Inhibition of cellobiohydrolases from Trichoderma reesei. Synthesis and evaluation of some glucose-, cellobiose-, and cellotriose-derived hydroximolactams and imidazoles

Article abstract of DOI:10.1002/(SICI)1522-2675(19990707)82:7<963::AID-HLCA963>3.0.CO;2-V

The lactam 16, the hydroximolactams 8, 20, 23, and 27, and the imidazole 32 were prepared following known methods. They were tested together with the known tetrazole 35 and the hydroximolactams 2 and 36 as inhibitors of the cellobiohydrolases Ce17A and Ce

Full text of DOI:10.1002/(SICI)1522-2675(19990707)82:7<963::AID-HLCA963>3.0.CO;2-V

Helvetica Chimica Acta ± Vol. 82 (1999)
963
Inhibition of Cellobiohydrolases from Trichoderma reesei.
Synthesis and Evaluation of Some Glucose-, Cellobiose-,
and Cellotriose-Derived Hydroximolactams and Imidazoles
by Stefan Vonhoffa), Kathleen Piensb)*, Muriel Pipeliera), Christophe Braetb), Marc Claeyssensb), and
Andrea Vasellaa)*
a) Laboratory of Organic Chemistry, ETH-Zentrum, Universitätstrasse 16, CH-8092 Zürich
b) Department of Biochemistry, Microbiology and Physiology, University of Gent, K.L.
Ledeganckstraat 35, B-9000 Gent
The lactam 16, the hydroximolactams 8, 20, 23, and 27, and the imidazole 32 were prepared following known
methods. They were tested together with the known tetrazole 35 and the hydroximolactams 2 and 36 as
inhibitors of the cellobiohydrolases Cel7A and Cel6A from Trichoderma reesei. Cel7A is only weakly inhibited
by these compounds. Comparing their inhibitory activity evidences the importance of occupying subsites ‡ 1
and ‡ 2. The results strongly suggest that the shape of none of the variants of the lactone-type inhibitor motif
embodied by these inhibitors is complementary to the subsite 1, i.e., analogous to the transition state. Cel6A
is rather strongly inhibited by the cellobiose analogues 20, 23, and 32, and by the cellotriose analogue 27. Their
relative inhibitory activities evidence that binding at subsite
2 depends upon the shape of the moiety
occupying subsite 1. There is only a small difference between the inhibition by the hydroximolactams 20 and
23, which may be (partially) protonated by the catalytic acid of either anti- or syn-protonating glycosidases, and
the imidazole 32, which can only be protonated by anti-protonating glycosidases. The results strongly suggest
that shape requirements must be met by glycosidase inhibitors before they can be used to characterize the
proton trajectory of glycosidases.
1. Introduction. ± Trichoderma reesei produces two cellobiohydrolases, Cel7A and
Cel6A (formerly CBH I and CBH II [1]), which degrade crystalline cellulose very
efficiently, releasing cellobiose from one or the other chain end [2]. X-Ray
crystallography revealed that both enzymes have extended tunnels containing the
active site [3][4].
For Cel7A, the ten structurally defined subsites for glucosyl units are designated
7 to ‡ 3 [3]. Hydrolysis occurs between subsites
1 and ‡ 1 with retention of
configuration at the anomeric C-atom [5][6]. The Cel6A active site tunnel contains
four well-defined binding sites ( 2 to ‡ 2) and two additional subsites (‡ 3, ‡ 4)
[4][7]. As with all family-6 enzymes, the b-1,4 linkages of cellulose are cleaved with
inversion of configuration [5][6].
On the basis of the crystal structure of endoglucanase I of Fusarium oxysporum
complexed to a nonhydrolysable thiooligosaccharide [8], and according to a recently
proposed classification for retaining glycosidases [9], enzymes of family 7 are expected
to be syn-protonators. Family-6 enzymes cannot be classified as yet according to the
syn- or anti-protonation trajectory. To distinguish between these is of obvious
importance for the design of glycosidase inhibitors.
Monosaccharide-derived lactam oximes and imidazoles are established, strong
inhibitors of anti-protonating exoglycosidases [9]. None of these inhibitors have been

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studied with inverting or syn-protonating glycosidases. They could be useful for
distinguishing syn- from anti-protonators. As far as proton transfer from the catalytic
acid is concerned, tetrahydropyridoimidazoles of type 1 are expected to be selective
inhibitors of anti-glycosidases, as illustrated in the Figure, while hydroximolactams like
2 may interact in an analogous way with either anti- or syn-protonators. Obviously, this
does not exclude other factors related to shape or electrostatic interactions.
Figure. anti- and syn-Protonation trajectory for imidazoles and for hydroximolactams
To address these issues, we prepared a number of mono- to trisaccharide-related
lactam oximes and a cellobiose-derived imidazole and tetrazole, and tested them as
inhibitors of T. reesei Cel7A and Cel6A.
2. Results and Discussion. ± 1. Synthesis of the Cellotrioside Analogue 8.
Alkylation under phase-transfer conditions [10] of the hydroximolactam 3 [11] with
the triflate 5, obtained in 91% yield from the lactoside 4 [12], yielded 70% of the
benzylated cellotrioside analogue 6 (Scheme 1). Debenzylation with Li in EtNH2
afforded the unprotected, crystalline analogue 8 in 62% yield after purification via
the acetate 7 [10].
Scheme 1
a) Tf2O, pyridine, CH2Cl2; 91%. b) NaOH, Et4NBr, H2O, toluene; 70%. c) Li, EtNH2, THF,
pyridine; 84%. d) NaOMe, MeOH; 75%.
608; Ac2O,

Helvetica Chimica Acta ± Vol. 82 (1999)
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The benzylated oxime ether 6 is characterized by an exchangeable NH signal at 5.49 ppm, by J(1,2) ˆ 7.5,
and J(1',2') ˆ 7.7 Hz. The imino group gives rise to a C(1'') s at 149.68 ppm, characteristic for the (Z)-
configuration [11], and a CˆN band at 1653 cm 1. J(2'',3'') ˆ 5.4, J(3'',4'') ˆ 5.6, and J(4'',5'') ˆ 9.3 Hz of the
acetylated cellotrioside analogue 7 indicate a B2,5 conformation (in CDCl3) of the hydroximolactam moiety, as
reported for acetylated d-gluconohydroximo-1,5-lactams [10][11], while J(2'',3'') ˆ 9.1, J(3'',4'') ˆ J(4'',5'') ˆ
4
9.2 Hz are in agreement with a C1 conformation (in D2O) of the hydroximolactam moiety of 8.
2. Synthesis of the Cellobionolactam 16. This lactam was prepared by analogy to d-
gluconolactam [13] from the benzylated cellobiose 9 [14] without purification of the
intermediates (Scheme 2). Oxidation of 9 with Dess-Martinꢀs periodinane [15] yielded
the known cellobionolactone 10 [16]1), which was treated with ammonia to give the
hydroxy amide 11. Oxidation of 11 with 4 equiv. of 1-hydroxy-1,2-benziodoxol-3(1H)-
one 1-oxide [15] gave the intermediate oxo amide2), which cyclized under workup
conditions to a mixture of the d-gluco- and l-ido-configured lactams 12 and 13.
Reductive deoxygenation of the mixture 12/13 with NaBH3CN and HCO2H in MeCN
yielded the d-gluconolactam 14 and the l-ido-epimer 15 in 38 and 7%, respectively,
from 9. In contrast to the reduction of the glucose-derived analogues, reduction of 12/
Scheme 2
a) Dess-Martin periodinane, CH2Cl2. b) NH3, CH2Cl2,
608. c) 1-Hydroxy-1,2-benziodoxol-3(1H)-one 1-
oxide, DMSO; H2O, Et2O. d) NaCNBH3, HCO2H, MeCN, 38% of 14 and 7% of 15 (from 9). e) Pd(OH)2, H2,
THF/MeOH/H2O 3 : 2 : 1, AcOH; 69%.
1
)
On a 20-g scale, the Dess-Martin oxidation proceeded in higher yields and more reproducibly than the
Swern oxidation.
Oxidation of 11 with DMSO/Ac2O, pyridine ´ SO3 complex, or pyridinium chlorochromate (PCC) was
incomplete. Swern oxidation gave a less polar product than 12 or 13, which was not isolated. Oxidation
with Dess-Martin periodinane led a product with a higher Rf value than 12 and 13.
2
)

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Helvetica Chimica Acta ± Vol. 82 (1999)
13 with NaBH4 in HCO2H [17] or with Et3SiH and BF3 ´ OEt2 [13] was lower yielding
and less diastereoselective3). The d-glucono- and l-idonolactams 14 and 15 were
separated by MPLC. Hydrogenolytic debenzylation of 14 in the presence of Pd(OH)2
yielded 69% of the cellobionolactam 164).
The d-gluco- and l-ido-configured hydroxylactams 12 and 13 are characterized by J(2,3) ˆ 6.7 and
J(3,4) ˆ 6.3, and J(2,3) ˆ 2.2 and J(3,4) ˆ 5.9 Hz, as well as by an exchangeable NH signal at 6.05 and 6.14 ppm,
respectively. For the d-glucono- and l-idonolactams 14 and 15, J(2,3) ˆ 5.6, J(3,4) ˆ 5.5, and J(4,5) ˆ 8.0, and
J(2,3) ˆ 5.2, J(3,4) ˆ 2.8, and J(4,5) ˆ 2.2 Hz, respectively, were observed for the lactam moiety. The J values of
the deprotected cellobionolactam 16 (J(2,3) ˆ 5.6, J(3,4) ˆ 5.5, J(4,5) ˆ 8.0, J(2',3') ˆ 9.3, J(3',4') ˆ 9.2, and
J(4',5') ˆ 9.4 Hz) in D2O are in agreement with a B2,5 and 4C1 conformation of the lactam and pyranosyl moiety,
respectively.
3. Synthesis of the Cellobionohydroximolactam 20 and the Phenylcarbamate 23.
Conversion of the lactam 14 into the thiolactam 17, followed by treatment with NH2OH
in boiling MeOH (cf. [11]) yielded 79% of the Bn-protected hydroximolactam 18
(Scheme 3). Debenzylation of 18 with Li in EtNH2 and acetylation afforded the
octaacetate 19, which was deprotected under Zemplen conditions to yield 66% of the
cellobionohydroximolactam 20.
Scheme 3
a) Lawessonꢀs reagent, toluene; 87%. b) NH2OH ´ HCl, NaHCO3, MeOH; 91%. c) Li, EtNH2, THF, 708;
Ac2O, pyridine; 76%. d) NaOMe, MeOH; 66%. e) N2H4 ´ AcOH, DMF. f) PhNCO, Et3N, THF; 59% from
17. g) NH3, MeOH; 64%.
The phenylcarbamate 23 was obtained in 38% overall yield from the octaacetate 19
by selective deacetylation of the acetoximo group with hydrazine acetate, treatment of
the resulting oxime 21 with PhNCO, and deacetylation of 22 with NH3 in MeOH [11]5).
3
)
Trialkylsilanes (R3SiH with R ˆ Me, Pr, or Bu) did not improve the diastereoselectivity [18]. Conversion
of the mixture of hydroxylactams 12 and 13 to the corresponding (ethylthio)lactams [19] followed by
radical reduction with Bu3SnH and AIBN [20] gave preferentially the l-ido-configured 15.
No reduction was observed with Pd-black in EtOH as catalyst.
4
5
)
)
Deacetylation with NaOMe in MeOH at 08 resulted in methanolysis of the carbamoyl group.

Helvetica Chimica Acta ± Vol. 82 (1999)
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4. Synthesis of the Cellotrioside Analogue 27. The benzylated cellotrioside analogue
25 (Scheme 4) was synthesized by a Hg(OAc)2-assisted condensation of the Bn-
protected cellobiothionolactam 17 with the hydroxylamine 24 [10]. Debenzylation of
25 with Li in EtNH2 afforded the cellotrioside analogue 27, which was purified via the
peracetylated derivative 26.
Scheme 4
a) (i-Pr)2EtN, Hg(OAc)2, THF; 78%. b) Li, EtNH2, THF, 608; Ac2O, pyridine; 42%. c) NaOMe, MeOH;
91%; d) (MeO)2CHCH2NH2, Hg(OAc)2, THF, 08; TsOH ´ H2O, toluene, 608; 69%. e) NIS, DMF, 708; 82%. f)
EtMgBr, then H2O (twice); 77% of 28 from 30, 66% of 29 from 31. g) Pd(OH)2, 6 bar of H2, AcOEt/MeOH/
AcOH; 69%.
The imino group of 25 gives rise to a C(1') s at 148.85 ppm, and a CˆN band at 1653 cm 1. The peracetate
26 is characterized by J(2',3') ˆ 4.3, J(3',4') ˆ 1.2, and J(4',5') ˆ 1.2 Hz and a W coupling of 2.6 Hz between
3
H
C(2') and H C(4'), which is in agreement with a E conformation of the hydroximolactam moiety. The J
values of the deprotected cellotrioside analogue 27 (J(2',3') ˆ J(3',4') ˆ 6.8, and J(4',5') ˆ 9.2 Hz) in CD3OD
indicate a B2,5 conformation of the hydroximolactam moiety.
5. Synthesis of the Cellobiose Analogue 32. We followed the procedure for the
preparation of the benzylated gluco-imidazole 1 [21]. Thionolactam 17 (Scheme 4) was

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treated with aminoacetaldehyde dimethyl acetal and Hg(OAc)2 in THFat 08. The crude
amidines were cyclized by treatment with TsOH ´ H2O in toluene6), yielding 69% of an
8 : 2 mixture of the gluco- and manno-imidazoles 28/29, the ratio of the isomers
depending upon the exact reaction conditions (compare [21]). Separation of these
isomers proved exceptionally difficult7). They were, however, separated via their
diiodo derivatives 30/31 obtained in 82% yield by treating 28/29 with excess N-
iodosuccinimide in DMF at 808, following a known procedure [21]. The diiodides were
separated by standard flash chromatography. The pure diiodides 30 and 31 were
deiodinated by repeated treatment with a ca. 1.5 molar excess of EtMgBr in THF at 08,
followed by workup with aqueous sat. NH4Cl solution, yielding 77 and 66% of the
imidazoles 28 and 298), respectively. Hydrogenolytic debenzylation of 28 in AcOH
under slightly elevated pressure (6 bar) in the presence of Pd(OH)2 yielded the desired
deprotected cellobioimidazole 32 in 69% yield.
The I substituents in 30, 31, 33, and 34 are evidenced by a strong upfield shift for the C(2) s at 81.6 ± 82.2 and
the C(3) s at 96.2 ± 96.7 ppm as compared to the unsubstituted imidazoles (C(2) and C(3) d of 28, 29, and 32 ± 34
at 118.5 ± 129.1 ppm). H C(2) and H C(3) of 28 and 29 in CDCl3 and of 32 in D2O resonating at 6.91 ±
1
7.20 ppm show small vicinal couplings (J ꢀ 1.4 Hz). Signal overlap in the H-NMR spectra of 28 ± 31 required
the measurement of 1H,1H-COSY spectra to allow an unambiguous assignment of H C(5) to H C(8) and of
most of the vicinal coupling constants. The coupling constants of the manno-imidazoles 29 (J(5,6) ˆ 2.5,
J(6,7) ˆ 6.7, and J(7,8) ˆ 3.3 Hz) and 32 (J(5,6) ˆ 1.0, J(6,7) ˆ 6.5, and J(7,8) ˆ 3.3 Hz) suggest a 7S5
conformation. The gluco-imidazoles 28 and 30 probably possess a similar conformation as indicated by J(7,8)
of 2.8 Hz for both. The values for J(5,6) and J(6,7) of 28 are 4.2 Hz. The corresponding couplings of 30 could
not be exactly determined due to signal overlap. Nevertheless, the signal widths suggest medium values. Thus,
the vicinal couplings of 28 and 30 suggest a ca. 2 : 1 equilibrium of the 6H7 and 7H6 conformers. The unprotected
cellobioimidazole 32, however, adopts a 7H6 conformation, as evidenced by the rather large coupling constants
(J(6,7) ˆ 8.4 and J(7,8) ˆ 8.2 Hz).
6. Inhibition Studies. Cel7A is subject to strong product inhibition by cellobiose (Ki
ca. 20 mm) [24]. As evidenced by crystal-structure analysis [25], this is probably due to
H-bonding between cellobiose and the product binding sites ‡ 1 and ‡ 2 and to ring-
stacking interactions with Trp 376 in the ‡ 1 site. As discussed in [9], the factors
relevant for the inhibition by transition-state analogues are shape, protonation
trajectory, and non-directional charge effects.
While the inhibition of Cel7A by the lactam 16, by the hydroximolactams 2 [11], 8,
20. 23, 27, and 36 [10], and by the azoles 32 and 35 [26] is always weaker than the one by
cellobiose, considerable differences in strength and inhibition type are observed (see
Table).
The poor inhibition by the hydroximolactam 2 [11] is in keeping with the
requirement for the substrate to fill subsites ‡ 1 and ‡ 2. It also indicates that 2 does
not meet the requirements of a transition-state analogue binding to subsite
1.
Surprisingly, the cellobiose analogue 36 does not inhibit; i.e., it binds neither at subsites
6
)
)
Treatment in aqueous toluene led to cleavage of the glycosidic bond.
7
The isomers were partially separated by HPLC on a CN phase; neither the unprotected isomers (obtained
by hydrogenolytic debenzylation) nor their acetylation products could be separated.
The first treatment with EtMgBr led selectively to the monoiodides 33 and 34 as evidenced by their
13C-NMR spectra. The regioselectivity is assumed by analogy to the selective dehalogenation of related
diiodides [22] and dibromides [23].
8
)

Helvetica Chimica Acta ± Vol. 82 (1999)
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Table. Inhibition Constants Ki [mm] of Cellobiose and Compounds 2, 8, 16, 20, 23, 27, 32, 35, and 36
Inhibitor
Cel7Aa)
Cel6Ab)
Ki
Ki
Type of inhibition
competitive
Cellobiose
2
20
600
ca. 1800c)
d
e
)
)
8
40
ca. 1300
190
740
ca. 6000
130
competitive
)
700
300
5
1
14
1
d
16
20
23
27
32
35
36
mixed-type (a ˆ 2.3)
competitive
d
)
non-competitive
d
1000
)
)
130
e
d
e
)
)
a) Activity measurements with 2-chloro-4-nitrophenyl b-lactoside at pH 5.7. b) Activity measurement on
cellotriose at pH 5; type of inhibition not determined. c) Determined in [29]. d) Not determined. e) No
inhibition at a concentration of 2 mm.
‡ 1/ ‡ 2, nor at
distance between the two monosaccharide moieties, impairing binding at ‡ 1/ ‡ 2, and
at 2/ 1; it again evidences the poor complementarity of the hydroximolactam
2/ 1, nor at
1/ ‡ 1. This may be rationalized by the greater
moiety to the 1 subsite. In keeping with this, 8 is a much better inhibitor than 36,
although it is only half as strong as cellobiose. This suggests that the cellobiosyl moiety

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Helvetica Chimica Acta ± Vol. 82 (1999)
of 8 binds to the ‡ 1/ ‡ 2 subsites, and that this binding is (weakly) impaired by the
hydroximolactam moiety. This interpretation is in keeping with the poor inhibition of
27, formally derived from 36 by attaching a glucosyl moiety to the hydroximolactam
ring. The assumption that the hydroximolactam moiety is a poor inhibition motif for
Cel7A, i.e., that it fits the 1 site poorly, is confirmed by the weak inhibition by the
lactam 16 and the hydroximolactam 20. Remarkably, 20 is a mixed type inhibitor,
meaning that it does not bind at subsites occupied by the substrate, i.e., 2, 1, and
‡ 1. In contradistinction to this, the phenylcarbamate 23, derived from 20, is a (weak)
competitive inhibitor. This is in agreement with the ring-stacking interaction, at the ‡ 1
site, of a glucosyl unit in the cellulose substrate, and of the aryl ring in the aryl-lactoside
substrate, meaning that 23 binds at the 2/ 1/ ‡ 1 subsites. Apparently, the stacking
interaction compensates partially for the poor fit of the hydroximoyl moiety at the
1
site. The similarity between the hydroximolactam and the tetrahydropyridoimidazole
moiety as inhibition motifs is expressed by the similar inhibition constants for 20 and 32,
and by their mode of inhibition (mixed for 20 and non-competitive for 32). The
tetrazole 35 is almost ten times weaker than the imidazole 32.
These results confirm the importance of binding to the subsites ‡ 1 and ‡ 2, as
expressed by the product inhibition. They also show that none of the variants of the
ꢁlactone-typeꢀ inhibitor motif [9] leads to a binding interaction at the
1 site. The
similarity of the inhibition by 20, which can be protonated by a syn-protonating
glycosidase, and by 32, which cannot, suggests that the main factor responsible for the
poor inhibition is the difference between the half-chair conformation common to the
inhibitors and the conformation at the 1 site of the transition state. This is in keeping
with the distortion of the glucosyl unit in the
1 subsite observed in the crystal
structure of one of the members of the family-7 glycosidases, EGI of Fusarium
oxysporum, in complex with a thioglucoside [8].
For Cel7A, the inhibition constant of 130 mm for the imidazole 32 compares to a Ki
of 88 mm for the inhibition by 32 of Cel5A [27], a retaining, anti-protonating
endoglucosidase for which a conformational change of the substrate moiety bound to
the 1 subsite has been strongly evidenced [28]. This means that the use of inhibitors
for the characterization of glycosidases as syn- or anti-protonators is restricted to
inhibitors that possess a shape close enough to that of the transition state.
That Cel6A cleaves cellulose from the non-reducing end [2] explains the weak
product inhibition by cellobiose [4][29]. The released cellobiose corresponds to the
cellobiosyl moiety of the substrate bound at subsites 2 and 1, where it must be
most strongly bound in the transition-state conformation. Neither 2 nor 36 inhibit this
enzyme, suggesting that the inhibition motif is not optimal. The trisaccharide analogue
8 is a weak inhibitor. Introduction of the additional glucosyl moiety that should bind at
the ‡ 2 site has a small but positive effect. A stronger effect is seen for the glucosyl
moiety that should bind at the 2 site, suggesting that the hydroximoyl moiety, while
not matching the requirements of the transition state, still has a positive effect on the
binding of the glucosyl moiety to the 2 site. Among the disaccharide analogues that
correspond the disaccharide moiety at the non-reducing end of 27, the more strongly
basic hydroximolactam 20, carbamate 23, and imidazole 32 are clearly stronger
inhibitors than the less basic lactam 16 and tetrazole 35. The inhibition constants for
these disaccharide analogues do not differ much. Although these results are more in

Helvetica Chimica Acta ± Vol. 82 (1999)
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keeping with the hypothesis that Cel6A is an anti-protonator, we are cautioned by the
results obtained in the inhibition of Cel7A and Cel5A, and the lack of inhibition by 2
and 36, suggesting that the shape requirements are not well met, and that non-
directional electrostatic interactions may be responsible for the stronger inhibition by
the basic disaccharide analogues. Apart from evidencing a dependence of the
interaction at the 2 site on the conformation of the glucosyl moiety at the 1 site,
these results show that the geometry of the transition state must differ significantly
from the one of lactone type inhibitors. A twist boat conformation and a (laterally)
protonable site that lies above the mean plane of the pyranosyl ring appear necessary
for strong and selective inhibitors that can be used to unambiguously characterize a
glycosidase as syn- or anti-protonator.
We thank the EU Biotechnology Programme (BBW Nr. 96.0375), the Swiss National Science Foundation,
the Belgium FWO, and F. Hoffmann-La Roche AG, Basel, for generous support, and Dr. B. Bernet for the critical
reading of the manuscript.
Experimental Part
General. Solvents were distilled before use. Moisture-sensitive reactions were run under Ar or N2 in dry
solvents. TLC: Merck silica gel 60 F254 plates; detection by heating with ꢁmostainꢀ (400 ml of aq. 10% H2SO4
soln., 20 g of (NH4)6Mo7O24 ´ H2O, 0.4 g of Ce(SO4)2) or 20% aq. H2SO4 soln. Flash chromatography (FC): silica
gel Merck 60 (0.040 ± 0.063 mm). HPLC: Spherisorb silica gel (5 mm) column (20 Â 250 mm). Reversed-phase
HPLC: RP-18 silica gel. M.p.: uncorrected. IR Spectra: Perkin Elmer 1600 FT-IR spectrometer. 1H-, 13C-, and
19F-NMR Spectra: at 200 ± 500 MHz on Varian instruments, chemical shifts d in ppm and coupling constants J in
Hz. FAB-MS: VG-SABSEQ instrument; 3-NOBA ˆ 3-nitrobenzyl alcohol. ESI-MS: Finnigan MAT TSQ 7000
instrument. The inhibition studies were carried out with purified Cel7A [30] and Cel6A [31] from Trichoderma
reesei.
Methyl 2,3,6-Tri-O-benzyl-4-O-{2,3,6-tri-O-benzyl-4-O-[(trifluoromethyl)sulfonyl]-b-d-galactopyranosyl}-
b-d-glucopyranoside (5). At 158, Tf 2O (2.0 ml, 12.8 mmol) was added dropwise within 20 min to a stirred
suspension of 4 (5.8 g, 6.4 mmol) [12], pyridine (1.0 ml, 12.8 mmol), and 3-Š molecular sieves (0.1 g) in CH2Cl2
(25 ml). The suspension was warmed to 08 within 3 h, poured into cold 1m aq. HCl (25 ml). After separation, the
org. layer was washed with H2O (3 Â 30 ml). Evaporation and FC (hexane/AcOEt 4 : 1) gave 5 (6.0 g, 91%).
Colourless oil. Rf (hexane/AcOEt 7 : 3) 0.75. IR (CH2Cl2): 3031w, 2880w, 1496w, 1454m, 1410s, 1212s, 1139s,
1099s, 1082s, 1022m, 925s, 631m. 1H-NMR (CDCl3, 300 MHz): 3.39 ± 3.52 (m, H C(2), H C(5), H C(2'),
H
C(3'), H C(5'), H C(6')); 3.62 (t, J ꢁ 8.7, H C(4)); 3.63 ± 3.65 (m, H' C(6')); 3.64 (s, MeO); 3.74 (dd,
J ˆ 10.3, < 1.0, H C(6)); 3.90 (dd, J ˆ 10.9, 3.1, H' C(6)); 4.02 (t, J ꢁ 9.3, H C(3)); 4.33 (d, J ˆ 11.5, PhCH);
4.37 (d, J ˆ 7.1, H C(1)); 4.38 (d, J ˆ 11.5, PhCH); 4.49 (d, J ˆ 8.1, H C(1')); 4.53 (d, J ˆ 11.5), 4.63 (d, J ˆ
12.4), 4.65 (d, J ˆ 12.4), 4.79 (d, J ˆ 11.5), 4.83 (d, J ˆ 11.5), 4.85 (d, J ˆ 10.2), 4.87 (d, J ˆ 10.9), 4.94 (d, J ˆ
11.2), 4.98 (d, J ˆ 10.9), 4.99 (d, J ˆ 10.6, 10 PhCH); 5.42 (d, J ˆ 2.5, H C(4')); 7.26 ± 7.42 (m, 30 H). 13C-NMR
(CDCl3, 75 MHz): 57.31 (q, MeO); 66.71, 68.15 (2t, C(6), C(6')); 70.93 (d, C(4')); 72.92, 73.32, 73.71 (3t, 3
PhCH2); 75.10 (d); 75.17, 75.68, 75.75 (3t, 3 PhCH2); 77.23 (d); 78.60 (d); 78.89 (d); 81.83 (d); 82.01 (d); 82.71
(d); 102.72 (d, C(1')); 105.03 (d, C(1)); 119.41 (q, J(C,F) ˆ 321, CF3); 127.73 ± 128.81 (several d); 137.52, 137.81,
138.47, 138.65, 139.00, 139.26 (6s). 19F-NMR (CDCl3, 282 MHz): 73.83. FAB-MS (3-NOBA): 1027 (15, [M
‡
H] ), 271(30), 181(80), 91(100).
Methyl {[(Z)-5-Amino-2,3,4,6-tetra-O-benzyl-5-deoxy-b-d-glucopyranosylidene]amino}-(1-N ! 4)-2,3,6-
tri-O-benzyl-b-d-glucopyranosyl-(1 ! 4)-2,3,6-tri-O-benzyl-b-d-glucopyranoside (6). A vigorously stirred mix-
ture of 3 [11] (3.0 g, 5.4 mmol), 5 (5.5 g, 5.4 mmol), Et4NBr (50 mg) in toluene (30 ml) and an aq. soln. of
NaOH (10.0 g in 30 ml) was heated to reflux for 16 h. The layers were separated, and the aq. layer was extracted
with CH2Cl2 (2 Â 25 ml). Drying of the combined org. layers (MgSO4), evaporation, and FC (hexane/AcOEt
4 : 1) afforded 6 (5.4 g, 70%). Colourless oil. Rf (hexane/AcOEt 7: 3) 0.80. IR (CH2Cl2): 3412w, 3068s, 2987s,
2925m, 2868m, 1653m, 1605w, 1550w, 1496m, 1453s, 1422s, 1389m, 1362m, 1288s, 1212m, 1094s, 1059s, 1028m,
896s. 1H-NMR (CDCl3, 200 MHz): 3.36 ± 3.55 (m, 7 H); 3.59 (s, MeO); 3.63 ± 3.68 (m, 1 H); 3.73 ± 3.78 (m, 2 H);
3.83 ± 3.86 (m, 1 H); 3.89 ± 3.96 (m, 3 H); 3.99 ± 4.09 (m, 4 H); 4.33 (d, J ˆ 7.5, H C(1)); 4.34 (d, J ˆ 11.2), 4.35
(d, J ˆ 11.6), 4.36 (d, J ˆ 12.0), 4.38 (d, J ˆ 11.2), 4.43 (d, J ˆ 12.0, 5 PhCH); 4.49 (AB, J ˆ 12.0, PhCH2); 4.52 (d,

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Helvetica Chimica Acta ± Vol. 82 (1999)
J ˆ 7.7, H C(1')); 4.56 (d, J ˆ 12.0), 4.57 (d, J ˆ 12.0), 4.58 (d, J ˆ 12.0, 3 PhCH); 4.59 (s, PhCH2); 4.64 (d, J ˆ
12.0), 4.73 (d, J ˆ 10.8), 4.77 (d, J ˆ 12.1), 4.78 (d, J ˆ 12.1, 4 PhCH); 4.80 (s, PhCH2); 4.88 (d, J ˆ 10.8), 5.09 (d,
J ˆ 11.2, 2 PhCH); 5.49 (br. s, NH); 7.15 ± 7.43 (m, 50 H). 13C-NMR (CDCl3, 50 MHz): 52.39 (d, C(5'')); 57.16
(q, MeO); 70.88 (t), 71.67 (2t, C(6), C(6'), C(6'')); 71.96 (2t), 72.83(2t), 73.72 (3t, 7 PhCH2); 73.83 (d); 74.42
(d); 74.20 (t), 75.18 (t), 76.09 (t, 3 PhCH2); 77.11 (d); 79.67 (2d); 81.40 (d); 82.39 (2d); 83.05(d); 83.43 (d);
83.90(d); 102.98 (d, C(1')); 104.31 (d, C(1)); 127.77 ± 129.20 (several d); 135.14 ± 139.52 (several s); 149.68 (s,
‡
C(1'')). FAB-MS (3-NOBA): 1432 (100, [M ‡ H] ), 1400(41), 1340(12), 1324(19). Anal. calc. for
C89H94N2O15 (1431.73): C 74.66, H 6.62, N 1.96; found: C 74.41, H 6.52, N 1.94.
Methyl {[(Z)-2,3,4,6-Tetra-O-acetyl-5-amino-5-deoxy-b-d-glucopyranosylidene]amino}-(1-N ! 4)-2,3,6-tri-
O-acetyl-b-d-glucopyranosyl-(1 ! 4)-2,3,6-tri-O-acetyl-b-d-glucopyranoside (7). At 608, a soln. of 6 (5.0 g,
3.5 mmol) in THF (12 ml) was added to a deep blue soln. of Li (0.73 g, 0.11 mol) in condensed EtNH2 (ca.
100 ml) within 10 min. The mixture was stirred at
608 for 10 min and treated with NH4Cl (5.0 g). After
evaporation, the residue was dried, dissolved in pyridine (40 ml), and treated with Ac2O (20 ml) at 08, and the
mixture stirred for 14 h at 208. After evaporation, the residue was dissolved in CH2Cl2 (100 ml) and washed with
sat. aq. NaHCO3 soln. (2 Â 50 ml). Drying of the org. phase (MgSO4), evaporation, FC (hexane/AcOEt 1 : 2)
and HPLC (hexane/AcOEt 2 : 3), afforded pure 7 (2.8 g, 84%). Colourless oil. Rf (hexane/AcOEt 2 : 3) 0.20. IR
(CH2Cl2): 3448w, 3068m, 2987m, 1754s, 1666m, 1518w, 1422m, 1370m, 1234s, 1230s, 1162m, 1043m, 896m.
1H-NMR (CDCl3, 300 MHz): 1.97, 1.98, 1.99, 2.00, 2.04, 2.05, 2.06, 2.07, 2.09, 2.10 (10s, 10 AcO); 3.44 (s, MeO);
3.56 (ddd, J ˆ 9.6, 4.7, 1.9, H C(5)); 3.58 (dddd, J ˆ 9.4, 6.1, 2.8, 1.3, H C(5'')); 3.73 (t, J ꢁ 9.0, H C(4)); 3.75
(ddd, J ˆ 10.0, 4.7, 2.2, H C(5')); 3.98 (dd, J ˆ 12.1, 6.7, H C(6'')); 3.99 (t, J ꢁ 9.7, H C(4')); 4.07 (dd, J ˆ 11.8,
5.0, H C(6)); 4.19 (dd, J ˆ 12.2, 2.2, H C(6')); 4.22 ± 4.25 (m, H' C(6')); 4.26 (dd, J ˆ 12.2, 2.8, H' C(6''));
4.34 (d, J ˆ 7.8, H C(1)); 4.45 (d, J ˆ 7.8, H C(1')); 4.49 (dd, J ˆ 12.1, 1.9, H' C(6)); 4.84 (dd, J ˆ 9.7, 7.9),
4.85 (dd, J ˆ 9.7, 7.8, H C(2), H C(2')); 4.94 (dd, J ˆ 9.3, 5.6, H C(4'')); 5.13 (t, J ꢁ 9.0, H C(3)); 5.17 (t, J ꢁ
5.6, H C(3'')); 5.25 (d, J ˆ 5.4, H C(2'')); 5.26 (t, J ꢁ 9.6, H C(3')); 5.32 (br. s, NH). 13C-NMR (CDCl3,
50 MHz): 20.58 ± 20.73 (several q); 52.21 (d, C(5'')); 57.03 (q, MeO); 61.98, 62.66, 63.03 (3t, C(6), C(6'), C(6''));
67.66 (d); 70.01 (d); 71.66 (d); 71.87 (d); 72.16 (2d); 72.27 (d); 72.64 (d); 72.85 (d); 76.49, 77.84 (2d, C(5),
C(5')); 101.59, 101.86 (2d, C(1), C(1')); 148.00 (s, C(1'')); 168.90, 169.53(2s); 169.74(2s); 169.97, 170.18, 170.34,
‡
‡
170.70, 170.86, 171.30(6s). FAB-MS (3-NOBA): 952 (39, [M ‡ H] ), 951 (100, M ), 631 (25).
Methyl {[(Z)-5-Amino-5-deoxy-b-d-glucopyranosylidene]amino}-(1-N ! 4)-b-d-glucopyranosyl-(1 ! 4)-
b-d-glucopyranoside (8). At 58, a soln. of 7 (2.4 g, 2.5 mmol) in MeOH (30 ml) was treated dropwise with a
freshly prepared 0.5n soln. of NaOMe in MeOH (ca. 1 ml). After 4 h, the mixture was stirred for 15 min with
‡
Amberlite IR-120 (H form), filtered, and evaporated. Reversed-phase HPLC (RP18 silica gel, H2O/MeOH
100 : 0 ! 20 : 80) and crystallization from MeOH afforded 8 (1.0 g, 75%). Colourless solid. Rf (AcOEt/MeOH/
H2O 4 : 2 : 1) 0.12. M.p. 159.5 ± 160.58 (MeOH). [a]D25 ˆ ‡ 103.6 (c ˆ 0.59, H2O). IR (KBr): 3440s, 3340s, 3200s,
2990m, 2940m, 1665s, 1640s, 1455m, 1330s, 1300s, 1150m, 1080s, 970s. 1H-NMR (D2O, 500 MHz): 3.18 (ddd, J ˆ
9.2, 4.9, 2.8, H C(5'')); 3.24 (m, 10 lines, J ˆ 9.4, 8.0, H C(2)); 3.32 (dd, J ˆ 9.2, 8.0, H C(2')); 3.51 (s, MeO);
3.53 (t, J ꢁ 9.2, H C(4'')); 3.54 ± 3.60 (m, H C(3), H C(4), H C(5)); 3.61 (t, J ꢁ 9.2, H C(3'')); 3.64 (dd,
J ˆ 12.2, 5.5, H C(6')); 3.65 (dd, J ˆ 11.9, 4.8, H C(6'')); 3.68 (ddd, J ˆ 9.2, 5.4, 1.9, H C(5')); 3.76 (dd, J ˆ
12.1, 4.8, H C(6)); 3.77 (dd, J ˆ 12.0, 2.8, H' C(6'')); 3.78 (t, J ꢁ 9.5, H C(4')); 3.79 (dd, J ˆ 12.1, 2.3,
H' C(6')); 3.84 (t, J ꢁ 9.3, H C(3')); 3.94 (dd, J ˆ 11.8, 1.9, H' C(6)); 4.09 (d, J ˆ 9.1, H C(2'')); 4.35 (d, J ˆ
8.0, H C(1)); 4.46 (d, J ˆ 8.0, H C(1')). 13C-NMR (CD3OD, 75 MHz): 58.17 (q, MeO); 58.66 (d, C(5'')); 61.62,
62.18, 63.19 (3t, C(6), C(6'), C(6'')); 70.58 (d); 70.71 (d); 74.56 (d); 74.60 (d); 75.22 (d); 76.13 (d); 76.24 (d);
76.37 (d); 76.74 (d); 80.61, 81.05 (2d, C(4), C(4')); 104.33, 104.99 (2d, C(1), C(1')); 155.13 (s, C(1'')). FAB-MS
‡
(3-NOBA): 531 (25, [M ‡ H] ), 460(70), 307(100), 154(60). Anal. calc. for C19H34N2O15 ´ H2O (548.48):
C 41.60, H 6.62, N 5.11; found: C 41.85, H 6.53, N 5.18.
2,3,6-Tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-b-d-glucopyranosyl)-d-glucono-1,5-lactone (10) [16]. At
238, a soln. of 9 [14] (22.2 g, 22.8 mmol) in CH2Cl2 (330 ml) was treated with 1,1,1-triacetoxy-1,1-dihydro-1,2-
benziodoxol-3(1H)-one (Dess-Martin periodinane) [15] (24.0 g, 57.0 mmol), and stirred for 5 h. The mixture
was diluted with Et2O (300 ml), poured into 200 ml of a 1:1 mixture of sat. aq. NaHCO3 soln. and Na2SO3 soln.
(20 g/100 ml H2O), and stirred at 238 for 20 min. After separation, the aq. layer was extracted with CH2Cl2 (2 Â
200 ml). Drying of the combined org. layers (MgSO4) and evaporation yielded crude 10 (21.7 g) which was
sufficiently pure to be used for the next step. Crystallization of a sample from EtOH gave colourless needles.
M.p. 110 ± 1128 ([16]: M.p. 114 ± 1158).
2,3,6-Tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-b-d-glucopyranosyl)-d-gluconamide (11). At 608, a soln.
of crude 10 (21.7 g, 23 mmol) in dry CH2Cl2 (280 ml) was added dropwise to condensed NH3 (ca. 40 ml). The
cooling bath was removed, and the soln. was kept under reflux for 2 h. Evaporation gave crude 11 (21.8 g) as an

Helvetica Chimica Acta ± Vol. 82 (1999)
973
oil which was used for the next step. A sample was purified by FC (hexane/AcOEt 6 : 1). Rf (hexane/AcOEt
4 : 1) 0.33. IR (CHCl3): 3517w, 3485w, 3400w, 3066w, 3007m, 2911w, 2869w, 1687s, 1497w, 1454m, 1359m, 1261s,
1091s, 1070s, 1024s. 1H-NMR (CDCl3, 500 MHz): 3.23 (br. s, OH); 3.35 (t, J ꢁ 8.6, H C(2')); 3.34 ± 3.37 (m,
H
C(5')); 3.50 (t, J ꢁ 9.1, H C(4')); 3.54 (t, J ꢁ 8.8, H C(3')); 3.55 (dd, J ˆ 10.0, 3.3, H C(6)); 3.54 ± 3.58 (m,
2 H C(6')); 3.64 (dd, J ˆ 10.1, 4.6, H' C(6)); 3.99 (m, H C(5)); 4.09 (dd, J ˆ 7.6, 3.6, H C(4)); 4.26 (dd, J ˆ
4.7, 3.6, H C(3)); 4.33 (d, J ˆ 11.9, PhCH); 4.38 (d, J ˆ 4.8, H C(2)); 4.41 (d, J ˆ 11.9), 4.42 (d, J ˆ 11.9), 4.44
(d, J ˆ 12.1, 3 PhCH); 4.45 (d, J ˆ 7.8, H C(1')); 4.52 (d, J ˆ 11.0), 4.57 (d, J ˆ 11.3), 4.62 (d, J ˆ 11.4), 4.66 (d,
J ˆ 11.2), 4.70 (d, J ˆ 11.1), 4.72 (d, J ˆ 11.2), 4.78 (d, J ˆ 11.1), 4.79 (d, J ˆ 11.0), 4.80 (d, J ˆ 11.0), 4.89 (d, J ˆ
11.0, 10 PhCH); 5.29 (br. s, NH); 6.69 (br. s, NH); 7.15 ± 7.33 (m, 35 H). 13C-NMR (CDCl3, 63 MHz): 69.14,
70.71 (2t, C(6), C(6')); 71.49 (d, C(5)); 73.37 (t), 73.44 (t), 73.58 (t), 74.77 (2t), 75.09 (2t, 7 PhCH); 75.77 (d);
76.82 (d); 77.90 (d); 79.37 (d); 80.13 (d); 82.30 (d); 84.84 (d); 103.54 (d, C(1')); 127.87 ± 128.76 (several d);
137.70, 138.16, 138.38, 138.41, 138.53 (5s); 138.83 (2s); 174.26 (s, C(1)). FAB-MS (3-NOBA): 988 (40, [M ‡
‡
H] ), 466(35), 181(95), 901(100).
5-Amino-2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-b-d-glucopyranosyl)-d-glucono-1,5-lactam (12) and
5-Amino-2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-b-d-glucopyranosyl)-l-idono-1,5-lactam (13). At 238, a
soln. of crude 11 (21.7 g, 22 mmol) in DMSO (90 ml) was added to a 1m soln. of 1-hydroxy-1,2-benziodoxol-
3(1H)-one 1-oxide [15] (25.0 g, 88 mmol) in DMSO (90 ml). After stirring for 5 h and the addition of H2O
(200 ml) and Et2O (300 ml) at 58, the mixture was stirred for 2 h at 238 and filtered through Celite. After
separation, the aq. layer was extracted with Et2O (3 Â 150 ml). The combined org. layers were dried and
evaporated to give 12/13 (21.3 g) as an oil which was dried at 0.01 Torr and used without further purification for
the next step. FC (hexane/AcOEt 6 : 1) gave pure samples of 12 and 13.
Data of 12: Rf (hexane/AcOEt 4 :1) 0.47. IR (CH2Cl2): 3350w, 3068s, 2986s, 1697m, 1550w, 1496w, 1422s,
1361m, 1288s, 1154m, 1070m, 1028m, 988w, 895s. 1H-NMR (CDCl3, 500 MHz): 3.36 ± 3.40 (m, H C(5')); 3.43 (d,
J ˆ 9.7, H C(6)); 3.46 (dd, J ˆ 9.2, 7.8, H C(2')); 3.52 (d, J ˆ 9.7, H' C(6)); 3.54 (dd, J ˆ 11.0, 5.0, H C(6'));
3.59 ± 3.61 (m, H C(3'), H C(4')); 3.65 (dd, J ˆ 11.0, 1.8, H C(6')); 3.79 (br. s, OH); 3.92 (d, J ˆ 6.3,
H
C(4)); 4.07 (d, J ˆ 6.7, H C(2)); 4.17 (t, J ꢁ 6.5, H C(3)); 4.32 (d, J ˆ 11.8), 4.39 (d, J ˆ 11.1), 4.40 (d, J ˆ
11.9), 4.42 (d, J ˆ 11.1, 4 PhCH); 4.43 (d, J ˆ 7.9, H C(1')); 4.55 (d, J ˆ 10.9), 4.62 (d, J ˆ 11.6), 4.66 (d, J ˆ
11.3), 4.70 (d, J ˆ 11.5, 4 PhCH); 4.80 (d, J ˆ 11.8, 2 PhCH); 4.84 (d, J ˆ 10.9), 4.85 (d, J ˆ 11.2), 4.91 (d, J ˆ
10.9), 4.97 (d, J ˆ 11.3, 4 PhCH); 6.05 (br. s, NH); 7.12 ± 7.41 (m, 35 H). 13C-NMR (CDCl3, 63 MHz): 68.89 (t,
C(6')); 72.47, 73.52, 73.59, 73.81, 74.09, 75.08, 75.20 (7t, C(6), 6 PhCH2); 75.39 (d, C(5')); 75.77 (t, PhCH2); 78.03
(d); 78.51 (2d); 78.89 (d); 82.28 (d); 82.73 (s, C(5)); 85.07 (d); 103.71 (d, C(1')); 127.77 ± 128.70 (several d);
‡
137.29, 137.70, 138.21, 138.27, 138.40 (5s); 138.56 (2s); 170.46 (s, C(1)). FAB-MS (Na): 1008 (35, [M ‡ Na] ),
‡
984 (55, [M 1] ), 860(69), 446(100).
Data of 13: Rf (hexane/AcOEt 4 :1) 0.38. IR (CH2Cl2): 3345w, 3070s, 2987s, 1697m, 1548w, 1497w, 1419s,
1
1360m, 1290s, 1152w, 1082m, 1028m, 896s. H-NMR (CDCl3, 500 MHz): 3.39 (t, J ꢁ 8.0, H C(2')); 3.40 ± 3.43
(m, H C(5')); 3.43 (d, J ˆ 9.1, H C(6)); 3.50 (d, J ˆ 9.1, H' C(6)); 3.56 ± 3.62 (m, H C(3'), H C(4'),
H
H
C(6')); 3.61 (dd, J ˆ 10.8, 4.6, H' C(6')); 3.72 (br. s, OH); 4.10 (d, J ˆ 2.2, H C(2)); 4.20 (dd, J ˆ 5.9, 2.8,
C(3)); 4.22 (s, PhCH2); 4.30 (d, J ˆ 5.9, H C(4)); 4.36 (d, J ˆ 11.1), 4.42 (d, J ˆ 12.3, 2 PhCH); 4.50 (d, J ˆ
7.8, H C(1')); 4.53 (d, J ˆ 10.9), 4.64 (d, J ˆ 11.8), 4.67 (d, J ˆ 11.5), 4.69 (d, J ˆ 11.4), 4.73 (d, J ˆ 11.7), 4.78 (d,
J ˆ 11.5), 4.80 (d, J ˆ 10.9), 4.83 (d, J ˆ 11.0), 4.91 (d, J ˆ 11.0), 5.02 (d, J ˆ 11.5, 10 PhCH); 6.14 (br. s, NH);
7.10 ± 7.45 (m, 35 H). 13C-NMR (CDCl3, 75 MHz): 68.95 (t, C(6')); 72.37, 73.21, 73.53, 73.57, 74.00, 75.09, 75.18
(7t, C(6), 6 PhCH2); 75.28 (d, C(5')); 75.79 (t, PhCH2); 77.91 (d); 78.09 (d); 78.40 (d); 82.26 (d); 82.43 (d); 83.90
(s, C(5)); 84.89 (d); 104.27 (d, C(1')); 127.77 ± 128.76 (several d); 137.29, 138.17 (2s); 138.26 (2s); 138.39, 138.57,
‡
138.63 (3s); 170.89 (s, C(1)). FAB-MS (Na): 1008 (22, [M ‡ Na] ), 860(50), 446(21), 181(23), 91(100).
5-Amino-2,3,6-tri-O-benzyl-5-deoxy-4-O-(2,3,4,6-tetra-O-benzyl-b-d-glucopyranosyl)-d-glucono-1,5-lactam
(14) and 5-Amino-2,3,6-tri-O-benzyl-5-deoxy-4-O-(2,3,4,6-tetra-O-benzyl-b-d-glucopyranosyl)-l-idono-1,5-lac-
tam (15). At 238, sodium cyanoborohydride (6.7 g, 107 mmol) was added to a soln. of crude 12/13 (21.3 g, ca.
22 mmol) in MeCN (310 ml) and HCO2H (80 ml). After 5 h at 358, the mixture was cooled to 58 and treated
slowly with a sat. aq. NaHCO3 soln. (300 ml). After separation, the aq. layer was extracted with CH2Cl2 (3 Â
200 ml). The combined org. layers were dried (MgSO4) and evaporated. FC (CH2Cl2/Et2O 95 : 5) and MPLC
(1 kg of silica gel, CH2Cl2/THF 97: 3, 10 ml/min) gave 14 (8.8 g, 38% from 9) and 15 (1.5 g, 7% from 9).
Data of 14: Colourless solid. Rf (hexane/AcOEt 3 :2) 0.29. M.p. 141 ± 1428 (EtOH). [a]2D5 ˆ ‡ 52.8 (c ˆ 0.5,
CHCl3). IR (CHCl3): 3392w, 3066w, 3007m, 2931w, 2867m, 1684s, 1497m, 1454s, 1361m, 1311w, 1070s, 1031m,
909w. 1H-NMR (CDCl3, 500 MHz): 3.35 (ddd, J ˆ 9.3, 4.5, 2.2, H C(5')); 3.41 ± 3.44 (m, H C(2'), H C(6));
3.56 ± 3.63 (m, H C(3'), H C(4'), H' C(6), 2 H C(6')); 3.80 (ddd, J ˆ 7.6, 3.4, 1.6, H C(5)); 3.87 (dd, J ˆ
8.0, 4.9, H C(4)); 3.95 (d, J ˆ 5.6, H C(2)); 4.08 (t, J ꢁ 5.5, H C(3)); 4.25 (d, J ˆ 11.8), 4.32 (d, J ˆ 11.7, 2

974
Helvetica Chimica Acta ± Vol. 82 (1999)
PhCH); 4.40 (d, J ˆ 7.8, H C(1')); 4.43 (s, PhCH2); 4.52 (d, J ˆ 10.9), 4.61 (d, J ˆ 11.7), 4.65 (d, J ˆ 11.4), 4.72
(d, J ˆ 11.7, 4 PhCH); 4.77 (s, PhCH2); 4.80 (d, J ˆ 10.9), 4.81 (d, J ˆ 11.0), 4.89 (d, J ˆ 11.5), 4.96 (d, J ˆ 10.9, 4
PhCH); 5.81 (br. s, NH); 7.16 ± 7.39 (m, 35 H). 13C-NMR (CDCl3, 75 MHz): 54.03 (d, C(5)); 68.85, 69.68 (2t,
C(6), C(6')); 73.20, 73.26, 73.39, 73.52 (4t, 4 PhCH2); 75.11 (2t, 2 PhCH2); 75.17 (d, C(5')); 75.84 (t, PhCH2);
77.87 (d); 78.29 (d); 79.07 (d); 81.30 (d); 82.41 (d); 84.95 (d); 104.12 (d, C(1')); 127.07 ± 128.72 (several d);
137.51, 137.89 (2s); 138.36 (2s); 138.49 (2s); 138.65 (s); 170.33 (s, C(1)). FAB-MS (3-NOBA): 1939 (18, [2 M ‡
‡
‡
H] ), 970 (80, [M ‡ H] ), 754(52), 448(50), 91(100). Anal. calc. for C61H63NO10 (970.17): C 75.52, H 6.55,
N 1.44; found: C 75.41, H 6.49, N 1.46.
Data of 15: Colourless solid. Rf (hexane/AcOEt 3 :2) 0.22. M.p. 114 ± 1158 (EtOH). IR (CHCl3): 3399w,
1
3007m, 2926m, 2867m, 1689s, 1469m, 1454s, 1360m, 1309w, 1070s, 1030m, 909w. H-NMR (CDCl3, 500 MHz):
3.40 (dd, J ˆ 9.1, 7.9, H C(2')); 3.40 ± 3.43 (m, H C(5')); 3.46 (t, J ꢁ 9.0, H C(6)); 3.53 (dd, J ˆ 9.0, 5.0,
H
C(6)); 3.55 (t, J ꢁ 9.5, H C(4')); 3.58 ± 3.64 (m, H C(3'), 2 H C(6')); 3.80 (ddd, J ˆ 9.1, 4.8, 2.2, H C(5));
3.96 (t, J ꢁ 2.1, H C(4)); 3.98 (d, J ˆ 5.2, H C(2)); 4.15 (d, J ˆ 12.0, PhCH); 4.16 (dd, J ˆ 5.3, 2.8, H C(3));
4.19 (d, J ˆ 11.8, PhCH); 4.37 (d, J ˆ 7.8, H C(1')); 4.41 (d, J ˆ 12.2), 4.44 (d, J ˆ 12.2), 4.53 (d, J ˆ 10.9), 4.58
(d, J ˆ 11.7), 4.63 (d, J ˆ 11.7), 4.71 (d, J ˆ 11.8), 4.77 (d, J ˆ 11.8), 4.79 (d, J ˆ 11.2), 4.80 (d, J ˆ 11.4), 4.81 (d,
J ˆ 10.9), 4.91 (d, J ˆ 11.0), 5.02 (d, J ˆ 11.8, 12 PhCH); 5.76 (br. s, NH); 7.17 ± 7.40 (m, 35 H). 13C-NMR
(CDCl3, 75 MHz): 52.10 (d, C(5)); 68.55, 69.21 (2t, C(6), C(6')); 72.79, 73.42 (2t, 2 PhCH2); 73.66 (t, 2 PhCH2);
75.25, 75.32 (2t, 2 PhCH2); 75.36 (d); 75.81 (d); 75.92 (t, PhCH2); 78.06 (d); 78.85 (d); 82.36 (2d); 84.94 (d);
104.48 (d, C(1')); 128.04 ± 128.81 (several d); 138.01, 138.38, 138.49, 138.60, 138.69, 138.81, 138.94 (7s); 171.78 (s,
‡
C(1)). FAB-MS (Na): 992 (2, [M ‡ Na] ), 448(20), 147(34), 91(100).
5-Amino-5-deoxy-4-O-(b-d-glucopyranosyl)-d-glucono-1,5-lactam (16). At 248, a soln. of 14 (0.30 g,
0.31 mmol) in THF/MeOH/H2O 3 :2 :1 (12 ml) and AcOH (3 ml) was treated with 20% Pd(OH)2/C (0.3 g) and
hydrogenated at 6 bar for 15 h. The suspension was filtered through Celite and the residue washed thoroughly
with MeOH and H2O. Evaporation of the combined filtrate and crystallization from H2O/EtOH 1:9 afforded 16
(58 mg, 69%). Colourless solid. Rf (AcOEt/MeOH/H2O 7:3 :2) 0.31. M.p. 125.3 ± 125.88 (H2O/EtOH 1:9).
[a]2D5 ˆ ‡ 16.1 (c ˆ 0.36, MeOH). IR (KBr): 3415s, 3340s, 3200s, 3030m, 2945m, 1662s, 1645s, 1540w, 1495m,
1385s, 1345s, 1320s, 1155s, 1075s, 1025s, 990m, 890m. 1H-NMR (D2O, 500 MHz): 3.29 (dd, J ˆ 9.3, 8.0, H C(2'));
3.37 (t, J ꢁ 9.2, H C(4')); 3.43 (ddd, J ˆ 9.7, 5.7, 2.2, H C(5')); 3.45 (t, J ꢁ 9.2, H C(3')); 3.50 (dt, J ˆ 8.2, 3.5,
H
C(5)); 3.69 (dd, J ˆ 12.4, 5.7, H C(6')); 3.75 ± 3.78 (AB, 2 H C(6)); 3.82 (t, J ꢁ 9.8, H C(3)); 3.86 (dd, J ˆ
12.4, 2.1, H' C(6')); 3.94 (dd, J ˆ 9.4, 8.4, H C(4)); 3.99 (d, J ˆ 9.9, H C(2)); 4.51 (d, J ˆ 7.8, H C(1')).
13C-NMR (D2O, 75 MHz): 58.95 (d, C(5)); 62.92, 63.45 (2t, C(6), C(6')); 72.32 (d); 73.03 (d); 74.98 (d); 76.07
‡
(d); 78.38 (d); 78.98 (d); 80.02 (d, C(4)); 105.74 (d, C(1')); 176.21 (s, C(1)). ESI-MS: 677 (60, [2M H] ),
428(8), 384(12), 374(18), 338(100, [M H] ). Anal. calc. for C12H21NO10 ´ H2O (411.35): C 40.34, H 6.49,
‡
N 3.91; found: C 40.55, H 6.48, N 3.93.
5-Amino-2,3,6-tri-O-benzyl-5-deoxy-4-O-(2,3,4,6-tetra-O-benzyl-b-d-glucopyranosyl)-1-thio-d-gluconolac-
tam (17). A mixture of 14 (1.40 g, 1.44 mmol) and Lawessonꢀs reagent (435 mg, 1.08 mmol) in toluene (25 ml)
was stirred at 408 for 2 h. Evaporation and FC (hexane/AcOEt 4 :1) gave 17 (1.24 g, 87%). Pale-yellow oil. Rf
(hexane/AcOEt 4 :1) 0.43. IR (CH2Cl2): 3367w, 3068s, 2987s, 1604w, 1550m, 1512m, 1497m, 1421s, 1362m, 1288s,
1153m, 1072m, 1028m, 988m, 896s. 1H-NMR (CDCl3, 500 MHz): 3.30 (ddd, J ˆ 9.7, 4.1, 1.9, H C(5')); 3.42 (dd,
J ˆ 9.0, 7.9, H C(2')); 3.47 (dd, J ˆ 9.9, 7.9, H-C(6)); 3.55 (dd, J ˆ 10.7, 2.1, H C(6')); 3.57 (t, J ꢁ 9.0,
H
C(3')); 3.61 (dd, J ˆ 10.2, 3.4, H' C(6)); 3.63 (dd, J ˆ 10.9, 4.1, H' C(6')); 3.64 (t, J ꢁ 9.2, H C(4')); 3.79
(dd, J ˆ 8.8, 2.2, H C(4)); 4.01 ± 4.05 (m, H C(5)); 4.19 (t, J ˆ 2.5, H C(3)); 4.24 (d, J ˆ 11.8), 4.31 (d, J ˆ
11.8, 2 PhCH); 4.37 (d, J ˆ 7.8, H-C(1')); 4.41 (d, J ˆ 12.1), 4.46 (d, J ˆ 12.1, 2 PhCH); 4.49 ± 4.51 (m, H C(2));
4.50 (d, J ˆ 11.7), 4.54 (d, J ˆ 10.9), 4.55 (d, J ˆ 11.8), 4.68 (d, J ˆ 11.7, 4 PhCH); 4.74 (s, PhCH2); 4.80 (d, J ˆ
10.9), 4.81 (d, J ˆ 10.9), 4.86 (d, J ˆ 11.7), 4.89 (d, J ˆ 11.0, 4 PhCH); 7.15 ± 7.39 (m, 35 H); 7.99 (br. s, NH).
13C-NMR (CDCl3, 75 MHz): 55.95 (d, C(5)); 68.45, 68.73 (2t, C(6), C(6')); 71.82, 72.21, 73.39, 73.58, 75.00 (5t, 5
PhCH2); 75.03 (d, C(5')); 75.10 (t, 2 PhCH2); 77.72 (d); 80.59 (d); 81.17 (d); 82.25 (d); 82.85 (d); 84.89 (d);
104.97 (d); 127.82 ± 18.88 (several d); 137.39, 137.84, 138.21 (3s); 138.39 (2s); 138.55, 138.74 (2s); 200.38 (s,
‡
C(1)). FAB-MS (3-NOBA): 1076(18), 986(100, M ), 878(15), 91(83).
(Z)-5-Amino-2,3,6-tri-O-benzyl-5-deoxy-4-O-(2,3,4,6-tetra-O-benzyl-b-d-glucopyranosyl)-d-gluconohydrox-
imo-1,5-lactam (18). A mixture of 17 (1.0 g, 1.0 mmol), NH2OH ´ HCl (175 mg, 2.53 mmol), and NaHCO3
(212 mg, 2.53 mmol) in MeOH (13 ml) was heated under reflux for 2.5 h. Filtration, evaporation, and FC
(hexane/AcOEt 4 : 1) afforded 18 (910 mg, 91%), which was further purified by HPLC (SiO2; hexane/Et2O 2 : 1,
10 ml/min). Colourless oil. Rf (hexane/AcOEt 4 : 1) 0.17. IR (CH2Cl2): 3588w, 3423w, 3068s, 2987s, 2867m,
1662w, 1550w, 1496w, 1421s, 1363m, 1288s, 1154m, 1071m, 1028m, 987w, 896s. 1H-NMR (CDCl3, 500 MHz): 3.31
(ddd, J ˆ 9.7, 4.0, 1.8, H C(5')); 3.43 (dd, J ˆ 9.1, 7.9, H C(2')); 3.55 (dd, J ˆ 10.8, 1.8, H C(6')); 3.56 (dd,

Helvetica Chimica Acta ± Vol. 82 (1999)
975
J ˆ 9.5, 6.7, H C(6)); 3.57 (t, J ꢁ 9.1, H C(3')); 3.64 (dd, J ˆ 10.7, 4.1, H' C(6')); 3.65 (t, J ꢁ 9.7, H C(4'));
3.68 (dd, J ˆ 9.5, 2.9, H' C(6)); 3.79 (dd, J ˆ 9.5, 2.2, H C(4)); 3.85 (m, H C(5)); 4.02 (br. s, H C(2)); 4.17 (t,
J ꢁ 2.5, H C(3)); 4.26 (d, J ˆ 12.0), 4.29 (d, J ˆ 11.9), 4.38 (d, J ˆ 12.1, 3 PhCH); 4.43 (d, J ˆ 7.8, H C(1')); 4.44
(d, J ˆ 11.1), 4.48 (d, J ˆ 11.9, 2 PhCH); 4.53 (d, J ˆ 11.6, 2 PhCH); 4.57 (d, J ˆ 12.3), 4.67 (d, J ˆ 11.9), 4.73 (d,
J ˆ 11.2), 4.79 (d, J ˆ 10.9, 4 PhCH); 4.80 (d, J ˆ 10.9, 2 PhCH); 4.89 (d, J ˆ 10.9, PhCH); 5.44 (s, NH); 6.52 (br.
s, OH); 7.11 ± 7.35 (m, 35 H). 13C-NMR (CDCl3, 75 MHz): 51.95 (d, C(5)); 68.08, 69.63 (2t, C(6), C(6')); 70.47,
71.96, 73.11, 73.58 (4t, 4 PhCH2); 74.52, 74.99 (2d, C(2), C(5')); 75.08, 75.12, 75.85 (3t, 3 PhCH2); 77.84 (d);
81.43 (d); 82.28 (d); 82.40 (d); 84.97 (d); 104.90 (d, C(1')); 127.71 ± 128.75 (several d); 138.10, 138.16, 138.25,
‡
138.51 (4s); 138.70 (2s); 138.89 (s); 149.71 (s, C(1)). FAB-MS (3-NOBA): 1970 (10, [2M ‡ H] ), 985 (100,
‡
[M ‡ H] ), 793(15), 91(67). Anal. calc. for C61H64N2O10 (985.12): C 74.37, H 6.55, N 2.84; found: C 74.35,
H 6.68, N 2.58.
(Z)-N1,2,3,6-Tetra-O-acetyl-5-amino-5-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-d-glucono-
hydroximo-1,5-lactam (19). At 708, a soln. of 18 (670 mg, 0.68 mmol) in THF (3.5 ml) was added dropwise to
a deep blue soln. of Li (0.17 g, 24.5 mmol) in condensed EtNH2 (ca. 20 ml). The mixture was stirred at 708 for
10 min and treated with NH4Cl (1.44 g). After evaporation, the residue was dried, dissolved in pyridine (10 ml),
treated with Ac2O (5 ml) at 158, and stirred for 4 h at 08. After evaporation, the residue was dissolved in
CH2Cl2 (25 ml), washed with brine (2 Â 25 ml), and dried (MgSO4). Evaporation and FC (CH2Cl2/acetone
17: 3) afforded 19 (590 mg, 76%). Colourless oil. Rf (hexane/AcOEt 17: 3) 0.24. IR (CHCl3): 3400w, 3008w,
1756s, 1656m, 1434w, 1368m, 1066m, 1042m, 841w, 818w. 1H-NMR (CDCl3, 500 MHz): 2.01, 2.02, 2.06, 2.07, 2.09,
2.10, 2.16, 2.19 (8s, 8 AcO); 3.64 (dt, J ˆ 8.8, 1.5, H C(4)); 3.75 (dddd, J ˆ 8.7, 6.0, 2.3, 0.9, H C(5)); 3.79 (ddd,
J ˆ 10.1, 4.5, 2.4, H C(5')); 4.04 (dd, J ˆ 12.0, 6.0, H C(6)); 4.08 (dd, J ˆ 12.4, 2.4, H C(6')); 4.27 (dd, J ˆ 12.4,
4.5, H' C(6')); 4.47 (dd, J ˆ 12.0, 2.5, H' C(6)); 4.79 (d, J ˆ 8.1, H C(1')); 4.96 (dd, J ˆ 9.6, 8.1, H C(2'));
5.07 (t, J ꢁ 10.1, H C(4')); 5.20 (t, J ꢁ 9.5, H C(3')); 5.38 (dt, J ˆ 3.8, 1.3, H C(2)); 5.65 (dd, J ˆ 3.7, 1.8,
H
C(3)); 5.79 (br. s, NH). 13C-NMR (CDCl3, 50 MHz): 19.63 (q, Me); 20.55 (q, 4 Me); 20.61, 20.77, 20.81 (3q,
3 Me); 53.01 (d, C(5)); 61.75, 62.96 (2t, C(6), C(6')); 67.48 (d); 68.08 (d); 69.63 (d); 71.57 (d); 72.14 (d); 72.92
(d); 79.69 (d, C(4)); 101.13 (d, C(1')); 151.36 (s, C(1)); 168.62, 169.52 (2s, 2 CˆO); 169.61 (s, 2 CˆO); 169.69,
‡
170.48, 170.91, 171.78 (4s, 4 CˆO). FAB-MS (3-NOBA): 691 (100, [M ‡ H] ), 649(25).
(Z)-4-O-(b-d-Glucopyranosyl)-d-gluconohydroximo-1,5-lactam (20). At 08,
a soln. of 19 (150 mg,
0.22 mmol) in MeOH (3.1 ml) was treated dropwise with a 1m soln. of MeONa in MeOH. After 6 h, the
‡
mixture was stirred with Amberlite IRC-50 (H form), filtered, and evaporated. Reversed-phase HPLC (RP18
silica gel, H2O/MeCN 100 :0 ! 10 : 90) and lyophilization afforded 20 (51 mg, 66%). Colourless solid. Rf
(AcOEt/MeOH/H2O 7: 3 : 2) 0.23. [a]2D5 ˆ ‡ 32.8 (c ˆ 0.36, MeOH). IR (KBr): 3390s, 3150s, 2980m, 2890w,
1
1655s, 1565m, 1365m, 1115s, 1020s, 955m. H-NMR (D2O, 500 MHz): 3.28 (dd, J ˆ 9.3, 8.0, H C(2')); 3.37 (t,
J ꢁ 9.2, H C(4')); 3.39 ± 3.45 (m, H C(5), H C(5')); 3.45 (t, J ꢁ 9.1, H C(3')); 3.69 (dd, J ˆ 12.4, 5.7,
H
C(6')); 3.74 (dd, J ˆ 12.0, 4.6, H C(6)); 3.79 ± 3.85 (m, H C(3), H C(4), H' C(6)); 3.86 (dd, J ˆ 12.4,
2.2, H' C(6')); 4.15 (d, J ˆ 8.0, H C(2)); 4.51 (d, J ˆ 7.9, H C(1')). 13C-NMR (D2O, 50 MHz): 58.66 (d,
C(5)); 63.48 (t, C(6), C(6')); 71.25 (d); 72.38 (d); 76.10 (d); 76.38 (d); 78.41 (d); 78.93 (d); 81.57 (d, C(4));
‡
105.90 (d, C(1')); 156.96 (s, C(1)). ESI-MS: 355 (100, [M ‡ H] ), 193(82). Anal. calc. for C12H22N2O10 ´ H2O
(372.31): C 38.71, H 6.49, N 7.52; found: C 38.75, H 6.36, N 7.43.
(Z)-2,3,6-Tri-O-acetyl-5-amino-5-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-d-gluconohydrox-
imo-1,5-lactam (21). At 228, a soln. of 19 (100 mg, 0.144 mmol) and hydrazine acetate (30 mg, 0.32 mmol) in
DMF (3 ml) was stirred for 30 min. After addition of AcOEt (20 ml) and washing with 0.1m aq. soln. of HCl, the
org. layer was dried (MgSO4) and evaporated. Drying at 0.01 mbar afforded 21 as a colourless oil, which was
used without further purification for the next step. Rf (hexane/AcOEt 4 :1) 0.43. IR (CHCl3): 3591w, 3402w,
1
3007w, 2928w, 1754s, 1671w, 1430w, 1370m, 1066m, 1041s, 908w. H-NMR (CDCl3, 300 MHz): 1.99, 2.01, 2.06,
2.07, 2.08, 2.09, 2.10, 2.13 (7s, 7 AcO); 3.66 ± 3.68 (m, H C(4), H C(5)); 3.76 (ddd, J ˆ 9.7, 4.1, 2.2, H C(5'));
3.98 (dd, J ˆ 11.2, 5.6, H C(6)); 4.07 (dd, J ˆ 12.5, 2.2, H C(6')); 4.28 (dd, J ˆ 12.5, 4.7, H' C(6')); 4.48 (dd,
J ˆ 11.0, 2.2, H' C(6)); 4.73 (d, J ˆ 8.1, H C(1')); 4.97 (dd, J ˆ 9.7, 8.1, H C(2')); 5.08 (t, J ꢁ 9.7, H C(4'));
5.19 (t, J ꢁ 9.7, H C(3')); 5.30 (br. d, J ˆ 4.4, H C(2)); 5.46 (br. s, NH); 5.50 (dd, J ˆ 4.4, 2.5, H C(3)); 7.08
(br. s, OH). 13C-NMR (CDCl3, 53 MHz): 20.61 (q, 3 Me); 20.70 (q, Me); 20.76 (q, 3 Me); 52.57 (d, C(5)); 61.74,
63.30 (2t, C(6), C(6')); 67.55 (d); 68.03 (d); 70.85 (d); 71.55 (d); 72.09 (d); 72.98 (d); 79.83 (d, C(4)); 101.33 (d,
C(1')); 147.99 (s, C(1)); 169.60 (s, 2 CˆO); 169.66 (s, 2 CˆO); 170.51, 170.90, 171.16 (3s, 3 CˆO). FAB-MS
‡
‡
(3-NOBA): 1297 (3, [2M ‡ H] ), 649 (100, [M ‡ H] ), 607(25).
(Z)-O-[2,3,6-Tri-O-acetyl-5-amino-5-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-d-glucopyra-
nosylidene]amino N-Phenylcarbamate (22). At 238, a soln. of crude 21 (68 mg, 0.104 mmol) in THF (1 ml) was
treated with Et3N (43 ml, 0.312 mmol) and PhNCO (12.5 ml, 0.156 mmol) and was stirred for 30 min.

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Helvetica Chimica Acta ± Vol. 82 (1999)
Evaporation and FC (CH2Cl2/acetone 9 :1) afforded 22 (80.5 mg, 59%). Colourless foam. Rf (hexane/AcOEt
1:2) 0.45. IR (CHCl3): 3448w, 3372w, 3007w, 1754s, 1669m, 1595w, 1521m, 1446w, 1434w, 1370m, 1067m, 1040m,
907w. 1H-NMR (CDCl3, 300 MHz): 1.99, 2.02, 2.07, 2.08, 2.10, 2.13, 2.14 (7s, 7 Me); 3.65 (br. d, J ˆ 8.7, H C(4));
3.75 ± 3.80 (m, H C(5), H C(5')); 3.99 (dd, J ˆ 11.8, 6.5, H C(6)); 4.09 (dd, J ˆ 12.5, 2.5, H C(6')); 4.26 (dd,
J ˆ 12.5, 4.4, H' C(6')); 4.39 (dd, J ˆ 11.8, 2.5, H' C(6)); 4.75 (d, J ˆ 8.1, H C(1')); 4.97 (dd, J ˆ 9.3, 8.1,
H
H
C(2')); 5.08 (t, J ꢁ 9.3, H C(4')); 5.19 (t, J ꢁ 9.3, H C(3')); 5.37 (d, J ˆ 3.7, H C(2)); 5.59 (dd, J ˆ 3.7, 1.9,
C(3)); 5.84 (br. s, NH); 7.11 (t, J ꢁ 7.5, 1 arom. H); 7.33 (t, J ꢁ 7.5, 2 arom. H); 7.48 (d, J ˆ 7.8, 2 arom. H);
8.41 (br. s, NHCO). 13C-NMR (CDCl3, 50 MHz): 20.47 (q, 3 Me); 20.59 (q, 2 Me); 20.69 (q, 2 Me); 52.24 (d,
C(5)); 62.63, 62.87 (2t, C(6), C(6')); 66.87 (d); 67.89 (d); 70.05 (d); 71.23 (d); 71.98 (d); 72.71 (d); 79.63 (d,
C(4)); 101.31 (d, C(1')); 119.66 (d, 2 C); 124.29 (d); 129.15 (d, 2 C); 137.06 (s); 149.49 (s, C(1)); 152.07 (s,
NCˆO); 168.73, 169.35 (2s, 2 CˆO); 169.53 (s, 2 CˆO); 170.29 (s, CˆO); 170.73 (s, 2 CˆO). FAB-MS (3-
‡
NOBA): 768 (65, [M ‡ H] ), 73(100).
(Z)-O-[5-Amino-5-deoxy-4-O-(b-d-glucopyranosyl)-d-glucopyranosylidene]amino N-Phenylcarbamate (23).
At 238, a soln. of 22 (86.7 mg, 0.113 mmol) in MeOH (4 ml) was treated with a sat. soln. of NH3 in MeOH
(0.4 ml) and stirred for 2 h. Evaporation at 288, FC (AcOEt/MeOH/H2O 9 :1:1), and lyophilization afforded 23
(34.2 mg, 64%). Colourless solid. Rf (AcOEt/MeOH/H2O 7:3 :2) 0.41. [a]D25
ˆ
11.2 (c ˆ 0.17, MeOH). IR
1
(KBr): 3480s, 3290s, 1720s, 1650s, 1610s, 1520m, 1450w, 1225m, 1120m, 1090m, 1035m, 960m. H-NMR (D2O,
500 MHz): 3.28 (dd, J ˆ 9.3, 8.0, H C(2')); 3.36 (t, J ꢁ 9.2, H C(4')); 3.43 (ddd, J ˆ 9.7, 5.7, 2.3, H C(5')); 3.45
(t, J ˆ 9.2, H C(3')); 3.50 (dt, J ꢁ 7.5, 4.1, H C(5)); 3.68 (dd, J ˆ 12.3, 5.8, H C(6')); 3.74 (dd, J ˆ 12.0, 4.5,
H
C(6)); 3.82 (dd, J ˆ 12.0, 3.3, H' C(6)); 3.84 (t, J ꢁ 7.8, H C(4)); 3.86 (dd, J ˆ 12.3, 2.2, H' C(6')); 3.93 (t,
J ꢁ 8.2, H C(3)); 4.20 (d, J ˆ 8.3, H C(2)); 4.50 (d, J ˆ 7.9, H C(1')); 7.16 ± 7.20 (m, 1 arom. H); 7.37 ± 7.38
(m, 4 arom. H). 13C-NMR (D2O, 75 MHz): 56.21(d, C(5)); 60.56, 60.72 (2t, C(6), C(6')); 68.43 (d); 69.61 (d);
73.07 (d); 73.29 (d); 75.64 (d); 76.18 (d); 78.53 (d, C(4)); 103.27 (d, C(1')); 121.54 (d, 2 C); 125.31 (d); 129.50 (d,
‡
‡
2 C); 136.84 (s); 155.69, 157.02 (2s, C(1), NCˆO). ESI-MS: 496(80), 475(23, [M ‡ H] ), 474(100, M ). Anal.
calc. for C19H27N3O11 ´ 1.5 H2O (500.45): C 45.60, H 5.43, N 8.39; found: C 45.29, H 5.43, N 8.05.
Methyl (2,3,4,6-Tetra-O-benzyl-b-d-glucopyranosyl)-(1 ! 4)-{[(Z)-5-amino-2,3,6-tri-O-benzyl-5-deoxy-d-
glucopyranosylidene]amino}-(1-N ! 4)-2,3,6-tri-O-benzyl-b-d-glucopyranoside (25). At 228, a soln. of 17
(0.40 g, 0.41 mmol) and 24 [10] (0.29 g, 0.62 mmol) in THF (15 ml, freshly distilled) was treated with (i-Pr)2EtN
(1.0 ml, 5.8 mmol) and Hg(OAc)2 (0.30 g, 0.94 mmol) and stirred for 8 h at 228. After filtration through Celite
and evaporation, the residue was dissolved in CH2Cl2 (25 ml) and washed with sat. aq. NaHCO3 soln. (2 Â
25 ml). Drying of the org. phase (MgSO4), evaporation, and FC (hexane/AcOEt 9 : 1) afforded 25 (0.43 g),
which was sufficiently pure to be used for the next step. Colourless oil. Rf (hexane/AcOEt 4 : 1) 0.25. IR
(CH2Cl2): 3400w, 3068s, 2986s, 2914m, 2868m, 1726w, 1653m, 1497m, 1454s, 1422s, 1361m, 1288s, 1212m, 1062s,
1028s, 896s. 1H-NMR (CDCl3, 200 MHz): 3.31 ± 3.36 (m, 1 H); 3.41 ± 3.56 (m, 4 H); 3.61 ± 3.77 (m, 6 H); 3.64 (s,
MeO); 3.88 ± 3.94 (m, 2 H); 4.03 ± 4.07 (m, 4 H); 4.21 (t, J ꢁ 3.0, H C(2')); 4.27 (d, J ˆ 12.0), 4.35 (d, J ˆ 12.1,
2 PhCH); 4.36 (d, J ˆ 8.8, H C(1)); 4.39 ± 4.44 (m, 4 PhCH); 4.50 ± 4.61 (m, H C(1''), 7 PhCH); 4.70 (d,
J ˆ 12.0), 4.75 (d, J ˆ 11.2), 4.77 (d, J ˆ 12.0), 4.85 (d, J ˆ 12.0), 4.86 (d, J ˆ 11.5), 4.93 (d, J ˆ 11.2), 4.95 (d,
J ˆ 10.8, 7 PhCH); 5.44 (br. s, NH); 7.15 ± 7.39 (m, 50 H). 13C-NMR (CDCl3, 50 MHz): 51.76 (d); 57.06 (q);
71.10 (t); 71.75 (t, 2 C); 72.96 (t, 2 C); 73.34 (t, 2 C); 73.50 (t, 3 C); 73.66 (d); 74.04 (d); 74.90 (t); 75.18 (t); 75.69
(t); 77.63 (d, 2C); 80.51 (d); 81.42 (d); 82.17 (d, 2 C); 82.78 (d); 83.36 (d); 84.77 (d); 104.67, 104.89 (2d, C(1),
C(1'')); 127.30 ± 128.64 (several d); 137.84 ± 139.33 (several s); 148.85 (s, C(1')). FAB-MS (3-NOBA): 1522(8),
‡
1432(100, [M ‡ H] ), 1400(23), 1340(19), 1324(10). Anal. calc. for C89H94N2O15 (1431.73): C 74.66, 6.62,
N 1.96; found: C 74.49, H 6.72, N 2.12.
Methyl (2,3,4,6-Tetra-O-acetyl-b-d-glucopyranosyl)-(1 ! 4)-{[(Z)-2,3,6-tri-O-acetyl-5-amino-5-deoxy-d-
glucopyranosylidene]amino}-(1-N ! 4)-2,3,6-tri-O-acetyl-b-d-glucopyranoside (26). At
608, a soln. of 25
(189 mg, 0.13 mmol) in THF (2 ml) was added to a deep blue soln. of Li (0.10 g, 14.3 mmol) in condensed
EtNH2 (ca. 10 ml) within 2 min. The mixture was stirred at 608 for 10 min and treated with NH4Cl (0.10 g).
After evaporation, the residue was dried, dissolved in pyridine (10 ml), treated with Ac2O (5 ml) at 08, and
stirred for 1 h at 208. After evaporation, the residue was dissolved in CH2Cl2 (25 ml) and washed with sat. aq.
NaHCO3 soln. (2 Â 25 ml). Drying of the org. phase (MgSO4), evaporation, and FC (hexane/AcOEt 1 : 2) gave,
after crystallization from Et2O, impure 26 (105 mg, 85%). HPLC (hexane/AcOEt 2 : 3) afforded pure 26 (0.39 g,
69%). Colourless solid. Rf (hexane/AcOEt 1 : 2) 0.22. M.p. 188 ± 1898 (Et2O/hexane). [a]D25
ˆ
50.1 (c ˆ 0.27,
CHCl3). IR (CH2Cl2): 3400w, 3068s, 2987s, 1755s, 1659w, 1551w, 1422s, 1369m, 1237s, 1161m, 1040m, 987w, 896s.
1H-NMR (CDCl3, 500 MHz): 2.00, 2.01, 2.02, 2.05, 2.06, 2.07, 2.08, 2.09, 2.10, 2.15 (10s, 10 AcO); 3.49 (s, MeO);
3.60 (ddd, J ˆ 10.5, 2.5, 1.6, H C(4')); 3.63 ± 3.67 (m, H C(5')); 3.77 (ddd, J ˆ 10.1, 4.5, 2.4, H C(5'')); 3.85
(ddd, J ˆ 10.0, 5.3, 2.2, H C(5)); 3.96 (dd, J ˆ 11.7, 6.5, H C(6')); 4.07 (dd, J ˆ 12.4, 2.3, H C(6'')); 4.08 (t,

Helvetica Chimica Acta ± Vol. 82 (1999)
977
J ꢁ 9.7, H C(4)); 4.25 (dd, J ˆ 12.3, 5.3, H C(6)); 4.28 (dd, J ˆ 12.2, 4.5, H' C(6'')); 4.35 (dd, J ˆ 12.0, 2.1,
H' C(6)); 4.40 (d, J ˆ 8.0, H C(1)); 4.40 (dd, J ˆ 11.5, 2.5, H' C(6')); 4.73 (d, J ˆ 8.1, H C(1'')); 4.94 (dd, J ˆ
8.0, 7.4); 4.96 (dd, J ˆ 8.1, 7.5, H C(2), H C(2'')); 5.07 (t, J ꢁ 9.5, H C(4'')); 5.19 (t, J ꢁ 9.6, H C(3'')); 5.20
(dt, J ꢁ 4.4, 1.2, H C(3')); 5.31 (br. s, NH); 5.36 (t, J ꢁ 9.5, H C(3)); 5.49 (dd, J ˆ 4.3, 2.6, H C(2')). 13C-NMR
(CDCl3, 50 MHz): 21.06 ± 21.38 (several q, Me); 53.39 (d, C(5')); 57.53 (q, MeO); 62.26, 63.34, 63.78 (3t, C(6),
C(6'), C(6'')); 67.88 (d); 68.54 (d); 70.67 (d); 72.00 (d); 72.39 (d); 72.48 (d); 72.58 (d); 72.73 (d); 73.43 (d); 78.73,
80.35 (2d, C(4), C(4')); 101.75, 102.22 (2d, C(1), C(1'')); 148.47 (s, C(1')); 169.36, 170.05, 170.12, 170.21, 170.34,
‡
170.78, 170.94, 171.36, 171.55, 171.77 (10s, 10 CˆO). FAB-MS (3-NOBA): 952 (100, [M ‡ H] ), 169(30),
109(38). Anal. calc. for C39H54N2O25 (950.85): C 49.26, H 5.72, N 2.95; found: C 49.19, H 5.78, N 3.01.
Methyl b-d-Glucopyranosyl-(1 ! 4)-{[(Z)-5-amino-5-deoxy-d-glucopyranosylidene]amino}-(1-N ! 4)-b-
d-glucopyranoside (27). At 58, a soln. of 26 (90 mg, 0.1 mmol) in MeOH (5 ml) was treated dropwise with a
freshly prepared 0.5n soln. of NaOMe in MeOH (ca. 0.1 ml). After 1 h, the mixture was treated with Amberlite
‡
IR-120 (H form), stirred for 15 min, and filtered. After evaporation, lyophilization gave 27 (46 mg, 91%).
Colourless solid. Rf (AcOEt/MeOH/H2O 4 : 2 : 1) 0.10. IR (KBr): 3425s, 3340s, 2990m, 2945m, 1662s, 1643s,
1
1455m, 1335s, 1090m, 1078s, 970s, 935m. H-NMR (CD3OD, 500 MHz): 3.21 ± 3.24 (m, H C(5')); 3.26 (t, J ꢁ
8.7), 3.30 (t, J ꢁ 8.8, H C(2), H C(2'')); 3.32 ± 3.36 (m), 3.36 (t, J ꢁ 8.8), 3.40 ± 3.44 (m, H C(3''), H C(4''),
H
H
C(5'')); 3.53 (s, MeO); 3.55 ± 3.58 (m, H C(5)); 3.63 (dd, J ˆ 9.2, 6.8, H C(4')); 3.64 (dd, J ˆ 11.8, 6.2,
C(6')); 3.71 (dd, J ˆ 12.3, 5.0, H C(6)); 3.73 ± 3.79 (m, H C(3), H C(4), H C(6'')); 3.82 (dd, J ˆ 12.3,
3.1, H' C(6)); 3.83 (t, J ꢁ 6.8, H C(3')); 3.88 (dd, J ˆ 11.5, 3.1, H' C(6'')); 3.89 (dd, J ˆ 11.8, 2.3, H' C(6'));
4.04 (d, J ˆ 6.8, H C(2')); 4.18 (d, J ˆ 7.8, H C(1)); 4.40 (d, J ˆ 7.8, H C(1'')). 13C-NMR (CD3OD, 50 MHz):
56.87 (d, C(5')); 57.32 (q, MeO); 62.52, 62.58, 62.71 (3t, C(6), C(6'), C(6'')); 70.84 (d); 71.63 (d); 75.06 (d, 2 C);
75.47 (d); 76.01 (d); 76.17 (d); 77.95 (d); 78.17 (d); 81.50, 81.85 (2d, C(4), C(4')); 105.18, 106.40 (2d, C(1),
‡
C(1'')); 154.60 (s, C(1')). FAB-MS (3-NOBA): 1084(3), 553(55), 531(55, [M ‡ H] ), 383(56), 307(100),
154(85).
Transformation of 17 into 28/29. A soln. of 17 (1.0 g, 1.01 mmol) in THF (6 ml, freshly distilled) was treated
with aminoacetaldehyde dimethyl acetal (570 ml, 5.23 mmol) and Hg(OAc)2 (450 mg, 1.37 mmol) and kept at 08
for 2 h. Normal workup and drying gave a crude product which was dissolved in toluene (36 ml), treated with
TsOH ´ H2O (250 mg, 1.3 mmol), and stirred overnight at 608. Normal workup and FC (hexane/AcOEt/Et3N
4 : 1 : 0.03) gave 28/29 4 : 1 (692 mg, 69%) which could not be separated by HPLC.
Iodination of 28/29. A soln. of 28/29 (350 mg, 0.353 mmol) in DMF (3.5 ml), was treated with N-
iodosuccinimide (793 mg, 3.53 mmol) and stirred at 708 for 24 h. The mixture was cooled, diluted with Et2O, and
washed with a sat. NH4Cl soln. The aq. phase was extracted several times with Et2O. The combined org. layers
were dried (MgSO4) and evaporated. FC (hexane/AcOEt 4 :1) gave 31 (133 mg, 30%), 30/31 (22 mg, 5%), and
30 (208 mg, 47%).
(5R,6R,7S,8S)-7,8-Bis(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydro-2,3-diiodo-6-(2,3,4,6-tetra-O-
benzyl-b-d-glucopyranosyloxy)imidazo[1,2-a]pyridine (30). Rf (hexane/AcOEt 4 :1) 0.29. IR (CHCl3): 3067w,
3007w, 2868m, 1610w, 1595w, 1497m, 1453m, 1432w, 1392w, 1361m, 1309w, 1261m, 1175s, 1070s, 1028m, 912w.
1H-NMR (500 MHz, CDCl3, assignment based on a DQFCOSY.GRASP spectrum): 3.33 (dd, J ˆ 8.8, 7.9,
H
C(2')); 3.45 (ddd, J ˆ 9.5, 4.0, 2.3, H C(5')); 3.57 (t, J ˆ 9.2, H C(4')); 3.62 (t, J ˆ 8.9, H C(3')); 3.64 ± 3.70
(m, 2 H C(6'), CH2 C(5)); 4.33 (d, J ꢁ 11.0, PhCH); 4.34 ± 4.35 (m, H C(7)); 4.41 (d, J ˆ 12.0, PhCH); 4.44
(d, J ˆ 11.8, PhCH); 4.47 (d, J ˆ 12.0, PhCH); 4.49 ± 4.56 (m, 4 PhCH, H C(5), H C(1')); 4.57 (d, J ˆ 11.7,
PhCH); 4.66 (d, J ˆ 2.6, H C(8)); 4.72 ± 4.77 (m, H C(6), 2 PhCH); 4.80 (d, J ˆ 10.8, PhCH); 4.89 (d, J ˆ 10.8,
PhCH); 4.91 (d, J ˆ 11.0, PhCH); 6.99 ± 7.01 (m, 2 arom. H); 7.15 ± 7.34 ( m, 33 arom. H). 13C-NMR (50.6 MHz,
CDCl3): 60.06 (d, C(5)); 68.95, 69.14 (2t, CH2 C(5), C(6')); 71.78, 71.90 (2d); 72.03, 72.60, 73.24, 73.52, 74.69,
75.01 (6t, 6 PhCH2); 75.01 (d, C(5')); 75.65 (t, PhCH2); 77.78 (d, C(4')); 79.04 (d); 81.55 (d, C(2')); 81.55 (s,
C(2)); 84.50 (d, C(3')); 96.16 (s, C(3)); 102.86 (d, C(1')); 127.64 ± 128.59 (several d); 137.58 (2s); 138.12 (s);
138.18 (2s); 138.24, 136.68 (2s); 148.91 (s, C(8a)).
(5R,6R,7S,8R)-7,8-Bis(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydro-2,3-diiodo-6-(2,3,4,6-tetra-O-
benzyl-b-d-glucopyranosyloxy)imidazo[1,2-a]pyridine (31). Rf (hexane/AcOEt 4 :1) 0.34. IR (CHCl3): 3066w,
3007m, 2867m, 1605w, 1497m, 1454m, 1435w, 1394w, 1361m, 1309w, 1262w, 1175s, 1070s, 1028m, 912w. 1H-NMR
(500 MHz, CDCl3, assignment based on a DQFCOSY.GRASP spectrum): 3.18 (dt, J ˆ 9.7, 2.5, H C(5')); 3.37
(dd, J ˆ 9.0, 7.9, H C(2')); 3.55 (dd, J ˆ 9.0, 5.1, CH C(5)); 3.59 (t, J ˆ 9.1, H C(3')); 3.61 ± 3.66 (m,
CH' C(5), 2 H C(6')); 3.66 (t, J ˆ 9.3, H C(4')); 3.75 (dd, J ˆ 6.5, 3.3, H C(7)); 4.35 (ddd, J ˆ 9.2, 5.1, 1.0,
H
C(5)); 4.39 (d, J ˆ 11.9, PhCH); 4.43 (d, J ˆ 11.9, PhCH); 4.50 (d, J ˆ 12.2, PhCH); 4.545 (d, J ˆ 12.0,
PhCH); 4.550 (d, J ˆ 12.2, PhCH); 4.58 (d, J ˆ 12.0, PhCH); 4.647 (s, PhCH2); 4.650 (d, J ˆ 12.1, PhCH); 4.654
(d, J ꢁ 12.2, PhCH); 4.660 (d, J ꢁ 8.0, H C(1')); 4.69 (d, J ˆ 11.4, PhCH); 4.73 (d, J ˆ 3.3, H C(8)); 4.74 (dd,

978
Helvetica Chimica Acta ± Vol. 82 (1999)
J ˆ 6.5, 1.1, H C(6)); 4.800 (d, J ˆ 10.9, PhCH); 4.805 (d, J ˆ 11.0, PhCH); 4.89 (d, J ˆ 10.9, PhCH); 7.16 ± 7.33
(m, 35 arom. H). 13C-NMR (50.6 MHz, CDCl3): 60.23 (d, C(5)); 69.21, 69.41 (2t, CH2 C(5), C(6')); 72.03,
72.16 (2d); 72.25, 72.84, 73.47, 73.74, 74.91, 75.21 (6t, 6 PhCH2); 75.28 (d, C(5')); 75.85 (t, PhCH2); 77.93 (d,
C(4')); 79.34 (d); 81.78 (d, C(2')); 81.78 (s, C(2)); 84.76 (d, C(3')); 96.66 (s, C(3)); 102.62 (d, C(1')); 127.84 ±
128.83 (several d); 138.86 (2s); 138.39 (s); 138.45 (2s); 138.52, 138.97 (2s); 149.18 (s, C(8a)).
Deiodination of 30. A soln. of 30 (130 mg, 0.106 mmol) in THF (2 ml) was treated at 08 with 1m EtMgBr in
Et2O (170 ml, 0.17 mmol), stirred for 15 min, and treated with sat. aq. NH4Cl soln. Extraction with CH2Cl2,
drying of the combined org. layers (MgSO4), and evaporation gave crude 33 (115 mg) which was completely
deiodinated by the repetition of this procedure. FC (hexane/AcOEt 7:3) gave 28 (80 mg, 77%).
(5R,6R,7S,8S)-7,8-Bis(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydro-2-iodo-6-(2,3,4,6-tetra-O-ben-
zyl-b-d-glucopyranosyloxy)imidazo[1,2-a]pyridine (33). Rf (hexane/AcOEt 7 :3) 0.22. 13C-NMR (50.6 MHz,
CDCl3): 58.09 (d, C(5)); 69.07, 69.83 (2t, CH2 C(5), C(6')); 71.27 (d); 71.85, 72.83, 73.48, 73.71 (4t, 4 PhCH2);
74.55 (d); 75.08, 75.23 (2t, 2 PhCH2); 75.32 (d, C(5')); 75.89 (t, PhCH2); 77.95 (d, C(4')); 79.16 (d); 82.13 (d,
C(2')); 82.20 (s, C(2)); 84.87 (d, C(3')); 102.63 (d, C(1')); 124.48 (d, C(3)); 127.82 ± 128.91 (several d); 137.58,
‡
137.93, 138.34, 138.42 (4s); 138.47 (2s); 138.88 (s); 145.17 (s, C(8a)). FAB-MS: 1119 ([M ‡ 1] ).
(5R,6R,7S,8S)-7,8-Bis(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydro-6-(2,3,4,6-tetra-O-benzyl-b-d-
glucopyranosyloxy)imidazo[1,2-a]pyridine (28). Rf (hexane/AcOEt/Et3N 1:1:0.03) 0.29. IR (CHCl3): 3066w,
1
3007m, 2961w, 2868m, 1495m, 1454m, 1362m, 1312w, 1262m, 1173s, 1070s, 1028m, 909w. H-NMR (500 MHz,
CDCl3; assignment based on a DQFCOSY.GRASP spectrum): 3.37 ± 3.40 (m, H C(2'), H C(5')); 3.56 ± 3.62
(m, 2 H C(6')); 3.62 ± 3.65 (m, H C(3'), H C(4')); 3.75 (d, J ˆ 5.6, CH2 C(5)); 4.318 (t, J ꢁ 3.5, H C(7));
4.337 (t, J ˆ 4.2, H C(6)); 4.38 (d, J ˆ 11.8, PhCH); 4.39 ± 4.43 (m, H C(5)); 4.41 (d, J ˆ 11.9, PhCH); 4.465 (d,
J ˆ 12.0, PhCH); 4.488 (d, J ꢁ 11.0, PhCH); 4.492 (d, J ꢁ 7.7, H C(1')); 4.51 (d, J ꢁ 12.1, PhCH); 4.54 (d, J ˆ
10.9, PhCH); 4.59 (s, PhCH2); 4.60 (d, J ꢁ 11.6, PhCH); 4.73 (d, J ˆ 2.8, H C(8)); 4.73 (d, J ˆ 11.7, PhCH); 4.78
(d, J ˆ 11.0, PhCH); 4.81 (d, J ˆ 10.9, PhCH); 4.84 (d, J ˆ 11.8, PhCH); 4.89 (d, J ˆ 11.0, PhCH); 6.96 (d, J ˆ
1.0), 7.10 (d, J ˆ 1.0, H C(2), H C(3)); 7.03 ± 7.06 (m, 2 arom. H); 7.15 ± 7.34 (m, 33 arom. H). 13C-NMR
(75.9 MHz, CDCl3): 57.90 (d, C(5)); 69.02, 69.87 (2t, CH2 C(5), C(6')); 71.65 (t, PhCH2); 71.70 (d); 72.75,
73.38, 73.63 (3t, 3 PhCH2); 74.91 (d); 74.94, 75.13 (2t, 2 PhCH2); 75.21 (d, C(5')); 75.78 (t, PhCH2); 77.88 (d,
C(4')); 79.73 (d); 82.06 (d, C(2')); 84.82 (d, C(3')); 102.86 (d, C(1')); 118.54 (d); 127.64 ± 128.74 (several d);
‡
128.97 (d); 137.69 (s); 138.41 (2s); 138.49 (2s); 138.52, 138.99 (2s); 143.03 (s, C(8a)). FAB-MS: 993 ([M ‡ 1] ).
Deiodination of 31. A soln. of 31 (208 mg, 0.167 mmol) in THF (2.8 ml) was treated at 08 with 1m EtMgBr
in Et2O (250 ml, 0.25 mmol), stirred for 15 min, and treated with a sat. aqueous NH4Cl soln. Extraction with
CH2Cl2, drying of the combined org. layers (MgSO4), and evaporation gave crude 34 (165 mg) which was
completely deiodinated by the repetition of this procedure. FC (hexane/AcOEt 7 :3) gave 29 (110 mg, 66%).
(5R,6R,7S,8R)-7,8-Bis(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydro-2-iodo-6-(2,3,4,6-tetra-O-ben-
zyl-b-d-glucopyranosyloxy)-imidazo[1,2-a]pyridine (34). 13C-NMR (50.6 MHz, CDCl3): 60.25 (d, C(5)); 68.82
(t, C(6')); 70.07 (d); 71.90, 71.93, 72.97, 73.43, 73.73, 75.12, 75.18 (7t, CH2 C(5), 6 PhCH2); 75.26 (d, C(5'));
75.66 (d); 75.86 (t, PhCH2); 77.59 (d); 77.87 (d, C(4')); 81.93 (s, C(2)); 82.30 (d, C(2')); 84.97 (d, C(3')); 102.07 (d,
C(1')); 125.37 (d, C(3)); 127.87 ± 128.79 (several d); 137.67, 138.79, 138.73, 138.41 (4s); 138.45 (2s); 138.72 (s);
145.67 (s, C(8a)).
(5R,6R,7S,8R)-7,8-Bis(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydro-6-(2,3,4,6-tetra-O-benzyl-b-d-
glucopyranosyloxy)imidazo[1,2-a]pyridine (29). Rf (hexane/AcOEt/Et3N 1:1:0.03) 0.29. IR (CHCl3): 3066w,
3008m, 2926w, 2868m, 1605w, 1595w, 1496m, 1454m, 1360m, 1309w, 1263m, 1175s, 1070s, 1028m, 909m. 1H-NMR
(500 MHz, CDCl3, assignment based on a DQFCOSY.GRASP spectrum): 3.26 (ddd, J ˆ 9.4, 3.9, 2.1, H C(5'));
3.37 (t, J ꢁ 8.4, H C(2')); 3.58 ± 3.64 (m, CH C(5), 2 H C(6')); 3.59 (t, J ˆ 9.1, H C(3')); 3.64 (t, J ˆ 9.2,
H
C(4')); 3.70 (dd, J ˆ 9.5, 6.1, CH' C(5)); 3.97 (dd, J ˆ 6.7, 3.3, H C(7)); 4.31 (d, J ˆ 11.9, PhCH); 4.32 ±
4.37 (m, H C(5)); 4.35 (d, J ˆ 11.9, PhCH); 4.47 (d, J ˆ 12.1, PhCH); 4.49 (dd, J ˆ 6.8, 2.5, H C(6)); 4.535 (d,
J ˆ 11.9, PhCH); 4.557 (d, J ꢁ 11.5, PhCH); 4.561 (d, J ꢁ 7.7, H C(1')); 4.595 (d, J ˆ 11.2, PhCH); 4.643 (d, J ˆ
12.1, PhCH); 4.652 (d, J ꢁ 12.4, 2 PhCH); 4.70 (d, J ˆ 12.3, PhCH); 4.802 (d, J ꢁ 11.0, PhCH); 4.805 (d, J ꢁ 11.3,
2 PhCH); 4.811 (d, J ˆ 3.3, H C(8)); 4.88 (d, J ˆ 11.0, PhCH); 6.91 (d, J ˆ 1.3), 7.07 (d, J ˆ 1.4, H C(2),
H
C(3)); 7.12 ± 7.31 (m, 35 arom. H). 13C-NMR (75.9 MHz, CDCl3): 60.10 (d, C(5)); 68.81 (t, C(6')); 70.17
(d); 71.56, 72.22, 72.75, 73.39, 73.71 (5t, CH2 C(5), 4 PhCH2); 75.08 (t, 2 PhCH2); 75.23 (d, C(5')); 75.83 (t,
PhCH2); 76.04 (d); 77.90 (d, C(4')); 78.16 (d); 82.27 (d, C(2')); 85.04 (d, C(3')); 102.07 (d, C(1')); 119.69 (d);
127.81 ± 128.71 (several d); 129.14 (d); 137.88 (s); 138.34 (2s); 138.45 (2s); 138.60, 138.81 (2s); 143.50 (s, C(8a)).
(5R,6R,7S,8S)-6-(b-d-Glucopyranosyloxy)-5,6,7,8-tetrahydro-5-[(hydroxy)methyl]imidazo[1,2-a]pyridine-
7,8-diol (32). A soln. of 28 (108 mg, 0.11 mmol) in AcOEt/MeOH/H2O 5 :17:3 (0.45 ml, 0.26m) was treated with
AcOH (0.1 ml) and 20% Pd(OH)2/C (110 mg) and hydrogenated at 6 bar for 2 d. The suspension was filtered

Helvetica Chimica Acta ± Vol. 82 (1999)
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through Celite (eluted with MeOH and H2O). Evaporation, FC (AcOEt/MeOH/H2O 7:3 :2), and lyophilization
gave 32 (27 mg, 69%). Rf (AcOEt/MeOH/H2O 7:3 :2) 0.14. 1H-NMR (500 MHz, D2O; assignment based on a
DQFCOSY.GRASP spectrum): 3.32 (dd, J ˆ 9.4, 7.9, H C(2')); 3.39 (t, J ꢁ 9.4, H C(4')); 3.45 (ddd, J ˆ 9.7,
5.5, 2.2, H C(5')); 3.485 (t, J ˆ 9.2, H C(3')); 3.71 (dd, J ˆ 12.4, 5.6, H C(6')); 3.87 (dd, J ˆ 12.4, 2.2,
H' C(6')); 3.90 (t, J ˆ 8.4, H C(7)); 4.09 (dd, J ˆ 12.6, 2.3, CH C(5)); 4.15 ± 4.22 (m, H C(5), H C(6),
CH' C(5)); 4.59 (d, J ˆ 7.9, H C(1')); 4.62 (d, J ˆ 8.2, H C(8)); 7.06, 7.20 (2 br. s, H C(2), H C(3)).
13C-NMR (75.9 MHz, D2O): 61.32 (t, C(6')); 63.44 (d, C(5)); 63.89 (t, CH2 C(5)); 70.08 (d); 72.28 (d); 74.95
(d); 76.01 (d); 78.35 (d); 79.08 (d); 79.14 (d); 105.89 (d, C(1')); 122.44, 125.46 (2d, C(2), C(3)); 147.99 (s,
C(8a)). Anal. calc. for C14H22N2O9 ´ 3 H2O (416.39): C 40.38, H 6.77, N 6.72; found: C 40.61, H 6.93, N 6.14. ESI-
‡
MS: 363 ([M ‡ 1] ).
Inhibition Studies. Determination of the inhibition constants (Ki) was performed in the presence of five
inhibitor concentrations which bracket the Ki value.
a) Inhibition of Cel7A. The inhibition constants were determined at 308 using a 50 mm KH2PO4/Na2HPO4
buffer (pH 5.7) and 2-chloro-4-nitrophenyl b-lactoside as substrate [32]. The release of 2-chloro-4-nitrophenol
was monitored continuously by measuring absorbance at 405 nm for 10 minutes. The Km value of this substrate is
460 mm [30]. The Ki values were determined classically either by Lineweaver-Burk plots or Dixon plots [33].
b) Inhibition of Cel6A. The inhibition experiments were performed at 278 in 10 mm NaOAc buffer (pH 5)
by following the hydrolysis of cellotriose, which has a Km value of 22 mm [31]. Samples were taken at 8 different
time points over 12 min and the amount of hydrolysed cellotriose was determined by high-performance anion-
exchange chromatography (HPAEC) equipped with a pulsed amperometric detector (PAD) and using the
conditions specified by the manufacture (Dionex, Sunnyvale, USA). The Ki values were obtained from Dixon
plots.
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Received March 18, 1999