Aminocyclopentitol inhibitors of α-L-fucosidases

Article abstract of DOI:10.1002/1522-2675(20010711)84:7<2119::AID-HLCA2119>3.0.CO;2-8

Aminocyclopentitol analogs of α-L-fucose were synthesized stercoselectively from D-ribose. Alkyl substituents were attached at the NH₂ group to mimic the glycosidic leaving group. The resulting (alkylamino)cyclopentitols inhibited α-L-fucosidases selectively with inhibition constants in the range of K_i = 10^-7 M. Comparisons with stereoisomers and acyclic analogs showed that this inhibition only occurs with N-alkyl substitution and proper configuration at the cyclopentane, as expected for transition-state-analog-type inhibition. These observations were supported by molecular-modeling comparisons between inhibitor and transition state.

Full text of DOI:10.1002/1522-2675(20010711)84:7<2119::AID-HLCA2119>3.0.CO;2-8

Helvetica Chimica Acta ± Vol. 84 (2001)
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Aminocyclopentitol Inhibitors of a-l-Fucosidases
by Adrian Blaser and Jean-Louis Reymond*
Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012 Bern
(fax: ‡‡ 41 31 631 80 57, e-mail: jean-louis.reymond@ioc.unibe.ch)
Aminocyclopentitol analogs of a-l-fucose were synthesized stereoselectively from d-ribose. Alkyl substituents
were attached at the NH2 group to mimic the glycosidic leaving group. The resulting (alkylamino)cyclopentitols
inhibited a-l-fucosidases selectively with inhibition constants in the range of Ki ˆ 10 7 m. Comparisons with
stereoisomers and acyclic analogs showed that this inhibition only occurs with N-alkyl substitution and proper
configuration at the cyclopentane, as expected for transition-state-analog-type inhibition. These observations
were supported by molecular-modeling comparisons between inhibitor and transition state.
Introduction. ± Glycosidase inhibitors can be used for treating diabetes, cancer,
viral (HIV, influenza) and bacterial infections, and as insecticides [1]. In conjunction
with our interest in raising glycosidase catalytic antibodies [2], we have been interested
in designing glycosidase inhibitors that are also transition-state analogs of the glycosidic
bond hydrolysis reaction. In particular, we have investigated the preparation of a-l-
fucosidase inhibitors [3]. Such inhibitors might be used as haptens to prepare a-l-
fucosidase catalytic antibodies [4], which could be useful to hydrolyze fucosylated cell-
surface glycoconjugates [5] such as Sialyl Lewis x in vivo, and thus serve as a novel
therapeutic agent for treating inflammation and cancer. Herein we report the synthesis
and properties of a-l-fucosidase inhibitors 1 ± 4 based on an aminocyclopentitol core
(Fig. 1). Inhibitor 4 can be conjugated to carrier proteins and used as a hapten for
immunizations.
Fig. 1. Aminocyclopentitol inhibitors of a-l-fucosidases
Results. ± Design. Glycosidase inhibitors were first isolated from natural sources,
mostly plants, where they apparently act either as antifeedants or as insecticides [6]. A

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large number of inhibitors have also been synthesized. All of these structures are
generally either considered to be or designed as stable mimics of the oxocarbonium
cation which occurs as an intermediate both in the acid-catalyzed and in the enzyme-
catalyzed glycosidic bond-cleavage reaction. These inhibitors include one or more of
the following structural features: a) a carbo- or heterocycle featuring the substituents of
the glycosidic substrate in the correct relative configuration; b) an sp2 center at the
position corresponding to C(1) of the glycoside substrate, which induces a half-chair
conformation resembling that of the oxocarbonium cation; c) a basic functional group
in a position either corresponding to or in proximity of one or both of the glycosidic O-
atoms. A nonbonding electron pair in this basic functional group, typically at a N-atom,
usually forms a H-bond or a salt bridge with one of the catalytic carboxy groups within
the enzymeꢀs active site. Typical examples include deoxy-nojirimycin 5, isofagomine 6,
and imidazole 7 (Fig. 2) [1c].
Fig. 2. Structure of oxocarbonium-cation intermediate for hydrolysis of d-glucosides and some glycosidase
inhibitors
Recently, we have shown that aminocyclopentitols, which may be considered as
analogs of protonated glycosides, such as ꢁb-galactoꢀ-aminocyclopentitol 8 or ꢁb-glucoꢀ-
aminocyclopentitol 9, are good and selective inhibitors of b-galactosidases and b-
glucosidases, with competitive inhibition constants in the range of Ki ˆ 10 7 m [7]. In
addition to displaying the substitution pattern of the glycoside substrate that other
inhibitors do, the aminocyclopentitols incorporate an additional stereogenic unit at the
NH2-substituted C-atom, whose relative configuration renders the a- or b-anomeric
configuration at the glycosidic bond. This suggests that one should be able to add an
alkyl substituent at that N-atom as a leaving-group mimic with specification of the
anomeric configuration. The resulting inhibitor would mimic the transition state of the
glycosidic bond-cleavage reaction, and not simply the oxocarbonium ion intermediate.

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We decided to test this hypothesis with the example of simple N-alkyl derivatives of an
ꢁa-l-fucoꢀ-configurated aminocyclopentitol, for which we had developed an efficient
synthesis [8].
Synthesis. The core ꢁa-l-fucoꢀ-aminocyclopentitol 10 (see below, Fig. 3) was
prepared from enone 11 according to a synthetic scheme similar to that used previously
for the b-configured analog and with the allylic epoxide 12 as key intermediate
(Scheme 1). Opening of the allylic epoxide 12 with LiBr in AcOH gave bromohydrin 13
Scheme 1. Stereoselective Synthesis of ꢁa-l-fucoꢀ-Aminocyclopentitols

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in good yield. This particular reagent combination was found after trying unsuccessfully
a series of published procedures for opening epoxides with bromide. It should be noted
that the acetonide protecting group of the diol moiety is stable towards AcOH, which
serves as a mild acid to activate the epoxide and as the solvent for the reaction. The OH
group in 13 was then converted to urethane 14 by reaction with benzyl isocyanate. The
CˆC bond was reduced stereoselectively from the sterically less-hindered a-face using
H2 and Wilkinsonꢀs catalyst. This catalyst was mild enough not to reduce the bromo and
N-benzyl functions. Reaction of the resulting intermediate 15 with NaH in THF then
gave tricyclic compound 16, now featuring the target ꢁa-l-fucoꢀ-aminocyclopentitol in
protected form. The carbamate was then saponified to give amine 17. Hydrogenation of
the benzyl group then gave 18.
Amine 17 was hydrolyzed in acid to give 1. Boc-protection of the amine 18 and
subsequent acidic hydrolysis gave the free ꢁa-l-fucoꢀ-aminocyclopentitol 10 (Fig. 3).
Reductive amination of benzeneacetaldehyde and benzenepropanal with amine 18 and
hydrolysis with acid gave the N-(2-phenylethyl) and N-(3-phenylpropyl)-substituted
ꢁa-l-fucoꢀ-aminocyclopentitols 2 and 3. Alternatively, reductive amination of the
functionalized aldehyde 21, prepared in two steps from 4-aminobenzyl alcohol, gave,
after hydrolysis, derivative 4, suitable for conjugation to a protein such as BSA or KLH.
N-Benzyl-substituted ꢁb-l-fucoꢀ-aminocyclopentitol 20 was also prepared by reductive
amination of 19 [3] for comparison purposes. Furthermore, we included (R)- and (S)-
2-aminobutanol (R)- and (S)-22, which we had discovered in a broad screen for
inhibition with commerically available compounds, and which represent truncated
acyclic analogs of 10 and 19, respectively, as well as the corresponding N-benzyl analogs
(R)- and (S)-23 as acyclic analogs of 1 and 20, respectively.
Fig. 3. Structures of aminocyclopentitols and related structures

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Enzyme Inhibition. All deprotected aminocyclopentitols were tested for inhibition
of four different a-l-fucosidases, three of mammalian origin and one of microbial
origin. Assays were conducted in aqueous HEPES buffer at pH 6.8 by means of the
hydrolysis of 4-nitrophenyl a-l-fucoside as a test reaction, under which conditions all
enzymes showed satisfactory activity. Competitive inhibition constants were deter-
mined by measuring the Michaelis-Menten parameter Vmax (in arbitrary absorbancy
units at 405 nm) and KM of each enzyme for the 4-nitrophenyl-a-l-fucoside substrate at
a series of inhibitor concentrations [I]. The Ki value was then obtained as the value of
the x intercept on the
[I] axis when reploting KM/Vmax as a function of inhibitor
concentration (Dixon replot, Fig. 4). The results are shown in the Table.
The free aminocyclopentitols 10 and 19 showed comparable inhibition potencies
with all enzymes. Their inhibition potency was approximately 10-fold stronger than that
of their acyclic analogs 22. The picture looked different with the N-alkylated
compounds. There, the properly configured N-benzyl-substituted ꢁa-l-fucoꢀ-amino-
cyclopentitol 1 showed a quite strong inhibition, with Ki as low as 3 ´ 10 7 m. By contrast,
its b-configured stereoisomer 20 was equally weak or weaker than 19 for all enzymes,
while the acyclic analogs did not inhibit the enzymes at all.
Variation of the spacer between the phenyl group and the aminocyclopentitol in the
a-series allowed further optimization of inhibition potency. The optimum was reached
with the N-(2-phenylethyl) derivative 2, which showed comparably strong potency on
all four enzymes, including the Fucosarium oxysporum 377 a-l-fucosidase, which
tended to be only weakly inhibited by the other derivatives in the series. Derivative 4,
suitable for conjugation to a protein, was also a potent inhibitor of all enzymes.
Fig. 4. Lineweaver-Burk plot and Dixon replot for inhibition of human-placenta a-l-fucosidase by inhibitor 2

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Table 1. Inhibition Data on a-l-Fucosidasesa)
Ki [mm]
bovine kidney
bovine epididymis
human placenta
Fucosarium oxysporum 377
1
2
3
4
0.7
0.4
0.4
0.4
15
0.6
0.3
0.3
0.7
12
0.3
0.09
0.14
0.19
14
6
0.3
3
50
125
10
19
28
18
22
60
20
110
28
18
180
(R)-22
(S)-22
(R)-23
(S)-23
400
200
(13%)
(26%)
350
125
(9%)
(24%)
200
100
(6%)
(26%)
1100
1300
(15%)
(22%)
a) Measured in 0.1m HEPES buffer at pH 6.8, 258, with 4-nitrophenyl a-l-fucoside as substrate. Competitive
inhibition constant Ki as determined by Dixon replot (Fig. 4). For weak inhibitors 23, the number in bracket is
the percent inhibition as measured with 1 mm of the inhibitor and 1 mm substrate.
Discussion. ± The occurrence of a 100-fold selectivity in inhibition of a-l-
fucosidases in the a-configured, N-substituted aminocyclopentitols 1 over its b-
configured stereoisomer 20 is striking. The reinforcing effect of the N-benzyl
substituent on inhibition indeed only occurs with the a-configured cyclopentitol, while
it diminishes the inhibition effect with the b-analog as well as with the closely related 2-
aminobutanols. These results suggest that 1, and perhaps only 1 (and its derivatives 2 ±
4), occupies the enzymeꢀs active site as an analog of the transition state, with the benzyl
group binding in a pocket normally occupied by the glycosidic leaving group.
Binding of the inhibitors to the fucosidase enzyme can be compared with transition-
state binding by means of Paulingꢀs analysis of enzymatic catalysis [9]. The general
chemical mechanism for glycosidic-bond cleavage involves pre-equilibrium protona-
tion of the leaving-group O-atom in a glycosidic substrate SOR (Scheme 2), followed
‡
by heterolytic bond cleavage to generate the oxocarbonium cation S , via a cationic
transition state (TS) [10]. The corresponding enzyme-catalyzed process starts with the
catalytic carboxy group as carboxylate, which is the predominant form at physiological
‡
pH, and binds H and the substrate. Glycosidic-bond cleavage follows, with the
bond scission preceeding leaving-group protonation, so that the transition state
features a partial negative charge on the leaving-group O-atom, with the catalytic
carboxy group still in protonated form next to it [11]. Transition-state binding by
the enzyme is formulated starting with the positively charged chemical transition
state and the enzyme with its catalytic carboxy group as carboxylate, accompanied
by a proton transfer between the leaving groupꢀs O-atom and this carboxylate.
Inhibitor binding should be similar to the transition-state binding event, the only
difference being that the bound inhibitor probably remains as an ion pair with
protonated amino group and negatively charged catalytic carboxy group, without
proton transfer.
This formulation of transition-state binding is reasonable for glycosidases because
the overall molecular geometry of the transition state, in particular the transition-state

Helvetica Chimica Acta ± Vol. 84 (2001)
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Scheme 2. Mechanisms of Acid-Catalyzed (upper row) and Enzyme-Catalyzed (lower row) Cleavage of
‡
Glycoside S
O
R to the Oxocarbonium-Cation Intermediate S and Aglycone ROH, and Inhibitor Binding
bond length and angles, do not change significantly between the chemical and the
enzymatic mechanism. In that situation it is interesting to compare the inhibitors with
the calculated transition state 24 for the acid-catalyzed hydrolysis of phenyl a-l-
fucoside, a model substrate with a leaving group matching the aromatic group chosen
for the inhibitors. A model transition-state can be generated by enforcing an elongated
glycosidic bond in the protonated glycoside. We chose a transition-state bond length of
1.9 Š, as suggested by the calculation of Bürgi and Dubler-Steudle [12]. To allow
structural comparisons, we searched for the most stable conformations in both
transition state 24 and the inhibitors by allowing free rotations of the bonds joining the
phenyl group to the carbohydrate part of the molecules. The most stable conformations
of protonated 1, 2, and 20 and of 24 are shown in Fig. 5, where the molecules are
displayed with their molecular surfaces colored according to the electrostatic potential.
‡
‡
While the weak inhibitor 20 ´ H clearly differs from 24, the potent inhibitors 1 ´ H , 2 ´
‡
H , and the transition state 24 present good similarities in terms of structure and charge
distribution. A good overlap between inhibitor and transition state, as demonstrated
here, does not often occur with glycosidase inhibitors, in particular since most inhibitors
do not incorporate a leaving-group mimic in conjunction with a glycosidic analog

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Fig. 5. Lowest-energy conformations for simulated transition state 24 and the protonated forms of inhibitors 1, 2,
and 20, as calculated with Spartan 5.1 (see Exper. Part). Electronic-density surfaces are colored according to
electrostatic potential ranging from ‡ 12 kcal/mol (red) to ‡ 140 kcal/mol (blue).

Helvetica Chimica Acta ± Vol. 84 (2001)
2127
allowing for an sp3 substitution at the position corresponding to C(1), with specification
of the anomeric configuration1).
Conclusion. ± The experiments above demonstrate a series of a-l-fucosidase
inhibitors based on an aminocyclopentitol framework and incorporating a leaving-
group mimic as an N-alkyl substituent. The structural analogies to the transition state
for a-l-fucoside cleavage, together with the finding that enzyme inhibition occurs
selectively only with the correct configurational pattern, and across four different
fucosidases, provides good qualitative support for the idea that these inhibitors are
transition-state analogs. It must be mentioned that the inhibition constants Ki ˆ 10 7 m
measured are not the best values available in the literature, where several inhibitors
have been reported with Ki ˆ 10 8 m for inhibition of a-l-fucosidases [14]. Derivatives
suitable for conjugation to a protein such as 4 might function as haptens to generate
catalytic antibodies with a-l-fucosidase activity. Immunization experiments are in
progress, and their results will be reported separately.
Ï
We would like to thank Prof. Dr. Vladimír Kren of the Institute of Microbiology, Academy of Sciences of
the Czech Republic, Prague, Czech Republic, for a sample of Fusarium oxysporum 377 a-l-fucosidase. This
work was supported by the University of Bern, the Swiss National Science Foundation, the Wander Stiftung, the
Â
Â
European COST program D13, and the Swiss ꢁOffice Federal de lꢀEducation et de la Scienceꢀ.
Experimental Part
1.
Syntheses.
(1S,2S,3S,4R)-1-Bromo-3,4-O-isopropylidene-5-methylidenecyclopentane-2,3,4-triol
(ˆ(3aS,4S,5S,6aR)-5-Bromotetrahydro-2,2-dimethyl-6-methylidene-4H-cyclopenta-1,3-dioxol-4-ol; 13). Allylic
epoxide 12 (220 mg, 1.31 mmol) and LiBr (568 mg, 6.54 mmol) were dissolved in AcoH (4 ml) and stirred for
4.5 h at r.t. FC (hexane/AcOEt 2 :1; Rf 0.45) and co-evaporation with toluene (3Â) yielded 13 (276 mg, 85%).
Colorless oil. [a]D20
ˆ
54.7 (c ˆ 0.51, CHCl3). IR (Film): 3418s (br.), 3012s, 2990s, 2936s, 1728w, 1456m, 1383s,
1374s, 1344w, 1311w, 1274m, 1243m, 1206s, 1156s, 1073s, 1047s, 997w, 967m, 940w, 909m, 865s, 853m. 1H-NMR
(500 MHz, CDCl3)2): 5.63 (dd, 4J(CH2ˆC(5),1) ˆ 1.6, 4J(CH2ˆC(5),4) ˆ 1.4, 1 H, CH2ˆC(5)); 5.62
(dd, 4J(CH2ˆC(5),1) ˆ 1.6, 4J(CH2ˆC(5),4) ˆ 1.4, 1 H, CH2ˆC(5)); 5.00 (dt, 3J(4,3) ˆ 6.4, 4J(4, CH2ˆC(5) ˆ
1.4, H C(4)); 4.52 ± 4.47 (m, H C(3), H C(1)); 4.38 (dd, 3J(2,1) ˆ 4.5, 3J(2,3) ˆ 2.5, H C(2)); 1.56 (s, 3 H,
Me2C); 1.35 (s, 3 H, Me2C). 13C-NMR (75 MHz, CDCl3)2): 146.94 (s, C(5)); 118.91 (t, CH2ˆC(5)); 112.96
(s, Me2C); 84.83 (d, C(3)); 82.57 (d, C(2)); 79.70 (d, C(4)); 52.22 (d, C(1)); 26.52 (q, Me2C); 24.87 (q, Me2C).
NMR Signals assigned by C,H correlation and homo-decoupling experiments. HR-EI-MS: 232.9813
‡
‡
(C8H10BrO3 , [M Me] ; calc. 232.9813).
(1S,2S,3R,4R)-2-O-(Benzylcarbamoyl)-1-bromo-3,4-O-isopropylidene-5-methylidenecyclopentane-2,3,4-
triol (ˆ(3aR,4S,5S,6aR)-5-Bromotetrahydro-2,2-dimethyl-6-methylidene-4H-cyclopenta-1,3-dioxol-4-yl Benzyl-
carbamate; 14). A soln. of 13 (54 mg, 0.22 mmol) in abs. CH2Cl2 (1.4 ml) was cooled in an ice bath to 08. After
adding Et3N (12 ml, 0.087 mmol) and benzyl isocyanate (31 ml, 0.36 mmol), the mixture was stirred at 08 for 5 h,
then diluted in Et2O (50 ml), and washed with H2O. The aq. phase was extracted (Et2O) and the combined org.
layer washed (brine) and evaporated. FC (hexane/AcOEt 2 :1; Rf 0.46) yielded 14 (85 mg, quant.). Yellow oil.
[a]2D0
ˆ
13.5 (c ˆ 0.46, CHCl3). IR (Film): 3381s (br.), 2989s, 2934s, 2360w, 1806w, 1727s, 1513s, 1455m, 1432w,
1379s, 1336m, 1286m, 1252s, 1232s, 1205s, 1162m, 1123m, 1085s, 1070s, 1035s, 1017s, 994m, 932m, 864m. 1H-NMR
(300 MHz, CDCl3)2): 7.4 ± 7.2 (m, PhCH2); 5.61 (d, 2J ˆ 4.1, CH2ˆC(5)); 5.31 (m, H C(2)); 4.99 (br. d, J ˆ 6.24,
H
C(3), NH); 4.60 (m, H C(4), H C(1)); 4.39 (d, 2J ˆ 5.9, PhCH2); 1.61 (s, 3 H, Me2C); 1.34 (s, 3 H, Me2C).
13C-NMR (75 MHz, CDCl3)2): 154.58 (s, CˆO); 147.99 (s, C(5)); 137.91 (s, arom. C); 128.75 (d, arom. C); 127.71
1
)
Vasella and co-workers find increased potency for inhibitors such as 7 by attaching aglycone mimics at the
imidazole ring. This leads to strong inhibitors of b-glycosidases. This design is not transition-state-analogy-
based and cannot be used to obtain a-selective inhibitors.
2
)
Arbitrary numbering; systematic names are given in parentheses after the nonsystematic names.

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Helvetica Chimica Acta ± Vol. 84 (2001)
(d, arom. C); 127.65 (d, arom. C); 119.44 (t, CH2ˆC(5)); 112.80 (s, Me2C); 83.99 (d, (2)); 83.44 (d, C(3)); 80.73
(d, C(4)); 48.11 (d, C(1)); 45.27 (t, PhCH2); 26.14 (q, Me2C); 24.52 (q, Me2C). HR-LSI-MS/MS: 382.0656
‡
(C17H21BrNO4 ; calc.: 382.0654). Anal. calc. for C17H20BrNO4: C 53.42, H 5.27; found: C 53.26, H 5.31.
(1S,2S,3R,4R,5S)-2-O-(Benzylcarbamoyl)-1-bromo-3,4-O-isopropylidene-5-methylcyclopentane-2,3,4-triol
(ˆ(3aR,4S,5S,6S,6aR)-5-Bromotetrahydro-2,2,6-trimethyl-4H-cyclopenta-1,3-dioxol-4-yl Benzylcarbamate;
15). To a soln. of 14 (46 mg, 0.12 mmol) in benzene (10 ml), a spatula of Wilkinsonꢀs catalyst (cat. amount)
was added. The flask was evacuated and filled with H2 (3Â). After stirring overnight, the mixture was
evaporated. FC (hexane/AcOEt 2 :1; Rf 0.4) yielded 15 (34 mg, 74%). White solid. M.p. 112 ± 1148. [a]D20
ˆ
‡28.0 (c ˆ 0.44, CHCl3). IR (KBr): 3331s (br.), 2977w, 2933s, 1726m, 1698s, 1540m, 1456m, 1380m, 1372m,
1310w, 1269m, 1206m, 1147m, 1077m, 1064m, 1046w, 1012m, 996m, 864m. 1H-NMR (300 MHz, CDCl3)2): 7.4 ±
7.2 ( m, PhCH2); 5.37 (s, NH); 4.97 (br. m, H C(4)); 4.63 (dd, 3J(2,3) ˆ 5.9, 3J(2,1) ˆ 5.5, H C(2)); 4.54
(d, 3J(3,2) ˆ 5.9, H C(3)); 4.38 (d, 2J ˆ 5.9, PhCH2); 4.19 (d, 3J(1,2) ˆ 5.5, H C(1)); 2.37 (m, H C(5)); 1.59
(s, 3 H, Me2C); 1.29 (s, 3 H, Me2C); 1.25 (d, 3J(3,5) ˆ 6.6, Me C(5)). 13C-NMR (75 MHz, CDCl3)2): 154.68
(s, CˆO); 138.04 (s, arom. C); 128.56 (d, arom. C); 127.48 (d, arom. C); 127.19 (d, arom. C); 111.64 (s, Me2C);
84.64 (d, C(2)); 83.84 (d, C(3)); 82.27 (d, C(4)); 55.32 (d, C(1)); 45.04 (t, PhCH2); 40.09 (d, C(5)); 25.27
‡
(q, Me2C); 23.54 (q, Me2C); 12.41 (q, Me C(5)). HR-LSI-MS/MS: 384.0812 (C17H23BrNO4 ; calc.: 384.0810).
Anal. calc. for C17H22BrNO4: C 53.14, H 5.77; found: C 53.19, H 5.78.
(1R,2R,3R,4R,5R)-4-(Benzylamino)-3-O-4-N-(oxomethylidene)-1,2-O-isopropylidene-5-methylcyclopen-
tane-1,2,3-triol (ˆ(3aR,3bR,6aR,7R,7aR)-6-Benzylhexahydro-2,2,7-trimethyl-5H-1,3-dioxolo[4,5:3,4]cyclo-
pent[1,2-d]oxazol-5-one; 16). To a soln. of 15 (140 mg, 0.37 mmol) in THF (15 ml), NaH (90 mg, 3.5 mmol)
was added. The mixture was stirred for 3 h, poured into H2O (15 ml) and extracted (Et2O, 3 Â ). The combined
org. layer was washed (brine) and evaporated. FC (hexane/AcOEt 2 :1; Rf 0.4) yielded 16 (88 mg, 80%).
Yellowish solid. M.p. 111 ± 1138. [a]D20
ˆ
42.0 (c ˆ 0.35, CHCl3). IR (KBr): 3434br, 2988w, 2931w, 2908w, 2360w,
1728s, 1444m, 1424m, 1369m, 1246m, 1222m, 1202w, 1163w, 1096m, 1035m, 1003w, 978w, 864m. 1H-NMR
(400 MHz, CDCl3)2): 7.4 ± 7.2 (m, PhCH2); 4.84 (d, 2J ˆ 15.2, 1 H, PhCH2); 4.68 (d, 3J(2,1) ˆ 5.1, H C(2)); 4.63
(d, 3J(3,4) ˆ 7.8, H C(3)); 4.56 (dd, 3J(1,2) ˆ 5.1, J(1,5) ˆ 4.7, H C(1)); 4.16 (d, 2J ˆ 15.2, 1 H, PhCH2); 3.74
3
(dd, 3J(4,3) ˆ 7.8, 3J(4,5) ˆ 7.5, H C(4)); 2.21 (m, H C(5)); 1.38 (s, 3 H, Me2C); 1.30 (s, 3 H, Me2C); 1.16
(d, 3J(3,5) ˆ 7.0, Me C(5)). NOE:
H C(4)/H C(3), H C(2)/H C(1), H
C(1)/H C(5). 13C-NMR
(75 MHz, CDCl3)2): 156.90 (s, CˆO); 135.76 (s, arom. C); 128.74 (d, arom. C); 127.86 (d, arom. C); 127.77
(d, arom. C); 111.31 (s, Me2C); 84.93 (d, C(1)); 83.82 (d, C(3)); 83.22 (d, C(2)); 64.19 (d, C(4)); 46.79
(t, PhCH2); 43.33 (d, C(5)); 26.79 (q, Me2C); 24.47 (q, Me2C); 13.63 (q, Me C(5)). HR-EI-MS: 303.1471
‡
(C17H21NO4 ; calc.: 303.1471). Anal. calc. for C17H21NO4: C 67.31, H 6.98, N 4.62; found: C 67.29, H 6.93, N 4.55.
(1R,2S,3R,4R,5R)-4-(Benzylamino)-1,2-O-isopropylidene-5-methylcyclopentane-1,2,3-triol (ˆ(3aS,4R,5R,
6R,6aR)-5-(Benzylamino)tetrahydro-2,2,6-trimethyl-4H-cyclopenta-1,3-dioxol-4-ol; 17). A soln. of 16 (77 mg,
0.25 mmol) in EtOH (12 ml) and 2m NaOH (12 ml) was heated for 3.5 h at 608 and evaporated. FC (hexane/
AcOEt 2 :1; Rf 0.15) yielded 17 (64 mg, 91%). White solid. M.p. 81 ± 838. [a]2D0 ˆ ‡55.0 (c ˆ 0.28, CHCl3). IR
(KBr): 3434 (br.), 3316s, 3284s, 2976s, 2938s, 2862m, 2360m, 1729w, 1498w, 1452s, 1377s, 1264s, 1205s, 1164m,
1134m, 1047s, 1033m, 999s, 883w, 864m. 1H-NMR (300 MHz, CDCl3)2): 7.4 ± 7.2 (m, PhCH2); 4.55 ± 4.49
(m, H C(1), H C(2)); 3.87 (m, H C(3)); 3.83 (d, 2J ˆ 12.9, 1 H, PhCH2); 3.74 (d, 2J ˆ 12.9, 1 H, PhCH2); 2.93
(dd, 3J(4,5) ˆ 11.1, 3J(4,3) ˆ 4.1, H C(4)); 1.96 (m, H C(5)); 1.41 (s, 3 H, Me2C); 1.30 (s, 3 H, Me2C); 1.16
(d, 3J(Me C(5),5) ˆ 7.0, Me C(5)). 13C-NMR (75 MHz, CDCl3)2): 139.52 (s, arom. C); 128.48 (d, arom. C);
128.02 (d, arom. C); 127.29 (d, arom. C); 109.55 (s, Me2C); 83.23 (d, C(1)); 81.01 (d, C(2)); 72.74 (d, C(3));
64.51 (d, C(4)); 52.70 (t, PhCH2); 39.45 (d, C(5)); 25.94 (q, Me2C); 23.66 (q, Me2C); 11.19 (q, Me C(5)). HR-
‡
EI-MS: 277.1673 (C16H23NO3 ; calc.: 277.1678). Anal. calc. for C16H23NO3: C 69.29, H 8.36, N 5.05; found:
C 69.15, H 8.26, N 5.01.
(1R,2S,3R,4R,5R)-4-Amino-1,2-O-isopropylidene-5-methylcyclopentane-1,2,3-triol (ˆ(3aS,4R,5R,6R,
6aR)-5-Aminotetrahydro-2,2,6-trimethyl-4H-cyclopenta-1,3-dioxol-4-ol; 18). To
a soln. of 17 (50 mg,
0.18 mmol) in EtOH (15 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 ꢂ and evaporated. FC (CH2Cl2/MeOH/
NH4OH 9 :1 :0.1; Rf 0.25) yielded 18 (26 mg, 78%). White solid. M.p. 101 ± 1038. [a]2D0 ˆ ‡47.1 (c ˆ 0.34,
CHCl3). IR (KBr): 3350s, 3286m, 2979s, 2940s, 2359w, 1592m, 1463m, 1384s, 1372s, 1263s, 1210s, 1166s, 1127m,
1094m, 1058s, 1043m, 1000s, 977m, 958m, 876m, 816w. 1H-NMR (300 MHz, CDCl3)2): 4.52 (dd, 3J(1,2) ˆ 5.9,
3J(1,5) ˆ 5.5,
H
C(1)); 4.32 (br. d, 3J(2,1) ˆ 5.9,H C(2)); 3.79 (br. d, 3J(3,4) ˆ 4.1,
H C(3)); 2.81
(dd, 3J(4,5) ˆ 11.4, 3J(4,3) ˆ 4.1, H C(4)); 2.40 (m, OH, NH2); 1.81 (m, H C(5)); 1.36 (s, 3 H, Me2C); 1.27
(s, 3 H, Me2C); 1.06 (d, 3J(Me C(5),5) ˆ 7.0, Me C(5)). 13C-NMR (75 MHz, CDCl3)2): 110.83 (s, Me2C);

Helvetica Chimica Acta ± Vol. 84 (2001)
2129
85.12 (d, C(1)); 82.01 (d, C(2)); 77.13 (d, C(3)); 58.71 (d, C(4)); 42.13 (d, C(5)); 26.27 (q, Me2C); 23.92
(q, Me2C); 11.28 (q, Me C(5)). Anal. calc. for C9H17NO3: C 57.73, H 9.15, N 7.48; found: C 57.68, H 9.06, N 7.39.
(1R,2R,3R,4R,5R)-4-(Benzylamino)-5-methylcyclopentane-1,2,3-triol (1). To a soln. of 17 (7 mg, 25 mmol)
in H2O (3 ml), 37% HCl soln. (1 ml) was added. The soln. was stirred for 5 h at 608. Lyophilization yielded 1 ´
HCl (7 mg, quant.). [a]2D0 ˆ ‡20.3 (c ˆ 0.335, MeOH). IR (KBr): 3385 (br.), 2933s, 2363w, 1718m, 1559m,
1499w, 1457s, 1210m, 1129m, 1050m, 1027m, 997w, 890w. 1H-NMR (300 MHz, CD3OD): 7.55 ± 7.4 (m, PhCH2);
3
4.31 (d, 2J ˆ 13.2, 1 H, PhCH2); 4.22 (dd, 3J(3,4) ˆ 7.0, J(3,2) ˆ 4.4, H C(3)); 4.16 (d, 2J ˆ 13.2, 1 H, PhCH2);
4.05 (dd, 3J(1,5) ˆ 5.2, 3J(1,2) ˆ 4.1, H C(1)); 3.96 (dd, 3J(2,3) ˆ 4.4, 3J(2,1) ˆ 4.1, H C(2)); 3.34 (m, H C(4));
2.20 (m, H C(5)); 1.15 (d, 3J(6,5) ˆ 7.0, Me). 13C-NMR (75 MHz, CD3OD): 132.74 (s, arom. C); 131.02
(d, arom. C); 130.63 (d, arom. C); 130.22 (d, arom. C); 80.36 (d, C(1)); 73.63 (d, C(3)); 73.50 (d, C(2)); 64.84
‡
(d, C(4)); 51.77 (t, PhCH2); 39.34 (d, C(5)); 13.34 (q, Me). EI-MS: 237 ([M HCl] ), 220, 208, 202, 190, 162,
106.
(1R,2R,3R,4R,5R)-4-Amino-5-methylcyclopentane-1,2,3-triol (10). To a mixture of impure 18 (22 mg,
0.12 mmol), (Boc)2O (61 mg, 0.28 mmol), and NaHCO3 (39 mg, 0.46 mmol), AcOEt (0.5 ml) and H2O (0.5 ml)
were added, and the mixture was stirred overnight at r.t. After adding more solvent (5 ml each), the aq. phase
was extracted with AcOEt (3 Â 8 ml) and evaporated. FC (hexane/AcOEt 2 :1; Rf 0.25) yielded N-[(tert-
butoxy)carbonyl]-protected 10 (13 mg, 40%). Colorless oil. 1H-NMR (300 MHz, CDCl3): 4.82 (d, 3J(NH, 4) ˆ
8.8, NH); 4.52 (dd, 3J(1,2) ˆ 5.9, 3J(1,5) ˆ 5.5, H C(1)); 4.36 (br. d, 3J(2,1) ˆ 5.9, H C(2)); 4.00 (br. m,
H
C(3)); 3.79 (br. m, H C(4)); 2.04 (m, H C(5)); 1.46 (s, tBu); 1.44 (s, 3 H, Me2C); 1.29 (s, 3 H, Me2C); 1.09
(d, 3J(Me, 5) ˆ 7.0, Me). 13C-NMR (75 MHz, CDCl3): 155.89 (s, CˆO); 110.06 (s, Me2C); 86.12 (d, C(1)); 83.66
(d, C(2)); 79.64 (d, C(3)); 79.10 (s, Boc); 60.48 (d, C(4)); 39.45 (d, C(5)); 28.34 (q, Boc); 25.95 (q, Me2C); 23.76
(q, Me2C); 10.75 (q, Me).
To a soln. of N-Boc-protected 10 (13 mg, 45 mmol) in H2O (3 ml), 37% HCl soln. (1 ml) was added. The
soln. was stirred for 5.5 h at 608 and evaporated. Lyophilization of the H2O-containing residue yielded free 10 ´
HCl (8 mg, quant.). [a]2D0 ˆ ‡14.6 (c ˆ 0.335, MeOH). IR (KBr): 3425 (br.), 3028m, 2927s, 2361m, 2341m,
1587w, 1506m, 1145m, 1120m, 1077m, 1048w, 983w, 945w. 1H-NMR (400 MHz, CD3OD): 4.11 (dd, 3J(3,4) ˆ 7.3,
3J(3,2) ˆ 5.1, H C(3)); 3.94 (dd, 3J(1,2) ˆ 4.5, 3J(1,5) ˆ 4.3, H C(1)); 3.89 (dd, 3J(2,3) ˆ 5.1, 3J(2,1) ˆ 4.5,
H
C(2)); 3.24 (dd, 3J(4,5) ˆ 9.3, 3J(4,3) ˆ 7.3, H C(4)); 2.05 (m, H C(5)); 1.16 (d, 3J(Me, H) ˆ 7.0, Me).
13C-NMR (75 MHz, CD3OD): 80.64 (d, C(3)); 74.80 (d, C(1)); 73.72 (d, C(2)); 57.77 (d, C(4)); 40.77 (d, C(5));
‡
12.80 (q, Me). HR-EI-MS: 148.0962 (C6H14NO3 ; calc.: 148.0974).
(1R,2R,3R,4R,5R)-5-Methyl-4-[(2-phenylethyl)amino]cyclopentane-1,2,3-triol (2). To a mixture of 18
(10 mg, 53 mmol) and benzeneacetaldehyde (15 mg, 125 mmol) in MeOH (ROMIL; 1 ml), NaCNBH3 (10.2 mg,
160 mmol) was added. The mixture was stirred for 24 h, then 1m HCl (0.5 ml) was added. After stirring overnight,
the crude was purified by prep. HPLC (RP-C18): 2 ´ CF3COOH (4.9 mg, 37%). [a]2D0 ˆ ‡13.3 (c ˆ 0.21,
MeOH). IR (KBr): 3422 (br.), 2360w, 2343m, 1677s, 1558w, 1456m, 1436w, 1384w, 1205s, 1137s, 840w, 801w.
1H-NMR (300 MHz, D2O): 7.4 ± 7.25 (m, PhCH2); 4.20 (dd, 3J(3,4) ˆ 7.7, 3J(3,2) ˆ 6.6, H C(3)); 3.98
(dd, 3J(1,5) ˆ 5.2, 3J(1,2) ˆ 4.4,
H
C(1)); 3.92 (dd, 3J(2,3) ˆ 6.6, 3J(2,1) ˆ 4.4,
H C(2)); 3.31 ± 3.24
(m, H C(4), CH2N); 2.98 (m, PhCH2); 2.16 (m, H C(5)); 1.07 (d, 3J(Me, 5) ˆ 7.0, Me). 13C-NMR (75 MHz,
CD3OD): 137.79 (s, arom. C); 129.99 (d, arom. C); 129.70 (d, arom. C); 128.27 (d, arom. C); 80.21 (d, C(3));
73.74 (d, C(2)); 73.53 (d, C(1)); 65.24 (d, C(4)); 49.17 (t, CH2N); 39.64 (d, C(5)); 33.44 (t, PhCH2); 13.83
‡
(q, Me). HR-LSI-MS: 252.1600 (C14H22NO3 ; calc.: 252.1600).
(1R,2R,3R,4R,5R)-5-Methyl-4-[(3-phenylpropyl)amino]cyclopentane-1,2,3-triol (3). As described for 2,
with 18 (10.3 mg, 55 mmol), benzenepropanal (16 mg, 120 mmol), MeOH (1 ml), NaCNBH3 (24 mg, 380 mmol),
and 1m HCl (0.5 ml): 3 ´ CF3COOH (2.8 mg, 20%). [a]2D0 ˆ ‡16.2 (c ˆ 0.105, MeOH). IR (KBr): 3440 (br.),
2925m, 2359w, 1682s, 1614s, 1558w, 1456w, 1204s, 1133s, 1061w, 840w, 801w. 1H-NMR (300 MHz, D2O): 7.4 ± 7.25
(m, PhCH2); 4.18 (dd, 3J(3,4) ˆ 7.7, 3J(3,2) ˆ 6.6, H C(3)); 3.98 (dd, 3J(1,5) ˆ 5.2, 3J(1,2) ˆ 4.1, H C(1)); 3.92
(dd, 3J(2,3) ˆ 6.6, 3J(2,1) ˆ 4.1,
H
C(2)); 3.22 (dd, 3J(4,5) ˆ 8.5, 3J(4,3) ˆ 7.7,
H C(4)); 3.00
(m, PhCH2CH2CH2); 2.68 (t, 3J(CH2 ,CH2) ˆ 7.7, PhCH2CH2CH2); 2.14 (m, H C(5)); 1.97 (quint.,
3J(CH2,CH2) ˆ 3J(CH2,CH2) ˆ 7.7, PhCH2Ch2CH2); 1.05 (d, 3J(Me, 5) ˆ 7.0, Me). 13C-NMR (75 MHz, CD3OD):
138.36 (s, arom. C); 129.69 (d, arom. C); 129.37 (d, arom. C); 127.46 (d, arom. C); 80.15 (d, C(3)); 73.73
(d, C(2)); 73.47 (d, C(1)); 65.28 (d, C(4)); 47.67 (t, PhCH2CH2CH2); 39.63 (d, C(5)); 33.63 (t, PhCH2CH2CH2);
‡
29.07 (t, PhCH2CH2CH2); 13.77 (q, Me). HR-LSI-MS: 266.1756 (C15H24NO3 ; calc.: 266.1756).
Methyl 5-[(4-Formylphenyl)amino]-5-oxopentanoate (21). To a soln. of Et3N (930 ml, 6.65 mmol) In CH2Cl2
(25 ml), 4-aminobenzyl alcohol (810 mg, 6.58 mmol) and methyl 5-chloro-5-oxopentanoate (1 ml, 7.24 mmol)
were added. After stirring for 2.5 h at r.t., CH2Cl2 (50 ml) was added, and the org. phase washed with 1m HCl
(2 Â 30 ml). Extraction of the aq. phase with CH2Cl2 (2 Â 30 ml), evaporation, and FC (hexane/AcOEt 1.4; Rf

2130
Helvetica Chimica Acta ± Vol. 84 (2001)
0.36) yielded methyl 5-{[4-(hydroxymethyl)phenyl]amino}-5-oxopentanoate (1.37 g, 83%). White solid. M.p.
77 ± 798. IR (KBr): 3301 (br.), 2950m, 1727s, 1655s, 1599m, 1538s, 1460m, 1412m, 1388w, 1317m, 1265m, 1206m,
1175m, 1076w, 1022m, 1012m, 994m, 893w, 820m. 1H-NMR (300 MHz, CDCl3): 7.50 (d, 3J ˆ 8.1, 2 H); 7.31
3
(d, 3J ˆ 8.1, 2 H); 4.65 (s, 2 H); 3.70 (s, 3 H); 2.45 (m, 4 H); 2.05 (quint. J ˆ 3J ˆ 7.0, 2 H). 13C-NMR (75 MHz,
‡
CD3OD): 173.9; 171.0, 137.2; 136.8; 127.7; 120.0; 64.8; 51.7; 36.27; 32.92; 20.72. EI-MS: 251 (M ), 235, 220, 218,
149, 132, 129, 123, 107, 101, 94.
To cooled (08) pyridine (18.2 ml), cromium(VI) trioxide (1.55 g, 15.5 mmol) was added in portions. The
above 4-(hydroxymethyl)phenyl derivative (1.30 g, 5.2 mmol) was dissolved in pyridine (10 ml) and added
dropwise to the chromium(VI) soln. After stirring for 1.5 h at r.t., the mixture was poured into H2O (120 ml),
extracted with AcOEt (3 Â 120 ml), washed with brine (200 ml), and evaporated. FC (hexane/AcOEt 1:1; Rf
0.25) yielded 21 (698 mg,54%). White solid. M.p. 118 ± 1198. IR (KBr): 3341s, 2954w, 1729s, 1701s, 1674m,
1598m, 1523s, 1435w, 1420w, 1388w, 1323m, 1299m, 1278m, 1248w, 1216m, 1160s, 1065w, 986w, 852w. 1H-NMR
(300 MHz, CDCl3): 9.92 (s, CHˆO)); 7.86 (br. d, 3J ˆ 8.1, 2 arom. H); 7.72 (br. d, 3J ˆ 8.1, 2 arom. H); 3.72
(s, MeO); 2.49 (m, CH2(2), CH2(4)); 2.08 (quint, 3J ˆ 3J ˆ 7.0, CH2(3)). 13C-NMR (75 MHz, CD3OD): 191.06
(d, CHO); 173.80 (s, C(1)); 171.00 (s, C(5)); 143.63 (s, arom. C); 132.13 (s, arom. C); 131.12 (d, arom. C); 119.20
‡
(d, arom. C); 51.71 (q, MeO); 36.43 (t, CH2(4)); 32.83 (t, CH2(2)); 20.22 (t, CH2(3)). EI-MS: 249 (M ), 218,
129, 121, 101.
5-Oxo-5-{{4-{{[(1R,2R,3R,4R,5R)-2,3,4-trihydroxy-5-methylcyclopentyl]amino}methyl}phenyl}amino}-
pentanoic Acid (4). To a mixture of 18 (21 mg, 112 mmol) and 21 (57 mg, 229 mmol) in MeOH (2 ml), NaCNBH3
(18.2 mg, 290 mmol) was added. The mixture was stirred for 18 h at r.t. To the crude ester, 1m NaOH (1 ml) was
added and stirred for 5 h at r.t. After evaporation, 1m HCl (2 ml) was added and stirred overnight. The crude was
purified by prep. HPLC (RP-C18): 4 ´ CF3COOH (23.8 mg, 46%). [a]2D0 ˆ ‡9.4 (c ˆ 0.16, MeOH). IR (KBr):
3422 (br.), 2926m, 2360w, 2343m, 1674s, 1612s, 1526m, 1413m, 1350w, 1310w, 1232w, 1204s, 1134s, 840w, 801w.
1H-NMR (300 MHz, CD3OD): 7.66 (d, 3J ˆ 8.6, 2 H); 7.45 (d, 3J ˆ 8.6, 2 H); 4.26 (d, 2J ˆ 12.9, 1 H); 4.21
3
(dd, 3J(1',2') ˆ 7.3, J(2',3') ˆ 4.4, 1 H, H C(2')); 4.12 (d, 2J ˆ 12.9, 1 H); 4.06 (dd, 3J(4',5') ˆ 5.9, 3J(3',4') ˆ 4.0,
1 H, H C(4')); 3.96 (dd, 3J(2',3') ˆ 4.4, 3J(3',4') ˆ 4.0, 1 H, H C(3')); 3.37 (dd, 3J(1',5') ˆ 8.6, 3J(1',2') ˆ 7.3, 1 H,
H
C(1')); 2.45 (t, 3J(4,3) ˆ 7.4, CH2(4)); 2.39 (t, 3J(2,3) ˆ 7.4, CH2(2)); 2.20 (m, H C(5')); 1.97 (quint., 3J ˆ 3J ˆ
7.4, CH2(3)); 1.15 (d, 3J(Me, 5') ˆ 7.0, Me). NOE: H C(2')/H C(3'), H C(2')/H C(1'). 13C-NMR (75 MHz,
CD3OD): 176.88 (s); 173.97 (s); 141.17 (s); 131.69 (d); 127.97 (s); 121.36 (d); 80.40 (d, C(3')); 73.66 (d, C(2'));
73.55 (d, C(4')); 64.67 (d, C(1')); 51.21 (t, ArCH2); 39.65 (d, C(5')); 36.89 (t, C(4)); 34.10 (t, C(2)); 22.00
‡
(t, C(3)); 13.57 (q, Me). HR-LSI-MS: 367.1872 (C18H27N2O6 ; calc.: 367.1869).
(1R,2R,3R,4S,5R)-4-(Benzylamino)-5-methylcyclopentane-1,2,3-triol (20). Free trans-amino alcohol 19
(6.3 mg, 34 mmol) was treated with benzaldehyde (4 ml, 39 mmol) and titanium(IV) isopropoxide (15 ml,
50 mmol) and stirred for 90 min. After addition of EtOH (40 ml) and NaCNBH3 (2 mg, 30 mmol), the soln. was
stirred over one weekend. Evaporation and prep. HPLC (RP-C18) yielded 20 ´ CF3COOH (3.2 mg, 27%).
[a]2D0
ˆ
6.2 (c ˆ 0.13, MeOH). IR (KBr): 3426 (br.), 2924w, 2360w, 2343m, 1684s, 1653m, 1558m, 1540w, 1506w,
1456m, 1207s, 1136s, 841w, 800m. 1H-NMR (300 MHz, CD3OD): 7.55 ± 7.4 (m, 5 H); 4.44 (d, 2J ˆ 13.2, 1 H,
PhCH2); 4.26 (dd, 3J(3,2) ˆ 6.6, J(3,4) ˆ 5.2, H C(3)); 4.22 (d, 2J ˆ 13.2, 1 H, PhCH2); 3.88 (dd, 3J(1,2) ˆ 4.4,
3
3J(1,5) ˆ 4.1, H C(1)); 3.82 (dd, 3J(2,3) ˆ 6.6, 3J(2,1) ˆ 4.4, H C(2)); 3.35 (dd, 3J(4,5) ˆ 8.2, 3J(4,3) ˆ 5.2,
H
C(1)); 2.48 (m, H C(5)); 1.12 (d, 3J(Me, 5) ˆ 7.4, Me). 13C-NMR (50 MHz, CD3OD): 132.74 (s, arom. C);
131.02 (d, arom. C); 130.63 (d, arom. C); 130.22 (d, arom. C); 80.89 (d, C(3)); 80.00 (d, C(1)); 75.15 (d, C(2));
‡
65.37 (d, C(4)); 51.37 (t, PhCH2); 37.34 (d, C(5)); 9.23 (q, Me). HR-EI-MS: 237.1354 (C13H19NO3 ; calc.:
237.1365).
2. Enzyme Measurements. The following glycosidases were assayed: a-l-fucosidase (EC 3.2.51) from
bovine kidney (Sigma), a-l-fucosidase from bovine epididymis (Sigma), a-l-fucosidase from human placenta
Ï
(Sigma), a-l-fucosidase from Fucosarium Oxysporum 377 (provided by Dr. V. Kren). All buffers and solns.
were prepared with MilliQ-deionized H2O. Enzymes were diluted with 0.1m HEPES buffer, pH 6.8. Substrate
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 pH 6.8, 258. The assays (100 ml) were followed in individual wells of flat-bottom 96-well half-area
polystyrene cell culture plates (Costar) by means of a UV Spectramax 250 instrument from Molecular Devices.
The release of 4-nitrophenol was followed at 405 nm over 15 min. The concentration of each enzyme in the
assays was adjusted such as to give ca. 0.2 to 0.5 OD increase as given by the instrument. The initial rate of
reaction of the first 10 min was linear and was used to calculate the rate. Rates were expressed in relative units as
mOD ´ min 1. The competitive inhibition constants Ki [mm] were determined from Dixon replots of inhibition
data.

Helvetica Chimica Acta ± Vol. 84 (2001)
2131
3. Molecular Modelling. Conformers of all stable transition-state analogs and of the model 24 were
calculated with the Monte Carlo method on a DEC-DPW500au workstation with Spartan 5.1. All conformers
were geometry-optimized by mechanics protocol MMFF 94 and further optimized with semi-empirical model
PM3. The electronic potentials were calculated on these optimized geometries and superimposed onto a surface
of constant electron density (0.002 e/au3) (blue ˆ positive, red ˆ negative). Only the minimum energy
conformer is shown.
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Received April 12, 2001