1,3-Dideoxynojirimycin-3-yl glycosides of β-(1→3)- and β-(1→6)-linked gluco-oligosaccharides

Article abstract of DOI:10.1016/j.carres.2006.05.004

Standard chemical methods involving the use of O-acetylated glycosyl trichloroacetimidates as glycosylating agents were used to prepare the five 1,3-dideoxynojirimycin-3-yl β-(1→3)-linked oligo-glucosides (1-5) and also the β-(1→6)-bonded glucobiose (gent

Full text of DOI:10.1016/j.carres.2006.05.004

Carbohydrate Research 341 (2006) 2115–2125
1,3-Dideoxynojirimycin-3-yl glycosides of b-(1!3)- and
b-(1!6)-linked gluco-oligosaccharides
Regine Blattner,a Richard H. Furneauxa,* and Zbigniew Pakulskia,b
aIndustrial Research Ltd, PO Box 31-310, Lower Hutt, New Zealand
bInstitute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka, 44/52, 01-224 Warszawa, Poland
Received 23 March 2005; received in revised form 7 April 2006; accepted 7 May 2006
Available online 5 June 2006
Abstract—Standard chemical methods involving the use of O-acetylated glycosyl trichloroacetimidates as glycosylating agents were
used to prepare the five 1,3-dideoxynojirimycin-3-yl b-(1!3)-linked oligo-glucosides (1–5) and also the b-(1!6)-bonded glucobiose
(gentiobiose)-based analogue 6 as potential fungicides. In the course of the work, the b-(1!6), b-(1!6)-linked analogue 8 of 6 and
6-O- and 4-O-b-glucopyranosyl-deoxynojirimycins 7 and 9, respectively, were also produced.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Fungicides; Glycosylation; Nojirimycin; 13C and 1H NMR; HR-MS/FAB; ES-TOF-MS
1. Introduction
synthesized on the plasma-side of the cell membrane,
branching as well as any putative cross-linking between
different polysaccharides take place in the cell wall
itself,1,2,5 and therefore the glycosylases and glycosyl
transferases which mediate the branching and cross-
linking processes may, in principle at least, be reached
by inhibitors which do not have to find means of trans-
port across the cell membranes as is the case with inhib-
itors of the b-(1!3) glucan synthases.1,2,4
Interest remains high in the development of new anti-
fungal agents, in particular for the treatment of the
increasing numbers of immuno-compromised patients
suffering from AIDS or coping with cancer chemothe-
rapy or recovery from organ transplant surgery. One
important strategy for developing suitable protection
against common infections depends on the use of com-
pounds that interfere specifically with the biosynthesis
of the key components of cell walls of the relevant path-
ogenic microorganisms.1–4 The efficacies of the penicil-
lins and cephalosporins as antibiotics depend on their
use in this approach directed against bacterial cell wall
biosynthesis; the search continues for fungicides which
act by similar mechanisms.
Together with a mannoprotein, a b-(1!3)-linked glu-
can with DP ꢀ 1500 and containing up to 10% b-(1!6)-
linked branching makes up the major part of fungal cell
walls. In addition, a b-(1!6) glucan with about 20% of
b-(1!3) branching, and also chitin, are minor compo-
nents. Whereas the linear part of the b-(1!3) glucan is
On the assumption that, in the course of the branch-
ing reactions, glucosyl and oligoglucosyl residues from
the non-reducing ends of b-(1!3)-glucans are trans-
ferred to 6-positions of non-terminal units of other
(1!3)-linked glucan chains,5,6 and that cross-linking
involves glycosidic bonding of the reducing centers of
some chains to primary positions of non-terminal units
of others,5–9 we have developed a strategy for the prep-
aration of potential inhibitors of the enzymes impli-
cated. This involves the specific O-glycosylation of
the well known glycosidase inhibitor 1-deoxynojiri-
mycin (1,5-dideoxy-1,5-imino-D-glucitol, DNJ)10 with
b-(1!3)- and b-(1!6)-linked glucose oligomers on the
principle that in such compounds the oligosaccharide
moieties would be recognized and bound by the relevant
enzymes, and thereby the inhibiting nitrogen-containing
moieties would be directed towards the active sites. The
*
Corresponding author. Tel.: +4 64 931 3168; fax: +4 64 931 3055;
e-mail: r.furneaux@irl.cri.nz
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.05.004

2116
R. Blattner et al. / Carbohydrate Research 341 (2006) 2115–2125
consequence could be selective interference in the pro-
duction of the branched glycans during the biosynthesis
of the major fungal cell wall polymers.
2. Results and discussion
For the purpose outlined above, D-glucose, its b-(1!3)-
linked dimer (laminaribiose), the corresponding b-
(1!3)-linked tetramer, hexamer and heptamer, and also
the b-(1!6)-linked glucobiose, gentiobiose, were sepa-
rately b-(1!3) bonded to DNJ to give compounds
1–6, respectively (Scheme 1). In addition, corresponding
glycosides having glucose and gentiobiose separately
linked b-(1!6) to the same amine (compounds 7 and
8, respectively) and glucose b-(1!4)-bonded to DNJ
(compound 9) were also synthesized (Scheme 2).
Previous studies encourage this approach. For exam-
ple, several bioactive glycosylated DNJ derivatives are
known, some having been isolated from natural sources
and some having been made by chemical or by enzymic
procedures.10–13 Not surprisingly, several of these com-
pounds inhibit carbohydrate-processing enzymes,10 as
do various nitrogen-containing analogues with some-
what modified structures,14–16 indicating that the area
could be fertile as a source of the enzyme inhibitors
required for the present work.
The procedure adopted (Scheme 1, for example, for
compounds 1–6) involved use of the appropriate O-acet-
ylated glycosyl trichloroacetimidates (1B–6B), made
from the corresponding sugar peracetates by selective
hydrolysis of the anomeric ester functions by use of
hydrazinium acetate in DMF,23 and treatment of the
derived free sugars (1A–6A) with trichloroacetonitrile in
dichloromethane, usually in the presence of potassium
carbonate.24 The glycosyl donors 1B–5B were coupled
with the monohydroxy DNJ derivative 10 (Scheme 1),
which was made from the corresponding 2,3-diol
together with the known 3-ester and the 2,3-diester, and
which was also used to make compound 6. For com-
pounds 7 and 8, selective glycosylation at the primary
position of the diol 11 was employed, and the (1!4)-
linked glucoside 9 was made using the 2,3,6-tribenzyl
ether 12.25 The products were purified by chromatogra-
phy and deprotected by standard deesterification fol-
lowed by hydrogenolysis, the alternative deprotection
sequence of hydrogenolysis followed by deesterification
being less successful.
More complex glycosylated DNJ compounds to have
been synthesized are analogues of the biologically
important sialyl-Lewis X17 and A18 tetrasaccharides
which have the N-heterocyclic compound replacing the
structurally related N-acetylglucosamine.
Specifically, as far as the strategy to be used in the
present work is concerned, homo-glucooligosaccharide
1-deoxynojirimycin glycosides are known to inhibit rel-
evant enzymes. Thus DNJ glycosides of maltooligosac-
charides, up to the heptaose, a-(1!4)-linked to the
aglycon inhibit some amylases with structure–activity
relationships which depend to a major extent on the
source of the enzymes.19 For example, a bacterial sac-
charifying a-amylase is not inhibited by DNJ itself,
but is by the 4-O-a-D-glucosyl and 4-O-a-maltosyl deriv-
atives to increasing extents; the activities however do not
increase further on subsequent elongation of the saccha-
ride chain. On the other hand, a microbial b-amylase is
inhibited by 4-O-a-D-glucosyl-DNJ, and to some extent
by the trisaccharide-linked analogue, but not by other
members of the series. Of special relevance to the present
work is the observation that the enzyme of Aerobacter
aerogenes that hydrolyzes the a-(1!3)- and a-(1!6)-
linked glucan pullulan is inhibited by 4-O-a-maltosyl-
DNJ and, increasingly, by higher members of the ser-
ies.19 In a similar fashion, 4-O-b-D-glucosyl- and -cello-
biosyl-DNJ inhibit cellulases of bacteria and fungi, the
latter being much the more potent, and 6-O-b-cellobio-
syl-DNJ also shows some cellulase inhibiting activity.20
In the course of that work it was reported that cellooli-
gosaccharide glycosides of DNJ had been made by use
of a cellodextrin phosphorylase of Clostridium thermo-
cellum expressed by E. coli, applied with glucose 1-phos-
phate as glucose donor and 4-O-D-glucosyl- and 6-O-
cellobiosyl-DNJ as acceptors.21
During glycosylation of diol 11 at O-6 with tetra-O-
acetyl-a,b-D-glucosyl trichloroacetimidate some selec-
tive deacetylation of the product occurred to give a
2,3,4-triacetate that afforded the same product (7D) on
deacetylation as did the tetraacetate (7C).
On the basis of the b-configurations assigned by
NMR coupling evidence to the glucosides 1, 7 and 9
(J1,2 = 8.0 0.1 Hz), and of expectations founded on
many analogous literature precedents,26 all of the glyco-
sidic bonds formed were assumed also to have this ano-
meric configuration. The couplings that were observed
1
in the anomeric doublet signals in the H NMR spectra
concurred, but not all such signals were visible—espe-
cially in the spectra of the more complex compounds.
Compound 7 has been reported previously,12 its forma-
tion establishing that diol 11 underwent preferential
substitution at the primary position in keeping with
normal experience.27 It is assumed that compound 8
was derived similarly.
Although, as already indicated, glycosylated DNJ
derivatives can inhibit various enzymes and show a
range of bioactivities, it is recognized that their potency
against glucosidases from several sources is not high.10
Although known in the first instance as a powerful
inhibitor of a-glucosidases, DNJ has also been shown
to display considerable activity against fungal b-glucosi-
dases, the b-enzyme of Aspergillus wentii, for example,
being inhibited with Ki=2.7 lM.22 DNJ and its deriva-
tives have also been recognized as being of potential
value as antidiabetic, antineoplastic and anti-HIV
agents.10

R. Blattner et al. / Carbohydrate Research 341 (2006) 2115–2125
2117
2
1
1
R O
R O
R O
3
AcO
AcO
1
NR
Cbz
2
AcO
AcO
AcO
O
O
1
b
O
R O
R O
O
Ph
R O
R O
N
O
O
O
O
+
1
O
HO
4
1
1
OR
OR
OR
n
OR
n
OAc
OAc
n
OCOCH Cl
2
n
R
H
1
2
3
4
10
R
R
R
R
0
0
1A
1B
a
(C=NH)CCl
1C
1D
1
2C
2D
2
3C
3D
3
4C
4D
4
5C
5D
5
0
Ac
H
H
Ac
H
H
Ac
H
H
Ac
H
H
PhCH Cbz COCH Cl
2
3
c
d
0
0
1
1
1
3
3
3
5
5
5
6
6
6
PhCH Cbz
H
H
H
H
1
1
H
2A
2B
a
(C=NH)CCl
PhCH Cbz COCH Cl
3
2
c
PhCH Cbz
H
d
H
H
H
3
3
H
3A
3B
a
a
PhCH Cbz COCH Cl
(C=NH)CCl
2
3
c
d
PhCH Cbz
H
H
H
H
5
5
4A
4B
H
PhCH Cbz COCH Cl
2
(C=NH)CCl
3
c
d
PhCH Cbz
H
H
PhH
H
H
H
H
H
6
6
5A
5B
Cbz
COCH Cl
2
H
H
Ac
H
H
a
(C=NH)CCl
c
3
Cbz
H
d
AcO
AcO
O
1
R O
3
O
AcO
AcO
2
AcO
b
R
1
O
R O
O
10
+
R O
N
1
2
O
OAc
R O
R O
1
O
R O
1
1
O
OR
OR
R O
OAc
4
OR
1
OR
4
1
2
2
3
R
R
R
R , R
R
H
6A
6B
6C
6D
6
Ac
H
H
PhCH Cbz COCH Cl
2
a
c
d
(C=NH)CCl
3
PhCH Cbz
H
H
H
H
Scheme 1. Reagents: (a) CCl3CN, K2CO3; (b) TMSOTf, CH2Cl2, molecular sieve; (c) MeONa, MeOH; (d) H2, Pd/C, H2O, pressure.
R1
O
O
R1O
HO
O
R1O
NCbz
OBn
HO
1 R1O
O
1B
+
R3
N
BnO
R1O
n
OR
O
1 HO
OR
R2O
a
11
OR2
n
R1
R2
R3
7C
7D
7
8C
8D
8
0
0
0
1
1
1
Ac
H
H
Ac
H
H
Bn
Bn
H
Bn
Bn
H
Cbz
Cbz
H
Cbz
Cbz
H
b
c
a
+
11
6B
b
c
R1O
R3
N
R2O
BnO
R1O
O
a
NCbz
OBn
R1O
HO
O
1B
+
BnO
R2O
OR1
OR2
12
R1
Ac
H
R2
Bn
Bn
H
R3
9C
9D
9
Cbz
Cbz
H
b
c
H
Scheme 2. Reagents: (a) TMSOTf, CH2Cl2, molecular sieve; (b) MeONa, MeOH; (c) H2, Pd/C, H2O, pressure.

2118
R. Blattner et al. / Carbohydrate Research 341 (2006) 2115–2125
For this reason, and because our primary interests were
in the effects of our products on whole organisms, they
(except compound 4) were tested against 20 organisms
as follows: nine fungi (four plant pathogens, Botrytis
cinerea, Colletotrichum acutatum, Diplodia pinea, Fusar-
ium oxysporum; three dermatophytes, Microsporum
canis, Trichophyton mentagrophytus, Trichophyton
rubrum; one yeast Candida albicans and one saprophyte,
Rhizopus stolonifer); three bacteria (Escherichia coli,
Pseudomonas aeruginosa and Staphylococcus aureus);
three insects [Lucilia cuprina (sheep blowfly), Heliothis
armigera (tomato fruitworm) and Spodoptera litura
(tropical armyworm)]; two algae (Chlorella, Dunaliella
salina); one nematode (Panagrelius redivivus); one plant
cell line (BY-2, tobacco cell line); and one crustacean
[Artemia salina (brine shrimp)].
Compounds were tested at a concentration of 1 mg/
mL, and in series of six threefold dilutions. One of the
dermatophyte fungi, Trichophyton rubrum, was suscepti-
ble to compound 1, but only at the highest concentra-
tion, and none of the other test organisms, except the
brine shrimp, reacted to any of the compounds at any
of the concentrations used. The exceptional shrimp
was vulnerable to compounds 1, 2 and 5–9, the first four
at concentrations >1 mg/mL, 5–8 at <1 mg > 0.33 mg/
mL and the 1,4-linked compound 9 was most potent
at < 0.33 mg > 0.11 mg/mL.
hexaose and laminariheptaose were purchased from Sei-
kagaku Corporation, and gentiobiose was purchased
from Sigma Chemical Co.
3.2. 4,6-O-Benzylidene-N-benzyloxycarbonyl-2-O-chloro-
acetyl-1,5-dideoxy-1,5-imino-D-glucitol (10), 4,6-O-benz-
ylidene-N-benzyloxycarbonyl-2,3-di-O-chloroacetyl-1,5-
dideoxy-1,5-imino-D-glucitol and 4,6-O-benzylidene-N-
benzyloxycarbonyl-3-O-chloroacetyl-1,5-dideoxy-1,5-
imino-D-glucitol
To a suspension of 4,6-O-benzylidene-N-benzyloxy-
carbonyl-1,5-dideoxy-1,5-imino-D-glucitol25,28 (771 mg,
2.0 mmol) in CH2Cl2 (20 mL) at 0 ꢁC were added 2,6-
lutidine (2.4 mL, 20 mmol), triethylamine (0.39 mL,
2.8 mmol) and chloroacetic anhydride (430 mg,
2.5 mmol), and the mixture was stirred at 0 ꢁC for
3.5 h, poured into ice-water (30 mL) and extracted with
CH2Cl2 (3 · 20 mL). The combined organic extracts
were washed with ice-cold HCl (2 M, 40 mL) and water
(20 mL), dried (Na2SO4) and evaporated to dryness.
Column chromatography (CH2Cl2/MeOH 300:1!
100:1) of the residue gave, in order of elution, the 2,3-
di-chloroacetate (165 mg, 15%), the required 2-ester 10
(231 mg, 25%) and the 3-ester (303 mg, 33%).
20
Compound 10: mp 50–52 ꢁC, ½aꢁD ꢂ13.8 (c 3.0,
CHCl3); 1H NMR (CDCl3): d 5.54 (s, 1H, CHPh),
5.13, 5.09 (2d, 2H, J 12.2 Hz, CH2Ph), 4.91 (m, 1H,
H-2), 4.82 (dd, 1H, J6a,6e 13.3, J5,6e 4.5 Hz, H-6e), 4.21
(dd, 1H, J5,6a 10.7 Hz, H-6a), 4.12 (dd, 1H, J1a,1e 13.9,
J1e,2 4.3 Hz, H-1e), 4.03, 3.96 (2d, 2H, J 14.9 Hz,
CH2Cl), 3.81 (m, 1H, H-3), 3.70 (t, 1H, J3,4, J4,5
9.5 Hz, H-4), 3.36 (ddd, 1H, H-5), 3.20 (dd, 1H, J1a,2
8.2 Hz, H-1a); 13C NMR (CDCl3): d 101.9, 80.0, 73.7,
73.7, 69.4 (CH2), 67.8 (CH2), 53.4, 45.2 (CH2), 40.6
(CH2). HR-MS/EI m/z calcd for C23H25ClNO7
(M+H)+: 462.1319, found: 462.1302.
3. Experimental
3.1. General methods
All glycosylation reactions were carried out under argon
in dry CH2Cl2. TLC was performed on Silica Gel HF-
254, and column chromatography on Silica Gel 230–
400 mesh (Merck). NMR spectra were recorded on solu-
tions in CDCl3, CD3OD or D2O (in the last case reso-
nances being referenced to acetone as a secondary
standard, dH 2.217, dC 33.17) with a Bruker Avance
NMR spectrometer at 300 MHz (for 1H) or 75 MHz
(for 13C). Chemical shifts were recorded in ppm from
tetramethylsilane as internal reference. Resonances for
ester and aromatic groups were observed as required
by the assigned structures but are not recorded. DEPT
experiments were conducted to identify the number of
hydrogen atoms bonded to carbon atoms; the methylene
group resonances are identified in the reported 13C
NMR spectral data. High resolution mass spectra
(HR-MS) were measured with a VG70-250S double
focusing magnetic sector mass spectrometer, a Mariner
8105 electrospray TOF mass spectrometer with ioniza-
tion effected by use of a caesium ion gun or a Mariner
5158 electrospray TOF instrument. Optical rotations
were measured with a Perkin–Elmer 241 automatic
polarimeter. Laminaribiose, laminaritetraose, laminari-
20
3-Ester: mp 146–148 ꢁC; ½aꢁD ꢂ7.6 (c 1.0, CHCl3),
20
1
lit.17 ½aꢁD ꢂ5.2 (CH2Cl2); H NMR (CDCl3): d 5.50 (s,
1H, CHPh), 5.12, 5.09 (2d, 2H, J 12 Hz, CH2Ph), 5.03
(dd, 1H, J2,3, 9.2, J3,4 7.7 Hz, H-3), 4.78 (dd, 1H, J6a,6e
10.7, J5,6e 4.4 Hz, H-6e), 4.31 (t, 1H, J5,6a 10.8 Hz, H-
6a), 4.16 (dd, 1H, J1a,1e 13.8, J1e,2 4.4 Hz, H-1e), 4.11
(s, 2H, CH2Cl), 3.8–3.65 (m, 2H, H-2, -4), 3.35 (ddd,
1H, J4,5 10.2 Hz, H-5), 2.98 (dd, 1H, J1a,2 9.2 Hz, H-
1a); 13C NMR (CDCl3): d 101.1, 79.6, 77.5, 69.4
(CH2), 69.1, 67.8 (CH2), 54.2, 48.9 (CH2), 40.8 (CH2);
HR-MS/EI m/z calcd for C23H25ClNO7 (M+H)+:
462.1319, found: 462.1334.
20
2,3-Diester: mp 132–135 ꢁC; ½aꢁD ꢂ22.5 (c 0.9,
CHCl3); 1H NMR (CDCl3): d 5.53 (s, 1H, CHPh),
5.25 (dd, 1H, J2,3 7.0, J3,4 9.6 Hz, H-3), 5.17–5.11 (2d,
2H, J 12.2 Hz, CH2Ph), 5.03 (ddd, 1H, J2,1a 8.5, J2,3
7.0, J2,1e 4.3 Hz, H-2), 4.83 (dd, 1H, J6a,6e 11.3, J5,6e
4.5 Hz, H-6e), 4.28 (t, 1H, J5,6a 10 Hz, H-6a), 4.20 (dd,
1H, J1a,1e 14.0 Hz, H-1e), 4.05 (s, 2H, CH2Cl), 3.99,

R. Blattner et al. / Carbohydrate Research 341 (2006) 2115–2125
2119
3.93 (2d, 2H, J 15.0 Hz, CH2Cl), 3.92 (t, 1H, J4,5 9.6 Hz,
H-4), 3.46 (dt, 1H, H-5), 3.30 (dd, 1H, H-1a); 13C NMR
(CDCl3): d 101.45, 77.3, 75.4, 71.4, 69.4 (CH2), 68.0
(CH2), 53.8, 45.6 (CH2), 40.5 (CH2), 40.4 (CH2). HR-
MS/EI m/z calcd for C25H26Cl2NO8 (M+H)+:
538.1035, found: 538.1055.
range ꢂ2.7 0.3 ppm with respect to lit. values.12
HR-MS/FAB m/z calcd for C12H24NO9 (M+H)+:
326.1451, found: 326.1442.
3.6. 2,4,6-Tri-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl)-a,b-D-glucopyranose (2A)
3.3. 3-O-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-
4,6-O-benzylidene-N-benzyloxycarbonyl-2-O-chloro-
acetyl-1,5-dideoxy-1,5-imino-D-glucitol (1C)
Laminaribiose (200 mg, 0.585 mmol) was peracetylated
under standard conditions (pyridine, acetic anhydride,
DMAP as catalyst) and after 24 h the solvents were
evaporated to dryness. The crude peracetates were dis-
solved in DMF (5 mL), hydrazinium acetate (1.3 mol
equiv)23 was added and the mixture was stirred at 20 ꢁC
for 3 h. Ethyl acetate (50 mL) was added, the organic
layer was washed with NaHCO3 (10 mL, satd aq) and
water (10 mL), dried (Na2SO4) and evaporated to dry-
ness. The residue was purified by column chromatogra-
phy (hexane/EtOAc 1:2) to give the title heptaacetate
(310 mg, 84%, mainly a-anomer): 13C NMR (CDCl3):
d 101.1, 90.6, 76.2, 73.4, 73.1, 72.1, 71.75, 68.5, 68.4,
67.9, 62.5 (CH2), 62.2 (CH2); HR-MS/FAB m/z calcd
for C26H35O17 (MꢂOH)+: 619.1874, found: 619.1857;
calcd for C26H36O18Na (M+Na)+: 659.1800, found:
659.1753.
˚
Powdered 4 A molecular sieves (300 mg) were added to
a stirred solution containing compound 10 (88 mg,
0.19 mmol) and 2,3,4,6-tetra-O-acetyl-a,b-D-glucopyr-
anosyl trichloroacetimidate 1B29,30 [(98 mg, 0.20 mmol),
made from tetraacetylglucose 1A] in dry CH2Cl2 (5 mL).
The mixture was stirred at room temperature for 30 min,
cooled in an ice bath and TMSOTf (30 lL) was added.
After 15 min at 0 ꢁC, triethylamine (0.5 mL) was added,
and the solvents were evaporated to dryness. Column
chromatography (hexane/EtOAc 2:1) of the residue gave
20
the title compound (107 mg, 71%): ½aꢁD ꢂ34.7 (c 0.21,
20
1
CHCl3), lit.25 ½aꢁD ꢂ24 (c 0.9, CHCl3); H NMR data
in agreement with lit. data;28 13C NMR (CDCl3): d
166.5, 156.0, 101.6, 100.7, 80.25, 79.0, 73.6, 73.2, 72.3,
71.8, 70.1 (CH2), 68.5, 68.2 (CH2), 62.1 (CH2), 52.9,
44.8 (CH2), 40.8 (CH2); HR-MS/FAB m/z calcd for
C37H41ClNO16 (MꢂH)+: 790.2114, found: 790.2133.
3.7. 2,4,6-Tri-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl)-a,b-D-glucopyranosyl trichloroacet-
imidate (2B)
3.4. 4,6-O-Benzylidene-N-benzyloxycarbonyl-1,5-di-
deoxy-3-O-b-D-glucopyranosyl-1,5-imino-D-glucitol (1D)
Anhydrous potassium carbonate (140 mg, 1 mmol) and
trichloroacetonitrile (0.5 mL) were added to a solution
of laminaribiose heptaacetate (2A, 141 mg, 0.22 mmol)
in CH2Cl2 (5 mL), the mixture was stirred at room tem-
perature overnight and solvents were evaporated to dry-
ness. Column chromatography (hexane/EtOAc 1:2) of
the residue gave the title trichloroacetimidates (175 mg,
Sodium methoxide in MeOH (0.1 mL containing 1.5 mg
of the alkoxide) was added to a solution of compound
1C (105 mg, 0.13 mmol) in MeOH (5 mL) and the mix-
ture was stirred at 20 ꢁC overnight. The solvent was
removed to afford the title pentaol as a white solid (70 mg,
1
20
100%) as a mixture of anomers (a,b 1:1.15): H NMR
100%): mp 155–157 ꢁC; ½aꢁD +1.8 (c 0.36, CHCl3/MeOH
20
(CDCl3): d 8.70, 8.68 (2s, 2 · 0.5H, NH), 6.46 (d,
0.5H, J 3.6 Hz, H-1a), 5.74 (d, 0.5H, J 8.3 Hz, H-1b);
13C NMR (CDCl3): d 161.3 (b), 160.65 (a), 101.0
(a,b), 95.6 (b), 93.2 (a), 78.7 (b), 72.9 (a), 72.8 (b),
71.8 (a), 71.6 (b), 71.5 (a), 71.15 (b), 70.3 (a), 68.2 (a),
68.1 (b), 67.9 (b), 67.3 (a), 61.8 (CH2), 61.6 (CH2),
60.4 (b). a,b-Assignments were made by use of the spec-
trum of the pure a-compound.31 HR-MS/FAB m/z
calcd for C28H36Cl3NO18Cs (M+Cs)+: 912.0052, found:
912.0016.
1:1), lit.25 ½aꢁD +0.2 (c 1.3, CHCl3/MeOH 1:1); 1H NMR
data in agreement with lit. data;25 13C NMR (CDCl3/
CD3OD 1:1): d 154.8, 103.1, 100.6, 84.4, 78.3, 75.9,
75.8, 73.6, 69.4, 68.8, 68.8 (CH2), 67.1 (CH2), 61.0
(CH2), 54.3, 48.3 (CH2); HR-MS/FAB m/z calcd for
C27H34NO11 (M+H)+: 548.2132, found: 548.2125.
3.5. 1,5-Dideoxy-3-O-b-D-glucopyranosyl-1,5-imino-D-
glucitol (1)
Pd/C (100 mg, 10%) was added to a solution of com-
pound 1D (59 mg, 0.11 mmol) in water (3 mL) and
hydrogenolysis was carried out under 55 psi for 3 days.
The catalyst was filtered off, and the solution was freeze
3.8. 3-O-[2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl-
(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyranosyl]-4,6-O-
benzylidene-N-benzyloxycarbonyl-2-O-chloroacetyl-1,5-
dideoxy-1,5-imino-D-glucitol (2C)
20
dried to afford compound 1 (32 mg, 91%): ½aꢁD +12.0 (c
20
0.2, H2O); lit.12 ½aꢁD +18.1 (c 0.74, H2O); The 1H NMR
(D2O) spectral data were identical to lit. data;25 13C
Compound 10 (120 mg, 0.260 mmol) was glycosylated
with the trichloroacetimidate 2B (192 mg) as in the
NMR (D2O) all 12 resonances had d values within the

2120
R. Blattner et al. / Carbohydrate Research 341 (2006) 2115–2125
preparation of compound 1C. Purification by flash chro-
3.12. 2,4,6-Tri-O-acetyl-3-O-[2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyr-
anosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyranosyl]-a-
D-glucopyranosyl trichloroacetimidate and 2,4,6-tri-O-
acetyl-3-O-[2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-
(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyranosyl-(1!3)-
2,4,6-tri-O-acetyl-b-D-glucopyranosyl]-b-D-glucopyran-
osyl trichloroacetimidate (3B)
matography (hexane/EtOAc 1:1) gave the title com-
20
1
pound (133 mg, 50%): ½aꢁD ꢂ38.2 (c 0.28, CHCl3); H
NMR (CDCl3): d 4.66 (d, 1H, J 8.1 Hz, H-10 or H-100),
4.55 (d, 1H, J 8.1 Hz, H-10 or H-100); 13C NMR (CDCl3):
d 166.5, 156.3; 101.7, 101.3, 100.6, 80.0, 79.2, 79.0, 73.9,
73.3, 72.2, 72.1, 71.4, 70.2 (CH2), 68.45, 68.3, 68.2
(CH2), 62.3 (CH2), 62.1(CH2), 52.0, 44.2 (CH2), 40.8
(CH2); HR-MS/FAB m/z calcd for C49H58ClNO24Na
(M+Na)+: 1102.2935, found: 1102.2824.
The 1-hydroxylaminaritetraose derivative 3A (285 mg)
was converted to the mixed trichloroacetimidates as
for the laminaribiose heptaacetate. Purification by col-
umn chromatography (hexane/EtOAc 1:4) gave the title
compounds (279 mg, 87%) as a 1:1 mixture of anomers
which were separated by further chromatography:
a-Anomer: 1H NMR (CDCl3): d 8.73 (s, 1H, NH), 6.45
(d, 1H, J 3.7 Hz, H-1); 13C NMR (CDCl3): d 160.9,
101.4, 101.1, 101.0, 93.45, 79.2, 78.6, 76.1, 73.5, 73.2,
72.9, 72.2, 72.1, 72.0, 71.2, 70.65, 68.8, 68.6, 68.4, 67.6,
62.5 (CH2), 62.3 (CH2), 62.0 (CH2), 62.0 (CH2), 60.7.
3.9. 4,6-O-Benzylidene-N-benzyloxycarbonyl-1,5-di-
deoxy-3-O-[b-D-glucopyranosyl-(1!3)-b-D-gluco-
pyranosyl]-1,5-imino-D-glucitol (2D)
The fully protected laminaribiosyl compound 2C (128
mg, 0.116 mmol) was deacylated in the usual way to give
20
the title compound (86 mg, 100%): ½aꢁD ꢂ13.0 (c 0.2,
1
MeOH); H NMR (CD3OD): d 4.63 (d, 1H, J 7.7 Hz,
H-10 or H-100), 4.55 (d, 1H, J 7.7 Hz, H-10 or H-100);
13C NMR (CD3OD): d 156.0, 105.8, 104.8, 102.8, 88.6,
86.1, 80.6, 78.6, 78.4, 78.1, 76.0, 75.6, 72.0, 71.4, 71.0,
70.2, 68.9, 63.1, 62.9, 55.6; HR-MS/FAB m/z calcd for
C33H44NO16 (M+H)+: 710.2660, found: 710.2677.
1
b-Anomer: H NMR (CDCl3): d 8.72 (s, 1H, NH),
5.75 (d, 1H, J 8.2 Hz, H-1); 13C NMR (CDCl3): d
161.4, 101.4, 101.1, 100.9, 95.85, 78.6, 78.35, 73.2,
73.15, 72.8, 72.3, 72.2, 72.0, 71.2, 68.7, 68.6, 68.4, 68.2,
62.45 (CH2), 62.3 (CH2), 62.15 (CH2), 62.05 (CH2),
60.7; HR-MS/FAB m/z calcd for C52H68Cl3NO34Cs
(M+Cs)+: 1488.1743, found: 1488.1748.
3.10. 1,5-Dideoxy-3-O-[b-D-glucopyranosyl-(1!3)-b-D-
glucopyranosyl]-1,5-imino-D-glucitol (2)
3.13. 3-O-[2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl-
(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyranosyl-(1!3)-
2,4,6-tri-O-acetyl-b-D-glucopyranosyl-(1!3)-2,4,6-tri-O-
acetyl-b-D-glucopyranosyl]-4,6-O-benzylidene-N-benzyl-
oxycarbonyl-2-O-chloroacetyl-1,5-dideoxy-1,5-imino-D-
glucitol (3C)
Compound 2D (86 mg, 0.121 mmol) was hydrogeno-
lyzed as for the preparation of compound 1 and gave
20
the title compound (44 mg, 75%): ½aꢁD +1.0 (c 0.29,
H2O); 13C NMR (D2O): d 103.2, 103.1, 87.3, 84.8,
76.4, 76.05, 76.0, 73.9, 73.7, 70.2, 70.0, 69.6, 68.5, 61.1
(CH2), 61.1 (CH2), 60.9 (CH2), 60.65, 48.4 (CH2); HR-
MS/FAB m/z calcd for C18H34NO14 (M+H)+:
488.1979, found: 488.1997.
Compound 10 (62 mg, 0.134 mmol) was glycosylated
with the trichloroacetimidates 3B (192 mg) derived from
laminaritetraose as in the preparation of compound 1C.
Chromatographic purification (hexane/EtOAc 1:2) gave
3.11. 2,4,6-Tri-O-acetyl-3-O-[2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyr-
anosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyranosyl]-a,b-
D-glucopyranose (3A)
20
the title compound (104 mg, 47%): ½aꢁD ꢂ36.1 (c 0.59,
CHCl3); 13C NMR (CDCl3): d 166.4, 156.2, 101.6,
101.3, 101.0, 100.8, 100.5, 80.0, 79.2, 78.9, 78.6, 78.4,
74.3, 73.9, 73.5, 73.15, 72.8, 72.2, 71.9, 71.1, 70.1
(CH2), 68.6, 68.5, 68.3, 68.1 (CH2), 62.2 (CH2), 62.2
(CH2), 62.0 (CH2), 62.0 (CH2), 52.0, 44.1 (CH2), 40.7
(CH2); HR-MS/FAB m/z calcd for C73H91ClNO40
(M+H)+: 1656.4806, found: 1656.4718.
Laminaritetraose was peracetylated and the ester
(460 mg, 0.380) specifically deacetylated at C-1 as de-
scribed for laminaribiose peracetate. Chromatographic
purification (hexane/EtOAc 1:5) gave the title com-
pound (395 mg, 91%, mainly a-anomer): 1H NMR
(CDCl3): d 4.54 (d, 1H, J 8.1 Hz, H-10 or H-100 or H-
1000b), 4.50 (d, J 8.0 Hz, H-10 or H-100 or H-1000), 4.38 (d,
1H, J 8.1 Hz, H-10 or H-100 or H-1000); 13C NMR (CDCl3):
d 101.1, 100.8, 100.5, 90.1, 78.9, 78.5, 75.4, 73.2, 72.9,
72.6, 71.65, 70.9, 68.3, 68.1, 67.45, 62.2 (CH2), 62.2
(CH2), 62.0 (CH2), 61.7 (CH2); HR-MS/FAB m/z calcd
for C50H68O34Cs (M+Cs)+: 1345.2646, found:
1345.2556.
3.14. 4,6-O-Benzylidene-N-benzyloxycarbonyl-1,5-di-
deoxy-3-O-[b-D-glucopyranosyl-(1!3)-b-D-glucopyranos-
yl-(1!3)-b-D-glucopyranosyl-(1!3)-b-D-glucopyranos-
yl]-1,5-imino-D-glucitol (3D)
The laminaritetraose compound 3C (115 mg, 0.175
mmol) was deacylated in the usual way to give the title
20
compound (73 mg, 100%): ½aꢁD ꢂ12.0 (c 0.05, DMSO);

R. Blattner et al. / Carbohydrate Research 341 (2006) 2115–2125
2121
13C NMR (DMSO-d6): d 156.0, 104.2, 103.5, 103.4,
3.18. 3-O-[2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl-
102.4, 100.5, 87.4, 87.1, 86.5, 83.3, 78.55, 77.3, 76.9,
76.7, 76.4, 74.2, 73.2, 73.1, 70.4, 69.4, 69.2 (CH2), 68.8,
68.3, 66.9 (CH2), 61.4 (CH2), 61.2 (CH2), 61.2 (CH2),
60.9 (CH2), 52.3, 47.3 (CH2); HR-MS/FAB m/z calcd
for C45H64NO26 (M+H)+: 1034.3717, found: 1034.3694.
(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyranosyl-(1!3)-
2,4,6-tri-O-acetyl-b-D-glucopyranosyl-(1!3)-2,4,6-tri-O-
acetyl-b-D-glucopyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-
glucopyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyr-
anosyl]-4,6-O-benzylidene-N-benzyloxycarbonyl-2-O-
chloroacetyl-1,5-dideoxy-1,5-imino-D-glucitol (4C)
3.15. 1,5-Dideoxy-3-O-[b-D-glucopyranosyl-(1!3)-b-D-
glucopyranosyl-(1!3)-b-D-glucopyranosyl-(1!3)-b-D-
glucopyranosyl]-1,5-imino-D-glucitol (3)
Compound 10 (36 mg, 0.078 mmol) was glycosylated
with the trichloroacetimidate 4B as for the preparation
of compound 1C. Chromatographic purification (hex-
ane/EtOAc/MeOH 1:3:0.2) gave the title compound
Compound 3D (46 mg, 0.044 mmol) was hydrogeno-
lyzed as for the preparation of compound 1 to give the
20
4C (92 mg, 52%): ½aꢁD ꢂ33.5 (c 0.23, CHCl3); 13C
20
title compound (25 mg, 69%): ½aꢁD ꢂ7.4 (c 0.27, H2O);
NMR (CDCl3): d 101.6, 101.3, 101.1, 100.9, 100.8,
100.4; HR-MS/FAB m/z calcd for C97H122ClNO56Cs
(M+Cs)+: 2364.5472, found: 2364.5339.
13C NMR (D2O): d 103.2, 103.1, 103.0, 103.0, 88.0,
84.75, 76.4, 76.1, 73.9, 73.7, 71.0, 70.4, 70.0, 68.5, 61.7
(CH2), 61.1 (CH2), 61.1 (CH2), 61.1 (CH2), 61.1
(CH2), 60.8, 49.0 (CH2); HR-MS/ESI m/z calcd for
C30H54NO24 (M+H)+: 812.3036, found: 812.3015.
3.19. 4,6-O-Benzylidene-N-benzyloxycarbonyl-1,5-dideo-
xy-3-O-[b-D-glucopyranosyl-(1!3)-b-D-glucopyranosyl-
(1!3)-b-D-glucopyranosyl-(1!3)-b-D-glucopyranosyl-
(1!3)-b-D-glucopyranosyl-(1!3)-b-D-glucopyranosyl]-
1,5-imino-D-glucitol (4D)
3.16. 2,4,6-Tri-O-acetyl-3-O-[2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl-(1!3)-O-2,4,6-tri-O-acetyl-b-D-gluco-
pyranosyl-(1!3)-O-2,4,6-tri-O-acetyl-b-D-glucopyran-
osyl-(1!3)-O-2,4,6-tri-O-acetyl-b-D-glucopyranosyl-
(1!3)-O-2,4,6-tri-O-acetyl-b-D-glucopyranosyl]-a,b-D-
glucopyranose (4A)
The above laminarihexaose derivative 4C (61 mg,
0.026 mmol) was deacylated in the usual manner to give
20
the title compound 4D (37 mg, 100%): ½aꢁD ꢂ18.0 (c 0.1,
H2O); HR-MS/FAB m/z calcd for C57H84NO36
Laminarihexaose was acetylated and the perester
(366 mg, 0.220 mmol) specifically deacetylated at C-1
as described for laminaribiose peracetate. Chromato-
graphic purification of the product (hexane/EtOAc/
MeOH 1:3:0.5) gave the title compound (360 mg, 99%,
mainly a-anomer): 13C NMR (CDCl3): d 101.4, 101.0,
90.4, 79.3, 78.6, 73.6, 73.3, 72.1, 71.25, 68.6, 68.45,
67.55, 62.4, 62.0 (CH2). HR-MS/ESI m/z calcd for
C74H100O50Cs (M+Cs)+: 1921.4337, found: 1921.4499.
(M+H)+: 1358.4773, found: 1358.4729.
3.20. 1,5-Dideoxy-3-O-[b-D-glucopyranosyl-(1!3)-b-D-
glucopyranosyl-(1!3)-b-D-glucopyranosyl-(1!3)-b-D-
glucopyranosyl-(1!3)-b-D-glucopyranosyl-(1!3)-b-D-
glucopyranosyl]-1,5-imino-D-glucitol (4)
Compound 4D (27 mg, 0.020 mmol) was hydrogeno-
lyzed as for the preparation of glycoside 1 to give the
20
deprotected compound 4 (18 mg, 78%): ½aꢁD +15.2 (c
3.17. 2,4,6-Tri-O-acetyl-3-O-[2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl-(1!3)-O-2,4,6-tri-O-acetyl-b-D-gluco-
pyranosyl-(1!3)-O-2,4,6-tri-O-acetyl-b-D-glucopyran-
osyl-(1!3)-O-2,4,6-tri-O-acetyl-b-D-glucopyranosyl-
(1!3)-O-2,4,6-tri-O-acetyl-b-D-glucopyranosyl]-a,b-D-
glucopyranosyl trichloroacetimidate (4B)
0.33, H2O); 13C NMR (D2O): d 103.2, 102.95, 84.6,
76.4, 76.05, 73.9, 73.7, 70.0, 68.5, 68.1, 67.2, 66.7, 61.1
(CH2), 60.2, 58.0, 46.2 (CH2); HR-MS/FAB m/z calcd
for C42H74NO34 (M+H)+: 1136.4092, found: 1136.4144.
3.21. 2,4,6-Tri-O-acetyl-3-O-[2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyr-
anosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyranosyl-
(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyranosyl-(1!3)-
2,4,6-tri-O-acetyl-b-D-glucopyranosyl-(1!3)-2,4,6-tri-O-
acetyl-b-D-glucopyranosyl]-a,b-D-glucopyranose (5A)
The 1-hydroxylaminarihexaose peracetate 4A (308 mg,
0.185 mmol) was converted to the mixed trichloroacet-
imidates as for laminaribiose heptaacetate. Chromato-
graphic purification (hexane/EtOAc/MeOH 1:3:0.2)
gave the title compounds (290 mg, 87%): 1H NMR
(CDCl3): d 8.7 (s, 1H, NH), 6.45 (d, 0.5H, J1,2 3.7 Hz,
H-1a), 5.75 (d, 0.5H, J1,2 8.2 Hz, H-1b), 5.30 (s, 2 H);
13C NMR (CDCl3): d 101.1, 100.7, 95.5, 93.1, 79.0,
78.2, 73.3, 72.9, 72.7, 72.5, 71.7, 70.9, 70.3, 68.45; 68.1,
67.2, 62.1, 61.7 (CH2), 60.4; HR-MS/FAB m/z calcd
for C76H100Cl3NO50Cs (M+Cs)+: 2064.3433, found:
2064.3605.
Laminariheptaose was acetylated and the perester
(184 mg, 0.082 mmol) specifically deacetylated at C-1
as described for laminaribiose peracetate. Chromato-
graphic purification of the product (hexane/EtOAc/
MeOH 2:6:1) gave the title compound (148 mg, 82%,
mainly a-anomer): 13C NMR (CDCl3): d 101.1, 101.0,
100.8, 90.2, 79.3, 78.9, 78.7, 76.0, 73.7, 73.2, 72.9, 72.6,

2122
R. Blattner et al. / Carbohydrate Research 341 (2006) 2115–2125
71.9, 71.2, 68.45, 67.2, 62.63 (CH2), 62.28, (CH2), 61.93
(CH2); HR-MS/FAB m/z calcd for C86H116O58Cs
(M+Cs)+: 2209.5182, found: 2209.5078.
(c 0.23, H2O); 13C NMR (D2O): d 157.0, 103.3, 103.1,
101.85, 84.8, 78.4, 76.45, 76.1, 73.9, 73.8, 70.0, 69.3
(CH2), 68.55, 61.1 (CH2), 53.4, 49.3 (CH2); HR-MS/
FAB m/z calcd for C63H94NO41 (M+H)+: 1520.5301,
found: 1520.5190.
3.22. 2,4,6-Tri-O-acetyl-3-O-[2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyr-
anosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyranosyl-
(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyranosyl-(1!3)-
2,4,6-tri-O-acetyl-b-D-glucopyranosyl-(1!3)-2,4,6-tri-
O-acetyl-b-D-glucopyranosyl]-a,b-D-glucopyranosyl
trichloroacetimidate (5B)
3.25. 1,5-Dideoxy-3-O-[b-D-glucopyranosyl-(1!3)-b-D-
glucopyranosyl-(1!3)-b-D-glucopyranosyl-(1!3)-b-D-
glucopyranosyl-(1!3)-b-D-glucopyranosyl-(1!3)-b-D-
glucopyranosyl-(1!3)-b-D-glucopyranosyl]-1,5-imino-
D-glucitol (5)
The 1-hydroxylaminariheptaose peracetate 5A (120 mg,
0.054 mmol) was converted to the mixed trichloroacet-
imidates as for laminaribiose heptaacetate. Chromato-
graphic purification (hexane/EtOAc/MeOH 5:15:1)
gave the title compounds (106 mg, 83%): 1H NMR
(CDCl3): d 8.68 (s, 1H, NH), 6.45 (d, 0.5 H, J 3.6 Hz,
H-1a), 5.75 (d, 0.5H, J 8.4 Hz, H-1b); 13C NMR
(CDCl3): d 161.35, 160.9, 101.4, 101.2, 101.0, 95.8,
93.4, 79.3, 78.7, 77.9, 76.2, 73.5, 73.25, 73.1, 72.75,
72.1, 71.2, 70.6, 68.6, 68.4, 62.4 (CH2), 62.0 (CH2),
Compound 5D (35 mg, 0.023 mmol) was hydrogeno-
lyzed as for the preparation of compound 1 to give the
20
title compound (17 mg, 57%): ½aꢁD +2.5 (c 0.28, H2O);
13C NMR (D2O): d 103.2, 103.0, 84.6, 76.5, 76.1, 73.9,
73.7, 70.0, 68.55, 68.1, 67.3, 66.7, 61.1 (CH2), 60.3,
58.0 (CH2), 46.2 (CH2); HR-MS/FAB m/z calcd for
C48H84NO39 (M+H)+: 1298.4620, found: 1298.4684.
3.26. 2,3,4-Tri-O-acetyl-6-O-(2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl)-a-D-glucopyranosyl trichloroacetimidate
(6Ba) and 2,3,4-tri-O-acetyl-6-O-(2,3,4,6-tetra-O-acetyl-
b-D-glucopyranosyl)-b-D-glucopyranosyl trichloroacet-
imidate (6Bb)
60.7
(CH2);
HR-MS/FAB
m/z
calcd
for
C88H116Cl3NO58Cs (M+Cs)+: 2352.4278, found:
2352.4318.
3.23. 3-O-[2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl-
(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyranosyl-(1!3)-
2,4,6-tri-O-acetyl-b-D-glucopyranosyl-(1!3)-2,4,6-tri-O-
acetyl-b-D-glucopyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-
glucopyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyr-
anosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-glucopyranosyl]-4,6-
O-benzylidene-N-benzyloxycarbonyl-2-O-chloroacetyl-
1,5-dideoxy-1,5-imino-D-glucitol (5C)
To a stirred solution of gentiobiose 2,20,3,30,4,40,60-
hepta-O-acetate (6A)23 (1.50 g, 2.37 mmol) in dry
CH2Cl2 (15 mL) at 0 ꢁC under Ar, trichloroacetonitrile
(1.5 mL, 15 mmol) was added, followed after 5 min by
sodium hydride (60%, 250 mg, 3.6 mmol). After 15 min
at 0 ꢁC, the reaction mixture was filtered through Celite
and applied to a column of silica gel (hexane/EtOAc 1:1)
to give the a-trichloroacetimidate as a foam (1.41 g,
76%) which crystallized from hexane: mp 156–158 ꢁC;
20
1
Compound 10 (28 mg, 0.061 mmol) was glycosylated
with the trichloroacetimidates 5B (112 mg) as in the
preparation of compound 1C. Chromatographic purifi-
cation (hexane/EtOAc/MeOH 5:15:1) gave the title
½aꢁD +51.6 (c 1.82, CH2Cl2); H NMR (CDCl3): d 8.64
(s, 1H, NH), 6.54 (d, 1H, J 3.6 Hz, H-1), 5.55 (t, 1H,
J 9.8 Hz), 5.18 (t, 1H, J 9.4 Hz), 5.0–5.1 (m, 3H, H-2,
-4, -40), 4.90 (dd, 1H, J 9.2, 8.0, Hz, H-20), 4.56 (d,
1H, J 7.9 Hz, H-10), 4.27 (dd, 1H, J 12.4, 4.5 Hz, H-6),
4.17 (ddd, 1H, J 10.5, 5.5, 2.0 Hz, H-50), 4.12 (dd, 1H,
J 12.4, 2.4 Hz, H-6), 3.96 (dd, 1H, J 11.3, 2.1 Hz, H-
60), 3.65 (ddd, 1H, J 9.9, 4.5, 2.4 Hz, H-5), 3.58 (dd,
1H, J 11.3, 5.4 Hz, H-60); 13C NMR (CDCl3): d 160.8,
100.5, 92.8, 72.8, 71.9, 71.2, 71.0, 69.9, 69.8, 68.3, 68.3,
67.3 (CH2), 61.8 (CH2); HR-MS/FAB m/z calcd for
C28H36Cl3NO18 (MꢂH)+: 778.0919, found: 778.0878.
This was followed by the b-isomer (0.33 g, 18%): mp
20
compound (58 mg, 45%): ½aꢁD ꢂ42.2 (c 0.23, CHCl3);
13C NMR (CDCl3): d 166.1, 155.9, 101.3, 101.0, 100.6,
100.2, 79.65, 78.9, 78.6, 78.2, 73.6, 72.9, 72.7, 72.4,
71.7, 70.8, 69.8 (CH2), 68.2, 68.0, 67.8 (CH2), 62.0
(3 · CH2), 61.6 (CH2), 60.3 (CH2), 43.8 (CH2), 40.4
(CH2); HR-MS/FAB m/z calcd for C109H138ClNO64Cs
(M+Cs)+: 2652.6317, found: 2652.6380.
3.24. 4,6-O-Benzylidene-N-benzyloxycarbonyl-1,5-dide-
oxy-3-O-[b-D-glucopyranosyl-(1!3)-b-D-glucopyranosyl-
(1!3)-b-D-glucopyranosyl-(1!3)-b-D-glucopyranosyl-
(1!3)-b-D-glucopyranosyl-(1!3)-b-D-glucopyranosyl-
(1!3)-b-D-glucopyranosyl]-1,5-imino-D-glucitol (5D)
20
180–181 ꢁC (from hexane/EtOAc); ½aꢁD ꢂ7.3 (c 1.50,
1
CH2Cl2); H NMR (CDCl3): d 8.81 (s, 1H, NH), 5.87
(d, J 7.3 Hz, H-1), 5.28, 5.24 and 5.02 (3t, 3H, J ꢀ 9.5 Hz),
5.15, 5.06 and 4.97 (3t, 3H, J ꢀ 9.5 Hz), 4.63 (d, 1H, J
8 Hz, 1H0), 4.26 (dd, 1H, J 12.4, 7.4 Hz, H-6), 4.11 (dd,
1H, J 12.4, 2.4 Hz, H-6), 3.87 (dd, 1H, J 12.2, 2.0 Hz,
H-60), 3.85 (m, 1H, H-5), 3.70 (dd, 1H, J 12.2, 7.1 Hz,
H-60), 3.63 (m, 1H, H-50); 13C NMR (CDCl3): d 160.8,
The above peracylated laminariheptaose derivative 5C
(32 mg, 0.012 mmol) was deacylated in the usual way
20
to give the title compound (20 mg, 100%): ½aꢁD ꢂ18.3

R. Blattner et al. / Carbohydrate Research 341 (2006) 2115–2125
2123
100.4, 95.4, 74.5, 72.8, 72.6 72.0, 71.0, 70.2, 68.6, 68.3, 67.6
(CH2), 61.8 (CH2); HR-MS/FAB m/z calcd for
C28H36Cl3NO18 (MꢂH)+: 778.0919, found: 778.0897.
(7.35 g, 13.0 mmol) in aqueous acetic acid (80%,
200 mL) was stirred at 25–30 ꢁC for 48 h. The solvents
were evaporated and column chromatography (hex-
ane/EtOAc 1:1) of the residue gave the title compound
20
3.27. 3-O-[2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl-
(1!6)-2,3,4-tri-O-acetyl-b-D-glucopyranosyl]-4,6-O-
benzylidene-N-benzyloxycarbonyl-2-O-chloroacetyl-1,5-
dideoxy-1,5-imino-D-glucitol (6C)
11 (5.58 g, 90%) as an oil: ½aꢁD +21.9 (c 1.1, CHCl3);
13C NMR (CDCl3): d 157.0, 75.35, 73.8, 72.8 (CH2),
71.1 (CH2), 67.55 (CH2), 67.4, 61.3 (CH2), 60.3, 38.5
(CH2). Anal. Calcd for C28H31NO6: C, 70.42; H, 6.54;
N, 2.93. Found: C, 70.19; H, 6.32; N, 3.03.
Compound 10 (138 mg, 0.300 mmol) was glycosylated
with the above gentiobiose-derived a-trichloroacetimid-
ate (6Ba) (240 mg, 0.310 mmol) as in the preparation of
compound 1C to give the title compound (147 mg,
44%): [a]D ꢂ11.4 (c 0.7, CHCl3); 13C NMR (CDCl3): d
166.1, 155.1, 100.9, 100.3, 100.3, 78.7, 78.6, 75.7, 73.2,
73.1, 72.2, 72.1, 71.0, 69.8, 69.1 (CH2), 68.7, 68.0 (CH2),
67.9 (CH2), 67.5, 61.15 (CH2), 54.55; 46.3 (CH2), 40.7
(CH2); HR-MS/FAB m/z calcd for C49H58ClNO24Na
(M+Na)+: 1102.2935, found: 1102.2934.
3.31. 6-O-[2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl]-
2,3-di-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-
imino-D-glucitol (7C) and 6-O-[2,3,4-tri-O-acetyl-b-D-
glucopyranosyl]-2,3-di-O-benzyl-N-benzyloxycarbonyl-
1,5-dideoxy-1,5-imino-D-glucitol
The dibenzyl derivative 11 (0.87 g, 1.82 mmol) was
glycosylated with tetra-O-acetyl-a,b-D-glucopyranosyl
trichloroacetimidates (1B) (0.50 g, 1.02 mmol) as
described for the preparation of compound 1C. The
crude product was fractionated by flash chromatogra-
phy (hexane/EtOAc 2:1!1:2) to give, in order of elu-
tion, the unreacted acceptor 11 (420 mg, 48%) and the
tetraacetylated title glycoside 7C (250 mg, 35%) as a
3.28. 4,6-O-Benzylidene-N-benzyloxycarbonyl-1,5-di-
deoxy-3-O-[b-D-glucopyranosyl-(1!6)-b-D-glucopyran-
osyl]-1,5-imino-D-glucitol (6D)
20
1
The above fully protected gentiobiose DNJ derivative
(6C) (137 mg, 0.124 mmol) was deacylated in the usual
way to give the title compound (87 mg, 97%):
foam: ½aꢁD ꢂ3.5 (c 0.68, CH2Cl2); H NMR (CDCl3):
d 4.8–5.1 (m, 5H), 4.3–4.7 (m, 6H), 4.15 (dd, 1H, J
12.3, 4.3 Hz), 4.0–4.15 (m, 3H), 3.4–3.8 (m, 5H), 3.08
(m, 1H); 13C NMR (CDCl3): d 155.6, 99.9, 73.8, 72.9,
71.8, 71.3 (CH2), 70.8, 70.1, 70.0 (CH2), 67.3, 66.5
(CH2), 65.8 (CH2), 64.5, 60.8 (CH2), 56.4, 36.1 (CH2);
HR-MS/FAB m/z calcd for C42H50NO15 (M+H)+:
808.3180, found: 808.3187.
20
½aꢁD ꢂ26.7 (c 0.15, MeOH/H2O 3:1); 1H NMR
(CD3OD/D2O 6:1): d 5.66 (s, 1H, CHPh), 5.14 (s, 2H,
CH2Ph), 4.63 (d, 1H, J 7.5 Hz, H-1 or H-10), 4.48 (d,
1H, J 7.8 Hz, H-1 or H-10); 13C NMR (CD3OD/D2O
6:1): d 157.7, 105.0, 104.2, 102.8, 84.5, 80.5, 78.1, 78.0,
77.9, 77.45, 75.8, 75.4, 71.7, 71.6, 71.2, 71.0 (CH2),
69.95 (CH2), 69.2 (CH2), 62.8 (CH2), 55.4, 49.7 (CH2);
HR-MS/FAB m/z calcd for C33H43NO16 (M+H)+:
710.2660, found: 710.2677.
This was followed by the title triacetate (108 mg,
20
1
14%): ½aꢁD +54 (c 0.62, CH2Cl2); H NMR (CDCl3): d
4.8–5.2 (m, 5H), 4.3–4.7 (m, 6H), 4.21 (dd, 1H, J 12.2,
3.9 Hz), 3.9–4.2 (m, 3H), 3.4–3.9 (m, 5H), 3.15 (m,
1H); 13C NMR (CDCl3): d 156.3, 102.6, 73.7, 73.1,
72.8, 72.3, 71.4 (CH2), 70.9, 70.0 (CH2), 69.5 (CH2),
67.4, 67.4 (CH2), 65.6, 61.0 (CH2), 57.1, 36.5 (CH2);
HR-MS/FAB m/z calcd for C40H48NO14 (M+H)+:
766.3075, found: 766.3148.
3.29. 1,5-Dideoxy-3-O-[b-D-glucopyranosyl-(1!6)-b-D-
glucopyranosyl]-1,5-imino-D-glucitol (6)
The above gentiobiose-DNJ octaol 6D (72 mg, 0.101
mmol) was hydrogenolyzed as for the preparation of
compound 1 to give the title compound (37 mg, 73%):
3.32. 2,3-Di-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-
6-O-b-D-glucopyranosyl-1,5-imino-D-glucitol (7D)
20
1
½aꢁD ꢂ2.2 (c 0.56, H2O); H NMR (D2O): d 4.49 (d,
1H, J 7.8 Hz, H-10 or H-100), 4.19 (dd, 1H, J 11.4,
1.7 Hz);13C NMR (D2O): d 103.5, 103.1, 87.75, 76.3,
76.0, 75.9, 75.25, 73.8, 73.6, 70.0, 69.9, 69.5, 69.35,
69.2 (CH2), 61.1 (CH2), 60.5, (CH2), 60.5, 48.0 (CH2);
HR-MS/FAB m/z calcd for C18H34NO14 (M+H)+:
488.1979, found: 488.1995.
Normal deacetylation of the 6-linked tetraacetate (7C)
and the 2,3,4-triacetate, followed by purification of the
crude products by flash chromatography (CH2Cl2/
EtOAc/MeOH 6:6:1) gave the title compound as a foam
20
1
(91%, combined yield): ½aꢁD +5.5 (c 1.31, CH2Cl2); H
NMR (CD3OD): d 5.15, 5.10 (2d, 2H, J 12.2 Hz,
CH2Ph), 4.68, 4.59 (2d, 2H, J 11.7 Hz, CH2Ph), 4.59,
4.40 (2d, 2H, J 11.6 Hz, CH2Ph), 4.2–4.3 (m, 3H), 4.13
(dd, 1H, J 10.6, 5.0 Hz), 4.04 (t, 1H, J 5.5 Hz), 3.91
(dd, 1H, J 11.9, 5.5 Hz), 3.82 (dd, 1H, J 11.9, 2.1 Hz),
3.55–3.7 (m, 3H), 3.1–3.4 (m, 5H); 13C NMR (CD3OD):
3.30. 2,3-Di-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-
1,5-imino-D-glucitol (11)
A suspension of 2,3-di-O-benzyl-4,6-O-benzylidene-N-
benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glucitol25

2124
R. Blattner et al. / Carbohydrate Research 341 (2006) 2115–2125
d 158.6, 104.8, 80.3, 78.1, 78.0, 78.0, 75.1, 73.7 (CH2),
71.9 (CH2), 71.5, 68.6 (CH2), 68.3 (CH2), 67.8, 62.8
(CH2), 59.6, 41.0 (CH2); HR-MS/FAB m/z calcd for
C34H42NO11 (M+H)+: 640.2758, found: 640.2772.
3.70 (dd, 1H, J 5.3, 12.4 Hz), 2.97 (t, 1H, J 11.9 Hz);
13C NMR (D2O): d 105.5, 105.1, 78.5, 78.3, 78.0, 77.3,
75.7, 75.5, 72.2, 71.7, 71.2 (CH2), 70.2, 69.5, 68.3
(CH2), 63.3 (CH2), 61.2, 48.5 (CH2); HR-MS/FAB
m/z calcd for C18H34NO14 (M+H)+: 488.1979, found:
488.2002.
3.33. 1,5-Dideoxy-6-O-b-D-glucopyranosyl-1,5-imino-D-
glucitol (7)
3.35. 4-O-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-2,
3,6-tri-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-
imino-D-glucitol (9C)
The above pentaol (7D) (72.5 mg, 0.113 mmol) was
hydrogenolyzed in MeOH (6 mL) and AcOH (1.5 mL)
in the presence of Pd/C catalyst (50 mg, 10%) at 50 psi
for 16 h, and the pale yellow syrup obtained after
removal of the solids and solvents was purified by
flash chromatography (CH2Cl2/MeOH/NH4OH 2:4:1)
Glycosylation of compound 1225 (539 mg) was carried
out with tetra-O-acetyl-a,b-D-glucopyranosyl trichloro-
acetimidate (1B) (492 mg) as for compound 1C. Column
chromatographic purification (hexane–EtOAc 2:1!1:1)
afforded the title compound (9C) (370 mg, 43%):
20
to give compound 7 (30 mg, 83%): ½aꢁD +8.1 (c 0.34,
1
H2O); the H NMR spectrum (D2O) was identical to
20
½aꢁD +4.4 (c 0.41, CHCl3); 13C NMR (CDCl3): d 156.2,
one previously published12 except that an additional
proton resonance (as required by the assigned structure)
was observed at d 3.26–3.40; 13C NMR (D2O), all d val-
ues were within 0.1 of literature values;25 HR-MS/FAB
m/z calcd for C12H24NO9 (M+H)+: 326.1451, found:
326.1459.
100.0, 79.1, 75.6, 74.35, 73.5 (CH2), 73.2, 72.7 (CH2),
72.2, 71.4, 71.2 (CH2), 68.8, 68.5 (CH2), 67.7 (CH2),
62.3 (CH2), 54.6, 41.05 (CH2); HR-MS/FAB m/z calcd
for C49H56NO15 (M+H)+: 898.3650, found: 898.3638.
3.36. 2,3,6-Tri-O-benzyl-N-benzyloxycarbonyl-1,5-di-
deoxy-4-O-b-D-glucopyranosyl-1,5-imino-D-glucitol (9D)
3.34. 1,5-Dideoxy-6-O-[b-D-glucopyranosyl-(1!6)-b-D-
glucopyranosyl]-1,5-imino-D-glucitol (8)
Usual deacetylation of the above 4-linked tetraacetate
(9C) (185 mg, 0.206 mmol) gave the title compound
20
Diol 11 (300 mg, 0.63 mmol) was glycosylated with the
gentiobiosyl a-trichloroacetimidate (6B) (96 mg,
0.12 mmol) as described for the preparation of com-
pound 1C. The crude product was fractionated by flash
chromatography (hexane/EtOAc 1:1!0:1). The main
fraction (325 mg), consisting of an inseparable mixture
of glycosylation product (8C) and unreacted acceptor
11, was dissolved in MeOH (15 mL) and treated with
methanolic MeONa at pH 11 for 18 h. Removal of the
solvent and purification of the oily residue by flash chro-
matography (CH2Cl2/EtOAc/MeOH 3.5:3.5:1) gave 2,3-
di-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-6-O-[b-
D-glucopyranosyl-(1!6)-b-D-glucopyranosyl]-1,5-imino-
(9D) as an oil (148 mg, 98%); ½aꢁD +3.3 (c 0.3, MeOH);
13C NMR (CD3OD): d 158.8, 104.9, 78.4, 78.1, 77.2,
76.05, 75.3, 74.3 (CH2), 74.1 (CH2), 72.4 (CH2), 72.1,
68.9 (CH2), 68.7 (CH2), 63.25 (CH2), 56.6, 41.3 (CH2);
HR-MS/FAB m/z calcd for C41H48NO11 (M+H)+:
730.3227, found: 730.3231.
3.37. 1,5-Dideoxy-4-O-b-D-glucopyranosyl-1,5-imino-D-
glucitol (9)
The above tetraol (9D) (159 mg, 0.218 mmol) was
hydrogenolyzed as described for compound 7D
[MeOH/AcOH 10:1 (3 mL), Pd/C (250 mg, 10%,
40 h)]. The catalyst was removed, the solvents were
evaporated and the product was stirred in water
(3 mL) with Amberlyst A26 (OHꢂ) resin for 4 h. Filtra-
tion and freeze drying afforded 9 (67 mg, 96%) as a
20
D-glucitol (8D) as a foam (58 mg, 59%): ½aꢁD ꢂ11.1 (c
1.8, CH2Cl2); 13C NMR (CD3OD): d 159.2, 105.9,
80.9, 79.0, 78.0, 76.1, 74.6 (CH2), 72.9, 72.6 (CH2),
72.5 (CH2), 70.8 (CH2), 70.8 (CH2), 69.6, 69.6, 68.6,
63.8 (CH2), 60.6, 41.8 (CH2). This octaol (50 mg) was
hydrogenolyzed as described for compound 7D in
MeOH (6 mL) and AcOH (1.5 mL) in the presence of
Pd/C catalyst (40 mg, 10% for 16 h), and the pale yellow
syrup obtained after removal of the solids and solvent
was purified by flash chromatography (CH2Cl2/
MeOH/NH4OH 2:4:1) to give the title compound 8
20
hygroscopic amorphous solid: ½aꢁD +20.7 (c 0.41,
H2O), lit.11 [a]D +25.0 (c 0.42, H2O); the H and 13C
1
NMR spectra were identical to previously reported spec-
tra.11 Anal. Calcd for C12H23NO9ÆH2O: C, 41.98;
H, 7.34; N, 4.08. Found: C, 42.36; H, 7.40; N, 3.57;
HR-MS/FAB m/z calcd for C12H24NO9 (M+H)+:
326.1451, found: 326.1444.
20
1
(29 mg, 95%) as a foam: ½aꢁD ꢂ7.3 (c 0.85, H2O); H
NMR (D2O): d 4.49 (d, 1H, J 7.8 Hz), 4.41 (d, 1H, J
7.7 Hz, 1H), 4.21 (dd, 1H, J 2.8, 11.9 Hz), 4.19 (dd,
1H, J 1.6, 11.7 Hz), 4.02 (dd, 1H, J 5.2, 11.9 Hz), 3.89
(dd, 1H, J 2.1, 12.4 Hz), 3.82 (dd, 1H, J 5.1, 11.7 Hz),
3.38. Biological testing
Testing was conducted by Michael Surrey and Peter
Wigley, BioDiscovery NZ Ltd.

R. Blattner et al. / Carbohydrate Research 341 (2006) 2115–2125
2125
9. Fonzi, W. A. J. Bacteriol. 1999, 181, 7070–7079.
Samples of compounds 1–3 and 5–9 were dissolved in
filter-sterilized deionized water at 2 mg/mL and the solu-
tions were serially diluted six times by a factor of three.
Double dilution then resulted on the addition of the
organisms in their nutrient media.
10. Iminosugars as Glycosidase Inhibitors; Stutz, A. E., Ed.;
¨
Wiley-VCH: Weinheim, 1999.
11. Asano, N.; Oseki, K.; Kaneko, E.; Matsui, K. Carbohydr.
Res. 1994, 258, 255–266.
12. Asano, N.; Oseki, K.; Tomioka, E.; Kizu, H.; Matsui, K.
Carbohydr. Res. 1994, 259, 243–255.
13. D’Andrea, F.; Catelani, G.; Mariana, M.; Vecci, B.
Tetrahedron Lett. 2001, 42, 1139–1142.
14. Look, G. C.; Fotsch, C. H.; Wong, C.-H. Acc. Chem. Res.
1993, 26, 182–190.
Acknowledgements
We thank Dr. H. Wong, Industrial Research Ltd, for
providing NMR services and Professor R. J. Ferrier
who assisted with the preparation of the manuscript.
15. Vonhoff, S.; Heightman, T. D.; Vasella, A. Helv. Chim.
Acta 1998, 81, 1710–1725.
16. Uchida, R.; Nasu, A.; Tokutake, S.; Kasai, K.; Tobe, K.;
Yamaji, N. Chem. Pharm. Bull. 1999, 47, 187–193.
17. Furui, H.; Kiso, M.; Hasegawa, A. Carbohydr. Res. 1992,
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Supplementary data
18. Kiso, M.; Furui, H.; Ando, K.; Hasegawa, A. J. Carbo-
hydr. Chem. 1993, 12, 673–677.
19. Arai, M.; Sumida, M.; Fukuhara, K.; Kainosho, M.;
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144–149.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2006.05.004.
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