A selective α-L-fucosidase inhibitor based on an aminocyclopentane framework

Article abstract of DOI:10.1002/(SICI)1522-2675(19990505)82:5<760::AID-HLCA760>3.0.CO;2-S

(1R,2R,3R,4S,5R)-4-amino-5-methylcyclopentane-1,2,3-triol (1) was prepared stereoselectively from D-ribose (Scheme). Aminocyclopentanetriol 1, which by its design may be considered an analog of the fucosyl cation, inhibits α-L-fucosidase selectively (K(i)

Full text of DOI:10.1002/(SICI)1522-2675(19990505)82:5<760::AID-HLCA760>3.0.CO;2-S

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Helvetica Chimica Acta ± Vol. 82 (1999)
A Selective a-l-Fucosidase Inhibitor Based
on an Aminocyclopentane Framework
by Adrian Blaser and Jean-Louis Reymond*
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern
(1R,2R,3R,4S,5R)-4-amino-5-methylcyclopentane-1,2,3-triol (1) was prepared stereoselectively from d-
ribose (Scheme). Aminocyclopentanetriol 1, which by its design may be considered an analog of the fucosyl
cation, inhibits a-l-fucosidase selectively (Ki ˆ 28 mm) over a- and b-glucosidase, a- and b-mannosidase, and a-
and b-galactosidase (Table).
Introduction. ± In conjunction with our efforts towards glycosidase catalytic
antibodies [1], we have become interested in raising a-l-fucosidase catalytic antibodies
[2]. a-l-Fucosidase catalytic antibodies might become useful therapeutic agents for
modifying fucosylated cell-surface glycoconjugates [3] such as Sialyl Lewis X in vivo,
and might serve as a novel therapeutic agent for treating inflammation and cancer.
A number of aza-sugars [4], such as deoxynojirimycin and castanospermine, which
occur as natural products in plants, are potent inhibitors of a variety of glycosidases
(Fig. 1). Inhibitors based on an aminocyclopentane framework have also been reported
[5], such as mannostatine [6] and the closely related chitinase inhibitor trehazoline and
related compounds [7]. Such glycosidase inhibitors have been investigated for treating
diabetes, cancer, viral (HIV) and bacterial infections, and as insecticides [8]. As to their
mechanism of action, these compounds most often act as competitive inhibitors of the
enzymes. Due to their close structural and, in their protonated form, electrostatic
resemblance to the oxycarbonium cation intermediate intervening in the acid-catalyzed
glycosidic cleavage, these compounds are generally believed to act as transition-state-
analog inhibitors of glycosidases.
A b-galactosidase catalytic antibody has recently been obtained by immunization
against a derivative of a pyrrolidine-based glycosidase inhibitor [9], and a b-glucosidase
catalytic antibody was obtained starting with a derivative of deoxynojirimycine [10].
These reports suggest that glycosidase inhibitors might be good starting points for
preparing immunogenic haptens aimed at inducing glycosidase catalytic antibodies. In
view of preparing a-l-fucosidase catalytic antibodies, we report the design, stereo-
selective synthesis, and inhibitory activity of aminocyclopentane 1, whose structure is
related to the known mannosidase inhibitor 2 [11] (Fig. 2). Compound 1 is shown to
inhibit selectively a-l-fucosidase over a series of other glycosidases.
Results. ± Aminocyclopentane 2 has been shown to strongly inhibit a-mannosidase
[10]. We reasoned that the related aminocyclopentane 1 might similarly inhibit a-l-
‡
fucosidase [12]. In essence, compound 1 ´ H mimicks the high-energy fucosyl cation
intervening in the hydrolytic cleavage of fucosides by its substitution pattern as well as

Helvetica Chimica Acta ± Vol. 82 (1999)
761
Fig. 1. Structures of glycosidase inhibitors
Fig. 2. Aminocyclopentanes 1 and 2 and the related glycosyl cations
by its positive charge, and may be considered, by design, a transition-state analog for
the reaction.
Retrosynthetic analysis suggested a stereoselective access to aminocyclopentane-
triol 1 from enone 3 (Scheme). This enone is accessible by a variety of routes [13], from
which we selected the three-step preparation from d-ribose as the most convenient
despite its moderate yield [14]. Epoxidation of 3 with basic hydrogen peroxide gave
epoxy ketone 4 as a single product (MeOH, 508, 1 h). Methylenation (Ph3PˆCH2,
THF, room temp., 1 h) gave epoxy-olefin 5 [15]. This sensitive and volatile product was

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Helvetica Chimica Acta ± Vol. 82 (1999)
immediately aminolyzed (NH3, EtOH, room temp., 3 h) to give amino alcohol 6 by
selective epoxide opening at the allylic position. The amino alcohol 6 was then
converted to the (tert-butoxycarbonyl)-protected (Boc) amino alcohol 7 (Boc2O, aq.
NaHCO3 soln./AcOEt, room temp. 18 h). Hydrogenation (1 atm H2, Pd/C, EtOH,
room temperature 15 h) proceeded stereoselectively to give 8, which was purified as its
benzyl ether 9 (NaH, BnBr, DMF, room temp.). Deprotection (3n aq. HCl, 408, 12 h)
gave the benzylated amino-alcohol 10. Alternatively, hydrogenation of 9 followed by
acidic deprotection gave the free amino alcohol 1.
Scheme. Synthesis of Aminocyclopentanetriol 1 and of 10 from d-Ribose
The structure and configuration of compounds 4 ± 10 are readily established from
the analytical data. In particular, the ꢀantiꢁ-relationship between the epoxy and
isopropylidenedioxy moieties relative to the cyclopentane ring in epoxy ketone 4, as
well as the position and relative configuration of the ring subsituents in amino-alcohols
1 and 10, are established by the 1H-NMR coupling constants and NOE effects between
ring protons (see Exper. Part).
Enzymes were assayed according to standard procedures by following the
hydrolysis of nitrophenyl glycosides spectrophotometrically. The results show that 1
inhibits a-l-fucosidase selectively (Ki ˆ 28 mm), with only a weak cross inhibition of

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763
Table. Inhibition of Glycosidases by Inhibitors 1 and 10a)
1
Inhibitor
Enzyme
U ´ ml
Ki
1
10
1
1
1
1
1
1
bovine-kidney a-l-fucosidase
bovine-kidney a-l-fucosidase
green-coffee-beans a-galactosidase
E. Coli b-galactosidase
yeast a-glucosidase
almond b-glucosidase
0.1
0.1
0.03
0.3
0.01
0.1
2.8 ´ 10 5 mb)
> 10 3 mc)
n.i.d)
n.i.d)
n.i.d)
ca. 10 3 me)
n.i.d)
jack-beans a-mannosidase
snail acetone-powder b-mannosidase
0.2
0.14
n.i.d)
a) 100-ml assays contained the indicated enzyme concentration, 1 mm of inhibitor
1 or 10, and the
corresponding nitrophenyl glycoside (1 mm) in 0.1m HEPES buffer at pH 6.8, at 258. b) Competitive inhibition
constant as determined by Dixon plot of inhibition data (see Fig. 3). c) 10% inhibition with 1 mm 10. d) n.i. ˆ no
inhibition observed with 1 mm 1.e) 50% inhibition with 1 mm 1.
Fig. 3. Dixon plot of inhibition data for aminocyclopentanetriol 1 with bovine-kidney a-l-fucosidase. Measured
~
*
in aq. 100 mm HEPES buffer pH 6.8 at 258 using (750 mm ( ) and 250 mm ( ) 4-nitrophenyl a-l-fucoside as
1
substrate. Vapp is given in milliOD ´ min at 405 nm (see Exper. Part). The two lines intersect on the horizontal line
crossing the vertical axis at 1/Vmax. The x-coordinate of the intersection point gives Ki (1). The measured Michaelis-
Menten constant KM for 4-nitrophenyl a-d-fucopyranoside was 750 mm.
almond b-glucosidase, and no measurable inhibition of five other glycosidases (Table
and Fig. 3). Inhibition of a-l-fucosidase is essentially abolished when the 2-OH group
is blocked as in benzyl ether derivative 10.
Discussion. ± Inhibition of a-l-fucosidase by compound 1 is competitive in nature,
which implies that this inhibitor competes with the substrate for occupancy of the
enzyme active site. As planned, the three OH substituents and the Me substituent of the
cyclopentane match the relative configuration and substitution pattern of the

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Helvetica Chimica Acta ± Vol. 82 (1999)
fucopyranose substrate. Selective inhibition of a-l-fucosidase over six other glyco-
sidases is probably mediated by selective recognition of these substituents by the
enzyme. This hypothesis is further supported by the fact that blocking the 2-OH group
to give benzyl ether 10 abolishes inhibition.
Compound 1 (Ki ˆ 28 mm) is very similar in its inhibition of a-l-fucosidase to
piperidinediol 11 (IC50 ˆ 26 mm), which is also very selective, and, similarily, only cross-
reacts with b-glucosidase (IC50 ˆ 0.5 mm) [12f] (Fig. 4). However both compounds are
much less potent than fuco-deoxynojirimycin 12 (Ki ˆ 2.7 nm) [12d] or its hydroxy-
methyl analog 15 (Ki ˆ 5.6 nm) [12e]. The latter amines are closer analogs of the
fucoside substrate than either 1 or 11. The reaction product l-fucose 13 (Ki ˆ 30 mm)
has been reported to inhibit a-l-fucosidase almost as well as 1, and its amino analog 14
is a micromolar inhibitor of this enzyme (Ki ˆ 0.75 mm) [12d]. Other analogs of a-l-
fucose, such as compounds 16 (Ki ˆ 6 mm) [12a] and 17 (Ki ˆ 5.9 mm) [12b], also inhibit
a-l-fucosidase, which confirms that matching the substitution and configuration of the
a-l-fucoside substrate leads to efficient inhibition of the enzyme. This might not be an
exclusive criterion for design, as suggested by the fact that compounds only distantly
related to a-l-fucose such as 18 also inhibit a-l-fucosidase (Ki ˆ 5.5 mm), albeit with
strongly reduced selectivity compared to other glycosidases (Ki ˆ 16.3 mm for b-
glucosidase) [12e].
‡
Is compound 1 ´ H a transition-state-analog inhibitor of bovine-kidney a-l-
fucosidase? As discussed in detail by Ermert et al. for the case of two micromolar
inhibitors of glucosidases and mannosidases [16], selective inhibition of one over other
glycosidases, as observed here for 1, is an important but insufficient condition for an
inhibitor to act as a transition-state analog. The formal proof requires quantitative
correlation of inhibition with catalytic potency for several diastereoisomeric inhibitor/
substrate pairs and their corresponding enzyme, which is not readily possible in the
‡
fucose series. In the present case, binding of 1 ´ H as transition-state analog would be
expected to involve electrostatic and/or H-bonding interactions between the positively
charged ammonio group in the inhibitor and the negatively charged carboxylate
residues responsible for catalysis in a-l-fucosidase [17], as is generally postulated for
glycosidase inhibitors that are carbohydrate mimics displaying a basic functionality
near the anomeric center [18]. Although it was implicitly planned in our design by
positioning of the primary ammonio group, the relatively weak inhibitory power of
compound 1 towards bovine-kidney a-l-fucosidase suggests that such an interaction
does not take place, or at least not in productive manner, and that 1 might not be a
transition-state-analog inhibitor of bovine-kidney a-l-fucosidase. This is, however, in
our view insufficient evidence to reject the possibility that a similar interaction leading
to productive antibody catalysis might occur in an antibody-antigen interaction
involving immunogenic derivatives of 1 and their corresponding antibodies.
Conclusion. ± In summary, we reported the design, synthesis, and evaluation of a
new selective a-l-fucosidase inhibitor. An efficient and stereoselective synthesis from
d-ribose was devised that might be expanded to produce related glycosidase inhibitors.
We are currently exploring the synthesis of further derivatives of 1, in particular N-
alkylated (4R)-stereoisomers of 1, to serve as immunogenic haptens for inducing a-l-
fucosidase catalytic antibodies.

Helvetica Chimica Acta ± Vol. 82 (1999)
765
Fig. 4. Structure and inhibition data for selected a-l-fucosidase inhibitors
This work was supported by the Swiss National Science Foundation, the Koordinationsgruppe für
Forschungsfragen der Basler chemischen Industrie (KGF), and the Wander Stiftung.
Experimental Part
(1aS,1bS,4aS,5aS)-Tetrahydro-3,3-dimethyl-5H-oxireno[3,4]cyclopenta[1,2-d][1,3]dioxol-5-one (4). To a
soln. of enone 3 (500 mg, 3.22 mmol) in MeOH (20 ml), 30% H2O2 soln. (0.7 ml, 6.44 mmol, 2 equiv.) was
added and the mixture was cooled to 508. Within 15 min, 2m NaOH (675 ml) was added. After 50 min at 408,
the mixture was poured into sat. NH4Cl soln. (30 ml) and extracted (CH2Cl2). The org. layer was washed
(brine), dried (Na2SO4), and evaporated. FC (hexane/AcOEt 5 : 1) yielded 4 (441 mg, 80%). Colourless oil. Rf
(hexane/AcOEt 2 : 1) 0.63. [a]2D0 ˆ‡ 32.8 (c ˆ 0.61, CHCl3). IR (Film): 3479(br.), 3028w, 2992m, 2938m, 1765s,
1456w, 1377s, 1231m, 1208m, 1156m, 1079s, 1005m, 969m, 865m. 1H-NMR (300 MHz, CDCl3): 4.91 (br. d,
J(1b,4a) ˆ 5.52, H C(4a)); 4.44 (br. d, J(1b,4a) ˆ 5.52, H C(1b)); 4.03 (br. d, J(1a,5a) ˆ 2.21, H C(5a)); 3.59
(br. d, J(1a,5a) ˆ 2.21, J(1b,1a) < 1, H C(1a)); 1.42, 1.38 (2 s, Me2C). NOE: H C(1a)/H C(5a), H C(1b)/
H
C(4a); no NOE between H C(1a) and H C(1b), and between H C(4a) and H C(5a). 13C-NMR
(75 MHz, CDCl3): 203.40(s); 114.16(s); 77.37(d); 75.36(d); 57.88(d); 54.29(d); 26.95(q); 24.93(q). EI-MS:
‡
‡
‡
170 (M ), 155 ([M 15] ), 113, 85. HR-EI-MS: 170.0575 (C8H10O4 ; calc. 170.0579). Anal. calc. for C8H10O4:
C 56.47, H 5.92; found: C 56.30, H 6.04.
(1aS,1bR,4aR,5aR)-Tetrahydro-3,3-dimethyl-5-methylidene-5H-oxireno[3,4]cyclopenta[1,2-d][1,3]dioxol
(5). A soln. of methyltriphenylphosphonium bromide/NaH (ꢀInstant Ylidꢁ 1.48 g, 2.4 mmol) in abs. THF (3 ml)

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Helvetica Chimica Acta ± Vol. 82 (1999)
in an oven-dried flask was stirred for 15 min at r.t. Epoxy ketone 4 (400 mg, 2.35 mmol) in abs. THF (2 ml) was
added. After stirring for 1 h at r.t., the mixture was poured into Et2O (60 ml), filtered over Celite, and
evaporated. FC (hexane/AcOEt 5 :1) yielded 5 (359 mg, 91%). Colourless oil. Rf (hexane/AcOEt 5 :1) 0.42.
[a]2D0 ˆ‡ 87.3 (c ˆ 0.395, CHCl3). IR (Film): 3055w, 2928m, 2856w, 1728w, 1585w, 1479w, 1435m, 1374w, 1270w,
1234w, 1158m, 1120m, 1071m, 1028w, 855w. 1H-NMR (300 MHz, CDCl3): 5.62 (d, J ˆ 1.83, 1 H), 5.52 (d, J ˆ
1.11, 1 H, ˆCH2); 4.72, 4.67 (2d, J ˆ 5.52, H C(1b), H C(4a')); 3.83, 3.79 (2d, J ˆ 2.22, H C(1a), H C(5a));
1.42, 1.37 (2s, Me2C). 13C-NMR (75 MHz, CDCl3): 146.75 (s); 117.17 (t); 112.79 (s); 80.32 (d); 78.99 (d); 59.19
‡
‡
(d); 58.97 (d); 27.41 (q); 25.44 (q). EI-MS: 153 ([M 15] ), 111, 81. HR-EI-MS: 153.0552 (C8H9O3
[M 15] ; calc. 153.0552).
,
‡
(3aS,4R,5S,6aR)-5-Amino-tetrahydro-2,2-dimethyl-6-methylidene-4H-cyclopenta-1,3-dioxol-4-ol (6). Ole-
fin 5 (330 mg, 1.96 mmol) was dissolved in EtOH (20 ml), and 25% NH3 soln. (50 ml) was added. The mixture
was stirred for 2 ± 3 h at r.t. and evaporated. FC (CH2Cl2/MeOH 9 :1) yielded 6 (280 mg, 77%). Yellow oil. Rf
(CH2Cl2/MeOH 9 :1) 0.26. [a]2D0 ˆ‡ 18.4 (c ˆ 0.75, CHCl3). IR (Film): 3361(br.), 2990s, 2935s, 1673w, 1590m,
1456m, 1374s, 1270m, 1222m, 1208s, 1157s, 1072s, 995m, 917m, 863s. 1H-NMR (300 MHz, CDCl3): 5.35 (dd, J ˆ
2.19, 1.47, 1 H), 5.26 (t, J ˆ 2.19, 1 H, ˆCH2); 4.90 (dt, J ˆ 7.0, 1.5, H C(6a)); 4.40 (dd, J ˆ 7.0, 4.0, H C(3a));
3.73 (dd, J ˆ 7.35, 3.66, H C(4)); 3.44 (dt, J ˆ 7.74, 2.22, H C(5)); 2.31 (br., NH2, OH); 1.46, 1.34 (2s, Me2C).
13C-NMR (75 MHz, CDCl3): 150.61 (s); 112.32 (s); 111.64 (t); 83.83 (d); 82.03 (d); 78.40 (d); 61.81 (d); 27.15
‡
‡
‡
(q); 24.92 (q). EI-MS: 185 (M ), 170 ([M 15] ), 127, 110, 98. HR-EI-MS: 185.1052 (C9H15NO3 ; calc.
185.1052).
(3aS,4R,5S,6aR)-5-{[(tert-Butoxy)carbonyl]amino}-tetrahydro-2,2-dimethyl-6-methylidene-4H-cyclopenta-
1,3-dioxol-4-ol (ˆ [(3aS,4R,5S,6aR)-Tetrahydro-4-hydroxy-2,2-dimethyl-6-methylidene-4H-cyclopenta-1,3-diox-
ol-5-yl]carbamic Acid tert-Butyl Ester; 7). To a mixture of 6 (320 mg, 1.73 mmol), (Boc)2O (640 mg,
2.9 mmol) and NaHCO3 (440 mg, 5.2 mmol), AcOEt (5 ml) and H2O (5 ml) were added, and the mixture was
stirred overnight at r.t. The aq. phase was extracted with AcOEt (80 ml) and the combined org. layer washed
(brine, 2 Â 15 ml) and evaporated. FC (hexane/AcOEt 2 : 1) yielded 7 (493 mg, quant.). White solid. M.p. 102 ±
1048. Rf (hexane/AcOEt 2 : 1) 0.17. [a]2D0 ˆ‡ 5.3 (c ˆ 0.48, CHCl3). IR (Film): 3443(br.), 2983s, 2935m, 1694s,
1504s, 1456m, 1368s, 1253m, 1207m, 1161s, 1055s, 1021m, 861m. 1H-NMR (300 MHz, CDCl3): 5.42, 5.34 (2s,
ˆCH2); 5.07 (br. d, J ˆ 5.88, NH); 4.90 (d, J ˆ 5.88, H C(6a)); 4.45 (dd, J ˆ 6.27, 1.47, H C(3a)); 4.26
(m, H C(5)); 3.99 (dd, J ˆ 4.05, 1.83, H C(4)); 3.81 (br., OH); 1.45, 1.32 (2s, Me2C); 1.42 (s, t-Bu). 13C-NMR
(75 MHz, CDCl3): 155.88 (s); 147.07 (s); 115.43 (t); 111.76 (s); 84.44 (d); 80.21 (d); 80.03 (s); 79.79 (d); 61.29
‡
‡
(d); 28.28 (q); 26.76 (q); 24.54 (q). EI-MS: 285 (M ), 270 ([M 15] ), 229, 214, 185, 171, 154, 127, 110, 98. HR-
‡
EI-MS: 285.1576 (C14H23NO5 ; calc. 285.1576). Anal. calc. for C14H23NO5: C 58.93, H 8.12, N 4.91; found:
C 58.80, H 8.15, N 4.94.
(3aS,4R,5S,6R,6aR)-5-{[(tert-Butoxy)carbonyl]amino}tetrahydro-2,2,6-trimethyl-4H-cyclopenta-1,3-dioxol-
4-ol (ˆ [(3aS,4R,5S,6R,6aR)-Tetrahydro-4-hydroxy-2,2,6-trimethyl-4H-cyclopenta-1,3-dioxol-5-yl]carbamic
Acid tert-Butyl Ester; 8). To a soln. of 7 (195 mg, 0.68 mmol) in EtOH (5 ml), 10% Pd/C (5 mg) was added.
The flask was evacuated and filled with H2 (3Â). After stirring overnight, the mixture was filtered over Celite
and evaporated. FC (hexane/AcOEt 2 : 1) yielded 8 (151 mg, 77%). Colourless oil. Rf (hexane/AcOEt 2 : 1)
0.26. [a]2D0
ˆ
40.3 (c ˆ 0.585, CHCl3). IR (Film): 3443(br.), 2983m, 2936m, 1697s, 1507s, 1456m, 1386m, 1376m,
1368m, 1251m, 1207m, 1162s, 1047s, 994w, 871w. 1H-NMR (300 MHz, CDCl3): 5.22 (d, J ˆ 9.18, NH); 4.58
(t, J ˆ 5.16, H C(6a)); 4.42 (dd, J ˆ 5.52, 1.47, H C(3a)); 4.05 (br. s, H C(4)); 3.76 (dd, J ˆ 9.18, 5.88,
H
C(5)); 3.28 (br. s, OH); 2.52 (m, H C(6)); 1.47, 1.30 (2s, Me2C); 1.42 (s, t-Bu); 1.11 (d, J ˆ 7.35,
Me C(6)). 13C-NMR (75 MHz, CDCl3): 155.84 (s); 110.21 (s); 86.15 (d); 83.15 (d); 79.47 (d); 79.34 (s); 61.50
‡
‡
(d); 37.46 (d); 28.33 (q); 25.85 (q); 23.14 (q); 8.69 (q). EI-MS: 287 (M ), 272 ([M 15] ), 231, 216, 173, 156,
155, 138, 129, 112, 103. HR-EI-MS: 287.1732 (C14H25NO5 ; calc. 287.1733).
‡
[(3aS,4R,5S,6R,6aR)-Tetrahydro-4-hydroxy-2,2,6-trimethyl-4H-cyclopenta-1,3-dioxol-5-yl]carbamic Acid
tert-Butyl Ester (9). A soln. of 8 (30 mg, 0.1 mmol) in abs. DMF (1 ml) was cooled to 148 (ice/acetone
bath). A 55% NaH dispersion (10 mg, 0.23 mmol) was added, and the mixture was stirred for 2 h at 148.
Benzyl bromide (14 ml, 19.7 mg, 0.115 mmol) was added and the mixture stirred for 3 h at r.t. Then, the mixture
was poured into H2O (15 ml) and extracted with AcOEt (3 Â 20 ml). The org. layer was washed with brine (3 Â
20 ml) and evaporated. FC (hexane/AcOEt 5 : 1) yielded 9 (28 mg, 70%). Colourless oil. Rf (hexane/AcOEt
5 : 1) 0.47. [a]D20ˆ 20.7 (cˆ1.015, CHCl3). IR (Film): 3446m, 2981m, 2932m, 1712s, 1498s, 1456m, 1385m,
1367m, 1270w, 1248m, 1207m, 1163s, 1062s, 981w, 871m. 1H-NMR (300 MHz, CDCl3): 7.35 ± 7.28 (m, Ph); 5.20
(d, Jˆ9.18, NH); 4.70 (d, Jˆ11.40, 1 H, PhCH2); 4.60 ± 4.50 (m, 3 H, PhCH2, H C(3a), H C(6a)); 4.04
(dd, Jˆ9.18, 5.88, H C(5)); 3.82 (s, H C(4)); 2.45 (m, H C(6)); 1.49, 1.31 (2s, Me2C); 1.45 (s, t-Bu); 1.15
(d, Jˆ7.35, Me C(6)). 13C-NMR (75 MHz, CDCl3): 155.52 (s); 137.96 (s); 128.34 (d); 127.71 (d); 127.61 (d);

Helvetica Chimica Acta ± Vol. 82 (1999)
767
110.37 (s); 86.98 (d); 84.89 (d); 83.18 (d); 79.03 (s); 71.47 (t); 58.05 (d); 38.01 (d); 28.40 (q); 25.92 (q); 23.22 (q);
‡
‡
8.77 (q). EI-MS: 377 (M ), 362 ([M15] ), 321, 276, 215, 170, 157, 139, 128, 112, 96. HR-EI-MS: 377.2201
‡
(C21H31NO5 ; calc. 377.2202). Anal. calc. for C21H31NO5: C 66.82, H 8.28, N 3.71; found: C 66.87, H 8.24, N 3.79.
(1R,2R,3R,4S,5R)-4-amino-5-methylcyclopentane-1,2,3-triol (1). To a soln. of 9 (40 mg, 0.1 mmol) in EtOH
(3 ml), 10% Pd/C (ca. 5 mg) was added. The flask was evacuated and filled with H2 (3Â). After stirring
overnight, the mixture was filtered over Celite and evaporated. FC (hexane/AcOEt 2 : 1) yielded 8 (27 mg, 88%)
as a colourless oil. The residue was dissolved in H2O (3 ml), and 37% HCl soln. (1 ml) was added. The soln. was
stirred overnight at 458 and evaporated. The product was redissolved in H2O and passed over a DOWEX column
(50 Â 8 cm, sulfonic acid): 1 ´ HCl (10.2 mg, 60%) eluted directly. After washing with H2O and MeOH, elution
with aq. NH3 soln. yielded a second fraction of free 1 (3.7 mg, 27%). Both compounds were white solids after
solvent evaporation and drying.
Data of 1 ´ HCl: [a]D20
ˆ
16.3 (c ˆ 0.51, MeOH).
Data of 1: IR (KBr): 3387s(br.), 3240s, 3156s, 2975s, 2921s, 2361w, 2343w, 1617m, 1578m, 1491s, 1450w,
1378w, 1330m, 1311m, 1292m, 1195w, 1114m, 1063s, 1043m, 992w, 968m. 1H-NMR (300 MHz, D2O): 4.00
(dd, Jˆ6.99, 4.05, H C(2)); 3.74 ± 3.87 (m, H C(1), H C(3)); 3.30 (dd, Jˆ8.10, 3.33, H C(4)); 2.37
(quint. d, Jˆ7.74, 3.33, H C(5)); 0.96 (d, J ˆ7.35, 3 H, Me C(5)). 13C-NMR (50 MHz, CD3OD): 86.15 (d);
‡
80.95 (d); 76.75 (d); 61.29 (d); 38.43 (d); 9.22 (q). EI-MS: 147 (M ), 130, 88, 86, 84, 73, 72. HR-EI-MS: 147.0889
‡
(C6H13NO3 ; calc. 147.0895).
(1R,2R,3R,4S,5R)-4-Amino-3-(benzyloxy)-5-methylcyclopentane-1,2-diol (10). To a soln. of 9 (15 mg,
40 mmol) in CH2Cl2 (5 ml), CF3COOH (5 ml) was added and stirred for 1.5 h at r.t. The solvent was evaporated
the residue dissolved in H2O (14 ml), 1m HCl (1 ml) added, and the mixture stirred for 4 h at r.t. and evaporated.
The product was purified over a DOWEX column (50  8 cm): free 10 (4.9 mg, 52%). White solid. [a]ˆ ‡ 6.0
(cˆ0.15, MeOH). IR (KBr): 3418s(br.), 2925s, 2361w, 2344w, 1616m, 1456w, 1404m, 1097m. 1H-NMR
(300 MHz, D2O): 7.36 (m, Ph); 4.66, 4.59 (2d, Jˆ10.65, PhCH2); 4.04 (m, H C(2), H C(3)); 3.93 (m,
H
C(1)); 3.53 (d, J(4,5)ˆ7.35, H C(4)); 2.38 (quint. d, J(4,5)ˆJ(5,Me)ˆ7.35, J(1,5)ˆ3.30, H C(5)); 1.03
(d, J(5,Me)ˆ7.35, Me C(5)). NOE: only
CD3OD): 129.38 (d); 128.90 (d); 128.80 (d); 90.40 (d); 80.51 (d); 77.36 (d); 73.06 (t); 59.42 (d); 37.61 (d);
9.05 (q). EI-MS: 237 (M ), 222 ([M 15] ), 161, 147, 132, 129, 107, 91. HR-EI-MS: 237.1356 (C13H19NO3
H C(1)/H C(5), H
C(4)/H C(5). 13C-NMR (75 MHz,
‡
‡
‡
;
calc. 237.1365).
Enzyme Measurements. Enzymes were purchased from Fluka and Sigma. The following glycosidases were
assayed: a-glucosidase (yeast, EC 3.2.1.20), b-glucosidase (almonds, EC 3.2.1.21), a-galactosidase (green coffee
beans, EC 3.2.1.22), b-galactosidase (E. coli, EC 3.2.1.23), a-mannosidase (jack beans, EC 3.2.1.24), b-
mannosidase (snail acetone powder, EC 3.2.1.25), and a-l-fucosidase (bovine kidney, EC 3.2.1.51). All buffers
and solns. were prepared using MilliQ-deionized H2O. Enzymes were diluted with 0.1m HEPES (4-(2-
hydroxyethyl)piperazine-1-sulfonic acid) buffer pH 6.8. Substrates and inhibitors were used as 10 mm stock
solns. in buffer.
Reactions were initiated by addition of substrate to a soln. containing enzyme and inhibitor in 0.1m HEPES
buffer: 100-ml assays were followed in individual wells of flat-bottom 96-well half-area polystyrene cell culture
plates (Costar) using a UV Spectramax 250 instrument from Molecular Devices. The release of nitrophenol was
followed at 405 nm over 1 h. The concentration of each enzyme in the assays was adjusted such as to give ca. 0.5
to 2.0 OD increase over 1 h as given by the instrument. The initial rate of reaction of the first 10 min was linear
1
and was used to calculate the rate. Rates were expressed in relative units as milliOD ´ min
.
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Received March 5, 1999