Synthesis of tetrahydropyridoimidazole-2-acetates: Effect of carboxy and methoxycarbonyl groups at C(2) on the inhibition of some β- and α-glycosidases

Article abstract of DOI:10.1002/hlca.200490273

The gluco- and manno-tetrahydropyridoimidazole-2-acetates and -acetic acids 16 and 17, and 20 and 21, respectively, were synthesized by condensation, in the presence of HgCl₂, of the known thionolactam 26 with the β-amino ester 25 that was obta

Full text of DOI:10.1002/hlca.200490273

Helvetica Chimica Acta ± Vol. 87 (2004)
3035
Synthesis of Tetrahydropyridoimidazole-2-acetates: Effect of Carboxy
and Methoxycarbonyl Groups at C(2) on the Inhibition of Some b- and
a-Glycosidases
by Miroslav Terinek and Andrea Vasella*
Laboratorium für Organische Chemie, ETH-Hönggerberg, Wolfgang-Pauli Strasse 10, CH-8093 Zürich
The gluco- and manno-tetrahydropyridoimidazole-2-acetates and -acetic acids 16 and 17, and 20 and 21,
respectively, were synthesized by condensation, in the presence of HgCl2, of the known thionolactam 26 with the
b-amino ester 25 that was obtained by addition of AcOMe to the imine 22, followed by debenzylation. The
resulting methyl esters 16 and 20 were hydrolyzed to the acetic acids 17 and 21. The (methoxycarbonyl)-
imidazole 14 and the acid 15 were obtained via the known aldehyde 29. The imidazoles 14 ± 17, 20, and 21 were
tested as inhibitors of the b-glucosidase from Caldocellum saccharolyticum, the a-glucosidase from brewerꢀs
yeast, the b-mannosidase from snail, and the a-mannosidase from Jack beans (Tables 1 ± 3). There is a similar
dependence of the Ki values on the nature of the C(2)-substituent in the gluco- and manno-series. With the
exception of 19, manno-imidazoles are weaker inhibitors than the gluco-analogues. The methyl acetates 16 and
20 are 3 ± 4 times weaker than the methyl propionates 5 and 11, in agreement with the hydrophobic effect. The
gluco-configured (methoxycarbonyl)-imidazole 14 is 20 times weaker than the methyl acetate 16, reflecting the
reduced basicity of 14, while the manno-configured (methoxycarbonyl)-imidazole 18 is only 1.2 times weaker
than the methyl acetate 20, suggesting a binding interaction of the MeOCO group and the b-mannosidase. The
carboxylic acids 6, 12, 15, 17, 19, and 21 are weaker inhibitors than the esters, with the propionic acids 6 and 12
being the strongest and the carboxy-imidazoles 15 and 19 the weakest inhibitors. The manno-acetate 21 inhibits
the b-mannosidase ca. 8 times less strongly than the propionate 12, but only 1.5 times more strongly than the
carboxylate 19, suggesting a compensating binding interaction also of the COOH group and the b-mannosidase.
The a/b selectivity for the gluco-imidazoles ranges between 110 for 15 and 13.4 ´ 103 for 6; the manno-imidazoles
are less selective. The methyl propionates proved the strongest inhibitors of the a-glucosidase (IC50 (5) ˆ 25 mm)
and the a-mannosidase (Ki(11) ˆ 0.60 mm).
Introduction. ± Nagstatin (1), a N-acetylgalactosamine-derived tetrahydroimida-
zopyridine possessing a carboxymethyl substituent at C(2) [1] was isolated from the
fermentation broth of Streptomyces amakusaensis in 1992 [2] and synthesized in 1995
by Tatsuta et al. [3][4]. It inhibits several glycosidases, most notably hog kidney N-
acetyl-b-d-glucosaminidase (IC50 ˆ 4 nm) [2][5]. The carboxymethyl group at C(2)
does not appear to be important for the inhibitory activity, since ꢁdebranchedꢀ nagstatin
analogues and related C(2)-unsubstituted glucose- and mannose-derived tetrahydro-
imidazopyridines are also potent inhibitors of b-glycosidases [5 ± 7]. These observa-
tions and the rationalisation of the inhibitory activity of these and analogous azoles ±
their similarity with the putative reaction intermediate, and the cooperative interaction
of the azole ring with the catalytic acid and nucleophile ± indicated a negligible role for
the substituents at C(2) [8 ± 10]. Subsequently, however, it was shown that C(2)-
substituents of tetrahydropyridoimidazoles (and corresponding substituents of related
azoles and pyrroles) can strongly affect the inhibitory properties, either indirectly, by
competing with the catalytic acid for H-bonding to the ꢁglycosidicꢀ heteroatom, and/or
ꢂ 2004 Verlag Helvetica Chimica Acta AG, Zürich

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Helvetica Chimica Acta ± Vol. 87 (2004)
directly, by interacting in their own right with the glycosidases [11 ± 14]. More recently,
we compared the effect of the nature of the C(2)-substituents of the gluco-configured
tetrahydroimidazopyridines 2 ± 7 on the inhibition of the b-glucosidases from Caldo-
cellum saccharolyticum (family 1) and from sweet almonds to the effect of the same
substituents of the manno-imidazoles 8 ± 13 on the inhibition of the b-mannosidase
from snail, arguing that the similar relative effect of these substituents evidences that
the reaction coordinates in the vicinity of the reactive intermediates for b-glucosidases
of family 1, and the snail b-mannosidase should not differ significantly1) [14]. Among
the C(2)-substituted imidazoles 2 ± 13, the gluco-configured methyl propionate 5 and its
manno-configured analogue 11 were two of the strongest inhibitors of b-glycosidases
[12][14]. The carboxylic acids 6, 12, and 19 are all weaker inhibitors than the
corresponding esters 5, 11, and 18. The manno-configured imidazole 18 possessing a
MeOCO group at C(2) group is a significantly weaker inhibitor than the methyl
propionate 11. It is not clear how much of this difference is due to the size and shape of
the substituents, and how much to the effect on the pKha value of the imidazoles (pKha
(11) ˆ 5.52; pKha (18) < 2.2 [14]). We wished, therefore, to compare the inhibition by
the gluco-configured methyl esters 14, 16, and 5 and of the manno-configured
analogues 18, 20, and 11 to each other, and to the corresponding gluco- and manno-
configured acids 15, 17, and 6, and 19, 21, and 12, respectively, to check for more-subtle
differences of these substituents on the inhibition of b-glucosidases and the b-
mannosidase. For this, we had to synthesize the methyl acetates 16 and 20 and the
corresponding acids 17 and 21.
So far, C(2)-substituted imidazopyridines were mostly synthesized via C(2)-iodo-
imidazoles [12][14] that are derived in two steps from the 2,3-unsubstituted imidazoles;
these were prepared by condensing the thionolactam 26 with 2-aminoacetaldehyde
1
)
Differences between the reaction coordinates preceding the transition state were expected on the basis of
the different interaction of the catalytic nucleophile with HOÀC(2) in b-glucosidases vs. b-mannosidases
[15] and evidenced by comparing gluco and manno isoquinuclidines [16][17].

Helvetica Chimica Acta ± Vol. 87 (2004)
3037
dimethyl acetal [7]. We planned to obtain 17 and 21 in a shorter, more-convergent way
from 26 and the b-amino ester 25 (Scheme). For a more-comprehensive struc-
tureÀactivity relationship of C(2)-substituted gluco- and manno-configured imidazole
esters and acids we also required the gluco-configured methyl imidazole-2-carboxylate
14 and the corresponding acid 15.
Scheme 1
a) AcOMe, BuLi, (i-Pr)2NH, BF3 ´ OEt2, THF, À 788; 63%. b) 1. BnNH2, AcOH, CH2Cl2; 2. NaBH(OAc)3;
3%. c) H2, Pd/C, MeOH; 64%. d) 1. HgCl2, THF; 2. TsOH ´ H2O, toluene, 608; 81 % of 27/28 56 :44. e) H2, Pd/C,
AcOEt/MeOH/AcOH; 91% of 16; 87% of 20; 95% of 14. f) KOH, EtOH/H2O, 508; 86% of 17; 81 % of 21;
80% of 15. g) MnO2, NaCN, AcOH/MeOH; 77%.
Synthesis. ± We planned to synthesize the gluco- and manno-configured methyl
esters 16 and 20 by condensing the gluconothio-1,5-lactam 26 [7] with the b-amino ester
25. While the synthesis of imidazoles by condensation of thionolactams with 2-
aminoacetaldehyde dimethyl acetal is well-precedented [7][18 ± 20], there are only
very few examples of the condensation with substituted amino acetals [21][22]. To
prepare the methyl ester 252) (Scheme), we started from benzyl[(2,2-dimethoxy)-
ethylidene]amine (22), which we recently described as useful precursor of a-amino-
2
)
The tert-butyl ester analogues of (S)-, (R)-, and (RS)-25 were synthesized in four steps and in overall yields
of 52 ± 68% from commercially available Z-Asp(OtBu)-H [23].

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Helvetica Chimica Acta ± Vol. 87 (2004)
phosphonates [22]. Treatment of 22 with the in situ generated lithium enolate of methyl
acetate3) in the presence of BF3 ´ OEt2 gave the benzylamine 234) (63%); 22 did not
react in the absence of BF3 ´ OEt2. The synthesis of 23 by reductive amination of the b-
keto ester 24 [42] with BnNH2 (AcOH, NaBH(OAc)3 [43 ± 45]) was not promising; 23
was isolated in only 3% from the complex crude product. The b-amino ester 25 was
obtained by hydrogenolysis (10% Pd/C) of 23 and isolated in 64% yield by bulb-to-
bulb distillation under reduced pressure.
In contradistinction to the condensation of 2-aminoacetaldehyde dimethyl acetal
with the thionolactam 26 [7], the one of the substituted amino acetal 25 required a
catalyst. Condensation of 25 with 26 in the presence of HgCl2, followed by treatment of
the crude with TsOH ´ H2O provided 57% (25-mg scale) of a 58 :42 mixture of the
gluco-imidazole 27 and the manno-isomer 28. Lower yields (22 ± 52%) of 27/28
resulted by replacing HgCl2 by HgO, HgBr2, HgCl2/Et3N, Hg(OAc)2, or Hg(OTf)2. The
ratio 27/28 varied between 68 :32 (HgO) and 45 :55 (Hg(OTf)2) (cf. Exper. Part). The
yield of 27 and 28 increased to 81% (27/28 56 :44) by conducting the reaction on a
larger scale (235 mg of 26) in the presence of HgCl2. The resulting imidazoles were
readily separated by chromatography to give 27 (44%), 27/28 50 :50 (2%), and 28
(35%). Hydrogenolysis (10% Pd/C, AcOH) of 27 and 28 provided the gluco- and
manno-esters 16 and 20, respectively, which were hydrolyzed with 0.4m KOH in EtOH/
H2O at 508 to the corresponding C(2)-carboxymethyl substituted imidazoles 17 (86%)
and 21 (81%). This synthesis afforded 17 and 21 in four steps from 26, and in overall
yields of 35 and 25%, respectively.
The gluco-configured methoxycarbonyl- and carboxy-imidazoles 14 and 15 were
prepared similarly to the manno-analogues [14]. Oxidation of the aldehyde 29 [12] with
MnO2 in the presence of NaCN and MeOH [46] provided the methyl ester 30 (77%)
that was debenzylated (H2, 10% Pd/C, AcOH) to the tetrol 14 (95%; 73% from 29).
Hydrolysis (KOH in EtOH/H2O, 508) yielded 80% of the carboxy-imidazole 15 (59%
from 29).
In agreement with previous observations [7][11][12][14], the C(2)-substituents do
not affect the solution conformation of the tetrahydroimidazopyridines. Thus, the
7
benzylated gluco- and manno-imidazoles 27, 28, and 30 are 2 :1mixtures of the H6 and
the 6H7 conformers5), while the unprotected imidazoles 14 ± 17, 20, and 21 adopt a 7H6
conformation (see Tables 4 and 6 in Exper. Part). The 13C signals of C(5)ÀC(8) of 27
and 28 (Table 5 in Exper. Part) were assigned on the basis of HSQC.GRASP spectrum;
those of the other imidazoles were assigned by analogy. The formation of the
[(methoxycarbonyl)methyl]-imidazoles 27 and 28 from 26 is confirmed by the
disappearance of the NH signal, by 13C sꢀs at 134.70 ppm (27 and 28), 13C dꢀs at
115.28 (27) and 117.18 ppm (28), and strong IR CˆO bands at 1737 and 1738 cmÀ1 for
27 and 28, respectively. The assignment of the gluco- and manno-configuration to 27
3
)
)
)
The addition of enolates of methyl acetate to imines [24][25], N-sulfinyl imines [26 ± 39], and N-acyl-
pyrazolines [40] are well-documented.
4
5
(R)-23 was prepared from 5-[(R)-menthyloxy]-furan-2(5H)-one in two steps in an overall yield of 45% and
used for the synthesis of b-lactams [41]. No spectroscopic data of (R)-23 were reported.
The direction of numbering of imidazopyridines (cf. 27 in Scheme) is opposite to that of pyranosides. Thus,
the sides above and below the plane of the imidazoles, as defined by the clockwise and counterclockwise
numbering, are interchanged relative to those defined by carbohydrate nomenclature.

Helvetica Chimica Acta ± Vol. 87 (2004)
3039
and 28, respectively, is based on the value of J(7,8) (5.3 and 3.1Hz, resp.; cf. Scheme).
The formation of the methoxycarbonyl-imidazole 30 is confirmed by the disappearance
of the CHO signal in the 1H-NMR spectrum, by the 1H s at 3.92 ppm (MeO), the 13C s
at 163.17 ppm, and a strong IR CˆO band at 1716 cmÀ1.
Enzymatic Tests and Discussion. ± The gluco- and manno-imidazopyridines 14 ± 17,
20, and 21 were tested as inhibitors of the b-glucosidase from Caldocellum
saccharolyticum (phosphate buffer, pH 6.8, 558), a-glucosidase from brewerꢀs yeast
(phosphate buffer, pH 6.8, 378), b-mannosidase from snail (acetate buffer, pH 4.5, 258),
and a-mannosidase from Jack beans (acetate buffer‡ ZnCl2, pH 4.5, 378), using the
corresponding 4-nitrophenyl glycopyranosides as substrates. The data for the inhibition
of the b-glucosidase and b-mannosidase are summarized in Table 1, and compared to
the inhibition of these enzymes by the known gluco-imidazoles 5 and 6, and the manno-
imidazoles 11, 12, 18, and 19. The data for the inhibition of the a-glucosidase, and the
selectivity of the inhibition of b- and a-glucosidases by gluco-imidazoles are compiled
in Table 2. The data for the inhibition of the a-mannosidase, and the selectivity of the
inhibition of the b- and a-mannosidases by manno-imidazoles are listed in Table 3.
Table 1. pKha Values of the C(2)-Substituted gluco- and manno-Imidazoles 5, 6, 11, 12, and 14 ± 21, and
Comparison of Their Inhibition of the b-Glucosidase from Caldocellum saccharolyticum and of the b-
Mannosidase from Snail
gluco-
Imidazoles
Inhibition of
manno-
Inhibition of
Comparison
b-glucosidasea)
Imidazoles b-mannosidaseb) of Ki values
R
No. pKha
Ki [nm]
No. pKha Ki [nm]
Ki (rel.)
b-gluco- b-manno-
sidase sidase
Esters
COOMe
CH2COOMe
CH2CH2COOMe
c
14
)
103
5.0e)
(a ˆ 3.4) 18d) ±
142
117
28
(a ˆ 4.3) 57.2
(a ˆ 3.4) 2.8
ꢀ1
5.1
4.2
ꢀ1
16 5.03
20
4.61
5f) 6.17
1.8 (a ˆ 2.5) 11d) 5.52
Acids
COOH
CH2COOH
CH2CH2COOH
15 4.95g)
17 6.40g)
6f) 4.06/7.05
2660
95
(a ˆ 2.7) 19d) 4.70 1210
(a ˆ 4.8) 295.6
10.6
12.1
8.1
(a ˆ 1.6) 21
5.08g) 810
9.0h)
12d) 4.15
100
(a ˆ 5.3) ꢀ1
ꢀ1
a) At 558 and pH 6.8. b) At 258 and pH 4.5. c) No inflection of the titration curve was observed between pH
values of 2.2 and 6.0. d) Data taken from [14]. e) Non-competitive inhibition. f) Data taken from [12]. g) No
additional pKha value was observed between pH values 2.2 ± 5.5 (15), 2.9 ± 7.9 (17), 2.6 ± 5.4 (21). h) IC50/2.
The data in Table 1 show qualitatively the same dependence of the Ki values on the
nature of the C(2)-substituent in the gluco- and manno-series. With the exception of 19,
the manno-imidazoles are weaker inhibitors than the corresponding gluco-imidazoles.
Considering the different pH optima of the b-glycosidases and the different extent to
which the imidazoles are protonated at the pH of the assay, the quantitative differences
between the relative Ki values for the gluco- and manno-imidazoles are small. On the

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Helvetica Chimica Acta ± Vol. 87 (2004)
Table 2. pKha Values of the C(2)-Substituted gluco-Imidazoles 5, 6, and 14 ± 17, and Comparison of Their
Inhibition of the a-Glucosidase from Brewerꢀs Yeast and of the b-Glucosidase from Caldocellum saccharolyticum
gluco-
Imidazoles
Inhibition of
Inhibition of
Comparison of Ki valuesc)
a-glucosidasea) b-glucosidaseb)
R
No. pKha
IC50
Ki
Ki (rel)
Ki (a-glucosidase)/
[mm]
[nm]
a-glucosidase Ki (b-glucosidase)
Esters
COOMe
CH2COOMe
CH2CH2COOMe
d
14
16
)
489
69
25
103
5.0
1.8
19.6
2.8
ꢀ16.94 ´ 1
2.37 ´ 103
5.03
6.90 ´ 103
5e) 6.17
0
3
Acids
COOH
CH2COOH
CH2CH2COOH
15
17
4.95f)
6.40f)
598
125
2660
95
9.0
2.5
0.5
ꢀ1
0.11 ´ 103
0.66 ´ 103
13.44 ´ 103
6e) 4.06/7.05 242
a) At 378 and pH 6.8. b) At 558 and pH 6.8; only Ki values shown (for the inhibition type see Table 1). c) Ki
Values for the a-glucosidase were calculated as IC50/2. d) No inflection of the titration curve was observed
f
between pH values of 2.2 and 6.0. e) Data taken from [12]. ) No additional pKha value was observed between
pH values 2.2 ± 5.5 (15) and 2.9 ± 7.9 (17).
basis of this observation, there should not be a significant difference between the
reaction coordinates, in the vicinity of the reactive intermediates, for b-glucosidases of
family 1and snail b-mannosidase [14].
The methyl propionates 5 and 11 are the strongest inhibitors of the b-glycosidases.
Weakest are the methyl carboxylates 14 and 18. The methyl acetates 16 and 20 inhibit
the b-glucosidase and the b-mannosidase 3 ± 4 times less strongly than the correspond-
ing methyl propionates 5 and 11. This difference corresponds to the increase of
hydrophobic surface area, amounting to a minimum of 0.68 kcal molÀ1 or a 3.2-fold
increase in binding constant for a Me and similarly for a CH2 group6). The gluco-
configured methoxycarbonyl-imidazole 14 is a 20 times weaker inhibitor of the b-
glucosidase than the methyl acetate 16. This difference is larger than expected from the
hydrophobic effect, and reflects the reduced basicity of the methoxycarbonyl-imidazole
14 (pKha < 2.2; see Table 1) and the weaker interaction with the catalytic acid. The
corresponding manno-configured methoxycarbonyl-imidazole 18 is, however, only a
1.2-times weaker inhibitor of the b-mannosidase than the methyl acetate 20. As the
pKha value of 18 is close to that of 14 [14], one has to postulate a compensating
interaction of the MeOCO substituent with the b-mannosidase.
The gluco- and manno-configured carboxylic acids 6, 12, 15, 17, 19, and 21 proved,
overall, weaker inhibitors of the b-glycosidases than the corresponding esters. One
finds a similar dependence of the inhibition of the b-glycosidases on the size of the
6
)
The contribution of hydrophobic interactions in the binding of biologically active compounds to their
receptors has been reviewed by Davis and Teague [47]. Recent examples of this effect on the inhibitory
activity of b-glycosidases include imidazopyridines of type 2 [12][14][48], d-gluconolactam oximes [49], b-
d-glucosylamines [50], d-gluconamidines [51][52], and 2,5-dideoxy-2,5-imino-d-mannitols [53 ± 55].

Helvetica Chimica Acta ± Vol. 87 (2004)
3041
Table 3. pKha Values of the C(2)-Substituted manno-Imidazoles 11, 12, and 18 ± 21, and Comparison of Their
Inhibition of the a-Mannosidase from Jack Beans and of the b-Mannosidase from Snail
manno-
Inhibition of
Inhibition of
Comparison of Ki values
Imidazoles a-mannosidasea) b-mannosidaseb)
R
No. pKha Ki
[mm]
Ki
[nm]
Ki (rel)
Ki (a-mannosidase)/
a-mannosidase Ki (b-mannosidase)
Esters
COOMe
18c)
±
12.5
0.91
0.60
142
117
28
20.8
1.5
ꢀ121
88.0
7.8
CH2COOMe
CH2CH2COOMe
Acids
COOH
CH2COOH
CH2CH2COOH
20
4.61
11c) 5.52
.4
19c) 4.70
21
12c) 4.15
1.33
1210
810
100
1.7
2.4
ꢀ17.8
1.1
2.3
5.08d) 1.85
0.78
a) At 378 and pH 4.5; competitive inhibition for all imidazoles. b) At 258 and pH 4.5; only Ki values shown (for
the inhibition type, see Table 1). c) Data taken from [14]. d) No additional pKha value was observed between pH
values of 2.6 and 5.4.
substituent at C(2) as for the ester analogues; the propionic acids 6 and 12 are the
strongest inhibitors and the imidazole-carboxylic acids 15 and 19 the weakest ones. The
gluco-configured carboxymethyl-imidazole 17 is a 10 times weaker inhibitor of the b-
glucosidase than 6, and a 28 times stronger inhibitor than 15. In both cases, the
differences are larger than expected from the difference of hydrophobicity of the
substituents at C(2). The pKha values of 6, 15, and 17 (see Table 1), and the pH value
(6.8) of the enzymatic assay suggest that the gluco-configured carboxylic acids are, at
least in part, bound as zwitterions. The lower inhibitory activity of the acids (as
compared to the esters) may reflect the impaired interaction of the partially protonated
imidazoles with the catalytic acid. The weak inhibition of the b-glucosidase by 15 may
be due to the reduced basicity of this imidazole (see Table 1). A disruptive interaction
of the glycosidases with the (solvated?) carboxylate anion cannot be excluded.
In agreement with the inhibition of the b-glucosidases by the propionate 6 and
acetate 17, respectively, the manno-configured acetate 21 inhibits the b-mannosidase
ca. 8 times less strongly than the propionate 12. However, 21 is only a 1.5-times stronger
inhibitor than the carboxylate 19. The pH value (4.5) of the enzymatic assay of the b-
mannosidase and the pKha values of the imidazoles 12, 19, and 21 (see Table 1) suggest
‡
‡
‡
that the inhibitors are bound as imidazolium salts 12 ´ H , 19 ´ H , and 21 ´ H . The
remarkably strong inhibition of the b-mannosidase by the carboxylic acid 19 supports a
binding interaction between the COOH group of 19 and the enzyme, similarly as
suggested above for the MeOCO group of 18.
Tables 2 and 3 show the data for the inhibition of the a-glucosidase from brewerꢀs
yeast (IC50 values) and the a-mannosidase from Jack beans (Ki values). There is no
clear correlation between the a/b selectivity and the strength of the inhibition of b-
glycosidases. The selectivity for the inhibition of the a- vs. b-glucosidases by gluco-
imidazoles ranges between 110 for the carboxylate 15 and 13.4 ´ 103 for the propionic
acid 6. The manno-imidazoles are less selective with the selectivity for the inhibition of

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Helvetica Chimica Acta ± Vol. 87 (2004)
the a- vs. b-mannosidases varying between 1.1 for the carboxylate 19 and 88 for the
methyl carboxylate 18. The gluco- and manno-configured methyl propionates 5 and 11
proved the strongest inhibitors of the a-glucosidase (IC50 ˆ 25 mm) and the a-
mannosidase (Ki ˆ 0.60 mm), respectively.
We thank M. Schneider for the pKha determinations, Dr. B. Bernet for checking the Exper. Part, and the
Swiss National Science Foundation and Oxford Glycosciences Ltd., Abingdon (UK), for generous support.
Experimental Part
General. Solvents were distilled: THF from Na and benzophenone, CH2Cl2 from P2O5, MeOH, (i-Pr)2NH,
and BnNH2 from CaH2, and AcOMe from MgSO4. Reactions were carried out under Ar, unless stated
otherwise. Qual. TLC: precoated silica-gel plates (Merck silica gel 60 F254); detection by heating with ꢁmostainꢀ
(400 ml of 10% H2SO4 soln., 20 g of (NH4)6 Mo7O24 ´ 6 H2O, 0.4 g of Ce(SO4)2). Flash chromatography (FC):
silica gel Fluka 60 (0.04 ± 0.063 mm). Optical rotations: 1-dm cell at 258, 589 nm. UV Spectra (ca. 0.2 mm solns.)
were taken in 1-cm cell at 258 in the range of 190 to 500 nm (log e values in parentheses). FT-IR spectra: KBr or
ca. 2% soln. in CHCl3, absorption in cmÀ1. 1H- and 13C-NMR spectra: chemical shifts d in ppm rel. to TMS as
external standard, and coupling constants J in Hz. HR-MALDI-MS: in gentisic acid (ˆ2,5-dihydroxybenzoic
acid, DHB) matrix. The pKha values were determined in H2O by potentiometric titration with HCl at 258. The b-
glucosidase from Caldocellum saccharolyticum (EC 3.2.1.21, as a lyophilized powder, Sigma G-6906), a-
glucosidase from brewerꢀs yeast (maltase, EC 3.2.1.20, as a lyophilized powder, Fluka 63412), b-mannosidase
from snail acetone powder (EC 3.2.1.25, as a suspension in 3.0m (NH4)2SO4 containing 10 mm AcONa, pH ca.
4.0, Sigma M-9400), a-mannosidase from Jack beans (EC 3.2.1.24, as a suspension in 3.0m (NH4)2SO4 and
0.1m m zinc acetate, pH ca. 7.5, Sigma M-7257), 4-nitrophenyl b-d-glucopyranoside (Fluka 73676), 4-
nitrophenyl a-d-glucopyranoside (Fluka 73673), 4-nitrophenyl b-d-mannopyranoside (Sigma N-1268), and 4-
nitrophenyl a-d-mannopyranoside (Sigma N-2127) were used without further purification.
Methyl 3-(Benzylamino)-4,4-dimethoxybutanoate (23). a) At À 788, a soln. of 22 (6.80 g, 35.2 mmol) in
THF (15 ml) was treated with BF3 ´ OEt2 (4.80 ml, 39.0 mmol) and stirred for 1h. In the meantime, a soln. of
LDA (prepared by addition of BuLi (1.6m, 44.5 ml, 71.2 mmol) to a soln. of (i-Pr)2NH (11.0 ml, 77.8 mmol) in
THF (50 ml) at À 788) was treated with freshly distilled AcOMe (11.8 ml, 0.148 mol), stirred for 35 min at
À 788, and treated with the THF soln. of 22 and BF3 ´ OEt2. The mixture was stirred for 150 min at À 788,
treated with H2O (50 ml), stirred for 15 min at 228, and diluted with Et2O (250 ml). After separation, the org.
layer was washed with H2O (100 ml), and the aq. layer was extracted with Et2O (2 Â 80 ml). The combined org.
layers were washed with brine (250 ml), dried (MgSO4), filtered, and evaporated. FC (hexane/AcOEt 3 :1 !
2 :1 ! 1:1) gave 22 (1.47 g, 22%) and 23 (5.94 g, 63%).
b) At 228, a soln. of 24 (540 mg, 3.07 mmol) in CH2Cl2 (30 ml) was successively treated with BnNH2
(0.38 ml, 3.48 mmol) and AcOH (0.19 ml, 3.32 mmol), stirred for 5 min, and treated with NaBH(OAc)3
(975 mg, 4.60 mmol). The mixture was stirred for 16 h at 228, treated with sat. NaHCO3 soln. (40 ml), diluted
with CH2Cl2 (50 ml), and washed with sat. NaHCO3 soln. (2 Â 50 ml). The combined aq. layers were extracted
with CH2Cl2 (2 Â 50 ml). The combined org. layers were washed with H2O (60 ml) and brine (60 ml), dried
(MgSO4), filtered, and evaporated. The 1H-NMR spectrum of the crude (816 mg) showed a complex mixture of
products from which 23 (22 mg, 3%) was isolated by FC (hexane/AcOEt 2 :1 ! 1:1).
Data of 23: Colourless oil. Rf (hexane/AcOEt 1:1) 0.46. IR (CHCl 3): 3337w, 3029m, 3011m, 2954m, 2837m,
1951w, 1877w, 1810w, 1731s, 1604w, 1495w, 1454m, 1439m, 1362m, 1285m, 1259m, 1167m, 1132s, 1115s, 1075s,
1029m, 1011m, 968m, 91 3m. 1H-NMR (CDCl3, 300 MHz): 1.72 ± 1.82 (br. s, exchange with CD3OD, NH); 2.46
(dd, J ˆ 6.5, 15.3, HÀC(2)); 2.59 (dd, J ˆ 5.6, 15.3, H'ÀC(2)); 3.18 ± 3.27 (m, HÀC(3)); 3.35, 3.40 (2s,
(MeO)2C(4)); 3.66 (s, CO2Me); 3.78, 3.87 (2d, J ˆ 13.1, PhCH2); 4.31( d, J ˆ 5.0, HÀC(4)); 7.18 ± 7.44 (m, 5
arom. H). 13C-NMR (CDCl3, 75 MHz): 34.94 (t, C(2)); 51.49 (q, CO2Me); 51.51 (t, PhCH2); 54.87, 55.67 (2q,
(MeO)2C(4)); 55.85 (d, C(3)); 106.18 (d, C(4)); 126.75 (d, C(4) of Ph); 127.97 (d, C(2) and C(6) of Ph); 128.15
‡
‡
(d, C(3) and C(5) of Ph); 140.16 (s, C(1) of Ph); 172.62 (s, C(1)). EI-MS: 267 (<1, M ), 236 (5, [M À MeO] ),
‡
‡
‡
‡
204 (<1 , [M À MeO À MeOH] ), 192 (60, [M À (MeO)2CH] ), 91(100, C 7H7 ), 75 (17, (MeO)2CH ), 65 (6),
28 (5). HR-ESI-MS: 1067.3520 (8), 1051.4210 (13), 957.3619 (27), 696.2766 (100), 529.2487 (21), 268.1550 (56,
‡
‡
[M ‡ H] , C14H22NO4 ; calc. 268.1549). Anal. calc. for C14H21NO4 (267.32): C 62.90, H 7.92, N 5.24; found: C
63.02, H 7.90, N 5.07.

Helvetica Chimica Acta ± Vol. 87 (2004)
3043
Methyl 3-Amino-4,4-dimethoxybutanoate (25). a) A suspension of 23 (710 mg, 2.66 mmol) and 10% Pd/C
(350 mg) in MeOH (17 ml) was hydrogenated at the atmospheric pressure and 228 for 5 h and filtered over
Celite. Evaporation and bulb-to-bulb distillation (1108/0.5 Torr) of the yellowish oil gave 25 (302 mg, 64%).
b) A suspension of 23 (4.10 g, 15.3 mmol) and 10% Pd/C (410 mg) in MeOH (100 ml) was hydrogenated at
6 bar and 228 for 1h and filtered over Celite. Evaporation (! yellowish oil) and distillation (micro-distillation
apparatus; 1108/0.5 Torr) gave 25 (1.54 g, 57%).
Data of 25: Colourless liquid. Rf (AcOEt/MeOH 10 :1) 0.10. B.p. 808/0.5 Torr. IR (CHCl3): 3387w, 3320w,
3028m, 2997m, 2955s, 2836m, 1731s, 1583m, 1439s, 1414w, 1371m, 1327m, 1297m, 1263m, 1168s, 1121s, 1072s,
1018m, 975m, 91 6m. 1H-NMR (CDCl3, 300 MHz): 1.42 ± 1.56 (br. s, exchange with CD3OD, NH2); 2.30 (dd, J ˆ
9.0, 16.2, HÀC(2)); 2.59 (dd, J ˆ 4.1, 16.2, HꢀÀC(2)); 3.24 ± 3.33 (m, HÀC(3)); 3.38, 3.39 (2s, (MeO)2C(4)); 3.66
(s, CO2Me); 4.12 (d, J ˆ 5.6, HÀC(4)). 13C-NMR (CDCl3, 75 MHz): 37.16 (t, C(2)); 49.92 (d, C(3)); 51.61 (q,
‡
CO2Me); 54.95, 55.25 (2q, (MeO)2C(4)); 107.15 (d, C(4)); 172.66 (s, C(1)). EI-MS: 178 (<1 , [M ‡ H] ), 160
‡
‡
‡
‡
(<1 , [M À NH3] ), 146 (10, [M À MeO] ), 129 (1, [M À NH3 À MeO] ), 114 (9, [M À MeO À MeOH] ), 102
‡
‡
(31, [M À (MeO)2CH] ), 86 (14), 75 (100, (MeO)2CH ), 70 (14), 60 (8), 47 (9), 43 (9), 28 (4). HR-ESI-MS:
‡
386.1794 (30), 364.1983 (25), 334.1874 (25), 307.1877 (74), 288.1428 (25), 200.0899 (100, [M ‡ Na] ,
‡
C7H15NNaO4 ; calc. 200.0899). Anal. calc. for C7H15NO4 (177.20): C 47.45, H 8.53, N 7.90; found: C 47.62, H
8.45, N 7.89.
Methyl (5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydroimidazo[1,2-a]pyri-
dine-2-acetate (27) and Methyl (5R,6R,7S,8R)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydro-
imidazo[1,2-a]pyridine-2-acetate (28). a) A suspension of 26 (25 mg, 45.1 mmol) and HgCl2 (17 mg, 62.6 mmol)
in THF (0.5 ml) was treated with 25 (48 mg, 0.271mmol), stirred for 18 h at 22 8, diluted with AcOEt (5 ml), and
filtered over Celite (the solid was washed with 45 ml of AcOEt). The combined filtrate and washing was washed
with brine (40 ml), dried (MgSO4), filtered, and evaporated. A soln. of the residue (61mg) in toluene (1.5 ml)
was treated with TsOH ´ H2O (40 mg, 0.210 mmol), stirred for 40 h at 608, cooled to 228, diluted with Et2O
(50 ml), and washed with sat. NaHCO3 soln. (3 Â 25 ml). The combined aq. layers were extracted with Et2O
(2 Â 30 ml). The combined org. layers were washed with H2O (50 ml) and brine (50 ml), dried (MgSO4),
filtered, and evaporated. FC (hexane/AcOEt 3 :1 ! 2 :1) gave 27 (9.1mg, 32%), 27/28 22 :78 (1.2 mg, 4%) and
28 (5.9 mg, 21%).
b) A suspension of 26 (25 mg, 45.1 mmol), HgCl2 (23 mg, 84.7 mmol), and Et3N (1 0ml, 71.7 mmol) in THF
(0.5 ml) was treated with 25 (48 mg, 0.271mmol), and stirred for 4 h at 22 8. Workup, treatment of the residue
(54 mg) with TsOH ´ H2O, and FC (as described in a) gave 27 (7.8 mg, 27%), 27/28 68 :32 (1.0 mg, 4%) and 28
(6.1mg, 21%).
c) A suspension of 26 (25 mg, 45.1 mmol) and HgO (18 mg, 83.1 mmol) in THF (0.5 ml) was treated with 25
(48 mg, 0.271mmol), and stirred for 96 h at 22 8. Workup, treatment of the residue (48 mg) with TsOH ´ H2O,
and FC (as described in a) gave 27 (3.5 mg, 12%), 27/28 74 :26 (5.5 mg, 19%) and 28 (2.2 mg, 8%).
d) A suspension of 26 (25 mg, 45.1 mmol) and Hg(OAc)2 (19 mg, 59.6 mmol) in THF (0.5 ml) was treated
with 25 (48 mg, 0.271mmol), and stirred for 2 h at 22 8. Workup, treatment of the residue (52 mg) with TsOH ´
H2O, and FC (as described in a) gave 27 (6.7 mg, 23%), 27/28 50 :50 (1.4 mg, 5%) and 28 (5.1mg, 18%).
e) A suspension of 26 (25 mg, 45.1 mmol) and HgBr2 (21mg, 58.3 mmol) in THF (0.5 ml) was treated with 25
(48 mg, 0.271mmol), and stirred for 24 h at 22 8. Workup, treatment of the residue (29 mg) with TsOH ´ H2O,
and FC (as described in a) gave 27 (2.4 mg, 8%), 27/28 55 :45 (1.7 mg, 6%) and 28 (2.3 mg, 8%).
f) A suspension of 26 (25 mg, 45.1 mmol) and Hg(OTf)2 (30 mg, 60.2 mmol) in THF (0.5 ml) was treated
with 25 (48 mg, 0.271mmol), and stirred for 24 h at 22 8. Workup, treatment of the residue (37 mg) with TsOH ´
H2O, and FC (as described in a) gave 27 (3.7 mg, 13%), 27/28 50 :50 (0.3 mg, 1%) and 28 (4.6 mg, 16%).
g) The experiment described in a was repeated on a large scale: a suspension of 26 (235 mg, 0.424 mmol)
and HgCl2 (160 mg, 0.589 mmol) in THF (5 ml) was treated with 25 (458 mg, 2.58 mmol), and stirred for 18 h at
228. Workup, treatment of the residue (461mg) with TsOH ´ H 2O (370 mg, 1.95 mmol) in toluene (10 ml), and
FC (as described in a) gave 27 (118 mg, 44%), 27/28 50 :50 (5 mg, 2%) and 28 (94 mg, 35%).
Data of 27: Rf (hexane/AcOEt 1:1) 0.58. [ a]D25 ˆ ‡48.1( c ˆ 1.00, CHCl3). UV (CHCl3): 285 (2.37), 264
(2.90), 258 (2.96), 240 (3.51). IR (CHCl3): 3090w, 3067w, 3032m, 3012m, 2955m, 2870m, 1952w, 1876w, 1814w,
1737s, 1633w, 1605w, 1562w, 1497m, 1455m, 1438m, 1362m, 1267m, 1152s, 1096s, 1028s, 91 0w. 1H-NMR (CDCl3,
300 MHz): see Table 4; additionally, 3.71(br. s, CH2ÀC(2)); 3.73 (s, MeO); 3.77 (irrad. at 4.18 ! d, J ˆ 10.3);
3.87 (irrad. at 3.77 ! change, irrad. at 4.18 ! change); 3.88 (irrad. at 4.11 ! change, irrad. at 4.18 ! change);
4.11 (irrad. at 4.75 ! d, J ˆ 7.5); 4.18 (irrad. at 3.77 ! dd, J ꢁ 2.2, 7.5); 4.46 (d, J ˆ 11.8, PhCH); 4.48 (d, J ˆ 11.2,
PhCH); 4.51( d, J ˆ 11.5, PhCH); 4.70 (d, J ˆ 11.2, PhCH); 4.75 (irrad. at 4.11 ! s); 4.827 (d, J ˆ 11.2, PhCH);
4.833 (d, J ˆ 11.8, PhCH); 4.84 (d, J ˆ 11.2, PhCH); 5.15 (d, J ˆ 11.5, PhCH); 7.19 ± 7.22 (m, 2 arom. H);

3044
Helvetica Chimica Acta ± Vol. 87 (2004)
Table 4. Selected 1H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] of the Protected and
Deprotected gluco-Imidazoles 14 ± 17, 27, and 30
14
15
16
17
27
30
Solvent
CD3OD
D2O
8.017.53
4.07 ± 4.12
3.96
3.83
4.64
4.09
4.26
8.4
D2O
CD3OD
D2O
CDCl3
CDCl3
HÀC(3)
HÀC(5)
HÀC(6)
HÀC(7)
HÀC(8)
CHÀC(5)
CH'ÀC(5)
J(5,6)
8.06
3.98 ± 4.04
3.85
3.78
4.58
3.96
4.20
7.8
7.22
7.03
7.03
7.76
4.18
3.83
4.11
4.75
3.77
3.87
7.8
3.86 ± 3.98
3.79
3.65
4.49
3.86 ± 3.98
4.10
3.84 ± 3.94
3.83
3.67
4.55
3.93
4.15
8.4
3.95 ± 3.99
3.913.88
3.75
4.56
4.02
4.19
9.0
4.27
4.16
4.80
3.77
3.87
7.8
9.7
J(6,7)
9.3
10.0
8.7
8.4
9.0
7.5
5.9
J(7,8)
7.8
4.4
2.2
11.5
9.0
3.5
3.4
7.5
)
2.8
14.0
8.4
3.7
1.9
11.8
8.4
2.8
2.8
12.2
5.3
5.3
2.8
10.3
4.1
5.6
2.8
10.6
a
J(5,CH)
J(5,CH')
J(CH,CH')
13.4
a) Not assigned.
Table 5. Selected 13C-NMR Chemical Shifts [ppm] of the Protected and Deprotected gluco-Imidazoles 14 ± 17, 27,
and 30, and of the Protected and Deprotected manno-Imidazoles 20, 21, and 28
Solvent
C(2)
C(3)
C(5)
CH2ÀC(5)
C(6)
C(7)
C(8)
C(8a)
gluco
14
15
CD3OD
D2O
CD3OD
D2O
CDCl3
CDCl3
133.04
137.30
134.97
137.58
134.70
133.04
125.71
121.37
117.08
114.69
115.28
124.16
63.44
60.96
62.88
60.12
57.89
58.05
61.13
58.66
61.18
58.52
68.09
67.67
69.64a)
67.17a)
69.85a)
67.93a)
75.92
75.78
74.40
76.26
74.64
81.83
81.00
68.89a)
67.83a)
69.16a)
67.09a)
74.04
149.60
146.61
147.59
145.38
143.04
144.54
16
17
27b)
30
76.27
72.82
manno
20
CD3OD
D2O
CDCl3
135.03
132.71
134.70
117.89
117.01
117.18
63.81
62.90
59.46
70.58
67.26
65.08
73.92
72.75
69.50
80.09
65.52
146.27
143.10
142.26
21
62.10a)
59.73
62.23a)
68.59
28b)
a) Assignment may be interchanged. b) Assignments based on HSQC-GRASP spectrum.
7.27 ± 7.45 (m, 18 arom. H). 13C-NMR (CDCl3, 75 MHz): see Table 5; additionally, 34.57 (t, CH2ÀC(2)); 51.87 (q,
MeO); 72.33, 73.14, 73.77, 74.02 (4t, 4 PhCH2); 127.32 ± 128.31 (several d); 137.15, 137.40, 137.62, 138.08 (4s);
‡
‡
171.48 (s, CˆO). HR-MALDI-MS: 655.2783 (58, [M ‡ Na] , C39H40N2NaO6 ; calc. 655.2784), 633.2964 (89,
‡
‡
‡
‡
[M ‡ H] , C39H41N2O6 ; calc. 633.2965), 525.2431(100, [ M À BnO] , C32H33N2O5 ; calc. 525.2389).
Data of 28: Rf (hexane/AcOEt 1:1) 0.43. [ a]D25 ˆ À29.8 (c ˆ 0.99, CHCl3). UV (CHCl3): 301(2.34), 264
(2.92), 259 (2.96), 240 (3.49). IR (CHCl3): 3090w, 3067w, 3032m, 3012m, 2955m, 2870m, 1952w, 1875w, 1810w,
1738s, 1608w, 1585w, 1563w, 1497m, 1455m, 1438m, 1407w, 1364m, 1267m, 1101s, 1027s, 91 0w. 1H-NMR (CDCl3,
300 MHz): see Table 6; additionally, 3.63 (irrad. at 4.11 ! d, J ˆ 10.3); 3.66 (br. s, CH2ÀC(2)); 3.72 (s, MeO);
3.76 (irrad. at 4.11 ! d, J ˆ 10.0); 3.90 (irrad. at 4.32 ! d, J ˆ 2.8, irrad. at 4.80 ! d, J ˆ 9.3); 4.11 (irrad. at
3.76 ! dd, J ˆ 5.3, 7.2); 4.32 (irrad. at 3.90 ! d, J ˆ 7.2); 4.45 (d, J ˆ 12.5, PhCH); 4.49 (d, J ˆ 12.8, PhCH); 4.62
(d, J ˆ 11.2, PhCH); 4.64 (d, J ˆ 11.8, PhCH); 4.68 (d, J ˆ 10.9, PhCH); 4.71( d, J ˆ 11.8, PhCH); 4.76 (d, J ˆ
12.1, PhCH); 4.80 (irrad. at 3.90 ! s); 5.02 (d, J ˆ 10.9, PhCH); 7.25 ± 7.41( m, 20 arom. H). 13C-NMR (CDCl3,
75 MHz): see Table 5; additionally, 34.62 (t, CH2ÀC(2)); 51.93 (q, MeO); 70.51, 71.65, 73.04, 74.82 (4t, 4
PhCH2); 127.23 ± 128.28 (several d); 137.34, 137.66, 137.83, 138.06 (4s); 171.39 (s, CˆO). HR-MALDI-MS:

Helvetica Chimica Acta ± Vol. 87 (2004)
3045
Table 6. Selected 1H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] of the Protected and
Deprotected manno-Imidazoles 20, 21, and 28
20
21
28
Solvent
CD3OD
D2O
CDCl3
HÀC(3)
HÀC(5)
HÀC(6)
HÀC(7)
HÀC(8)
CHÀC(5)
CHꢀÀC(5)
J(5,6)
7.26
3.86 ± 3.93
4.12
3.83
4.82
3.89
4.08 ± 4.18
7.5
7.38
4.18
4.34
4.12
5.13
4.10
4.25
6.9
7.13
4.11
4.32
3.90
4.80
3.63
3.76
6.8
J(6,7)
8.7
9.0
9.3
J(7,8)
3.7
3.4
3.1
J(5,CH)
J(5,CH')
J(CH,CH')
5.6
)
13.7
3.7
2.8
12.5
6.8
3.1
10.3
a
a) Not assigned.
‡
‡
‡
‡
655.2784 (42, [M ‡ Na] , C39H40N2NaO6 ; calc. 655.2784), 633.2965 (100, [M ‡ H] , C39H41N2O6 ; calc.
‡
‡
633.2965), 525.2404 (71, [M À BnO] , C32H33N2O5 ; calc. 525.2389). Anal. calc. for C39H40N2O6 (632.75): C
74.03, H 6.37, N 4.43; found: C 73.99, H 6.41, N 4.57.
Methyl (5R,6R,7S,8S)-5,6,7,8-Tetrahydro-6,7,8-trihydroxy-5-(hydroxymethyl)imidazo[1,2-a]pyridine-2-ace-
tate (16). A soln. of 27 (110 mg, 0.174 mmol) in AcOEt/MeOH/AcOH 1 :1:1 (2.4 ml) was treated with 10% Pd/
C (75 mg), hydrogenated at 6 bar and 228 for 40 h, and filtered over Celite (washing with 30 ml of MeOH/H2O
9 :1). Evaporation of the combined filtrate, washing, and FC (AcOEt/MeOH/H2O 20 :1 :1! 1 0 :1 :1! 7:1:1)
gave crude 16 (50 mg) as a brownish solid. A soln. of crude 16 in MeOH (3 ml) was treated with activated
charcoal and stirred for 20 min. Filtration, evaporation, and lyophilisation yielded 16 (43.2 mg, 91%). White
solid. Rf (AcOEt/MeOH/H2O 7:1 :1) 0.11. a[ ]2D5 ˆ À20.6 (c ˆ 1.02, MeOH). pKha ˆ 5.03. UV (MeOH): 221
(3.63). IR (0.6% in KBr): 3600 ± 2400s (br.), 2955m, 2902m, 1716s, 1638m, 1571w, 1440m, 1407m, 1359m, 1312m,
1226m, 1183m, 1105m, 1066m, 1024m, 940w, 906w, 870w. 1H-NMR (CD3OD, 300 MHz): see Table 4;
additionally, 3.66 (br. s, CH2ÀC(2)); 3.67 (irrad. at 3.83 ! change, irrad. at 4.45 ! d, J ꢁ 8.4); 3.73 (s, MeO);
3.83 (irrad. at 3.67 ! d, J ꢁ 8.7); 3.84 ± 3.94 (irrad. at 4.15 ! change); 3.93 (irrad. at 4.15 ! d, J ꢁ 3.4); 4.15
(irrad. at 3.93 ! d, J ꢁ 2.0); 4.55 (irrad. at 3.67 ! s). 13C-NMR (CD3OD, 75 MHz): see Table 5; additionally,
‡
33.67 (t, CH2ÀC(2)); 52.98 (q, MeO); 174.19 (s, CˆO). HR-MALDI-MS: 295.0898 (89, [M ‡ Na] ,
‡
‡
‡
C11H16N2NaO6 ; calc. 295.0906); 273.1078 (100, [M ‡ H] , C11H17N2O6 ; calc. 273.1086); 255.0967 (6, [M À
‡
‡
OH] , C11H15N2O5 ; calc. 255.0981).
(5R,6R,7S,8S)-5,6,7,8-Tetrahydro-6,7,8-trihydroxy-5-(hydroxymethyl)imidazo[1,2-a]pyridine-2-acetic Acid
(17). A soln. of 16 (20 mg, 73.5 mmol) in 0.4m soln. of KOH in EtOH/H2O 4 :1(1ml) was heated at 50 8 for
1h and evaporated. The residue was taken up in H 2O (3 ml) and applied to ion-exchange chromatography
‡
(Amberlite CG-120, H form, elution with 0.1m aq. NH3). Evaporation and lyophilisation gave 17 (16.3 mg,
86%). Colourless hygroscopic resin. Rf (AcOEt/MeOH/H2O 3 :1:1) 0.09. [ a]2D5 ˆ À27.8 (c ˆ 0.76, H2O). pKha
ˆ
6.40 (no additional pK value was observed in the pH range 2.9 ± 7.9). UV (H2O): 224 (3.62). IR (0.3% in KBr):
3600 ± 2400s (br.), 2953m, 2920m, 2852w, 1575s, 1506m, 1459m, 1390s, 1327m, 1273m, 1197w, 1178w, 1106m,
1070m, 1019m, 904w, 873w, 737m. 1H-NMR (D2O, 300 MHz): see Table 4; additionally, 3.43 (br. s, CH2ÀC(2)).
13C-NMR (D2O, 75 MHz): see Table 5; additionally, 36.56 (t, CH2ÀC(2)); 179.47 (s, CˆO). HR-MALDI-MS:
‡
‡
‡
‡
281.0743 (45, [M ‡ Na] , C10H14N2NaO6 ; calc. 281.0750), 259.0927 (100, [M ‡ H] , C10H15N2O6 ; calc.
‡
‡
259.0930), 227.0656 (30, [M À CH2OH] , C9H11N2O5 ; calc. 227.0668).
Methyl (5R,6R,7S,8R)-5,6,7,8-Tetrahydro-6,7,8-trihydroxy-5-(hydroxymethyl)imidazo[1,2-a]pyridine-2-
acetate (20). A soln. of 28 (90 mg, 0.142 mmol) in AcOEt/MeOH/AcOH 1:1 :1 (2.1 ml) was treated with
10% Pd/C (60 mg), hydrogenated at 6 bar and 228 for 40 h, and filtered over Celite (washing with 30 ml of
MeOH/H2O 9 :1). Evaporation of the combined filtrate, washing, FC (AcOEt/MeOH/H2O 1:0 :0 ! 1 5 :1 :1!

3046
Helvetica Chimica Acta ± Vol. 87 (2004)
7:1:1), and lyophilisation gave 20 (33.5 mg, 87%). White solid. Rf (AcOEt/MeOH/H2O 7:1 :1) 0.13.
[a]2D5 ˆ À19.0 (c ˆ 1.01, MeOH). pKha ˆ 4.61. UV (MeOH): 221 (3.67). IR (0.3% in KBr): 3600 ± 2400s (br.),
2954m, 2924m, 2852w, 1731s, 1636w, 1568w, 1510w, 1457m, 1439m, 1407m, 1354m, 1263m, 1215m, 1177m, 1092m,
1063m, 1019m, 902w, 834w. 1H-NMR (CD3OD, 300 MHz): see Table 6; additionally, 3.61(br. s, CH2ÀC(2)); 3.69
(s, MeO); 3.83 (irrad. at 4.12 ! d, J ꢁ 3.4, irrad. at 4.82 ! d, J ˆ 8.7); 3.86 ± 3.93 (irrad. at 4.12 ! change); 4.12
(irrad. at 3.83 ! d, J ꢁ 6.8); 4.82 (irrad. at 3.83 ! s). 13C-NMR (CD3OD, 75 MHz): see Table 5; additionally,
‡
34.14 (t, CH2ÀC(2)); 52.58 (q, MeO); 173.13 (s, CˆO). HR-MALDI-MS: 295.0899 (100, [M ‡ Na] ,
‡
‡
‡
C11H16N2NaO6 ; calc. 295.0906); 273.1086 (94, [M ‡ H] , C11H17N2O6 ; calc. 273.1086).
(5R,6R,7S,8R)-5,6,7,8-Tetrahydro-6,7,8-trihydroxy-5-(hydroxymethyl)imidazo[1,2-a]pyridine-2-acetic Acid
(21). A soln. of 20 (15 mg, 55.1 mmol) in 0.4m soln. of KOH in EtOH/H2O 4 :1(1ml) was heated at 50 8 for 1h,
evaporated. The residue was taken up in H2O (3 ml) and applied to ion-exchange chromatography (Amberlite
‡
CG-120, H form, elution with 0.1m aq. NH3). Evaporation and lyophilisation gave 21 (11.5 mg, 81%) as a
colourless hygroscopic resin containing substantial amounts of H2O. The sample for microanalysis was dried for
8 d at 10À4 Torr. Rf (AcOEt/MeOH/H2O 3 :1:1) 0.07. [ a]D25 ˆ À20.2 (c ˆ 0.52, H2O). pKha ˆ 5.08 (no additional
pK value was observed in the pH range 2.6 ± 5.4). UV (H2O): 225 (3.76). IR (0.4% in KBr): 3600 ± 2400s (br.),
2918m, 1583s, 1458m, 1385s, 1271m, 1168m, 1096m, 1069s, 1015m, 903m, 827w, 757m. 1H-NMR (D2O, 300 MHz):
see Table 6; additionally, 3.62 (br. s, CH2ÀC(2)). 13C-NMR (D2O, 75 MHz): see Table 5; additionally, 34.05 (t,
‡
‡
CH2ÀC(2)); 176.77 (s, CˆO). HR-MALDI-MS: 281.0743 (57, [M ‡ Na] , C10H14N2NaO6 ; calc. 281.0750),
‡
‡
259.0926 (100, [M ‡ H] , C10H15N2O6 ; calc. 259.0930). Anal. calc. for C10H14N2O6 ´ H2O (276.24): C 43.48, H
5.84, N 10.14; found: C 43.16, H 5.60, N 10.20.
Methyl (5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-5,6,7,8-tetrahydroimidazo[1,2-a]pyri-
dine-2-carboxylate (30). A suspension of 29 (144 mg, 0.245 mmol), MnO2 (550 mg, 6.33 mmol, Aldrich
21,764 ± 6) and NaCN (70 mg, 1.43 mmol) in MeOH (8 ml) was treated with AcOH (40 ml, 0.699 mmol), stirred
at 228 for 3 h, and filtered through Celite (washing with 20 ml of MeOH and 20 ml of AcOEt). After
evaporation, a soln. of the residue in AcOEt (30 ml) was washed with H2O (20 ml) and brine (20 ml), dried
(Na2SO4), filtered, and evaporated. FC (hexane/AcOEt 2 :1 ! 1:1) gave 29 (8.8 mg, 6%) and 30 (116 mg,
77%).
Data of 29: see [12].
Data of 30: Colourless oil. Rf (hexane/AcOEt 1:1) 0.31. [ a]2D5 ˆ ‡40.0 (c ˆ 1.03, CHCl3). UV (CHCl3): 244
(4.00). IR (CHCl3): 3159w, 3090w, 3067w, 3031m, 3012m, 2952m, 2914w, 2869m, 1952w, 1876w, 1810w, 1716s,
1602w, 1585w, 1549m, 1497w, 1454m, 1424w, 1361m, 1329m, 1250m, 1167m, 1096s, 1069s, 1028m, 1011m, 942w,
1
910w. H-NMR (CDCl3, 300 MHz): see Table 4; additionally, 3.77 (irrad. at 4.27 ! d, J ˆ 10.3); 3.83 (irrad. at
4.16 ! d, J ꢁ 6.5, irrad. at 4.27 ! d, J ꢁ 5.6); 3.87 (irrad. at 4.27 ! d, J ˆ 10.6); 3.92 (s, MeO); 4.16 (irrad. at
3.83 ! d, J ˆ 4.4); 4.456 (d, J ˆ 11.2, PhCH); 4.459 (d, J ˆ 12.8, PhCH); 4.51( d, J ˆ 12.1, PhCH); 4.59 (d, J ˆ
11.5, PhCH); 4.74 (d, J ꢁ 11.2, PhCH); 4.75 (d, J ˆ 11.5, PhCH); 4.78 (d, J ˆ 11.5, PhCH); 4.80 (irrad. at 4.16 !
s); 5.02 (d, J ˆ 11.5, PhCH); 7.14 ± 7.20 (m, 2 arom. H); 7.24 ± 7.40 (m, 18 arom. H). 13C-NMR (CDCl3, 75 MHz):
see Table 5; additionally, 51.64 (q, MeO); 72.11, 73.14, 73.25, 73.47 (4t, 4 PhCH2); 127.44 ± 128.39 (several d);
‡
136.81, 137.13, 137.19, 137.69 (4s); 163.17 (s, CˆO). HR-MALDI-MS: 641 .2629 (1 00M, [‡ Na] ,
‡
‡
‡
C38H38N2NaO6 ; calc. 641.2627), 619.2818 (73, [M ‡ H] , C38H39N2O6 ; calc. 619.2808), 511.2213 (52, [M À
‡
‡
‡
‡
BnO] , C31H31N2O5 ; calc. 511.2233), 479.1958 (22, [M À BnO À MeOH] , C30H27N2O4 ; calc. 479.1971). Anal.
calc. for C38H38N2O6 (618.72): C 73.77, H 6.19, N 4.53; found: C 73.57, H 6.45, N 4.31.
Methyl (5R,6R,7S,8S)-5,6,7,8-Tetrahydro-6,7,8-trihydroxy-5-(hydroxymethyl)imidazo[1,2-a]pyridine-2-car-
boxylate (14). A soln. of 30 (98 mg, 0.158 mmol) in AcOEt/MeOH/AcOH 1:1:1(2.4 ml) was treated with 10%
Pd/C (100 mg), hydrogenated at 6 bar and 228 for 46 h, and filtered over Celite (washing with 20 ml of MeOH/
H2O 9 :1). Evaporation of the combined filtrate and washing, co-evaporation with toluene (3 Â 5 ml), and FC
(AcOEt/MeOH/H2O 1 :0 :0! 10 :1:1) gave crude 14 (43 mg) as a brownish solid. A soln. of crude 14 in MeOH
(3 ml) was treated with activated charcoal and stirred for 10 min. Filtration and evaporation yielded 14
(39.0 mg, 95%). White solid. Rf (AcOEt/MeOH/H2O 10 :1:1) 0.15. [a]2D5 ˆ À37.7 (c ˆ 1.01, MeOH). UV
(MeOH): 239 (3.77), 202 (3.51). IR (0.5% in KBr): 3600 ± 2400s (br.), 2949w, 2923w, 1693m, 1640m, 1561w,
1447w, 1373w, 1340w, 1233m, 1104m, 1066w, 1012m, 943w, 904w, 864w, 772w. 1H-NMR (CD3OD, 300 MHz): see
Table 4; additionally, 3.78 (irrad. at 3.85 ! change, irrad. at 4.58 ! d, J ˆ 9.3); 3.85 (irrad. at 3.78 ! change);
3.91( s, MeO); 3.96 (irrad. at 4.20 ! change); 3.98 ± 4.04 (irrad. at 3.85 ! change, irrad. at 4.20 ! dd, J ꢁ 4.4,
7.5); 4.58 (irrad. at 3.78 ! s). 1H-NMR (D2O, 300 MHz): see Table 4; additionally, 3.83 (irrad. at 3.96 ! change,
irrad. at 4.64 ! d, J ˆ 9.7); 3.89 (s, MeO); 3.96 (irrad. at 3.83 ! change); 4.07 ± 4.12 (irrad. at 3.96 ! change,
irrad. at 4.26 ! change); 4.09 (irrad. at 4.26 ! change); 4.64 (irrad. at 3.83 ! s). 13C-NMR (CD3OD, 75 MHz):

Helvetica Chimica Acta ± Vol. 87 (2004)
3047
‡
see Table 5; additionally, 52.89 (q, MeO); 166.05 (s, CˆO). HR-MALDI-MS: 281.0744 (44, [M ‡ Na] ,
‡
‡
‡
C10H14N2NaO6 ; calc. 281.0750), 259.0926 (100, [M ‡ H] , C10H15N2O6 ; calc. 259.0930).
(5R,6R,7S,8S)-5,6,7,8-Tetrahydro-6,7,8-trihydroxy-5-(hydroxymethyl)imidazo[1,2-a]pyridine-2-carboxylic
Acid (15). A soln. of 14 (20 mg, 77.5 mmol) in 0.4m soln. of KOH in EtOH/H2O 4 :1(1ml) was kept at 50 8 for
210 min and evaporated. The residue was taken up in H2O (3 ml) and applied to ion-exchange chromatography
‡
(Amberlite CG-120, H form, elution with 0.1m aq. NH3). Evaporation and lyophilisation gave 15 (15.2 mg,
80%). Colourless hygroscopic resin. Rf (AcOEt/MeOH/H2O 3 :1:1) 0.08. [ a]2D5 ˆ À72.3 (c ˆ 0.59, H2O). pKha
ˆ
4.95 (no additional pK value was observed in the pH range 2.2 ± 5.5). UV (H2O): 226 (3.94), 192 (4.02). IR
(0.2% in KBr): 3600 ± 2400s (br.), 2918m, 2852m, 1620m, 1574s, 1544m, 1401s, 1331m, 1269m, 1180w, 1106m,
1070m, 1029m, 908w, 869w, 782m. 1H-NMR (D2O, 300 MHz): see Table 4. 13C-NMR (D2O, 75 MHz): see
‡
‡
Table 5; additionally, 169.46 (s, CˆO). HR-MALDI-MS: 245.0768 (100, [M ‡ H] , C9H13N2O6 ; calc. 245.0774),
‡
‡
290.0412 (31, [M ‡ 2 Na] , C9H12N2Na2O6 ; calc. 290.0491).
Inhibition Studies. Determination of the inhibition constants (Ki) or the IC50 values was performed with a
range of inhibitor concentrations (typically 4 ± 7 concentrations) that bracket the Ki or IC50 value, and substrate
concentrations that bracket the KM of each enzyme (for Ki, typically 5 ± 7 concentrations), or correspond to it
(for IC50).
a) Inhibition of Caldocellum saccharolyticum b-Glucosidase. KM ˆ 0.73 ± 0.81m m. ([56]: KM ˆ 0.51m m;
[57]: KM ˆ 0.51± 0.78 m m). Inhibition constants (Ki) and IC50 values were determined at 558 at an enzyme
concentration of 0.005 units/ml, with a 0.08m KH2PO4/K2HPO4 buffer (pH 6.8) and 4-nitrophenyl b-d-
glucopyranoside as the substrate. The enzymatic reaction was started after incubation of the enzyme (150 ml) in
presence of the inhibitor (40 ml) during 1h at 55 8 by the addition of the substrate (10 ml). The enzyme reaction
was quenched by addition of 0.2m borate buffer (pH 9.0, 100 ml) after 30 min, and the absorption at 405 nm was
taken as rate for the hydrolysis of the substrate after subtraction of the absorption of a blank probe (H2O, buffer,
substrate). IC50 Values were determined by plotting the reciprocal value of the rate of substrate hydrolysis vs.
the inhibitor concentration. After fitting a straight line to the data by linear regression, the negative [I]-intercept
of this plot provided the appropriate IC50 value. Ki Values were determined by taking the slopes from the
Lineweaver ± Burk plots [58] and plotting them vs. the inhibitor concentrations [59]. After fitting a straight line
to the data by linear regression, the negative [I]-intercept of this plot provided the appropriate Ki value. a
Values were determined by plotting the 1/v axis intercepts from the Lineweaver ± Burk plots vs. the inhibitor
concentrations [59]. After fitting a straight line to the data by linear regression, the negative [I]-intercept of this
plot provided the appropriate a ´ Ki value.
b) Inhibition of Brewerꢀs Yeast a-Glucosidase. KM ˆ 0.21m m. ([57]: KM ˆ 0.18 ± 0.25 mm). Inhibition
constants (Ki) and IC50 values were determined at 378 at an enzyme concentration of 0.13 units/ml, with a 0.08m
KH2PO4/K2HPO4 buffer (pH 6.8) and 4-nitrophenyl a-d-glucopyranoside as the substrate. The enzymatic
reaction was started after incubation of the enzyme (150 ml) in presence of the inhibitor (40 ml) during 1h at 37 8
by the addition of the substrate (10 ml). The increase of absorption per min at 405 nm was taken as rate for the
hydrolysis of the substrate. The increase was linear during 10 min. IC50 Values were determined by plots as
described in a.
c) Inhibition of Snail b-Mannosidase. KM ˆ 0.55 ± 0.57 mm ([14]: KM ˆ 0.42 ± 0.80 mm). IC50 and Ki values
were determined at 258 at an enzyme concentration of 0.048 units/ml, with a 0.04m acetate buffer (pH 4.5) and
4-nitrophenyl b-d-mannopyranoside as the substrate. The enzymatic reaction was started after incubation of the
enzyme (100 ml) in presence of the inhibitor (50 ml) during 1h at 25 8 by the addition of the substrate (50 ml). The
enzyme reaction was quenched by addition of 0.2m borate buffer (pH 9.0, 100 ml) after 5 min, and the
absorption at 405 nm was taken as rate for the hydrolysis of the substrate after subtraction of the absorption of a
blank probe (H2O, buffer, substrate). IC50, Ki , and a values were determined by plots as described in a.
d) Inhibition of Jack Beans a-Mannosidase. KM ˆ 2.11 ± 2.13 mm ([60]: KM ˆ 2.5 mm; [1 4]:KM ˆ 1.8 ±
2.8 mm). As described in c, inhibition studies were carried out at 378 at an enzyme concentration of 0.086
units/ml, with a 0.04m acetate buffer (pH 4.5), containing 1.5 mmol of ZnCl2 and 4-nitrophenyl a-d-
mannopyranoside as the substrate. The enzymatic reaction was started after the incubation at 378 for 1h.
REFERENCES
[1] T. Aoyama, H. Naganawa, H. Suda, K. Uotani, T. Aoyagi, T. Takeuchi, J. Antibiot. 1992, 45, 1557.
[2] T. Aoyagi, H. Suda, K. Uotani, F. Kojima, T. Aoyama, K. Horiguchi, M. Hamada, T. Takeuchi, J. Antibiot.
1992, 45, 1404.

3048
Helvetica Chimica Acta ± Vol. 87 (2004)
[3] K. Tatsuta, S. Miura, Tetrahedron Lett. 1995, 36, 6721.
[4] K. Tatsuta, S. Miura, H. Gunji, Bull. Chem. Soc. Jpn. 1997, 70, 427.
[5] K. Tatsuta, S. Miura, S. Ohta, H. Gunji, J. Antibiot. 1995, 48, 286.
[6] K. Tatsuta, S. Miura, S. Ohta, H. Gunji, Tetrahedron Lett. 1995, 36, 1085.
[7] T. Granier, N. Panday, A. Vasella, Helv. Chim. Acta 1997, 80, 979.
[8] T. D. Heightman, A. Vasella, K. E. Tsitsanou, S. E. Zographos, V. T. Skamnaki, N. G. Oikonomakos, Helv.
Chim. Acta 1998, 81, 853.
[9] T. D. Heightman, A. T. Vasella, Angew. Chem., Int. Ed. 1999, 38, 750.
[10] F. Gessier, T. Tschamber, C. Tarnus, M. Neuburger, W. Huber, J. Streith, Eur. J. Org. Chem. 2001, 4111.
[11] N. Panday, A. Vasella, Synthesis 1999, 1459.
[12] N. Panday, Y. Canac, A. Vasella, Helv. Chim. Acta 2000, 83, 58.
[13] T. Granier, F. Gaiser, L. Hintermann, A. Vasella, Helv. Chim. Acta 1997, 80, 1443.
[14] M. Terinek, A. Vasella, Helv. Chim. Acta 2003, 86, 3482.
[15] A. Vasella, G. J. Davies, M. Böhm, Curr. Opin. Chem. Biol. 2002, 6, 619.
[16] M. Böhm, E. Lorthiois, M. Meyyappan, A. Vasella, Helv. Chim. Acta 2003, 86, 3787.
[17] M. Böhm, E. Lorthiois, M. Meyyappan, A. Vasella, Helv. Chim. Acta 2003, 86, 3818.
[18] N. Panday, M. Meyyappan, A. Vasella, Helv. Chim. Acta 2000, 83, 513.
[19] S. J. Williams, R. Hoos, S. G. Withers, J. Am. Chem. Soc. 2000, 122, 2223.
[20] A. Peer, A. Vasella, Helv. Chim. Acta 1999, 82, 1044.
Â
Â
Â
Â
Â
[21] G. Abraham, S. Solyom, E. Csuzdi, P. Berzsenyi, I. Ling, I. Tarnawa, T. Hamori, I. Pallagi, K. Horvath, F.
Â
Â
Â
Â
Andrasi, G. Kapus, L. G. Harsing Jr., I. Kiraly, M. Patthy, G. Horvath, Bioorg. Med. Chem. 2000, 8, 2127.
[22] M. Terinek, A. Vasella, Helv. Chim. Acta 2004, 87, 719.
[23] T. Mindt, U. Michel, F. Dick, Helv. Chim. Acta 1999, 82, 1960.
[24] S. Saito, K. Hatanaka, H. Yamamoto, Org. Lett. 2000, 2, 1891.
[25] S. Saito, K. Hatanaka, H. Yamamoto, Tetrahedron 2001, 57, 875.
[26] F. A. Davis, R. T. Reddy, R. E. Reddy, J. Org. Chem. 1992, 57, 6387.
Â
[27] J. J. Jiang, K. K. Schumacher, M. M. Joullie, F. A. Davis, R. E. Reddy, Tetrahedron Lett. 1994, 35, 2121.
[28] F. A. Davis, R. E. Reddy, J. M. Szewczyk, J. Org. Chem. 1995, 60, 7037.
[29] F. A. Davis, J. M. Szewczyk, Tetrahedron Lett. 1998, 39, 5951.
[30] T. P. Tang, J. A. Ellman, J. Org. Chem. 1999, 64, 1 2.
[31] Y. Lee, R. B. Silverman, Org. Lett. 2000, 2, 303.
[32] F. A. Davis, T. N. Fang, B. Chao, D. M. Burns, Synthesis 2000, 2106.
[33] F. A. Davis, B. Chao, T. N. Fang, J. M. Szewczyk, Org. Lett. 2000, 2, 1041.
[34] Y. Lee, R. B. Silverman, Tetrahedron 2001, 57, 5339.
[35] T. P. Tang, J. A. Ellman, J. Org. Chem. 2002, 67, 7819.
[36] K. Lee, M. C. Zhang, D. J. Yang, T. R. Burke, Bioorg. Med. Chem. Lett. 2002, 12, 3399.
[37] L. Huang, L. S. Brinen, J. A. Ellman, Bioorg. Med. Chem. 2003, 11, 21 .
[38] F. A. Davis, Y. Zhang, G. Anilkumar, J. Org. Chem. 2003, 68, 8061.
[39] F. A. Davis, B. Yang, J. H. Deng, J. Org. Chem. 2003, 68, 5147.
[40] F. M. Guerra, M. R. Mish, E. M. Carreira, Org. Lett. 2000, 2, 4265.
[41] M. Lubben, B. L. Feringa, Tetrahedron: Asymmetry 1991, 2, 775.
[42] J. A. Secrist III, C. J. Hickey, R. E. Norris, J. Org. Chem. 1977, 42, 525.
[43] A. F. Abdel-Magid, C. A. Maryanoff, K. G. Carson, Tetrahedron Lett. 1990, 31, 5595.
[44] A. F. Abdel-Magid, C. A. Maryanoff, Synlett 1990, 537.
[45] A. F. Abdel-Magid, K. G. Carson, B. D. Harris, C. A. Maryanoff, R. D. Shah, J. Org. Chem. 1996, 61, 3849.
[46] E. J. Corey, N. W. Gilman, B. E. Ganem, J. Am. Chem. Soc. 1968, 90, 5616.
[47] A. M. Davis, S. J. Teague, Angew. Chem., Int. Ed. 1999, 38, 736.
[48] E. Dubost, T. Tschamber, J. Streith, Tetrahedron Lett. 2003, 44, 3667.
[49] S. Vonhoff, T. D. Heightman, A. Vasella, Helv. Chim. Acta 1998, 81, 1710.
[50] P. Greenberg, A. H. Merrill, D. C. Liotta, G. A. Grabowski, Biochim. Biophys. Acta 1990, 1039, 1 2.
[51] G. Legler, M.-T. Finken, S. Felsch, Carbohydr. Res. 1996, 292, 91 .
[52] G. Legler, M.-T. Finken, Carbohydr. Res. 1996, 292, 103.
[53] T. M. Wrodnigg, S. G. Withers, A. E. Stütz, Bioorg. Med. Chem. Lett. 2001, 11, 1063.
[54] A. Hermetter, H. Scholze, A. E. Stütz, S. G. Withers, T. M. Wrodnigg, Bioorg. Med. Chem. Lett. 2001, 11,
1339.

Helvetica Chimica Acta ± Vol. 87 (2004)
3049
[55] T. M. Wrodnigg, F. Diness, C. Gruber, H. Häusler, I. Lundt, K. Rupitz, A. J. Steiner, A. E. Stütz, C. A.
Tarling, S. G. Withers, H. Wölfler, Bioorg. Med. Chem. 2004, 12, 3485.
[56] A. R. Plant, J. E. Oliver, M. L. Patchett, R. M. Daniel, H. W. Morgan, Arch. Biochem. Biophys. 1988, 262,
181.
[57] C. Blüchel, C. V. Ramana, A. Vasella, Helv. Chim. Acta 2003, 86, 2998.
[58] H. Lineweaver, D. Burk, J. Am. Chem. Soc. 1934, 56, 658.
[59] I. H. Segel, ꢁEnzyme Kineticsꢀ, Wiley, New York, 1975.
[60] Y. T. Li, J. Biol. Chem. 1967, 242, 5474.
Received September 30, 2004