4)-â-D-
sor bitol (20): 13C NMR (CDCl3, 75 MHz) δ 103.51 (C-1), 88.65
(C-6′), 79.78 (C-4′), 77.19 (C-5), 71.32, 72.64, 72.87 (C-2, C-2′,
C-3, C-5′), 69.76 (C-3′), 68.67 (C-4), 63.00, 62.45 (C-1′, C-6′).
B. Con d en sa tion w ith F or m a ld eh yd e. The filtrate of
the crude aldehyde solution obtained in A was combined with
37% aqueous formaldehyde solution (10 mL) and 50% NaOH
(144 mL). A cooling bath was applied during addition of NaOH
since an exothermic reaction ensued upon hydroxide addition.
Alternatively, the anion exchange resin IRA-400(OH-) (180
mL) can be used instead of NaOH. With the resin, the
exothermic reaction has not been detected. The reaction
mixture was stirred at room temperature for 8-16 h, heated
to 55 °C, and deionized using sequentially ion-exchange
columns (2.5 cm × 100 cm) containing resins IR-120(H+) and
IRA-400(OH-) followed by IRA-400(HSO3-) to remove form-
aldehyde. The product was obtained by evaporation of the
solvent followed by drying the residue over P2O5 under
vacuum.
Meth yl 5-C-(h yd r oxym eth yl)-r-L-a r a bin oh exop yr a n o-
sid e (21): yield 60%; TLC (Whatman K5F) Rf ) 0.54, MeCN:
H2O ) 80:20; [R]D ) -37.44 (c, 2.94, H2O, 23.0 °C); 13C NMR
(CDCl3, 75 MHz) 100.40 (C-1), 79.63 (C-5), 70.70, 70.55 (C-2,
C-3), 68.40 (C-4), 60.82 (C-6), 57.86 (C-7), 57.14 (CH3).
Ben zyl 5-C-(h yd r oxym eth yl)-r-L-a r a bin oh exop yr a n o-
sid e (22): yield 57%; TLC (Whatman K5F) Rf ) 0.44, MeCN:
H2O ) 80:20; [R]D ) -56.1 (c, 1.60 H2O, 23.O °C); 13C NMR
(CDCl3, 75 MHz) 137.26, 129.08, 128.79 (phenyl), 98.90 (C-1),
80.01 (C-5), 71.26, 71.08 (C-2, C-3, CH2), 68.90 (C-4), 61.64
(C-6), 58.59 (C-7).
(D + L)-Glycer yl 5-C-(h yd r oxym eth yl)-r-L-a r a bin o-h ex-
op yr a n osid e (23) was prepared from 2′,3′-isopropylidene-(D
+ L)-glyceryl â-D-galacto-hexodialdo-1,5-pyranoside (19). The
isopropylidene group was cleaved during the workup of the
condensation product with IR-120(H+): yield 36%; TLC (What-
man K5F) Rf ) 0.19, MeCN:H2O ) 75:25; [R]D ) -31.39 (c,
1.77, H2O, 23.0 °C); 13C NMR (CDCl3, 75 MHz) 99.97, 99.75
(C-1), 70.94, 70.80, 70.68 (C-2, C-3, C-4, C-2′), 68.34 (C-1′),
62.49, 62.34, 60.80, 57.80 (C-6, C-7, C-3′).
4-0-[5′-C-(Hydr oxym eth yl)-r-L-a r a bin o-h exopyr an osyl]-
D-sor bitol (24): yield 62%; TLC (Whatman K5F) Rf ) 0.40,
MeCN:H2O ) 70:30; [R]D ) +20.48 (c, 0.53, H2O, 23.0 °C); 13
C
NMR (CDCl3, 75 MHz) 99.94 (C-1), 80.24 (C-5′), 79.61 (C-4),
72.52, 71.37, 70.68, 69.53 (C-2, C-2′, C-3, C-3′, C-5), 68.84 (C-
4), 62.91 (C-6′), 62.39 (C-6), 61.30 (C-1), 58.19 (C-7).
Ben zyl 4-0-[5′-C-(h yd r oxym eth yl)-r-L-a r a bin o-h exop y-
r a n osyl]-(1f4)-â-D-glu cop yr a n osid e (25): yield 44%; TLC
(Analtech GF) Rf ) 0.11, CHCl3:CH3OH ) 8:2; [R]D ) -15.61
(c, 6.45, H2O, 23.4 °C); 13C NMR (CDCl3, 67.8 MHz) 136.97,
129.08, 128.79 (Ph), 101.43 (C-1′), 100.22 (C-1′), 80.59 (C-5′),
79.03 (C-4), 75.06, 74.82, 73.16, 71.16, 71.32, 70.74 (C-5, C-3,
C-3′, C-2, C-2′, CH2), 68.72 (C-4′), 61.29, 60.55 (C-6, C-6′), 58.85
(C-7′).
P r ep a r a tion of 5-C-(Hyd r oxym eth yl)h exoses fr om Un -
p r otected Ca r boh yd r a tes. A. Oxid a tion w ith Ga la ctose
Oxid a se (GOa se). To a solution of carbohydrate substrate
(250 mM) in 100 mM sodium phosphate buffer at pH 7 (400
mL) containing CuSO4 (5 mM) and histidine (10 mM) at 4 °C
were added catalase (Sigma C-40, 83 000 units) and galactose
oxidase (8300 units). The reaction mixture was stirred and
aerated at this temperature until completion (14-24 h).
Galactose oxidase was recovered in the retentate by ultrafil-
tration of the reaction mixture using an Amicon ultrafiltration
stired cell equipped with 10 000 or 30 000 MWCO membrane.
The retantate was washed with the fresh buffer to remove the
Su p p or tin g In for m a tion Ava ila ble: NMR spectra for
2-4, 8-12, 14, 15a ,b, 16a ,b, 18-21, 21a , 22, 23, and 25 (32
pages). This material is contained in libraries on microfiche,
immediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
J O970232P