Synthesis of oxidized methyl 4-O-methyl-β-D-glucopyranoside and methyl β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside derivatives as substrates for fluorescence labeling reactions

Article abstract of DOI:10.1016/S0008-6215(02)00048-4

The synthetic cellulose model compounds methyl 4-O-methyl-β-D-glucopyranoside and methyl 4-O-methyl-β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside and related 6-O-protected intermediates were oxidized in good to fair yields using Swern-conditions or bromine/bis(tributyltin) oxide, respectively, to afford compounds containing 6-aldehyde, 3-keto, and 2,3-diketo groups. Cellobiose and oxidized monosaccharides were then labeled with the carbonyl-selective fluorescence marker 9-(7-amino-1,4,7-trioxaheptyl)-9H-carbazolecarboxamide (CCOA). The labeled derivatives serve as model compounds for the determination of minute amounts of carbonyl groups in cellulosic polysaccharides.

Full text of DOI:10.1016/S0008-6215(02)00048-4

Carbohydrate Research 337 (2002) 691–700
www.elsevier.com/locate/carres
Synthesis of oxidized methyl 4-O-methyl-b-
methyl b- -glucopyranosyl-(1“4)-b-
as substrates for fluorescence labeling reactions
D-glucopyranoside and
D
D-glucopyranoside derivatives
Ju¨rgen Ro¨hrling, Antje Potthast, Thomas Lange, Thomas Rosenau,
Immanuel Adorjan, Andreas Hofinger, Paul Kosma*
Christian-Doppler-Laboratory, Institute of Chemistry, Uni6ersity of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria
Received 28 November 2001; accepted 4 February 2002
Abstract
The synthetic cellulose model compounds methyl 4-O-methyl-b-
D
-glucopyranoside and methyl 4-O-methyl-b-D-glucopyranosyl-
(1“4)-b- -glucopyranoside and related 6-O-protected intermediates were oxidized in good to fair yields using Swern-conditions
D
or bromine/bis(tributyltin) oxide, respectively, to afford compounds containing 6-aldehyde, 3-keto, and 2,3-diketo groups.
Cellobiose and oxidized monosaccharides were then labeled with the carbonyl-selective fluorescence marker 9-(7-amino-1,4,7-tri-
oxaheptyl)-9H-carbazolecarboxamide (CCOA). The labeled derivatives serve as model compounds for the determination of
minute amounts of carbonyl groups in cellulosic polysaccharides. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Oxidation; Cellulose; Cellobioside; Fluorescence labelling
producibility and insufficient yields due to restrained
accessibility of the polymeric substrate.2,3 The problem
of accurate quantitation becomes even more serious
taking into account the small amount of carbonyls
usually found in pulps, which is in the range of a few
mmols per gram.
1. Introduction
The reliable and accurate determination of oxidized
groups in cellulosic materials still represents a largely
unsolved, but fundamental problem in the pulp and
paper industry. Carbonyl and carboxylic acid groups,
which are introduced into cellulosic fibers by a number
of bleaching procedures, constitute ‘hot spots’ along the
cellulosic chain. Carbonyl groups are implicated in
various detrimental effects such as loss in fiber strength
due to peeling reactions and b-elimination, as well as
decrease of brightness in cellulosic products induced by
thermal or photochemical yellowing reactions.1
The determination and quantitation of carbonyl
groups (aldehyde groups, reducing-end groups, 2-keto-,
3-keto-, and 2,3-diketo groups) has to meet a number
of experimental shortcomings. Traditional methods rely
on heterogeneous derivatization of carbonyls with hy-
droxylamine or cyanide, which suffer from limited re-
We have previously reported on the synthesis of a
fluorescence label, 9-(7-amino-1,4,7-trioxaheptyl)-9H-
carbazolecarboxamide (CCOA), which permits a homo-
geneous derivatization reaction of carbonyl groups in
the common cellulose solvent system N,N-dimethylac-
etamide–LiCl.4 The precolumn derivatization with the
fluorescence label is followed by a subsequent chro-
matographic separation of the labeled polysaccharide
and the simultaneous determination of the molecular
mass using multi-angle laser-light scattering (MALLS)
detection. Hence, the label to be used must not interfere
with the spectral characteristics of the laser light system
(at 488 or 633 nm for the commercial GPC-MALLS
equipment). The fluorescence label was attached to a
spacer group to provide similar spectral characteristics
of the labeled material independently of the type and
position of the carbonyl groups. In order to develop the
method into a reliable procedure for analytical use,
* Corresponding author. Tel.: +43-1-360066055; fax: +
43-1-360066059.
E-mail address: pkosma@edv2.boku.ac.at (P. Kosma).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00048-4

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J. Ro¨hrling et al. / Carbohydrate Research 337 (2002) 691–700
model compounds were needed as reference materials.
Related oxidized 4-O-ethyl derivatives of methyl b-
urated carbonyl moiety was assigned on the basis of the
13C NMR data observed for C-2, C-3, and C-4 at
186.27, 138.14, and 151.01 ppm, respectively, whereas
the 13C NMR signal of C-3 in 9 was measured at 205.18
ppm. Assignments were confirmed by HMQC and
HMBC measurements. While 8 was too unstable for
subsequent deprotection, the methyl hex-3-ulopyran-
oside derivative 9 was subjected to hydrogenolysis in
D
-
glucopyranoside have been used as O-methyloxime
derivatives for GC-MS analysis.5 Herein we report on
the chemical synthesis and subsequent fluorescence la-
beling of oxidized 4-O-methyl glucopyranoside deriva-
tives as soluble model compounds for similar structural
entities occurring in oxidized cellulosic substrates. Vali-
dation of the method and application for the determi-
nation of carbonyl group profiles in oxidized pulps will
be published elsewhere.
MeOH which afforded known methyl 4-O-methyl-b-D-
ribo-hex-3-ulopyranoside (10) in 98% yield. The com-
pound had previously been obtained in 2.6% yield upon
chromium trioxide treatment of 7.12 Alternatively, 10
was much more efficiently prepared by reaction of
methyl 4-O-methyl glucopyranoside (7) with bis(tri-n-
butyltin) oxide followed by oxidation with bromine in
CHCl3,13 which gave a nearly quantitative yield (98%)
of 10. In aqueous solution, 10 occurred as a mixture of
the keto- and hydrate form in a 5:3 ratio, which was
deduced from the integration values of the anomeric
protons, which were correlated—based on HMBC
measurements—to the 13C NMR signals at C-3 at
206.81 and 95.78 ppm, respectively.
2. Results and discussion
For the synthesis of the hemi-protected derivatives 6
and 7, known methyl 4,6-O-benzylidene-b-D-glucopy-
ranoside (1)6 was converted into the 2,3-di-O-methoxy-
benzyl derivative 2 in 76% yield by reaction with
p-methoxybenzyl chloride–NaH in DMF.7 Subsequent
reductive ring opening with HCl–NaCNBH3 in THF–
diethyl ether8 afforded preferentially the 6-O-benzyl
Swern oxidation of 4 afforded a good yield (82%) of
the 6-aldehydo derivative 11, which was deprotected by
hydrogenolysis with 10% Pd–C to furnish known14
ether derivative 3 in 56% yield, together with a small
proportion of the 4-O-benzyl ether derivative 4 (11%),
which were separated by column chromatography. Sub-
sequent O-methylation of 3 with MeI–NaH in THF
produced the 4-O-methyl derivative 5 in 96% yield. For
selective oxidation at the C-2 and C-3 positions, the
p-methoxybenzyl groups were removed by DDQ oxida-
tion in 82% yield to furnish 6. Further deblocking of
the 6-O-benzyl group was achieved by hydrogenolysis
in the presence of 10% Pd–carbon in methanol in 92%
methyl b- -gluco-hexodialdo-1,5-pyranoside (12) in
D
27% yield after chromatography. Optical rotation val-
ues were not in agreement with published values;
NMR-data indicated that the aldehyde group at C-6
was fully hydrated.14
In addition to the monosaccharide derivatives, the
previously described15 cellulose model disaccharide
derivative 13 was subjected to oxidative modification.
Tritylation using trityl chloride–pyridine at 100 °C af-
forded the 6,6%-bis-O-trityl ether derivative 14 in 74%
yield. Swern oxidation with Me2SO–trifluoroacetic acid
anhydride at −60 °C produced the fully oxidized disac-
charide derivative 18 as the major product in 50% yield
containing keto groups at C-2 and C-3%, a hydrated
keto group at C-2%, and a keto group at C-3 being
present in the enol form. The structural assignments
yield to give known methyl 4-O-methyl-b-D-glucopy-
ranoside (7).9 Swern oxidation of 6 with Me2SO–tri-
fluoroacetic anhydride10,11 was best performed using
three equivalents of oxidant, which allowed recovery of
educt 6 (56%) and furthermore, provided a better chro-
matographic separation of the oxidized products. Thus,
the enol form of the labile 2,3-diketo derivative 8 was
obtained (34%) in addition to a small amount of the
3-oxo derivative 9 (3%). The presence of the a,b-unsat-

J. Ro¨hrling et al. / Carbohydrate Research 337 (2002) 691–700
693
and differentiation between the two units were achieved
following HMBC assignment of the two methoxyl
groups linked to C-1 and C-4%, respectively, which then
provided the distinction between the C-4 and C-4% as
well as C-1 and C-1% signals, respectively. As a minor
byproduct, the 3%-O-methylthiomethyl ether derivative
15 was isolated in 12% yield,16,17 and the disaccharide
derivative 16 was obtained in 7% yield. Similar to 18,
the dicarbonyl group in the reducing unit of disaccha-
ride 16 occurred as the conjugated enone, the terminal
residue, however, had only one hydrated keto group at
C-2%. Removal of the trityl ether groups of 16 and 18
was accomplished by hydrogenolysis with 10% Pd–C in
methanol, which furnished the disaccharide model com-
pounds 17 and 19 in 30% and 68% yield, respectively.
All assignments were confirmed by HMQC and HMBC
measurements. Thus, the HMBC spectrum (Fig. 1) of
19 showed the connectivity of the O-methyl group
signal at 3.63 ppm to the carbon signal at 78.73 ppm
(C-4%), whereas the signal of the glycosidic O-methyl
group was correlated to the anomeric carbon at 99.91
ppm (C-1), which in turn displayed the connectivity to
the H-5 signal at 4.91 ppm. The signal of H-5 gave
correlations with C-1, C-6 and the olefinic carbons at
127.58 (C-3) and 152.14 ppm (C-4), respectively. The
latter signals are comparable with the shifts observed
for 8 as holds true for the carbonyl signal of C-2 at
186.06 ppm (compound 8: C-2 at 186.27 ppm). The
remaining carbonyl groups of the nonreducing unit at
95.19 and 92.74 ppm are consistent with the presence of
hydrated keto groups. The compounds will be used for
the development of other fluorescence labels which
might differentiate between mono- and dicarbonyl
moieties.
Derivatization with CCOA 20 was tested using the
6-O-benzyl derivative 9 and compound 10, which gave
the corresponding oxime derivatives 21 and 22 as mix-
Fig. 1. Two-dimensional plot of the HMBC-spectrum of 19 in
D2O (hydrated form).

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J. Ro¨hrling et al. / Carbohydrate Research 337 (2002) 691–700
695
tures of E/Z isomers in good yields and with a slight
preference for the formation of the Z-isomers. To
ensure that the steric influence of the E/Z—arrange-
ment does not translate into different spectral proper-
ties of the fluorescence label, the isomers were separated
by chromatography. Excitation and emission wave-
lengths of the isomers proved to be identical in the
cellulose solvent system N,N-dimethylacetamide–LiCl.
The same held true for the labeled aldehyde derivative
23, which was obtained as the E-configured isomer in
93% yield together with a small proportion of the
Z-derivative. Finally, reducing end groups also reacted
smoothly with the oxyamine 20 as shown for cellobiose
24, which afforded derivative 25 in 75% yield, although
the reaction time was significantly longer compared to
the monosaccharide model compounds.
stirred at rt for 1 h. Then 4-methoxybenzyl chloride
(7.40 mL, 54.6 mmol) was added dropwise during 10
min. The mixture was kept at rt for 60 h. Another
portion of 4-methoxybenzyl chloride (5.00 mL, 36.8
mmol) was added and the mixture was stirred for 20 h.
After addition of MeOH (5 mL) and stirring for 1 h,
the mixture was poured onto satd aq NH4Cl solution.
After extraction with EtOAc, the organic layer was
dried (MgSO4) and concentrated. The crude product
was chromatographed (toluene“9:1 toluene–EtOAc)
to give 2 as colorless crystals (9.89 g, 76%): mp 110–
1
112 °C (MeOH); [h]D20 −24° (c 0.7, CHCl3); H NMR
(CDCl3): l 7.53–7.22 (m, 9 H, Ph), 6.90–6.79 (m, 4 H,
Ph), 5.56 (s, 1 H, CHPh), 4.85–4.65 (m, 4 H, 4
CH2Ph), 4.39 (d, 1 H, J1,2 7.5 Hz, H-1), 4.36 (dd, 1 H,
J5,6b 4.9, J6a,6b 11.2 Hz, H-6b), 3.80 (s, 3 H, OMe), 3.79
(s, 3 H, OMe), 3.76 (dd, 1 H, J5,6a 1.7 Hz, H-6a), 3.71
(dd, 1 H, J2,3 9.2, J3,4 9.2 Hz, H-3), 3.65 (dd, 1 H, J4,5
9.2 Hz, H-4), 3.58 (s, 3 H, 1-OMe), and 3.43–3.34 (m,
2 H, H-2, H-5). Anal. Calcd for C30H34O8: C, 68.95; H
6.56. Found: C, 68.71; H, 6.50.
Further optimization of the labeling reaction and
application for the carbonyl quantification in oxidized
pulps will be presented in a forthcoming publication.
3. Experimental
Methyl 6-O-benzyl-2,3-di-O-(4-methoxybenzyl)-i-
glucopyranoside (3) and methyl 4-O-benzyl-2,3-di-O-(4-
methoxybenzyl)-i- -glucopyranoside (4).—A solution
of HCl in Et2O (2 M, 2 mL) was added dropwise to a
suspension of 2 (115 mg, 0.22 mmol), NaCNBH3 (207
mg, 3.3 mmol), and powdered molecular sieves (0.2 g, 3
D-
General methods.—Melting points were determined
with a hot stage and are uncorrected. Optical rotations
were measured with a Perkin–Elmer 243 B polarimeter.
1H NMR spectra were recorded at 297 K with a Bruker
D
1
,
Avance DPX instrument operating at 300 MHz for H
A) in dry THF (15 mL) at −20 °C until the evolution
using CDCl3 as solvent and tetramethylsilane as inter-
nal standard, unless stated otherwise. Coupling con-
stants are given in Hz (first order values). 13C NMR
spectra were measured at 75.47 MHz and referenced to
1,4-dioxane (l 67.40). Homo- and heteronuclear 2D
NMR spectroscopy was performed with Bruker stan-
dard software. TLC was performed on E. Merck pre-
coated plates (5×10 cm, layer thickness 0.25 mm,
Silica Gel 60F254); detection was effected by spraying
with anisaldehyde–H2SO4. For column chromatogra-
phy, silica gel (0.040–0.063 mm) was used. Concentra-
tion of solutions was performed at reduced pressure
and B40 °C. Elemental analyses were provided by Dr
J. Theiner, Mikroanalytisches Laboratorium, Institut
fu¨r Physikalische Chemie, University of Vienna.
MALDI-TOF mass spectra were obtained on a Dy-
namo (Thermo BioAnalysis) instrument in the positive
ion mode using 2% 2,5-dihydroxybenzoic acid as ma-
trix, by Dr F. Altmann, Institut fu¨r Chemie, University
of Agricultural Sciences, Vienna. LC-MS measurements
were performed by Dr E. Rosenberg, Institut fu¨r Ana-
lytische Chemie, TU Vienna, using HP1100 HPLC/
MSD with APCI interface.
of gas ceased. The solution was stirred for 1 h at
−20 °C and for 30 min at 5 °C. For complete conver-
sion, NaCNBH3 (207 g, 3.3 mmol) and then a solution
of HCl in Et2O (2 M, 2 mL) were added dropwise at
5 °C until the evolution of gas ceased. The mixture was
stirred for 15 min, Et3N (3 mL) was added and the
mixture was poured onto water–EtOAc (50 mL).
Molecular sieves were removed by filtration. After ex-
traction of the filtrate with EtOAc, the organic layer
was dried (MgSO4) and concentrated. The crude mate-
rial was chromatographed (60:30:1 n-hexane–EtOAc–
Et3N) to give 3 as colorless crystals (65 mg, 56%): mp
60–61 °C (MeOH); [h]2D0 −9° (c 0.9, CHCl3); Rf 0.31
1
(3:1 n-hexane–EtOAc); H NMR (CDCl3): l 7.34–7.22
(m, 9 H, Ph), 6.90–6.80 (m, 4 H, Ph), 4.89–4.55 (m, 6
H, 6 CH2Ph), 4.31 (d, 1 H, J1,2 7.3 Hz, H-1), 3.80 (m,
6 H, 2 OMe), 3.78 (dd, 1 H, J5,6b 3.2, J6a,6b 10.1 Hz,
H-6b), 3.69 (dd, 1 H, J5,6a 4.5 Hz, H-6a), 3.60–3.50 (m,
4 H, 1-OMe, H-4), 3.45–3.30 (m, 3 H, H-2, H-5, H-3),
and 2.44 (br. s, 1 H, OH). Anal. Calcd for C30H36O8: C,
68.69; H 6.92. Found: C, 68.47; H, 6.84.
Further elution gave 4 as colorless crystals (13.2 mg,
11%): mp 79–81 °C (MeOH); [h]2D0 +15° (c 0.3,
CHCl3); Rf 0.14 (3:1 n-hexane–EtOAc); 1H NMR
(CDCl3): l 7.35–7.18 (m, 9 H, Ph), 6.88–6.80 (m, 4 H,
Ph), 4.88–4.58 (m, 6 H, 6 CH2Ph), 4.32 (d, 1 H, J1,2 7.8
Hz, H-1), 3.86 (dd, 1 H, J5,6b 2.8, J6a,6b 12.1 Hz, H-6b),
3.79 (m, 3 H, OMe), 3.78 (m, 3 H, OMe), 3.70 (dd, 1 H,
Methyl 4,6-O-benzylidene-2,3-di-O-(4-methoxyben-
zyl)-i-
99.2 mmol) was added gradually to a solution of methyl
4,6-O-benzylidene-b- -glucopyranoside (7.00 g, 24.8
mmol) in dry DMF (100 mL). The suspension was
D
-glucopyranoside (2).—Powdered NaH (2.38 g,
D

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J. Ro¨hrling et al. / Carbohydrate Research 337 (2002) 691–700
J5,6a 4.8 Hz, H-6a), 3.62 (dd, 1 H, J2,3 9.0, J3,4 9.0 Hz,
H-3), 3.57 (s, 3 H, 1-OMe), 3.52 (dd, 1 H, J4,5 9.0 Hz,
H-4), 3.39–3.30 (m, 2 H, H-2, H-5), and 1.70 (br. s, 1
H, OH). Anal. Calcd for C30H36O8: C, 68.69; H 6.92.
Found: C, 68.41; H, 6.93.
Methyl 6-O-benzyl-4-O-methyl-i-
eno-2-ulopyranoside (8) and methyl 6-O-benzyl-4-O-
methyl-i- -ribo-hex-3-ulopyranoside (9).—Dry Me2SO
D-erythro-hex-3-
D
(476 mL, 6.70 mmol) in dry CH2Cl2 (50 mL) was treated
with trifluoroacetic anhydride (703 mL, 5.03 mmol) at
−60 °C. The solution was stirred for 10 min, then a
solution of 6 (500 mg, 1.68 mmol) in dry CH2Cl2 (5 mL)
was added. The mixture was stirred at −60 °C for 1.5
h. After the addition of Et3N (1.39 mL, 10.0 mmol) in
dry CH2Cl2 (5 mL), the reaction mixture was stirred at
−60 °C for a further 1.5 h. The solution was allowed to
warm up to −40 °C. After addition of 19:1 MeOH–
water (3 mL), the solution was evaporated and the
residue was chromatographed (34:15:1 CHCl3–n-hex-
ane–MeOH) to afford 8 as a colorless syrup (169 mg,
34%): [h]2D0 −139° (c 0.2, CHCl3); Rf 0.76 (49:1
CH2Cl2–MeOH); IR (KBr): 1747 (CꢀO), and 1694
Methyl 6-O-benzyl-2,3-di-O-(4-methoxybenzyl)-4-O-
methyl-i- -glucopyranoside (5).—Powdered NaH (175
D
mg, 7.31 mmol) was added gradually to a solution of 3
(1.92 g, 3.66 mmol) in dry THF (50 mL). The suspen-
sion was stirred at rt for 1 h. Then methyl iodide (455
mL, 7.31 mmol) was added. After 20 h MeOH (5 mL)
was added and the mixture was stirred for 1 h. Satd aq
NH4Cl was added and the mixture was extracted with
EtOAc (199 mL). The organic layer was dried (MgSO4)
and concentrated to give pure 5 as colorless crystals
(1.89 g, 96%): mp 61–62 °C (MeOH); [h]2D0 +44° (c 0.4,
1
CHCl3); H NMR (CDCl3): l 7.48–7.24 (m, 9 H, Ph),
1
6.90–6.85 (m, 4 H, Ph), 4.87–4.55 (m, 6 H, 6 CH2Ph),
4.28 (d, 1 H, J1,2 7.5 Hz, H-1), 3.83 (m, 6 H, 2 OMe),
3.78 (dd, 1 H, J5,6b 2.0, J6a,6b 11.7 Hz, H-6b), 3.70 (dd,
1 H, J5,6a 4.5 Hz, H-6a), 3.60 (s, 3 H, 4-OMe), 3.52–3.48
(m, 4 H, H-3, 1-OMe), 3.42–3.33 (m, 2 H, H-2, H-5),
and 3.27 (dd, 1 H, J3,4 9.6, J4,5 9.6 Hz, H-4). Anal. Calcd
for C31H38O8: C, 69.13; H 7.11. Found: C, 69.10; H,
7.13.
(CꢀC); H NMR (CDCl3): l 7.40–7.26 (m, 5 H, Ph),
5.57 (s, 1 H, OH), 4.95 (s, 1 H, H-1), 4.66 (dd, 1 H, J5,6a
3.3, J5,6b 8.5 Hz, H-5), 4.63 (s, 2 H, CH2Ph), 4.16 (s, 3
H, 4-OMe), 3.96 (dd, 1 H, J6a,6b 9.0 Hz, H-6a), 3.77 (dd,
1 H, H-6b), and 3.57 (s, 1 H, 1-OMe); 13C NMR
(CDCl3): l 186.27 (C-2), 151.01 (C-4), 138.14 (C-3),
129.64–128.30 (Ph), 98.80 (C-1), 74.37 (C-5), 73.87
(CH2Ph), 73.06 (C-6), 60.77 (4-OMe), and 57.01 (1-
OMe); MALDI-TOF MS: m/z 317.67 [M+Na]+.
Further elution gave 9 as a colorless solid (13.2 mg,
3%): [h]2D0 +30° (c 0.5, CHCl3); Rf 0.45 (49:1 CH2Cl2–
Methyl 6-O-benzyl-4-O-methyl-i-D-glucopyranoside
(6).—A solution of 5 (1.75 g, 3.25 mmol) and 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (2.21 g, 9.75
mmol) in 18:1 CH2Cl2–water (30 mL) was stirred at rt
for 40 min. The mixture was concentrated and chro-
matographed (1:5 toluene–EtOAc) to give 5 as colorless
crystals (793 mg, 82%): mp 76–78 °C (EtOAc); [h]D20
1
MeOH); IR (KBr): w 1726 (CꢀO); H NMR (CDCl3): l
7.38–7.26 (m, 5 H, Ph), 4.72 (d, 1 H, J 12.2 Hz, CH2Ph),
4.59 (d, 1 H, CH2Ph), 4.19 (d, 1 H, J1,2 7.7 Hz, H-1),
4.12 (d, 1 H, H-2), 4.08 (d, 1 H, J4,5 9.8 Hz, H-4), 3.85
(dd, 1 H, J5,6b 2.0, J6a,6b 11.3 Hz, H-6b), 3.78 (dd, 1 H,
J5,6a 3.7 Hz, H-6a), 3.62 (s, 3 H, 1-OMe), 3.57 (br. s, 1
H, OH), and 3.54–3.47 (m, 4 H, H-5, 4-OMe); 13C
NMR (CDCl3): l 205.18 (C-3), 137.89 (Ph), 128.40–
127.76 (Ph), 105.49 (C-1), 80.51 (C-4), 77.30 (C-2), 75.00
(C-5), 73.66 (CH2Ph), 68.29 (C-6), 59.52 (4-OMe), and
57.31 (1-OMe). Anal. Calcd for C15H20O6: C, 60.80; H
6.80. Found: C, 60.99; H, 6.81. Finally, starting material
6 (282 mg, 56%) was isolated.
1
−18° (c 0.8, CHCl3); H NMR (CDCl3): l 7.37–7.25
(m, 5 H, Ph), 4.66 (d, 1 H, J 12.2 Hz, CH2Ph), 4.57 (d,
1 H, CH2Ph), 4.16 (d, 1 H, J1,2 7.7 Hz, H-1), 3.77 (dd,
1 H, J5,6b 2.2, J6a,6b 10.9 Hz, H-6b), 3.70 (dd, 1 H, J5,6a
6.0 Hz, H-6a), 3.62 (dd, 1 H, J2,3 9.0, J3,4 9.0 Hz, H-3),
3.56 (s, 3 H, OMe), 3.52 (s, 3 H, OMe), 3.43–3.35 (m,
2 H, H-2, H-5), 3.26 (dd, 1 H, J4,5 9.0 Hz, H-4), 2.60 (br.
s, 1 H, OH), and 2.46 (br. s, 1 H, OH). Anal. Calcd for
C15H22O6: C, 60.39; H 7.43. Found: C, 60.53; H, 7.38.
Methyl 4-O-methyl-i-
D
-glucopyranoside (7).—Com-
Methyl
4-O-methyl-i-D-ribo-hex-3-ulopyranoside
pound 6 (70.0 mg, 0.235 mmol) was hydrogenolyzed in
the presence of 10% Pd–C (12.0 mg) in dry MeOH (20
mL) at atmospheric pressure for 15 h at rt. The catalyst
was filtered off, and the mixture was concentrated to
give 7 as a colorless solid (45.0 mg, 92%): [h]2D0 −18° (c
0.7, water), lit. −18° (c 1.0, water);9 1H NMR (D2O):
l 4.34 (d, 1 H, J1,2 8.0 Hz, H-1), 3.90 (dd, 1 H, J5,6b 2.3,
J6a,6b 12.3 Hz, H-6b), 3.73 (dd, 1 H, J5,6a 5.5 Hz, H-6a),
3.56 (dd, 1 H, J2,3 9.3, J3,4 9.3 Hz, H-3), 3.54 (s, 3 H,
OMe), 3.53 (s, 3 H, OMe), 3.43 (ddd, 1 H, J4,5 9.3 Hz,
H-5), 3.24 (dd, 1 H, H-2), and 3.17 (dd, 1 H, J4,5 9.3 Hz,
H-4); 13C NMR (D2O): l 103.97 (C-1), 80.14 (C-4),
76.25 (C-3), 75.74 (C-5), 73.90 (C-2), 61.20 (C-6), 60.87
(4-OMe), and 58.00 (1-OMe).
(10).—Hydrogenolysis of 9 (15.0 mg, 0.05 mmol) was
performed as described for 7. The crude product was
chromatographed (19:1 CH2Cl2–MeOH) to give 10 as a
colorless solid (10.1 mg, 98%): mp 149–152 °C (MeOH),
lit. 152–153 °C (PrOH);12 [h]2D3 +9° (c 0.3, water), lit.
+11° (c 0.5, water);12 IR (KBr): w 1731 (CꢀO); 1H
NMR (D2O): l keto form: 4.41 (d, 1 H, J1,2 8.1 Hz,
H-1), 4.20 (dd, 1 H, J2,4 1.6 Hz, H-2), 4.12 (dd, 1 H, J4,5
10.1 Hz, H-4), 3.92 (dd, 1 H, J5,6b 2.2, J6a,6b 12.8 Hz,
H-6b), 3.76 (dd, 1 H, J5,6a 4.6 Hz, H-6a), 3.56 (s, 3 H,
1-OMe), 3.53–3.43 (m, 4 H, 4-OMe, H-5); hydrate
form: 4.35 (d, 1 H, J1,2 8.0 Hz, H-1), 3.92 (dd, 1 H, J5,6b
2.2, J6a,6b 12.8 Hz, H-6b), 3.67 (dd, 1 H, J5,6a 5.4 Hz,
H-6a), and 3.53–3.43 (m, 7 H, 1-OMe, 4-OMe, H-5);

J. Ro¨hrling et al. / Carbohydrate Research 337 (2002) 691–700
697
13C NMR (D2O): l keto form: 206.81 (C-3), 105.19
(C-1), 81.45 (C-4), 77.64 (C-2), 75.73 (C-5), 61.16 (C-6),
60.22 (4-OMe), and 58.20 (1-OMe); hydrate form:
103.08 (C-1), 95.78 (C-3), 80.64 (C-4), 75.29 (C-5),
74.68 (C-2), 62.03 (4-OMe), 61.45 (C-6), and 57.86
(1-OMe); 5:3 ketone–hydrate.
(1“4)-b-D-glucopyranoside (400 mg, 1.08 mmol) and
triphenylchloromethane (723 mg, 2.59 mmol) were dis-
solved in dry pyridine (10 mL) and stirred at 100 °C for
2 h. For complete conversion, another portion of
triphenylchloromethane (300 mg, 1.07 mmol) was
added and the mixture was kept at 100 °C for a further
2 h. The suspension was concentrated and the residue
was chromatographed (1:5 toluene–EtOAc“EtOAc)
to afford 14 as a colorless solid (674 mg, 74%): [h]D20
Alternati6e synthesis of 10.—A suspension of 7 (300
mg, 1.44 mmol), (n-Bu3Sn)2O (1.48 mL, 2.90 mmol)
,
and molecular sieves 3 A (300 mg) in CHCl3 (25 mL)
1
+2° (c 0.6, CHCl3); H NMR (CDCl3): l 7.50–7.15
were heated under reflux for 3 h. The suspension was
cooled to 0 °C and bromine (170 mL, 3.32 mmol) was
added until a faint coloration was observed. The sus-
pension was applied onto a column of silica gel and
washed with CHCl3. Elution with 19:1 CHCl3–MeOH
furnished 10 as a solid. Yield: 290 mg (98%).
(m, 30 H, Ph), 4.33 (d, 1 H, J1,2 7.6 Hz, H-1), 4.27 (d,
1 H, J3,OH 1.7 Hz, 3-OH), 4.17 (d, 1 H, J1%,2% 7.8 Hz,
H-1%), 3.75–3.45 (m, 9 H, H-4, 1-OMe, H-3, H-5, H-6b,
H-6b, H-2), 3.37–3.10 (m, 9 H, H-3%, H-5%, H-6a,
4%-OMe, H-4%, H-2%, H-6a%), 2.49 (d, 1 H, J3%,OH 2.2 Hz,
3%-OH), 2.47 (d, 1 H, J2,OH 1.4 Hz, 2-OH), and 1.54 (br.
s, 1 H, 2%-OH). Anal. Calcd for C52H54O11·H2O: C,
71.54; H 6.47. Found: C, 71.61; H, 6.52.
Methyl 4-O-benzyl-2,3-di-O-(4-methoxybenzyl)-i-D-
gluco-hexodialdo-1,5-pyranoside (11).—Dry Me2SO
(14.9 mL, 0.210 mmol) in dry CH2Cl2 (10 mL) was
treated with trifluoroacetic anhydride (26.6 mL, 0.191
mmol) at −60 °C. The solution was stirred for 10 min
and then 4 (20 mg, 0.0381 mmol) in dry CH2Cl2 (5 mL)
was added. The mixture was stirred at −60 °C for 1.5
h. After addition of Et3N (52.8 mL, 0.379 mmol) in dry
CH2Cl2 (5 mL), the reaction mixture was stirred at
−60 °C for 1.5 h. The solution was allowed to warm
up to −40 °C. A solution of 19:1 MeOH–water (3
mL) was added, the solution was concentrated and the
residue was chromatographed (3:1 toluene–EtOAc) to
afford 11 as a colorless solid (16.5 mg, 82%): [h]2D0 +7°
Methyl
phenylmethyl-i-
drate-(1“4)-6-O-triphenylmethyl-i-
eno-2-ulopyranoside (15), methyl 6-O-triphenylmethyl-4-
O-methyl-i- -arabino-hex-2-ulopyranosyl-2-hydrate-
(1“4)-6-O-triphenylmethyl-i- -erythro-hex-3-eno-2-
ulopyranoside (16), and methyl 4-O-methyl-6-O-
triphenylmethyl-i- -erythro-hex-2,3-diulopyranosyl-2-
hydrate-(1“4)-6-O-triphenylmethyl-i- -erythro-hex-
4-O-methyl-3-O-methylthiomethyl-6-O-tri-
-arabino-hex-2-ulopyranosyl-2-hy-
-erythro-hex-3-
D
D
D
D
D
D
3-eno-2-ulopyranoside (18).—Dry Me2SO (329 mL, 4.63
mmol) in dry CH2Cl2 (50 mL) was treated with trifl-
uoroacetic anhydride (588 mL, 4.21 mmol) at −60 °C.
The solution was stirred for 10 min and then 14 (600
mg, 0.702 mmol) in dry CH2Cl2 (5 mL) was added. The
mixture was stirred at −60 °C for 1.5 h. After the
addition of Et3N (1.28 mL, 9.26 mmol) in dry CH2Cl2
(5 mL), the reaction mixture was stirred at −60 °C for
1.5 h. The solution was allowed to warm up to −
40 °C. After addition of MeOH (5 mL) and water (0.5
mL), the solution was concentrated and the residue was
chromatographed (34:15:1 CHCl3–n-hexane–MeOH)
to afford first 15 as a colorless solid (77.8 mg, 12%):
[h]2D0 −12° (c 0.4, CHCl3); Rf 0.90 (19:1 CHCl3–
1
(c 0.5, CHCl3); H NMR (CDCl3): l 9.61 (d, 1 H, J5,6
1.0 Hz, H-6), 7.38–7.15 (m, 9 H, Ph), 6.90–6.80 (m, 4
H, Ph), 4.86–4.57 (m, 6 H, 6 CH2Ph), 4.40 (d, 1 H, J1,2
7.3 Hz, H-1), 3.85–3.77 (m, 7 H, H-5, 2 OMe), 3.73–
3.63 (m, 2 H, H-3, H-4), 3.58 (s, 3 H, 1-OMe), and 3.40
(dd, 1 H, J2,3 9.0 Hz, H-2); 13C NMR (CDCl3): l 197.13
(C-6), 159.47 (Ph), 159.44 (Ph), 137.59 (Ph), 128.19–
130.57 (Ph), 113.98 (Ph), 104.76 (C-1), 83.53 (C-3),
81.52 (C-2), 78.39 (C-5), 77.32 (C-4), 75.40 (CH2Ph),
74.96 (CH2Ph), 74.47 (CH2Ph), 57.47 (1-OMe), and
55.42 (2 OMe); MALDI-TOF MS: m/z 545.12 [M+
Na]+.
1
MeOH); H NMR (CDCl3): l 7.50–7.05 (m, 30 H, 6
Methyl i-D-gluco-hexodialdo-1,5-pyranoside-6-hy-
Ph), 5.10 (s, 1 H, H-1%), 4.87 (d, 1 H, J 11.0 Hz,
OCH2S), 4.86 (s, 1 H, H-1), 4.78 (d, 1 H, J3%,4% 3.4 Hz,
H-3%), 4.72 (d, 1 H, J 11.0 Hz, OCH2S), 4.58 (dd, 1 H,
J5,6a 4.3, J5,6b 4.3 Hz, H-5), 3.85–3.75 (m, 2 H, H-5%,
H-4%), 3.65–3.55 (m, 2 H, H-6b, H-6a), 3.46 (s, 3 H,
1-OMe), 3.41–3.37 (m, 2 H, H-6b%, H-6a%), 3.36 (s, 3 H,
4%-OMe), and 2.16 (s, 3 H, SCH3); 13C NMR (CDCl3):
l 183.19 (C-2), 151.60 (C-4), 143.91 (Ph), 129.17–
127.46 (Ph, C-3), 99.52 (C-1), 98.17 (C-2%), 95.37 (C-1%),
87.49 (2 CPh3), 84.53 (C-3%), 79.13 (C-4%), 78.07 (C-5%),
72.41 (C-5), 70.01 (SCH2O), 63.79 (C-6), 62.76 (C-6%),
58.95 (4%-OMe), 56.80 (1-OMe), and 14.99 (SCH3); LC-
MS: m/z 243.1 [Ph3C]+.
drate (12).—Hydrogenolysis of 11 (300 mg, 0.574
mmol) was performed as described for 7. The crude
product was chromatographed (4:1 CH2Cl2–MeOH) to
give 12 as a colorless solid (33.0 mg, 27%): [h]2D0 −47°
(c 0.3, water), lit. [h]2D2 −71° (c 0.5, water);14 1H NMR
(D2O): l 5.25 (d, 1 H, J5,6 2.3 Hz, H-6), 4.37 (d, 1 H,
J1,2 7.9 Hz, H-1), 3.57 (s, 3 H, 1-OMe), 3.52–3.47 (m,
2 H, H-3, H-4), 3.40 (m, 1 H, H-5), and 3.26 (dd, 1 H,
J2,3 9.7 Hz, H-2); 13C NMR (D2O): l 104.23 (C-1),
88.74 (C-6), 77.30 (C-5), 76.43 (C-3), 73.77 (C-2), 70.94
(C-4), and 58.06 (1-OMe).
Methyl 4-O-methyl-6-O-triphenylmethyl-i-
pyranosyl-(1“4)-6-O-triphenylmethyl-i- -glucopyran-
oside (14).—Methyl 4-O-methyl-b- -glucopyranosyl-
D-gluco-
D
Subsequent elution gave 16 as a colorless solid (46.7
D
mg, 7%): [h]2D0 −4° (c 0.2, CHCl3); Rf 0.55 (19:1

698
J. Ro¨hrling et al. / Carbohydrate Research 337 (2002) 691–700
CHCl3–MeOH); 1H NMR (CDCl3): l 7.60–7.05 (m, 30
H, Ph), 5.15 (s, 1 H, H-1%), 4.93 (s, 1 H, H-1), 4.85 (dd,
1 H, J5,6a 4.3, J5,6b 8.0 Hz, H-5), 3.85–3.79 (m, 2 H,
H-6b, H-6a), 3.76–3.32 (m, 6 H, H-6b%, H-6a%, H-4%,
H-3%, H-5, H-5%), 3.31 (s, 3 H, 1-OMe), 3.25 (s, 3 H,
4%-OMe), and 3.14 (dd, 1 H, J5%,6a% 3.5, J6a%,6b% 10.0 Hz,
H-6a%); 13C NMR (CDCl3): l 184.19 (C-2), 149.97
(C-4), 144.09 (Ph), 143.91 (Ph), 127.28–129.29 (Ph),
126.19 (C-3), 100.29 (C-1), 93.90 (C-1%), 91.77 (C-2%),
87.63 (CPh3), 86.61 (CPh3), 78.39 (C-4%), 75.84 (C-3% or
C-5%), 73.46 (C-5), 72.59 (C-5% or C-3%), 66.53 (C-6),
61.52 (C-6%), 61.12 (4%-OMe), and 57.02 (1-OMe). Anal.
Calcd for C52H50O12·1.3 H2O: C, 70.15; H 5.95. Found:
C, 70.19; H, 5.71.
was treated as described for 7 to give 19 as a colorless
solid (35.9 mg, 68%): [h]2D0 +31° (c 0.2, water); UV:
umax 274 nm (m 1.0×105 dm2/mol, MeOH); IR (KBr): w
1755 and 1691 (CꢀO), and 1651 (CꢀC); 1H NMR
(D2O): l 5.43 (s, 1 H, H-1%), 5.18 (s, 1 H, H-1), 4.91
(dd, 1 H, J5,6a 5.5, J5,6b 3.8 Hz, H-5), 4.10–3.95 (m, 2
H, H-6b, H-6a), 3.91–3.83 (m, 1 H, H-6b%), 3.76–3.60
(m, 2 H, H-5%, H-6a%), 3.63 (s, 3 H, 1-OMe), 3.61 (s, 3
H, 4%-OMe), and 3.52 (d, 1 H, J4%,5% 9.7 Hz, H-4%); 13C
NMR (D2O): l 186.06 (C-2), 152.14 (C-4), 127.58
(C-3), 99.91 (C-1), 95.19 (C-3% or C-2%), 93.55 (C-1%),
92.74 (C-2% or C-3%), 78.73 (C-4%), 76.07 (C-5%), 74.79
(C-5), 62.90 (C-6), 62.08 (4%-OMe), 61.07 (C-6%), and
57.69 (1-OMe); LC-MS: m/z 379.1 [M–H]−. Anal.
Calcd for C14H20O12: C, 44.22; H 5.30. Found: C,
44.09; H, 5.12.
General procedure for labeling of keto sugars.—The
keto compound (0.03–0.05 mmol) and a fivefold molar
excess of 20 were dissolved in water (1 mL) and stirred
overnight at rt. The solution was treated with satd aq
NaHCO3, and extracted with EtOAc (50 mL). The
organic layer was dried (MgSO4) and concentrated.
Column chromatography of the residue afforded the
labeled compounds (E- and Z-isomer) and unreacted
20. In case of highly polar products, the extraction step
was omitted and the reaction mixture was freeze-dried
prior to chromatography.
Finally, 18 was obtained as a colorless solid (302 mg,
50%): [h]2D0 −12° (c 0.8, CHCl3); Rf 0.45 (19:1 CHCl3–
1
MeOH); H NMR (CDCl3): l 7.48–7.09 (m, 30 H, Ph),
5.02 (s, 1 H, H-1%), 4.89 (s, 1 H, H-1), 4.77 (dd, 1 H,
J5,6a 4.6, J5,6b 7.1 Hz, H-5), 4.69 (d, 1 H, J4%,5% 9.5 Hz,
H-4%), 3.80–3.72 (m, 2 H, H-6b, H-6a), 3.65–3.49 (m, 2
H, H-6b%, H-5%), 3.27 (s, 3 H, 1-OMe), 3.25 (s, 3 H,
4%-OMe), and 3.16 (dd, 1 H, J5%,6a% 3.6, J6a%,6b% 11.0 Hz,
H-6a%); 13C NMR (CDCl3): l 199.20 (C-3%), 182.77
(C-2), 149.20 (C-4), 143.92 (Ph), 143.85 (Ph), 129.15–
127.44 (Ph), 126.41 (C-3), 100.40 (C-1), 95.01 (C-1%),
91.43 (C-2%), 87.73 (CPh3), 86.81 (CPh3), 79.38 (C-4%),
75.64 (C-5%), 73.27 (C-5), 66.43 (C-6), 61.23 (C-6%), 60.28
(4%-OMe), and 57.12 (1-OMe). Anal. Calcd for
C52H48O12·1.3 H2O: C, 70.25; H 5.75. Found: C, 70.26;
H, 5.86.
Methyl
6-O-benzyl-4-O-methyl-i-D-ribo-hex-3-
ulopyranoside
{7-[(9H-9-carbazolylcarbonyl)-amino]-
1,4,7-trioxaheptyl}imine (21).—Reaction of 9 (10.0 mg,
0.034 mmol) and 20 (89 mg, 0.24 mmol) was performed
according to the general procedure. Final purification
by chromatography (1:1“1:2 n-hexane–EtOAc) gave
21 (E-isomer) as a colorless oil (7.3 mg, 36%): [h]D20
−20° (c 0.6, CHCl3); Rf 0.35 (1:1 n-hexane–EtOAc);
UV: umax 230 nm (m 5.6×105 dm2/mol, MeOH); Fluo-
rescence: uexc 290 nm, uem 346 nm [DMAc/LiCl 0.9%
Methyl 4-O-methyl-i-D-arabino-hex-2-ulopyranosyl-
2-hydrate-(1“4)-i- -erythro-hex-3-eno-2-ulopyran-
D
oside (17).—A suspension of 16 (67.0 mg, 0.078 mmol)
and 10% Pd–C (12.0 mg) in dry MeOH (20 mL) was
hydrogenolyzed at atmospheric pressure for 15 h at rt.
The catalyst was filtered off, and the mixture was
concentrated. The residue was chromatographed (9:1
CH2Cl2–MeOH) to give 17 as a colorless solid (8.8 mg,
30%): [h]2D0 +39° (c 0.3, water); UV: umax 274 nm (m
9.0×104 dm2/mol, MeOH); IR (KBr): w 1686 (CꢀO),
1
(m/v)]; IR (KBr): w 1715 (CꢀN); H NMR (CDCl3): l
8.81 (br. s, 1 H, NH), 8.11 (d, 2 H, J4,5 8.3 Hz, H-5),
8.00 (d, 2 H, J2,3 7.7 Hz, H-2), 7.48 (ddd, 2 H, J2,4 1.0,
J3,4 7.9 Hz, H-4), 7.35 (dd, 2 H, H-3), 7.30–7.20 (m, 5
H, Ph), 4.75 (m, 1 H, H-4%), 4.65 (d, 1 H, J1,2 1.8 Hz,
H-1%), 4.44 (s, 2 H, CH2Ph), 4.33 (t, 2 H, J 4.6 Hz,
CH2ON), 4.25 (t, 2 H, J 4.4 Hz, CH2ON), 4.14 (m, 1 H,
H-5%), 3.90–3.75 (m, 5 H, CH2OCH2, H-2%), 3.52–3.42
(m, 2 H, H-6a%, H-6b%), 3.35 (s, 3 H, 4%-OMe), and 3.34
(s, 3 H, 1%-OMe); 13C NMR (CDCl3): l 154.16 (CꢀO),
149.44 (C-3%), 138.30, 138.16 (C-1, Ph), 128.77 (Ph),
128.14 (Ph), 127.98 (Ph), 127.54 (C-4), 125.83 (C-6),
123.25 (C-3), 120.44 (C-2), 114.90 (C-5), 104.73 (C-1%),
76.35 (C-5%), 76.11 (CH2ON), 73.73 (CH2ON, CH2Ph),
71.62 (C-4%), 71.27 (C-2%), 70.83 (CH2OCH2), 70.35
(CH2OCH2), 70.11 (C-6%) 58.06 (4%-OMe), and 56.75
(1%-OMe); MALDI-TOF MS: m/z 631.05 [M+Na]+.
Further elution gave 21 (Z-isomer) as a colorless oil
(9.6 mg, 47%): [h]D20 −44° (c 1.0, CHCl3); Rf 0.25 (1:1
1
and 1652 (CꢀC); H NMR (D2O): l 5.37 (s, 1 H, H-1%),
5.16 (s, 1 H, H-1), 4.91 (dd, 1 H, J5,6a 4.8, J5,6b 4.8 Hz,
H-5), 4.05–3.95 (m, 2 H, H-6b, H-6a), 3.88 (dd, 1 H,
J5%,6b% 2.1, J6a%,6b% 12.4 Hz, H-6b%), 3.85 (d, 1 H, J3%,4% 9.3
Hz, H-3%), 3.74 (dd, 1 H, J5%,6a% 5.3 Hz, H-6a%), 3.63–3.58
(m, 7 H, H-5%, 1-OMe, 4%-OMe), and 3.42 (dd, 1 H, J3%,4%
9.3, J4%,5% 9.3 Hz, H-4%); 13C NMR (D2O): l 186.28
(C-2), 152.14 (C-4), 127.61 (C-3), 99.94 (C-1), 94.21
(C-1%), 92.41 (C-2%), 78.44 (C-4%), 76.64 (C-5%), 76.41
(C-3%), 73.79 (C-5), 62.91 (C-6), 61.23 (4%-OMe), 60.89
(C-6%), and 57.72 (1-OMe); LC-MS: m/z 364.1 [M–wa-
ter]−. Anal. Calcd for C14H22O12: C, 43.98; H 5.80.
Found: C, 44.16; H, 5.60.
Methyl 4-O-methyl-i-
osyl-2-hydrate-(1“4)-i-
D
-erythro-hex-2,3-diulopyran-
-erythro-hex-3-eno-2-ulopy-
D
ranoside (19).—Compound 18 (120.0 mg, 0.139 mmol)

J. Ro¨hrling et al. / Carbohydrate Research 337 (2002) 691–700
699
n-hexane–EtOAc); UV: umax 230 nm (m 5.8×105 dm2/
mol, MeOH); Fluorescence: uexc 290 nm, uem 344 nm
[DMAc/LiCl 0.9% (m/v)]; IR (KBr): w 1724 (CꢀN); H
H-4%), 3.57 (dd, 1 H, J5%,6b% 5.3, J6a%,6b% 11.4 Hz, H-6b%),
3.46 (dd, 1 H, J5%,6a% 7.0, H-6a%), 3.42 (s, 3 H, 1%-OMe),
and 3.29 (s, 3 H, 4%-OMe); 13C NMR (CD3OD): l
156.09 (CꢀO), 155.03 (C-3%), 139.39 (C-1), 127.94 (C-4),
126.47 (C-6), 123.64 (C-3), 120.96 (C-2), 115.37 (C-5),
103.80 (C-1), 80.75 (C-5%), 79.04 (C-4%), 76.92 (CH2ON),
74.79 (CH2ON), 70.77 (CH2OCH2), 70.71 (CH2OCH2),
66.51 (C-2%), 63.10 (C-6%), 57.10 (4%-OMe), and 56.52
(1-OMe); MALDI-TOF MS: m/z 540.25 [M+Na]+.
Anal. Calcd for C25H31N3O9: C, 58.02; H, 6.04; N, 8.12.
Found: C, 57.72; H, 5.93; N, 7.88.
1
NMR (CDCl3): l 9.17 (br. s, 1 H, NH), 8.13 (d, 2 H,
J4,5 8.3 Hz, H-5), 8.00 (d, 2 H, J2,3 7.7 Hz, H-2), 7.49
(ddd, 2 H, J2,4 1.2, J3,4 7.9, J4,5 8.3 Hz, H-4), 7.34 (dd,
2 H, J3,5 0.9 Hz, H-3), 7.30–7.20 (m, 5 H, Ph), 4.52–
4.42 (m, 4 H, H-1%, H-2%, CH2Ph), 4.36–4.27 (m, 4 H, 2
CH2ON), 4.13 (ddd, 1 H, J4%,5% 1.5, J5%,6a% 8.4, J5%,6b% 4.8
Hz, H-5%), 3.90–3.69 (m, 6 H, CH2OCH2, H-4%), 3.51
(dd, 1 H, J6a%,6b% 9.9 Hz, H-6b%), 3.42 (dd, 1 H, H-6b%),
3.32 (s, 3 H, 4%-OMe), and 3.14 (s, 3 H, 1%-OMe); 13C
NMR (CDCl3): l 153.99, 153.76 (CꢀO, C-3), 137.94,
137.84 (C-1, Ph), 128.36 (Ph), 127.69 (Ph), 127.60 (Ph),
127.12 (C-4), 125.38 (C-6), 122.73 (C-3), 119.95 (C-2),
114.55 (C-5), 101.81 (C-1%), 77.56 (C-4%), 77.16 (C-5%),
75.85 (CH2ON), 73.35 (CH2Ph), 72.89 (CH2ON), 69.99
(CH2OCH2), 69.70, 69.60 (C-6%, CH2OCH2), 65.53 (C-
2%) 56.54 (4%-OMe), and 56.38 (1%-OMe); MALDI-TOF
MS: m/z 631.54 [M+Na]+. Anal. Calcd for
C32H39N3O9: C, 63.04; H, 6.45; N, 6.89. Found: C,
63.20; H, 6.29; N, 6.68.
Methyl i-D-gluco-hexodialdo-1,5-pyranoside {7-
[(9H-9-carbazolylcarbonyl)amino]-1,4,7-trioxaheptyl}-
imine (23).—Compound 12 (10.0 mg, 0.048 mmol) and
20 (87.0 mg, 0.24 mmol) were reacted according to the
general procedure. Final purification by chromatogra-
phy (93:7 CH2Cl2–MeOH) gave 23 (E-isomer) as color-
less crystals (22.2 mg, 93%): mp 170–172 °C (MeOH);
[h]2D0 −29° (c 0.3, MeOH); Rf 0.27 (93:7 CH2Cl2–
MeOH); UV: umax 231 nm (m 5.0×105 dm2/mol,
MeOH); Fluorescence: uexc 290 nm, uem 346 nm
1
[DMAc/LiCl 0.9% (m/v)]; IR (KBr): w 1674 (CꢀN); H
Methyl 4-O-methyl-i-D-ribo-hex-3-ulopyranoside {7-
NMR (d6-Me2SO): l 11.50 (br. s, 1 H, NH), 8.18 (d, 2
H, J2,3 7.7 Hz, H-2), 8.00 (d, 2 H, J4,5 8.4 Hz, H-5), 7.51
(dd, 2 H, J3,4 8.4 Hz, H-4), 7.36 (dd, 2 H, H-3), 7.36 (d,
1 H, J5%,6% 7.3 Hz, H-6%), 5.14 (d, 2 H, J 4.9 Hz, 2 OH),
5.08 (d, 1 H, J 4.1 Hz, OH), 4.19–4.13 (m, 5 H, H-1%,
2 CH2ON), 3.80–3.66 (m, 5 H, CH2OCH2, H-5%), 3.36
(s, 3 H, OMe), 3.23–3.10 (m, 2 H, H-3%, H-4%), and
3.05–2.94 (m, 1 H, H-2%); 13C NMR (d6-Me2SO): l
152.82 (CꢀO), 148.71 (C-6%), 137.66 (C-1), 126.76 (C-4),
124.13 (C-6), 122.06 (C-3), 120.18 (C-2), 114.09 (C-5),
104.03 (C-1%), 75.94 (C-3%), 75.01 (CH2ON), 73.18 (C-5%,
C-2%), 72.73 (CH2ON), 71.57 (C-4%), 68.75 (CH2OCH2),
68.60 (CH2OCH2), and 56.18 (1%-OMe); MALDI-TOF
MS: m/z 527.69 [M+Na]+.
[(9H-9-carbazolylcarbonyl)-amino]-1,4,7-trioxaheptyl}-
imine (22).—Compound 10 (7.0 mg, 0.034 mmol) and
20 (62.0 mg, 0.17 mmol) were reacted according to the
general procedure. Final purification by chromatogra-
phy (19:1 CH2Cl2–MeOH) gave 23 (E-isomer) as a
colorless oil (4.8 mg, 27%): [h]2D0 −25° (c 0.4, MeOH);
Rf 0.35 (19:1 CH2Cl2–MeOH); UV: umax 230 nm (m
5.9×105 dm2/mol, MeOH); Fluorescence: uexc 290 nm,
uem 346 nm [DMAc/LiCl 0.9% (m/v)]; IR (KBr): w 1701
1
(CꢀN); H NMR (CD3OD): l 8.10–8.04 (m, 4 H, H-2,
H-5), 7.49 (ddd, 2 H, J3,4 7.9, J4,5 8.3, J2,4 1.2 Hz, H-4),
7.34 (ddd, 2 H, J2,3 7.7, J2,4 1.0 Hz, H-3), 4.73 (dd, 1 H,
H-4%), 4.71 (d, 1 H, J1%,2% 2.9 Hz, H-1%), 4.35–4.29 (m, 2
H, CH2ON), 4.26–4.21 (m, 2 H, CH2ON), 3.96–3.80
(m, 6 H, CH2OCH2, H-2%, H-5%), 3.53 (d, 2 H, J5%,6% 6.3
Hz, H-6%), 3.41 (s, 3 H, 1%-OMe), and 3.38 (s, 3 H,
4%-OMe); 13C NMR (CD3OD): l 156.09 (CꢀO), 153.31
(C-3%), 139.47 (C-1), 127.94 (C-4), 126.46 (C-6), 123.61
(C-3), 120.96 (C-2), 115.36 (C-5), 105.34 (C-1%), 79.30
(C-5%), 76.94 (CH2ON), 74.68 (CH2ON), 72.04 (C-2%),
71.15 (C-4%), 70.77 (CH2OCH2), 63.12 (C-6%), 57.93
(4%-OMe), and 56.76 (1%-OMe); MALDI-TOF MS: m/z
540.73 [M+Na]+.
Further elution gave 23 (Z-isomer) as a colorless
solid: (0.9 mg, 4%): Rf 0.20 (93:7 CH2Cl2–MeOH); UV:
1
umax 231 nm (m 4.5×105 dm2/mol, MeOH); H NMR
(CD3OD): l 8.08 (d, 2 H, J4,5 8.5 Hz, H-5), 8.07 (d, 2
H, J2,3 7.9 Hz, H-2), 7.48 (dd, 2 H, J3,4 8.5 Hz, H-4),
7.33 (dd, 2 H, H-3), 6.70 (d, 1 H, J5%,6% 6.8 Hz, H-6%),
4.55 (dd, 1 H, J4%,5% 9.2 Hz, H-5%), 4.28 (t, 2 H, J 4.5 Hz,
CH2ON), 4.23 (t, 2 H, J 4.5 Hz, CH2ON), 4.14 (d, 1 H,
J1%,2% 7.8 Hz, H-1%), 3.89 (t, 2 H, CH2OCH2), 3.83 (t, 2
H, CH2OCH2), 3.46 (s, 3 H, OMe), 3.34–3.29 (m, 1 H,
H-3%), 3.24 (dd, 1 H, J3%,4% 9.2 Hz, H-4%), and 3.12 (dd, 1
H, J2%,3% 8.1 Hz, H-2%); 13C NMR (CD3OD): l 153.25
(CꢀO), 149.33 (C-6%), 127.78 (C-4), 126.23 (C-6), 123.18
(C-3), 120.90 (C-2), 115.24 (C-5), 105.48 (C-1), 77.45
(C-3%), 76.32 (CH2ON), 74.93 (C-2%), 74.69 (CH2ON),
73.79 (C-4%), 70.88 (CH2OCH2), 70.71 (CH2OCH2),
69.06 (C-5%), and 57.47 (1-OMe); MALDI-TOF MS:
m/z 527.53 [M+Na]+. Further elution (17:3 CH2Cl2–
MeOH) gave unchanged 20 (50 mg, 57%).
Further elution gave 22 (Z-isomer) as a colorless oil
(8.6 mg, 49%): [h]2D0 +8° (c 0.4, MeOH); Rf 0.29 (19:1
CH2Cl2–MeOH); UV: umax 230 nm (m 5.9×105 dm2/
mol, MeOH); Fluorescence: uexc 290 nm, uem 346nm
1
[DMAc/LiCl 0.9% (m/v)]; IR (KBr): w 1706 (CꢀN); H
NMR (CD3OD): l 8.10–8.04 (m, 4 H, H-5, H-2), 7.48
(ddd, 2 H, J3,4 7.4, J4,5 7.4, J2,4 1.2 Hz, H-4), 7.35 (ddd,
2 H, J2,3 8.0, J3,5 1.0 Hz, H-3), 4.59 (d, 1 H, J1%,2% 6.1 Hz,
H-1%), 4.47 (d, 1 H, H-2%), 4.34 (m, 2 H, CH2ON), 4.25
(m, 2 H, CH2ON), 3.95–3.79 (m, 6 H, CH2OCH2, H-5%,

700
J. Ro¨hrling et al. / Carbohydrate Research 337 (2002) 691–700
i-D
-Glucopyranosyl-(1“4)-i-
D
-glucose {7-[(9H-9-
References
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—Cellobiose (20.0 mg, 0.058 mmol) and 20 (107 mg,
0.29 mmol) were dissolved in water (3 mL) and stirred
for 7 days at rt. A satd aq NaHCO3 solution (0.1 M,
0.5 mL) was added and the solution was freeze-dried.
The solid residue was purified by chromatography
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less solid (28.5 mg, 75%): [h]2D0 −3° (c 0.4, MeOH);
UV: umax 230 nm (m 5.8×105 dm2/mol, MeOH); Fluo-
rescence: uexc 290 nm, uem 344 nm [DMAc/LiCl 0.9%
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(m/v)]; IR (KBr): w 1696 (CꢀN); H NMR (CD3OD):
l 8.18–8.06 (m, 6.9 H, H-2, H-5), 7.64 (d, 1 H,
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Technical assistance by Maria Hobel is gratefully
acknowledged. Financial support by the Christian-
Doppler Research Society and Lenzing AG is grate-
fully acknowledged. Thomas Lange has been
supported by the European Commission within the
Programme ‘Improving Human Research Potential
and the Socio-economic base’ MC-fellowship HPMF-
CT-2000-00531. The authors also thank Dr. Michael
Puchberger for recording NMR spectra.