Synthesis and biological evaluation of α-L-fucosidase inhibitors: 5a-carba-α-L-fucopyranosylamine and related compounds

Article abstract of DOI:10.1002/1099-0690(200103)2001:5<967::AID-EJOC967>3.0.CO;2-J

5a-Carba-α-L-fucopyranosylamine (5), an α-glucosidase inhibitor validamine analog possessing an α-L-fucose-type structure, and four related compounds (4 and 6-8) were synthesized and their glycosidase inhibitory potential determined. Carbafucosylamine has already been shown to possess a specific and very strong inhibitory activity against α-L-fucosidase (K_i = 1.2 × 10^-8 M, bovine kidney). Judging from the activity of the other analogs prepared, this amine might be expected to be a lead compound for development of a new type of α-L-fucosidase inhibitor.

Full text of DOI:10.1002/1099-0690(200103)2001:5<967::AID-EJOC967>3.0.CO;2-J

FULL PAPER
-Fucosidase Inhibitors:
Synthesis and Biological Evaluation of α-
L
5a-Carba-α-
L
-fucopyranosylamine and Related Compounds[‡]
Seiichiro Ogawa,*[a] Ayako Maruyama,[a] Takashi Odagiri,[a] Hideya Yuasa,[b] and
Hironobu Hashimoto[b]
Keywords: Carbohydrates
/
Cyclitols
/
Carba sugars / 5a-Carba-α--fucopyranosylamine / Enzyme inhibitors /
α--Fucosidase inhibitors
5a-Carba-α-
hibitor validamine analog possessing an α-
L
-fucopyranosylamine (5), an α-glucosidase in-
sess a specific and very strong inhibitory activity against α-
-fucosidase (Ki = 1.2 × 10−8
, bovine kidney). Judging from
L-fucose-type
L
M
structure, and four related compounds (4 and 6−8) were syn-
thesized and their glycosidase inhibitory potential deter-
the activity of the other analogs prepared, this amine might
be expected to be a lead compound for development of a
mined. Carbafucosylamine has already been shown to pos- new type of α-L-fucosidase inhibitor.
Introduction
Our interest has been focused on the development of new
types of inhibitors of α--fucosidase, which plays important
roles in the turnover of glycoproteins by lysosomal degrada-
tion[2] and in fertilization in some organisms by triggering
acrosome reactions.[3] α-Fucosidase inhibitors are potential
research tools for these fucosidase-relevant events and even
as potential drugs for cancer and HIV, through inhibition
of the contribution of turnover processes and/or invasion
of the extracellular matrix secreted fucosidases.[4]
1-Deoxyfuconojirimycin[5] (1, Scheme 1) is the most
powerful inhibitor of α--fucosidase known to date, and is
therefore an important biological tool in glycobiology. Re-
cently, many pseudo-sugars, aza-sugar-type compounds[6]
mimicking 1, and thia-sugars Ϫ including 5-thio-5-deoxy-
α--fucopyranose[7] (2) Ϫ have been designed and synthe-
sized as prospective, effective α--fucosidase inhibitors.
Some 5a-carbahexopyranosylamines and their analogs Ϫ
validamine (3), valienamine, and valiolamine Ϫ first isol-
ated by degradation of validamycins,[8] are important
classes of α-glucosidase inhibitors, and their syntheses and
chemical modifications have been extensively examined,
and biochemical studies[9] made. In fact, acarbose,[10] the
pseudo-tetrasaccharide composed of the imino-linked vali-
enamine, and voglibose,[11] N-(1,3-dihydroxyprop-2-yl)vali-
olamine, have been widely used clinically for treatment of
diabetes. Experience gained indicates that 5a-carba-sugar
derivatives have comparatively low toxicities, commensurate
Scheme 1. α--Fucosidase inhibitors deoxyfuconojirimycin (1), 5-
deoxy-5-thio-α--glucopyranose (2), and some validamine-type α-
-fucosidase inhibitors synthesized in this study
with further clinical uses in mammals. Therefore, several
validamine analogs possessing α-galacto,[12] β-gluco,[13] and
α-manno-type[13] structures were synthesized and their gly-
cosidase inhibitory activity tested. These analogs, however,
were found to be only rather weak or moderate glycosidase
inhibitors, in comparison with the potential exhibited by
the α-gluco-type validamine.
In a preceding paper,[1] the first synthesis of 5a-carba-α-
-fucopyranosylamine was described, and its specific and
very strong α--fucosidase inhibition demonstrated. As a
next step, it is important both to determine the activity of
the pure -antipode, corresponding to the -fucopyranose
residue contained in cell surface oligosaccharide chains, and
to estimate its scope for structural modification. We here
report[14] details of the synthesis and evaluation of fucosid-
ase inhibitory activity of 5a-carba-α--fucopyranosylamine,
[‡]
Pseudosugars, 42. Ϫ Part 41: Ref.[1]
[a]
Department of Applied Chemistry, Faculty of Science and
Engineering, Keio University,
Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan
Fax: (internat.) ϩ 81-45/566-1551
E-mail: ogawa@applc.keio.ac.jp
[b]
Department of Life Science, Faculty of Bioscience and
Biotechnology, Tokyo Institute of Technology,
Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
Fax: (internat.) ϩ 81-45/924-5805
E-mail: hhashimo@bio.titech.ac.jp
Eur. J. Org. Chem. 2001, 967Ϫ974
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/0305Ϫ0967 $ 17.50ϩ.50/0
967

S. Ogawa, A. Maruyama, T. Odagiri, H. Yuasa, H. Hashimoto
FULL PAPER
along with two carbafucopyranose derivatives possessing
branched aminomethyl functions at their C-1 positions.
Results and Discussion
As a starting compound for the preparation of 5a-carba-
-fucopyranose derivatives, we chose 2,3,4-tri-O-acetyl-6-
bromo-6-deoxy-5a-carba-β--glucopyranosyl
bromide[15]
(9), obtained from the optically resolved endo adduct[16] of
furan and acrylic acid.
Compound 9 was selectively dehydrobrominated by treat-
ment with 2 mol-equiv. of silver fluoride[17] in pyridine, con-
verting it into the crystalline exo-methylene derivative 10 in
83% yield. Inversion of the configuration at C-4 was then
attempted by means of an intramolecular nucleophilic sub-
stitution reaction of the 4-sulfonate of 10, assisted by
neighboring participation of the 3-acetoxy group
(Scheme 2). Thus, compound 10 was de-O-acetylated with
4  hydrochloric acid in aqueous THF, and the resulting
triol was treated with 2,2-dimethoxypropane (10 mol-
equiv.) in DMF in the presence of TsOH, giving the 2,3-
and 3,4-O-isopropylidene derivatives 11 and 12 in 73 and
23% yield, respectively, after chromatography on silica gel.
Compound 11 was successively treated with mesyl chloride
in pyridine, de-O-isopropylidenated with 60% aqueous
acetic acid, and acetylated with acetic anhydride in pyridine
to give the crystalline mesylate 13 in an overall yield of 73%.
Compound 13 smoothly underwent direct displacement in
60% aqueous acetic acid through an intermediate acetoxon-
ium ion to give, after conventional acetylation, the 4-epimer
14 of 10 in 98% yield. Compound 14 was readily converted
into the 3,4-O-isopropylidene derivative 15 (85%) by con-
ventional means.
Furthermore, de-O-acetylation with 4  hydrochloric
acid, followed by treatment with N,N-diisopropylethylam-
ine, gave the epoxide, which was hydrogenated without be-
ing isolated in the presence of Wilkinson catalyst, affording
the α--fucose-type epoxide 16 (58%) and its 5-epimer 17
(14%). The former is a versatile intermediate[18] for the pre-
paration of carbafucopyranose derivatives.
To provide compound 14 (Scheme 3) with protecting
groups stable to basic conditions, methoxymethyl groups
were introduced in place of the acetyl functions in the usual
Scheme 2. Preparation of intermediates 14 and 16 for the synthesis
of 5a-carbafucopyranose derivatives; reagents and conditions: (a)
AgF (2 equiv.), pyridine, 5 h, room temp; (b) 4  HCl/THF, 3 d,
60 °C; 2,2-dimethoxypropane (10 equiv.), TsOH (0.2 equiv.), DMF,
7 h, 60 °C; (c) MsCl/pyridine, 0 °C; 60% aq. AcOH, room temp;
Ac2O/pyridine, overnight, room temp; (d) 60% aq. AcOH, 3 h, 60
°C; Ac2O/pyridine; (e) 4  HCl/THF; 2,2-dimethoxypropane (4
equiv.), TsOH, DMF, 4 h, room temp; (f) 4  HCl/THF; N,N-diiso-
propylethylamine (6 equiv.), CH2Cl2, overnight, 40 °C; H2, Wilkin-
son catalyst (0.1 equiv.), benzene, 1 drop of MeOH, 12 h, room
temp.
Treatment of 19 with potassium acetate (10 mol-equiv.)
manner, with conversion into the tris(methoxymethyl) ether in DMF at 100 °C gave[20] the substitution product 22
18 (84%). Catalytic hydrogenation of 18 with Pt/C or Pd/C (Scheme 4, 50%) and the elimination product 21 (46%),
catalyst gave an approximately 3Ϫ4:1 ratio of the carbafu- demonstrating that 19 is a poor substrate for moderate nu-
cosyl and -altrosyl bromides 19 and 20. However, hydro- cleophiles. Deprotection of 22 with 4  hydrochloric acid in
genation in benzene with use of Wilkinson catalyst was aqueous THF gave free 5a-carba-α--fucopyranose[21,22]
4
shown to improve the selectivity, to afford 19 reproducibly (65%). Azidolysis of 19 with sodium azide (5 mol-equiv.),
in 80% yield, together with a trace of 20 (ca. 10%). In the however, proceeded very smoothly in DMF at 90 °C to af-
presence of DBU in toluene, compound 19 produced the ford the azide 23 selectively, in 86% yield. Deprotection of
expected alkene 21, together with an appreciable amount of 23 with 4  hydrochloric acid at room temperature and sub-
isomerized alkenes. However, it was subsequently found sequent reduction of the azido group with triphenylphos-
that treatment of 19 with sodium hydride in the presence of phane in THF gave, after chromatography on Dowex 50 W
a trace of methanol in DMF[19] selectively gave 21 in 91% ϫ 2 (Hϩ) resin with 1% aqueous ammonia, the free carbaf-
yield. Compound 21 may be utilized for synthesis of carbaf- ucopyranosylamine 5 in 66% yield. Treatment of 19 with a
ucopyranose derivatives.
large excess of sodium cyanide (15 mol-equiv.) in dry DMF
968
Eur. J. Org. Chem. 2001, 967Ϫ974

Synthesis and Biological Evaluation of α-L-Fucosidase Inhibitors
FULL PAPER
Scheme 3. Preparation of intermediates 19 and 21 for synthesis of
5a-carbafucopyranose derivatives; reagents and conditions: (a) 4 
HCl/THF; chloromethoxymethane (6 equiv.), diisopropylethylam-
ine (6 equiv.), 14 h, 40 °C; (b) H2, Wilkinson catalyst (0.2 equiv.),
benzene/1 drop of MeOH, 16 h, room temp; (c) NaH (6 equiv.),
DMF, 6 h, 60 °C
Scheme 5. Synthesis of spiro epoxides 29 and 30, and their conver-
sion into 1-aminomethyl derivatives 7 and 8 of 5a-carbafucopyr-
anose; reagents and conditions: (a) DBU (6 equiv.), toluene, 80 °C,
4 h; (b) mCPBA (1.5 equiv.), CH2Cl2, 5.5 h, room temp; (c) 4 
HCl/THF; (d) NaN3 (3 equiv.), DMF, 15-crown-5 ether, 20 h, 80
°C; (e) 4  HCl/THF, 10 h, room temp; H2, 5%Pd/C, EtOH, 3 d,
60 °C
Scheme 4. Synthesis of 5a-carba-α--fucopyranose (4) and -fucopy-
ranosylamine (5), and nitrile 24; reagents and conditions: (a) AcOK
(10 equiv.), 20 h, 100 °C; (b) 4  HCl/THF, 3 h, 50 °C; (c) NaN3
(5 equiv.), DMF, 9 h, 90 °C; (d) Ph3P, 5% aq THF, 3 d, 60 °C; (e)
NaCN (15 equiv.), DMF, 20 h, 100 °C; (f) 1  DIBAL (2 equiv.)/
toluene, 4 h, Ϫ78 Ǟ Ϫ60 °C; NaBH4 (1 equiv.), MeOH; MsCl,
Et3N
at 100 °C afforded the nitrile 24 (90%), along with a trace
of the alkene 21 (5%). Reduction of 24 with 1  DIBAL (2
mol-equiv.) in toluene at Ϫ60 °C gave an approximately 2:1
mixture of epimeric aldehydes, which was treated (without
separation) with sodium borohydride in methanol. The re-
sulting alcohols were isolated as the mesylates 25 (47%) and
26 (38%).
Scheme 6. NOE experiments on compounds 29 and 30
Compound 25 was treated with DBU in toluene at 80 °C
to give the exo-methylene compound 27 (Scheme 5, 85%)
and the alcohol 28 (18%). Oxidation of 27 with mCPBA in thyloxy group at C-3 appeared to be preferable. Azidolysis
CH2Cl2 at room temperature afforded epimeric spiro epox- of 29 and 30 in DMF at 80 °C afforded the azides 31 and
ides 29 and 30 in 77 and 21% yield (Scheme 6), respectively; 32 in 94 and 88% yield, respectively. Compounds 31 and 32
their structures were established by NOE experiments. were transformed quantitatively into the respective amines 7
Rear-side attack of the peracid away from the methoxyme- and 8 by hydrolysis with 4  hydrochloric acid and successive
Eur. J. Org. Chem. 2001, 967Ϫ974
969

S. Ogawa, A. Maruyama, T. Odagiri, H. Yuasa, H. Hashimoto
FULL PAPER
hydro-genation with Pd/C in ethanol. The amines 7 and 8
were purified on silica gel and subjected directly to biolo-
gical assays.
Experimental Section
General: Melting points: Mel-Temp capillary melting-point appar-
atus, uncorrected values. Ϫ Specific rotations: Jasco DIP-370 po-
larimeter, 1-dm cells. Ϫ IR spectra: Jasco A-202 or FT-IR-200. H
1
NMR spectra: Jeol JNM GSX-270 f.t. (270 MHz) and Jeol
Lambda-300 (300 MHz); solvent CDCl3, internal standard tetra-
methylsilane (TMS), CD3OD external acetone. Ϫ Mass spectra:
positive ion electrospray ionization with a Jasco GC-Mass GC-
Mate, and Perseptive Biosystems Mariner LC Mass. Ϫ TLC: 60
GF silica gel (E. Merck, Darmstadt); detection by charring with
concd. H2SO4. Ϫ Column chromatography: 60 K070 silica gel (Ka-
tayama Chemicals, Osaka) and Wakogel C-300 (silica gel, 300
mesh, Wako Chemical, Osaka). Ϫ Organic solutions, after drying
with anhydrous Na2SO4, were concentrated at Ͻ 50 °C and re-
duced pressure.
Biological Assay
Compounds 4Ϫ8 were assayed[1] for inhibitory activity
against α--fucosidase (bovine kidney), and their Ki values
are listed in Table 1. Compound 4 (Ki ϭ 4.3 ϫ 10Ϫ5 )
showed moderate inhibitory activity comparable to that of
5-thiofucopyranose[7] 2 (Ki ϭ 8.4 ϫ 10Ϫ5 ). These results
appeared to be due simply to hydrophobic interaction be-
tween the ring methylene group and the enzymes, as has
been proposed for the cases of the sulfur atoms of 5-thio-
glucose[23] and 5-thiofucose.[7]
2,3,4-Tri-O-acetyl-6-bromo-6-deoxy-5a-carba-β-L-glucopyranosyl
Bromide (9): (1R)-2-exo,3-endo-Diacetoxy-5-endo-acetoxymethyl-7-
oxabicyclo[2.2.1]heptane (3 g), prepared conventionally from (2S)-
7-endo-oxabicyclo[2.2.1]hept-5-ene-2-carboxylic acid,[16] was heated
with 20% hydrobromic acid/acetic acid in a sealed tube for 22 h at
80 °C. The reaction mixture obtained from fourteen sealed tubes
(total amount of the triacetate used: 42.0 g, 0.147 mol) was poured
onto cold ethyl acetate (1.2 L), and the solution was washed with
brine (400 mL ϫ 3) and saturated aqueous sodium hydrogen car-
bonate (400 mL ϫ 2), dried, and concentrated. The residual prod-
uct (ca. 70 g) was eluted from a silica gel column (400 g) with ethyl
acetate/hexane (1:3 Ǟ 1:1) to give 9 (ca. 44 g, 77%) as a colorless
syrup; [α]2D0 ϭ ϩ2.8 (c ϭ 1.4, CHCl3) [ref.[16] -bromide, crystals,
m.p. 99Ϫ100 °C, [α]D16 ϭ Ϫ3.6 (c ϭ 1.1, CHCl3)]. Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 1.98Ϫ2.08 [m, 2 H, 4-H, 5a(ax)-H], 2.00,
2.05, and 2.08 (3 s, each 3 H, 3 ϫ Ac), 2.61 [ddd, J1,5a(eq) ϭ 4.4,
Table 1. Inhibitory activity of compounds 2 and 4Ϫ8 against α--
fucosidase (α--fucosidase, bovine kidney, and p-nitrophenyl α--
fucopyranoside were purchased from Sigma)
Compound
Inhibitory activity (Ki, )[a]
2
4
5
6
7
8
8.4 ϫ 10Ϫ5
4.3 ϫ 10Ϫ5
1.2 ϫ 10Ϫ8
NI[b]
2.8 ϫ 10Ϫ6
3.0 ϫ 10Ϫ7
[a]
0.54Ϫ1.37 µ, p-nitrophenyl α--fucopyranoside, 17 µ citrate
buffer, pH ϭ 6.0, ref.[1]
Ϫ
NI: No inhibitory activity (Ͻ10Ϫ4).
[b]
J5,5a(eq) ϭ 1.7, J5agem ϭ 13.4 Hz, 1 H, 5a(eq)-H], 3.26 (dd, J5,6a
6.6, J6gem ϭ 10.7 Hz, 1 H, 6a-H), 3.40 (dd, J5,6b ϭ 3.1 Hz, 1 H,
and very strong inhibitor (Ki ϭ 1.2 ϫ 10Ϫ8 ). Replacement 6b-H), 3.96 [ddd, J1,2 ϭ 10.2, J1,5a(ax) ϭ 12.2 Hz, 1 H, 1-H], 5.02
ϭ
However, compound 5 was demonstrated to be a specific
(dd, J3,4 ϭ 9.8, J4,5 ϭ 9.9 Hz, 1 H, 4-H), 5.03 (dd, J2,3 ϭ 9.8 Hz,
1 H, 3-H), 5.21 (dd, 1 H, 2-H). Ϫ C13H18Br2O6 (430.1): calcd. C
36.30, H 4.22; found C 36.48, H 4.39.
of the 1-hydroxy group of 4 by an amino function surpris-
ingly resulted in a more than 3000-fold increase in its activ-
ity. Also, with regard to binding to enzymes, fucopyranosyl-
amine is apparently overwhelmed by 4, indicating the im-
portance of its hydrophobic 5a-ring methylene portion.
This compound has been shown to possess an inhibitory
potency approaching that of the potent 1-deoxyfuconojiri-
mycin[5] (1), which has promise as a therapeutic agent for
cancer and AIDS.
The fact that both the epimers 7 and 8, possessing amino-
methyl branching groups at C-1, were shown to possess
strong activity (Ki ϭ 2.8 and 0.3 ϫ 10Ϫ6 , respectively)
suggests that carbafucosylamine 4 is a potential lead com-
pound, with possible improvement in potency achievable by
chemical modification around the anomeric positions. Fur-
thermore, 5a-carbafucopyranosyl residues could easily be
introduced into the oligosaccharide chains by way of imino
linkages, as shown[14] by the attempted preparation of
2,3,4-Tri-O-acetyl-5a-carba-β-L-xylo-hex-5-enopyranosyl Bromide
(10): Compound 9 (7.81 g, 18 mmol) in dry pyridine (50 mL) was
stirred with silver fluoride (2 mol-equiv.) for 5 h at room temper-
ature. The mixture was diluted with ethyl acetate (300 mL), and
insoluble material was removed by filtration. The organic solution
was washed with 1  hydrochloric acid, saturated aqueous sodium
hydrogen carbonate, and water, dried, and concentrated. Crystal-
lization from ethanol gave 10 (5.26 g, 83.0%) as needles; m.p.
1
121Ϫ124 °C (from EtOH), [α]2D6 ϭ ϩ14 (c ϭ 0.40, CHCl3). Ϫ H
NMR (300 MHz, CDCl3): δ ϭ 2.01, 2.11, and 2.17 (3 s, each 3 H,
3 ϫ Ac), 2.97 [dd, J1,5a(ax) ϭ 11.6, J5agem ϭ 13.9 Hz, 1 H, 5a(ax)-
H], 3.01 [dd, J1,5a(eq) ϭ 4.7 Hz, 1 H, 5a(eq)-H], 3.83 (ddd, J1,2
ϭ
9.6 Hz, 1 H, 1-H), 4.96 (dd, J2,3 ϭ 9.6, J3,4 ϭ 9.8 Hz, 1 H, 3-H),
5.02 and 5.09 (2 br. s, each 1 H, ϭCH2), 5.32 (dd, 1 H, 2-H), 5.43
(d, 1 H, 4-H). Ϫ C13H17BrO6 (349.2): calcd. C 44.72, H 4.91; found
C 44.64, H 4.89.
imino-linked
5a-carba--fucopyranosyl-α-(1Ǟ4)-2-acet-
2,3-O-Isopropylidene-5a-carba-β-
ide (11) and 3,4-O-Isopropylidene-5a-carba-β-
L
-xylo-hex-5-enopyranosyl Brom-
-xylo-hex-5-enopyr-
amido-2-deoxy--glucopyranose derivatives designed for
fucosyltransferase inhibition. Since the enantiomeric 5a-
carba-α--fucopyranosylamine[14] presumably has pre-
served activity to some extent, the structureϪactivity rela-
tionship of inhibitors of this kind seems to be rather flexible
and not rigidly determined.
L
anosyl Bromide (12): Compound 10 (13.3 g, 38.0 mmol) was dis-
solved in a mixture of 4  hydrochloric acid (25 mL) and THF
(75 mL), and heated for 3 d at 60 °C. The mixture was concentrated
to dryness and the residue was crystallized from ethanol. The crys-
talline triol (ca. 7.8 g) was treated with 2,2-dimethoxypropane
970
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Synthesis and Biological Evaluation of α-L-Fucosidase Inhibitors
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(46 mL, 10 mol-equiv.) in DMF (150 mL) in the presence of TsOH
stirred for 20 h at 60 °C, and then concentrated to dryness. The
hydrate (1.5 g, 0.20 mol-equiv.) for 7 h at 60 °C. After neutraliza-
residue was treated with chloromethoxymethane (4.8 mL, 6 mol-
tion with triethylamine, the mixture was diluted with ethyl acetate equiv.) in the presence of diisopropylethylamine (11 mL, 6 mol-
(600 mL), and the solution was washed with water and saturated
aqueous sodium hydrogen carbonate, dried, and concentrated. The
residual syrup was chromatographed on a silica gel column (300 g)
with ethyl acetate/toluene (1:13) as the eluent to give crystalline 11
(7.29 g, 72.2%) and syrupy 12 (2.31 g, 23.0%).
equiv.) for 14 h at 40 °C. The mixture was then diluted with chloro-
form (300 mL), and the solution was washed thoroughly with
water, dried, and concentrated. The residue was eluted from a col-
umn of silica gel (200 g) with ethyl acetate/hexane (1:5 Ǟ 1:3) to
give 18 (3.2 g, 84%) as a syrup; [α]2D6 ϭ ϩ59 (c ϭ 1.00, CHCl3). Ϫ
1H NMR (300 MHz, CDCl3): δ ϭ 2.72 [dd, 1 H, J1,5a(eq) ϭ 5.4,
J5agem ϭ 13.1 Hz, 5a(eq)-H], 2.86 [dd, J1,5a(ax) ϭ 12.5 Hz, 1 H,
5a(ax)-H], 3.38 (m, 1 H, 2-H), 3.40, 3.44, and 3.52 (3 s, each 3 H,
Compound 11: M.p. 96Ϫ98 °C (from EtOH), [α]2D8 ϭ ϩ64 (c ϭ 0.10,
1
MeOH). Ϫ H NMR (300 MHz, CDCl3): δ ϭ 1.47 and 1.51 (2 s,
each 3 H, CMe2), 2.57 [dd, J1,5a(a) ϭ 1.1, J5agem ϭ 14.4 Hz, 1 H,
5a(a)-H], 3.02 [dd, J1,5a(b) ϭ 4.8 Hz, 1 H, 5a(b)-H], 3.28 (dd, J2,3 ϭ
9.1, J3,4 ϭ 9.8 Hz, 1 H, 3-H), 3.70 (dd, J1,2 ϭ 10.5, Hz, 1 H, 2-H),
3.88 (ddd, 1 H, 1-H), 4.30 (d, 1 H, 4-H), 5.13 and 5.35 (2 br. s,
each 1 H, ϭCH2). Ϫ C10H15BrO3 (265.1): calcd. C 45.65, H 5.75;
found C 45.87, H 6.12.
3 ϫ OMe), 3.76 [ddd, J1,2 ϭ 9.7 Hz, 1 H, 1-H], 4.08 (dd, J2,3
ϭ
9.5 Hz, 1 H, 2-H), 4.35 (d, J3,4 ϭ 3.0 Hz, 1 H, 4-H), 4.60 and 4.66
(ABq, Jgem ϭ 6.6 Hz), 4.78 (s, 2 H), and 4.84 and 4.97 (ABq,
Jgem ϭ 6.1 Hz) (3 ϫ CH2OMe), 5.09 and 5.11 (2 br. s, each 1 H, ϭ
CH2). Ϫ C13H23BrO6 (355.2): calcd. C 43.96, H 6.53; found C
44.11, H 6.60.
Compound 12: [α]2D6 ϭ ϩ84 (c ϭ 0.12, MeOH). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 1.49 and 1.50 (2 s, each 3 H, CMe2), 2.65
[dd, J1,5a(a) ϭ 1.3, J5agem ϭ 14.4 Hz, 1 H, 5a(a)-H], 2.92 [dd,
3,4-O-Isopropylidene-5a-carba-β-L-arabino-hex-5-enopyranosyl
Bromide (15): A solution of the triacetate 14 (0.32 g, 0.90 mmol) in
a mixture of 4  hydrochloric acid (7 mL) and THF (21 mL) was
maintained for 20 h at 60 °C, and then concentrated. Crude triol
obtained was dissolved in dry DMF (6 mL) and the solution was
treated with 2,2-dimethoxypropane (0.45 mL, 4 mol-equiv.) and
TsOH hydrate (34 mg) for 4 h at room temperature. After neutral-
ization with triethylamine, the mixture was concentrated and the
residue was chromatographed on a silica gel column (18 g) with
ethyl acetate/hexane (1:6) to give 15 (0.20 g, 85%) as a syrup;
J1,5a(b) ϭ 5.4, Hz, 1 H, 5a(b)-H], 3.30 (dd, J2,3 ϭ 9.3, J3,4 ϭ 9.5 Hz,
1 H, 3-H), 3.80 (ddd, J1,2 ϭ 9.3 Hz, 1 H, 1-H), 3.98 (dd, 1 H, 2-
H), 3.99 (d, 1 H, 4-H), 4.96 and 5.10 (2 br. s, each 1 H, ϭCH2). Ϫ
C10H15BrO3 (265.1): calcd. C 45.65, H 5.75; found C 46.04, H 6.09.
2,3-Di-O-acetyl-4-O-mesyl-5a-carba-β-L-xylo-hex-5-enopyranosyl
Bromide (13): To a solution of 11 (3.8 g, 14.5 mmol) in pyridine
(30 mL) was added mesyl chloride (2.2 mL, 2 mol-equiv.) at 0 °C,
and the mixture was stirred for 2 h at the same temperature. After
addition of a small amount of methanol, the mixture was diluted
with ethyl acetate (200 mL) and the solution was washed with 1 
hydrochloric acid, saturated aqueous sodium hydrogen carbonate,
and water, dried, and concentrated. The crystalline residue was
treated with 60% aqueous acetic acid (100 mL) for 4 h at room
temperature, concentrated to dryness, and acetylated overnight at
room temperature with acetic anhydride (8 mL) and pyridine
(20 mL). The mixture was diluted with ethyl acetate (200 mL), and
the solution was washed with 1  hydrochloric acid, saturated
aqueous sodium hydrogen carbonate, and water, dried, and concen-
trated. Crystallization from ethanol gave 13 (4.0 g, 73%) as needles:
m.p. 119Ϫ121 °C (from EtOH), [α]2D1 ϭ ϩ71 (c ϭ 0.10, MeOH). Ϫ
1H NMR (300 MHz, CDCl3): δ ϭ 2.07 and 2.09 (2 s, each 3 H, 2
ϫ Ac), 2.64 [dd, J1,5a(ax) ϭ 12.8, J5agem ϭ 14.3 Hz, 1 H, 5a(ax)-H],
3.04 [dd, J1,5a(eq) ϭ 4.8 Hz, 1 H, 5a(eq)-H], 3.06 (s, 3 H, Ms), 3.82
(ddd, J1,2 ϭ 10.6 Hz, 1 H, 1-H), 4.98 (dd, J2,3 ϭ 9.5, J3,4 ϭ 9.9 Hz,
1 H, 3-H), 5.14 (d, 1 H, 4-H), 5.29 (dd, 1 H, 2-H), 5.34 and 5.22
(2 br. s, each 1 H, ϭCH2). Ϫ C12H17BrO7S (385.2): calcd. C 37.41,
H 4.45; found C 37.60, H 4.63.
1
[α]2D4 ϭ Ϫ30 (c ϭ 0.55, CHCl3). Ϫ H NMR (300 MHz, CDCl3):
δ ϭ 1.42 and 1.57 (2 s, each 3 H, CMe2), 2.82Ϫ2.95 (m, 2 H, 5a,5a-
H), 3.78 [ddd, J1,2 ϭ 10.5, J1,5a(a) ϭ 5.1, J1,5a(b) ϭ 4.8 Hz, 1 H, 1-
H], 3.83 (dd, J2,3 ϭ 7.0 Hz, 1 H, 2-H), 4.00 (dd, J3,4 ϭ 5.3 Hz, 1
H, 3-H), 4.56 (d, 1 H, 4-H), 5.27 and 5.33 (2 s, each 1 H, ϭCH2). Ϫ
C10H15BrO3 (263.1): calcd. C 45.65, H 5.75; found C 45.43, H 5.96.
1,2-Anhydro-3,4-di-O-(methoxymethyl)-5a-carba-α-
(16) and 1,2-Anhydro-6-deoxy-3,4-di-O-(methoxymethyl)-5a-carba-
β- -altropyranose (17): Compound 14 (0.44 g, 1.26 mmol) was de-
L-fucopyranose
D
O-acetylated as in the preparation of 10 to give the crude triol,
which, without purification, was treated with N,N-diisopropylethyl-
amine (1.3 mL, 6 mol-equiv.) in CH2Cl2 (6 mL) and maintained
overnight at 40 °C. The mixture was diluted with chloroform
(200 mL) and the solution was washed thoroughly with water,
dried, and concentrated. The residue was eluted from a column of
silica gel (15 g) with ethyl acetate/hexane (1:4), to give a major frac-
tion as a homogeneous syrup. The product was hydrogenated in
benzene (4 mL) containing 1 drop of methanol in the presence of
Wilkinson catalyst (120 mg, ca. 0.1 mol-equiv.) for 12 h at room
temperature. The products were chromatographed on a silica gel
column (15 g) with ethyl acetate/toluene (1:8) to give syrupy 16
(0.17 g, 58%) and 17 (0.04 g, 14%).
2,3,4-Tri-O-acetyl-5a-carba-β-L-arabino-hex-5-enopyranosyl Brom-
ide (14): Compound 13 (3.9 g, 10.1 mmol) was mixed with 60%
aqueous acetic acid (100 mL), maintained for 3 h at 60 °C, and
then concentrated to dryness. The residual product was acetylated
in the usual manner to give 14 (3.5 g, 98%) as crystals; m.p.
Compound 16: [α]2D2 ϭ ϩ82 (c ϭ 1.10, CHCl3). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 1.01 (d, J5,Me ϭ 6.8 Hz, 3 H, CMe), 1.53
[br. ddd, J5,5a(a) ϭ 11.5, J5,5a(b) ϭ 5.5 Hz, 1 H, 5-H], 1.76 [ddd,
1
137Ϫ138 °C (from EtOH), [α]2D6 ϭ ϩ14 (c ϭ 0.34, CHCl3). Ϫ H
J1,5a(a) ϭ 2.1, J5agem ϭ 14.7 Hz, 1 H, 5a(a)-H], 1.92 [ddd, J1,5a(b)
ϭ
NMR (300 MHz, CDCl3): δ ϭ 2.01, 2.10, and 2.14 (3 s, each 3 H,
3 ϫ Ac). 2.81Ϫ2.97 [m, 2 H, 5a(ax)-H, 5a(eq)-H], 3.85 [ddd, J1,2 ϭ
11.5, Hz, 1 H, 5a(b)-H], 3.09 (dd, J2,3 ϭ 2.2, J3,4 ϭ 3.6 Hz, 1 H,
3-H), 3.31 (br. ddd, J1,2 ϭ 1.7, Hz, 1 H, 1-H), 3.42 and 3.43 (2 s,
each 3 H, 2 ϫ OMe), 3.63 (dd, 1 H, 2-H), 3.74 (d, 1 H, 4-H), 4.67
and 4.76, and 4.80 and 4.86 (2 ABq, Jgem ϭ 6.8 Hz, 2 ϫ OCH2-
OMe). ؊ HRMS [C10H17O4, Mϩ Ϫ OCH3]: calcd. 201.1127;
found 201.1140.
10.3, J1,5a(ax) ϭ 9.6, J1,5a(eq) ϭ 1.7 Hz, 1 H, 1-H], 4.83 (dd, J2,3
ϭ
10.2, J3,4 ϭ 3.2 Hz, 1 H, 3-H), 5.58 (dd, 1 H, 2-H), 5.68 (d, 1 H,
4-H). Ϫ C13H17BrO6 (349.2): calcd. C 44.72, H 4.91; found C 44.80,
H 5.07.
2,3,4-Tri-O-(methoxymethyl)-5a-carba-β-L-arabino-hex-5-
enopyranosyl Bromide (18): A solution of compound 14 (3.71 g) in
a mixture of 4  hydrochloric acid (25 mL) and THF (75 mL) was
Compound 17: [α]2D1 ϭ ϩ49 (c ϭ 0.48, CHCl3). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 0.94 (d, J5,Me ϭ 6.6 Hz, 3 H, CMe), 1.62
Eur. J. Org. Chem. 2001, 967Ϫ974
971

S. Ogawa, A. Maruyama, T. Odagiri, H. Yuasa, H. Hashimoto
FULL PAPER
[br. dd, J5,5a(a) ϭ 10.5, J5agem ϭ 14.7 Hz, 1 H, 5a(a)-H], 2.06 [dddd,
J5,5a(b) ϭ 7.3, Hz, 1 H, 5-H], 2.19 [ddd, J1,5a(b) ϭ 4.2, 1 H, 5a(b)-
(17 mg, 46%) and 22 (22 mg, 50%); [α]2D6 ϭ Ϫ1 (c ϭ 0.6, CHCl3).
Ϫ 1H NMR (300 MHz, CDCl3): δ ϭ 0.99 (d, J5,Me ϭ 4.9 Hz, 3 H,
H], 3.17 (br. dd, J1,2 ϭ 3.9 Hz, 1 H, 1-H), 3.35 (dd, J2,3 ϭ 2.9 Hz, CMe), 1.26Ϫ1.68 [m, 2 H, 5a(ax)-H, H-5a(eq)-H], 1.96 (m, 1 H, 5-
1 H, 2-H), 3.43 and 3.40 (2 s, each 3 H, 2 ϫ OMe), 3.47 (dd, J3,4 ϭ H), 2.09 (s, 3 H, Ac), 3.36, 3.41, and 3.42 (3 s, each 3 H, 3 ϫ OMe),
3.1, J4,5 ϭ 9.8 Hz, 1 H, 4-H), 4.28 (dd, 1 H, 3-H), 4.60 and 4.73, 3.85 (br. s, 1 H, 4-H), 3.97 (dd, J1,2 ϭ 3.1, J2,3 ϭ 9.6 Hz, 1 H, 2-
and 4.77 and 4.84 (2 ABq, Jgem ϭ 6.7 Hz, 2 ϫ OCH2OMe). ؊ H), 4.68 and 4.72 (ABq, Jgem ϭ 6.8 Hz), 4.73 and 4.77 (ABq,
HRMS [C10H17O4, Mϩ Ϫ OCH3]: calcd. 201.1127; found 201.1140. Jgem ϭ 6.9 Hz), and 4.83 and 4.91 (ABq, Jgem ϭ 6.6 Hz) (3 ϫ
OCH2OMe), 5.34 [br. dd, J1,5a(ax) ഠ 0, J1,5a(eq) ϭ 6.0 Hz, 1 H, 1-
2,3,4-Tri-O-(methoxymethyl)-5a-carba-β-
(19) and 6-Deoxy-2,3,4-tri-O-(methoxymethyl)-5a-carba-α-
L
-fucopyranosyl Bromide
H]. ؊ HRMS [C13H26O6, Mϩ Ϫ OAc ϩ H]: calcd. 278.1729;
found 278.1729.
D-altro-
pyranosyl Bromide (20): To a solution of 18 (0.82 g, 2.3 mmol) in
dry benzene (6.0 mL) were added Wilkinson catalyst (430 mg, 0.2
mol-equiv.) and 2 drops of methanol. The mixture was vigorously
stirred under hydrogen for 16 h at room temperature, and then con-
centrated to dryness. The residue was chromatographed on a silica
gel column (60 g) with ethyl acetate/toluene (1:8 Ǟ 1:6) to give
syrupy 19 (0.66 g, 80%) and 20 (0.12 g, 15%).
5a-Carba-α-L-fucopyranose (4): Compound 22 (21 mg, 62 µmol)
was treated with a mixture of 4  hydrochloric acid (0.5 mL) and
THF (0.5 mL) over 3 h at 50 °C. The product was eluted from a
column of silica gel (1 g) with methanol/chloroform (1:3) to give 4
(6.4 mg, 65%) as a syrup; [α]2D8 ϭ Ϫ9 (c ϭ 0.10, MeOH). Ϫ 1H
NMR (300 MHz, CDCl3): δ ϭ 0.83 (d, J5,Me ϭ 7.0 Hz, 3 H, Me),
1.23 [ddd, J1,5a(eq) ϭ 5.4, J5,5a(eq) ϭ 2.5, J5a gem ϭ 12.0 Hz, 1 H,
5a(eq)-H], 1.49Ϫ1.65 [m, 2 H, 5-H, 5a(ax)-H], 3.24Ϫ3.40 (m, 3 H,
Compound 19: [α]2D1 ϭ ϩ104 (c ϭ 0.90, CHCl3). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 1.02 (d, J5,Me ϭ 6.6 Hz, 3 H, CMe), 1.64
(dddd, J4,5 ϭ 2.0 Hz, 1 H, 5-H), 2.01 [ddd, J1,5a(eq) ϭ 4.7, J5,5a(eq) ϭ
5.0, J5agem ϭ 12.4 Hz, 1 H, 5a(eq)-H], 2.07 [ddd, J1,5a(ax) ϭ 10.4,
J5,5a(ax) ϭ 9.0 Hz, 1 H, 5a(ax)-H], 3.44 (dd, J3,4 ϭ 2.2 Hz, 1 H, 3-
H), 3.41, 3.42, and 3.51 (3 s, each 3 H, 3 ϫ OMe), 3.83 (br. s, 1 H,
4-H), 3.86 (ddd, J1,2 ϭ 9.3 Hz, 1 H, 1-H), 3.93 (dd, J2,3 ϭ 9.1 Hz,
1 H, 2-H), 4.73 (s, 2 H), 4.65 and 4.90 (ABq, Jgem ϭ 6.6 Hz), and
4.81 and 4.84 (ABq, Jgem ϭ 6.4 Hz) (3 ϫ CH2OMe). ؊ HRMS
[C13H25O6Br, Mϩ Ϫ OCH3]: 325.0650; found 325.0658.
1-H, 2-H, 3-H), 3.64 (br. s, 1 H, 4-H). ؊ HRMS [C7H14O4, Mϩ
ϩ
Na]: calcd. 162.1130; found 162.1130.
2,3,4-Tri-O-(methoxymethyl)-5a-carba-α-L-fucopyranosyl
Azide
(23): A mixture of 19 (45 mg, 0.13 mmol), sodium azide (40 mg, 5
mol-equiv.), and DMF (2.5 mL) was stirred for 9 h at 90 °C. The
mixture was diluted with ethyl acetate (30 mL) and the solution
was washed thoroughly with brine, dried, and concentrated. The
residue was eluted from a column of silica gel (30 g) with ethyl
acetate/toluene (1:8) to give 23 (34 mg, 86%) as a syrup; [α]2D3
ϭ
Compound 20: [α]2D6 ϭ ϩ89 (c ϭ 0.18, CHCl3). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 1.02 (d, J5,Me ϭ 6.5 Hz, 3 H, CMe), 2.01
[ddd, J1,5a(eq) ϭ 5.4, J5,5a(eq) ϭ 7.1, J5agem ϭ 13.0 Hz, 1 H, 5a(eq)-
1
ϩ12 (c ϭ 0.90, CHCl3). Ϫ H NMR (300 MHz, CDCl3): δ ϭ 1.84
(br. s, 3 H, CMe), 3.38, 3.41, and 3.42 (3 s, each 3 H, 3 ϫ OMe),
4.06 (dd, J2,3 ϭ 8.1, J3,4 ϭ 3.4 Hz, 1 H, 3-H), 4.12Ϫ4.13 (m, 1 H,
1-H), 4.17 (dd, J1,2 ϭ 4.1 Hz, 1 H, 2-H), 4.23 (br. d, 1 H, 4-H),
H], 2.18 [dddd, 1 H, 5-H], 2.39 [ddd, J1,5a(ax) ϭ 12.5, J5,5a(ax)
ϭ
4.5 Hz, 1 H, 5a(ax)-H], 3.40, 3.42, and 3.49 (3 s, each 3 H, 3 ϫ
OMe), 3.71 (dd, J2,3 ϭ 8.6, J3,4 ϭ 3.0 Hz, 1 H, 3-H), 3.76 (ddd,
J1,2 ϭ 8.8 Hz, 1 H, 1-H), 3.76 (dd, J4,5 ϭ 3.7 Hz, 1 H, 4-H), 3.96
(dd, 1 H, 2-H), 4.08 [ddd, J1,5a(ax) ϭ 11.3, J1,5a(eq) ϭ 4.9 Hz, 1 H,
1-H], 4.08 (dd, 1 H, 2-H), 4.35 (d, 1 H, 4-H), 4.70 and 4.72 (ABq,
Jgem ϭ 6.6 Hz), 4.72 and 4.76 (ABq, Jgem ϭ 6.9 Hz), and 4.80 and
4.89 (ABq, Jgem ϭ 6.4 Hz) (3 ϫ CH2OMe).
4.67 and 4.88, 4.70 and 4.78, and 4.74 and 4.77 (3 ABq, Jgem
ϭ
6.6 Hz, 3 ϫ CH2OMe). Ϫ HRMS [C13H26O6, Mϩ Ϫ N3 ϩ H]:
calcd. 278.1729; found 278.1729.
5a-Carba-α-L-fucopyranosylamine (5): A mixture of 23 (20 mg, 63
µmol), 4  hydrochloric acid (0.5 mL), and THF (0.5 mL) was
stirred for 10 h at room temperature, and concentrated. The residue
was treated with triphenylphosphane (0.1 mL) in 5% aqueous THF
(1 mL) over 3 d at 60 °C. The mixture was concentrated and the
residue was chromatographed on a Dowex 50 W ϫ 2 (Hϩ) resin
with 1% aqueous ammonia as eluent to give 5 (6.6 mg, 66%) as a
syrup; [α]2D0 ϭ Ϫ38 (c ϭ 0.15, MeOH). Ϫ 1H NMR (300 MHz,
CD3OD): δ ϭ 1.43 [ddd, J1,5a(eq) ϭ J5,5a(eq) ഠ 3, J5agem ϭ 12.9 Hz,
1 H, 5a(eq)-H], 1.68 [ddd, J1,5a(ax) ϭ 3.7, J5,5a(ax) ϭ 12.9 Hz, 1 H,
5a(ax)-H], 1.72Ϫ1.97 (m, 1 H, 5-H), 3.15 (ddd, J1,2 ϭ 4.4 Hz, 1 H,
1-H), 3.59 (dd, J2,3 ϭ 9.5, J3,4 ϭ 2.9 Hz, 1 H, 3-H), 3.71 (dd, 1 H,
2-H), 3.72 (br. s, 1 H, 4-H). Ϫ HRMS [C7H15NO3, Mϩ ϩ H]:
calcd. 162.1130; found 162.1106.
(1S,2S,3R,6R)-1,2,3-Tri-O-(methoxymethyl)-4-methylcyclohex-5-
ene-1,2,3-triol (21): To a solution of 19 (110 mg, 3.10 mmol) in
DMF (1 mL) were added sodium hydride (74 mg, 6 mol-equiv.) and
1 drop of methanol, and the mixture was stirred for 6 h at 60 °C.
After neutralization with Dowex 50 W ϫ 2 (Hϩ) resin, the mixture
was concentrated and the residue was eluted from a column of
silica gel (8 g) with ethyl acetate/toluene (1:9 Ǟ 1:6) to give 21
(78 mg, 91%) as a syrup; [α]2D8 ϭ ϩ12 (c ϭ 1.2, CHCl3). Ϫ 1H
NMR (300 MHz, CDCl3): δ ϭ 1.12 (d, 3 H, J4,Me ϭ 7.3 Hz, CMe),
2.55 (dddd, J3,4 ϭ 1.7, J4,6 ϭ 1.9 Hz, 1 H, 4-H), 3.40, 3.41, and
3.42 (3 s, each 3 H, 3 ϫ OMe), 3.80 (dd, J1,2 ϭ 7.9, J2,3 ϭ 2.0 Hz,
1 H, 2-H), 4.01 (br. dd, 1 H, 3-H), 4.39 (ddd, J1,5 ϭ 2.5, J1,6
ϭ
2,3,4-Tri-O-(methoxymethyl)-5a-carba-α-L-fucopyranosylcarbo-
3.1 Hz, 1 H, 1-H), 4.67 and 4.73 (ABq, Jgem ϭ 6.8 Hz), 4.75 and
4.79 (ABq, Jgem ϭ 6.6 Hz), and 4.83 and 4.96 (ABq, Jgem ϭ 6.6 Hz)
(3 ϫ OCH2OMe), 5.47 (ddd, J5,6 ϭ 10.0 Hz, 1 H, 6-H), 5.68 (ddd,
1 H, 5-H). ؊ HRMS [C13H24O6, Mϩ]: calcd. 276.1573; found
276.1573.
nitrile (24): A mixture of 19 (67 mg, 0.19 mmol), sodium cyanide
(137 mg, 15 mol-equiv.) and dry DMF (0.7 mL) was stirred for 20 h
at 100 °C. Toluene (10 mL) was added to the mixture, and an insol-
uble material was removed by filtration. The filtrate was concen-
trated and the residue was chromatographed on silica gel (4 g) with
acetone/hexane (1:9 1:5) to give 24 (51 mg, 90%) as a syrup, to-
gether with a trace of 21 (3 mg, 5%); [α]2D0 ϭ ϩ41 (c ϭ 0.19,
2,3,4-Tri-O-(methoxymethyl)-5a-carba-α-L-fucopyranosyl Acetate
(22): A mixture of 19 (45 mg, 0.13 mmol), potassium acetate
(93 mg, 10 mol-equiv.), and DMF (1 mL) was stirred for 20 h at
100 °C. After cooling, the mixture was diluted with ethyl acetate
(30 mL), and the solution was washed thoroughly with brine, dried,
and concentrated. The products were chromatographed on a silica
gel column (3 g) with ethyl acetate/toluene (1:8) to give syrupy 21
CHCl3). Ϫ 1H NMR (300 MHz, CDCl3): δ ϭ 1.05 (s, J5,Me
ϭ
6.6 Hz, 3 H, CMe), 1.68 [ddd, J1,5a(eq) ϭ 7.9, J5,5a(eq) ϭ 2.9,
J5agem ϭ 13.0 Hz, 1 H, 5a(eq)-H], 1.79 [ddd, J1,5a(ax) ϭ 3.9,
J5,5a(ax) ϭ 12.2 Hz, 1 H, 5a(ax)-H], 2.05 (m, 1 H, 5-H), 3.34 (ddd,
J1,2 ϭ 5.1 Hz, 1 H, 1-H), 3.46, 3.42 and 3.41 (3 s, each 3 H, 3 ϫ
972
Eur. J. Org. Chem. 2001, 967Ϫ974

Synthesis and Biological Evaluation of α-
L-Fucosidase Inhibitors
FULL PAPER
OMe), 3.80 (dd, J2,3 ϭ 9.8, J3,4 ϭ 2.5 Hz, 1 H, 3-H), 3.87 (br. s, 1 H, 7a-H), 5.01 (br. s, 1 H, 7b-H). Ϫ HRMS [C14H26O6, Mϩ]: calcd.
H, 4-H), 3.91 (dd, 1 H, 2-H), 4.64 and 4.72, 4.73 and 4.79, and
4.82 and 4.90 (3 ABq, Jgem ϭ 6.6 Hz, 3 ϫ CH2OMe). Ϫ HRMS
[C14H25NO6, Mϩ Ϫ OCH3]: calcd. 272.1499; found 272.1498.
290.1729; found 290.1710.
Compound 28: [α]2D0 ϭ Ϫ32 (c ϭ 0.10, CHCl3). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 1.02 (d, J ϭ 6.8 Hz, 3 H, CMe), 1.43Ϫ1.58
(m, 2 H, 5,5-H), 1.85Ϫ1.90 (m, 1 H, 6-H), 2.28Ϫ2.35 (m, 1 H, 4-
H), 3.41, 3.45, and 3.49 (3 s, each 3 H, 3 ϫ OMe), 3.45 (br. d,
J2,3 ϭ 11.0 Hz, 1 H, 2-H), 3.55 (dd, J3,4 ϭ 5.1 Hz, 1 H, 3-H), 3.82
(br. s, 1 H, 1-H), 3.83 (br. s, 1 H, OH), 3.91 (dd, J4,7a ϭ 9.5, J7gem ϭ
10.3 Hz, 1 H, 7a-H), 4.11 (dd, J4,7b ϭ 4.6, 1 H, 7b-H), 4.49 and
4.81 (ABq, Jgem ϭ 6.8 Hz), 4.66 and 4.74 (ABq, Jgem ϭ 6.7 Hz),
and 4.68 and 4.77 (ABq, Jgem ϭ 6.6 Hz) (3 ϫ CH2OMe). Ϫ MS
{C14H28O7 (308.37); m/z (%): 309 (12) [Mϩ].
(1R,2R,3R,4S,6R)- and (1R,2R,3R,4R,6R)-4-(Methanesulfonyloxy-
methyl)-1,2,3-tri-O-(methoxymethyl)-6-methyl-1,2,3-cyclohexane-
triol (25 and 26): To a solution of 24 (50 mg, 160 µmol) in toluene
(1 mL) was added 1  DIBAL/toluene (320 µL, 2 mol-equiv.) at
Ϫ78 °C, and then the mixture was stirred for 4 h at Ϫ60 °C. It was
then diluted with ethyl acetate (1 mL) and stirred with acetic acid
(1 mL) and silica gel (0.30 g) for 1 h at room temperature. Insoluble
material was removed by filtration and the filtrate was concen-
trated. The residue was treated with sodium borohydride (6.2 mg,
1 mol-equiv.) in methanol (0.5 mL) for 20 min at 0 °C. The mixture
was diluted with ethyl acetate (30 mL), and the solution was
washed with water thoroughly, dried, and concentrated. The res-
idue was treated with 2 drops of mesyl chloride and triethylamine
for 2 h at 0 °C, and the reaction mixture was diluted with chloro-
form (20 mL), washed with water, dried, and concentrated. The
residual products were chromatographed on silica gel (3 g) with
butanone/hexane (1:6) to give syrupy 25 (30 mg, 47%) and 26
(24 mg, 38%).
(1R,2R,3R,4S,6R)-4-(Hydroxymethyl)-6-methyl-1,2,3-cyclohexane-
triol (6): Compound 28 (6 mg, 19 µmol) was deprotected as in the
preparation of 4, and the crude product was eluted from a silica
gel column (1 g) with methanol/chloroform (1:5) to give 6 (3 mg,
94%) as a syrup; [α]2D0 ϭ ϩ27 (c ϭ 0.25, MeOH). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 0.99 (d, 3 H, J6,Me ϭ 6.8 Hz, CMe),
1.45Ϫ1.61 (m, 2 H, 5,5-H), 1.78Ϫ1.82 (m, 1 H, 6-H), 2.10Ϫ2.16
(m, 1 H, 4-H), 3.49 (dd, J1,2 ϭ 2.8, J2,3 ϭ 10.0 Hz, 1 H, 2-H), 3.53
(dd, J3,4 ϭ 10.0 Hz, 1 H, 3-H), 3.86Ϫ3.92 (m, 2 H, 7,7-H). Ϫ
HMRS [C8H16O4, Mϩ ϩ H]: calcd. 177.1125; found 177.1125.
Compound 25: [α]1D9 ϭ ϩ4.7 (c ϭ 0.48, CHCl3). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 1.04 (d, J6,Me ϭ 7.1 H, 3 H, CMe),
1.46Ϫ1.64 [m, 2 H, 5a(ax)-H, 5a(eq)-H], 1.96 (m, 1 H, 6-H), 2.45
[ddddd, J3,4 ϭ 4.7, J4,5a(ax) ϭ 4.4, J4,5a(eq) ϭ 6.3, J4,7a ϭ 6.1, J4.7b ϭ
8.9 Hz, 1 H, 4-H], 3.00 (s, 3 H, Ms), 3.36 and 3.37 (2 s, 3 and 6 H,
(1R,2S,3S,4R,5R)- and (1S,2R,3R,4S,6R)-1,7-Anhydro-1-(hydroxy-
methyl)-2,3,4-tri-O-(methoxymethyl)-5-methyl-1,2,3,4-cyclohexane-
tetraol (29 and 30): A mixture of 27 (20 mg, 69 µL), mCPBA (18 mg,
1.5 mol-equiv.), and CH2Cl2 (2 mL) was stirred for 5.5 h at room
temperature. After treatment with aqueous sodium thiosulfate
(2 mL), the mixture was extracted with chloroform (20 mL), and
the solution was washed with saturated aqueous sodium hydrogen
carbonate and water, dried, and concentrated. The residue was
chromatographed on a silica gel column (2 g) with ethyl acetate/
hexane (1:4) to give 29 (22 mg, 77%) and 30 (6 mg, 21%). Both
compounds 29 and 30 were labile and did not give satisfactory
HRMS data.
3 ϫ OMe), 3.69 (br. d, J2,3 ϭ 7.8 Hz, 1 H, 2-H), 3.98 (dd, J3,4
ϭ
4.7 Hz, 1 H, 3-H), 4.17 (dd, J7gem ϭ 11.2 Hz, 1 H, 7a-H), 4.39 (dd,
1 H, 7b-H), 4.64 and 4.68, 4.64 and 4.69, and 4.67 and 4.76 (3
ABq, Jgem ϭ 6.6 Hz, 3 ϫ CH2OMe). Ϫ HRMS [C13H24O7S, Mϩ
Ϫ OCH3 ϫ 2]: calcd. 324.1242; found 324.1242.
Compound 26: [α]1D9 ϭ ϩ84 (c ϭ 0.77, CHCl3). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 1.01 (d, J6,Me ϭ 6.9 Hz, 3 H, CMe),
1.45Ϫ1.70 [m, 3 H, 5a(ax)-H, 5a(eq)-H, 6-H], 1.83 (m, 1 H, 4-H),
3.03 (s, 3 H, Ms), 3.40, 3.41, and 3.43 (3 s, each 3 H, 3 ϫ OMe),
Compound 29: [α]2D2 ϭ ϩ43 (c ϭ 0.28, CHCl3). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 1.02 (d, J5,Me ϭ 6.1 Hz, 3 H, CMe), 2.06
(br. d, J6gem ϭ 9.8 Hz, 6a-H), 2.63 (d, J7gem ϭ 5.0 Hz, 1 H, 7a-H),
3.09 (d, 1 H, 7b-H), 3.40, 3.41, and 3.43 (3 s, each 3 H, 3 ϫ OMe),
3.81 (dd, J2,3 ϭ 10.0, J3,4 ϭ 2.3 Hz, 1 H, 3-H), 4.18 (d, 1 H, 2-H),
3.52 (dd, J1,2 ϭ 2.4, J2,3 ϭ 9.4 Hz, 1 H, 2-H), 3.67 (dd, J3,4
ϭ
10.7 Hz, 1 H, 3-H), 3.82 (br. d, 1 H, 1-H), 4.21 (dd, J4,7a ϭ 6.6,
J7gem ϭ 9.4 Hz, 1 H, 7a-H), 4.43 (dd, J4,7b ϭ 3.2 Hz, 1 H, 7b-H),
4.64 and 4.67, 4.68 and 4.76, and 4.85 and 4.87 (3 ABq, Jgem
ϭ
4.64 and 4.87 (ABq, Jgem ϭ 6.6 Hz), 4.70 and 4.92 (ABq, Jgem
6.8 Hz), and 4.72 and 4.73 (ABq) (3 ϫ CH2OMe).
ϭ
6.6 Hz, 3 ϫ OCH2OMe). Ϫ HRMS [C13H24O7S, Mϩ Ϫ OCH3 ϫ
2]: calcd. 324.1242; found 324.1213.
Compound 30: [α]2D2 ϭ ϩ77 (c ϭ 0.13, CHCl3). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 1.04 (d, J5,Me ϭ 6.8 Hz, 3 H, CMe), 1.06
(dd, J5,6a ϭ 4.3, J6gem ϭ 12.8 Hz, 1 H, 6a-H), 1.73Ϫ1.82 (m, 1 H,
5-H), 2.13 (ddd, J5,6b ϭ 12.8, J6b,7a ϭ 2.0 Hz, 1 H, 6b-H), 2.44 (d,
J7gem ϭ 5.6 Hz, 1 H, 7b-H), 3.04 (dd, 1 H, 7a-H), 3.38, 3.42, and
3.43 (3 s, each 3 H, 3 ϫ OMe), 3.56 (dd, J2,3 ϭ 10.0, J3,4 ϭ 2.4 Hz,
1 H, 3-H), 3.86 (br. d, 1 H, 4-H), 4.12 (d, 1 H, 2-H), 4.42 and 4.76
(ABq, Jgem ϭ 6.9 Hz), 4.67 and 4.95 (ABq, Jgem ϭ 6.6 Hz), and
4.63Ϫ4.65 (ABq) (3 ϫ OCH2OMe).
(1R,2R,3R,6R)-1,2,3-Tri-O-(methoxymethyl)-6-methyl-4-methylene-
1,2,3-cyclohexanetriol (27) and (1R,2R,3R,4S,6R)-4-(Hydroxy-
methyl)-1,2,3-tri-O-(methoxymethyl)-6-methyl-1,2,3-yclohexanetriol
(28): A mixture of 25 (9 mg, µmol), DBU (1.8 mL, 6 mol-equiv.),
and toluene (1 mL) was stirred for 4 h at 80 °C, and then diluted
with ethyl acetate (15 mL). The solution was washed with 1  hy-
drochloric acid, saturated sodium hydrogen carbonate, and water,
dried, and concentrated. The residue was chromatographed on a
silica gel column (1 g) with ethyl acetate/hexane (1:3) to give 27
(5 mg, 80%) and 28 (1.3 mg, 18%).
(1S,2S,3R,4S,6R)-1-(Azidomethyl)-2,3,4-tri-O-(methoxymethyl)-5-
methyl-1,2,3,4-cyclohexanetetrol (31): A mixture of 29 (20 mg, 65
µmol), sodium azide (13 mg, 3 mol-equiv.), 15-crown-5 ether (13
Compound 27: [α]2D0 ϭ ϩ21 (c ϭ 0.17, CHCl3). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ 1.00 (d, J6,Me ϭ 6.6 Hz, 3 H, CMe), 1.64 µL), and DMF (0.4 mL) was stirred for 20 h at 80 °C, and then
(m, 1 H, 6-H), 2.06 (dd, J5eq,6 ϭ 4.4, J5gem ϭ 13.1 Hz, 1 H, 5eq- diluted with ethyl acetate (30 mL). The solution was washed thor-
H), 2.16 (dd, J5ax,6 ϭ 12.0, 1 H, 5ax-H), 3.39, 3.40, and 3.41 (3 s, oughly with brine, dried and concentrated. The residue was eluted
each 3 H, 3 ϫ OMe), 3.45 (dd, J1,2 ϭ 2.6, J2,3 ϭ 9.4 Hz, 1 H, 2-
H), 3.84 (br. s, 1 H, 1-H), 4.34 (br. d, 1 H, 3-H), 4.68 and 4.90
(ABq, Jgem ϭ 7.1 Hz), 4.69 and 4.76 (ABq, Jgem ϭ 6.3 Hz), and
4.73 and 4.79 (ABq, Jgem ϭ 6.4 Hz) (3 ϫ CH2OMe), 4.82 (br. s, 1
from a column of silica gel (2 g) with acetone/hexane (1:6) to give
1
31 (21 mg, 94%) as a syrup; [α]1D5 ϭ ϩ88 (c ϭ 0.75, CHCl3). Ϫ H
NMR (300 MHz, CDCl3): δ ϭ 1.46 (dd, J5,6eq ϭ 4.3, J6gem
ϭ
13.5 Hz, 1 H, 6eq-H), 1.55 (ddd, J5,6ax ϭ 12.2, J6ax,OH ϭ 2.0 Hz,
Eur. J. Org. Chem. 2001, 967Ϫ974
973

S. Ogawa, A. Maruyama, T. Odagiri, H. Yuasa, H. Hashimoto
FULL PAPER
[1]
6ax-H), 1.92Ϫ2.00 (m, 1 H, 5-H), 2.32 (d, 1 H, OH), 3.34 and 3.38
(2 s, 6 and 3 H, 3 ϫ OMe), 3.25 (d, J7gem ϭ 11.5 Hz, 1 H, 7a-H),
3.35 (d, 1 H, 7b-H), 3.73 (br. s, 1 H, 4-H), 3.78 (dd, J2,3 ϭ 9.8,
J3,4 ϭ 2.0 Hz, 3-H), 4.58 and 4.67 (ABq, Jgem ϭ 6.8 Hz), 4.59 and
4.79 (ABq, Jgem ϭ 6.7 Hz), and 4.65 and 4.86 (ABq, Jgem ϭ 6.4 Hz)
(3 ϫ CH2OMe). Ϫ HRMS [C14H27N3O7, Mϩ ϩ H]: calcd.
350.1927; found 350.1932.
S. Ogawa, R. Sekura, A. Maruyama, H. Yuasa, H. Hashimoto,
Eur. J. Org. Chem. 2000, 2089Ϫ2093.
[2]
[3]
B. Winchester, G. W. Fleet, Glycobiology 1992, 2, 199Ϫ210.
R. DeSantis, M. R. Pinto, Mechanism of Fertilization: Plant to
Humans (Ed.: B. Dale), Springer-Verlag, Berlin, 1990, pp.
297Ϫ304.
R. J. Bernacki, M. J. Niedbala, W. Korytnyk, Cancer Meta-
stasis Rev. 1985, 4, 81Ϫ102.
G. W. Fleet, A. N. Shaw, S. V. Evans, L. E. Fellows, J. Chem.
Soc., Chem. Commun. 1985, 841Ϫ842; H. Paulsen, M. Matzke,
B. Ortheb, R. Nuck, W. Reutter, Liebigs Ann. Chem. 1990,
953Ϫ963.
E. Shitara, Y. Nishimura, F. Kojima, T. Takeuchi, Bioorg. Med.
Chem. 2000, 8, 343Ϫ352; S. M. Andersen, C. Ekhart, I. Lundt,
A. E. Stutz, Carbohydr. Res. 2000, 326, 22Ϫ33 and references
therein.
[4]
[5]
(1R,2S,3S,4R,6R)-1-(Azidomethyl)-2,3,4-tri-O-(methoxymethyl)-5-
methyl-1,2,3,4-cyclohexanetetrol (32): Compound 30 (6 mg, 20
µmol) was treated with sodium azide (4 mg, 3 mol-equiv.) as in the
preparation of 31, and the product was eluted from a column of
silica gel (1 g) with ethyl acetate/hexane (1:4) to give 32 (6 mg, 88%)
as a syrup; [α]1D5 ϭ ϩ60 (c ϭ 0.33, CHCl3). Ϫ 1H NMR (300 MHz,
CDCl3): δ ϭ 1.03 (d, J5,Me ϭ 6.1 Hz, 3 H, CMe), 1.56Ϫ1.60 (m, 2
H, 5-H, 6a-H), 1.86 (br. d, J6gem ϭ 10.5 Hz, 1 H, 6b-H), 3.16 (d,
J7gem ϭ 12.8 Hz, 1 H, 7a-H), 3.39, 3.40, and 3.47 (3 s, each 3 H, 3
ϫ OMe), 3.49 (dd, J2,3 ϭ 10.5, J3,4 ϭ 2.7 Hz, 1 H, 3-H), 3.56 (d,
1 H, 7b-H), 3.76 (br. s, 1 H, 4-H), 3.78 (dd, 1 H, 2-H), 3.99 (s, 1
H, OH), 4.66 and 4.76 (ABq, Jgem ϭ 6.1 Hz), 4.68 and 4.87 (ABq,
Jgem ϭ 6.8 Hz), and 4.67 (s, 2 H) (3 ϫ OCH2OMe). Ϫ HRMS
[C14H27N3O7, Mϩ ϩ H]: calcd. 350.1927; found 350.1932.
[6]
[7]
[8]
O. Tsuruta, H. Yuasa, H. Hashimoto, Bioorg. Med. Chem. Lett.
1996, 6, 1989Ϫ1992.
S. Horii, T. Iwasa, E. Mizuta, Y. Kameda, J. Antibiot. 1971, 24,
59Ϫ63; Y. Kameda, N. Asano, M. Yoshikawa, M. Takeuchi, T.
Yamaguchi, K. Matsui, S. Horii, H. Fukase, J. Antibiot. 1984,
37, 1301Ϫ1307.
Y. Kameda, N. Asano, M. Yoshikawa, K. Matsui, J. Antibiot.
1980, 33, 1575Ϫ1576; Y. Kameda, N. Takai, N. Asano, Y. Ka-
meda, K. Matsui, Chem. Pharm. Bull. 1990, 38, 1970Ϫ1972;
M. Takeuchi, K.Kamata, M. Yoshida, Y. Kameda, K. Matsui,
J. Biochem. 1990, 108, 42Ϫ46; T. Suami, S. Ogawa, Adv. Car-
bohydr. Chem. Biochem. 1990, 48, 21Ϫ90; and references
therein.
D. D. Schmidt, W. Frommer, B. Junge, L. Müller, W. Wing-
ender, E. Truscheit, D. Schafer, Naturwissenschaften 1977, 64,
535Ϫ536.
S. Horii, H. Fukase, T. Matsuo, Y. Kameda, N. Asano, K. Mat-
sui, J. Med. Chem. 1986, 29, 1038Ϫ1046.
[9]
(1S,2S,3R,4S,6R)-1-(Aminomethyl)-5-methyl-1,2,3,4-cyclohexane-
tetrol (7): Compound 31 (10 mg, 29 µmol) was deprotected and
reduced as described in the preparation of 5, and the product was
purified by elution from a column of silica gel (1 g) with methanol/
[10]
chloroform (1:4) to give 7 (4.6 mg, ca. 100%) as a syrup; [α]2D0
ϭ
[11]
[12]
[13]
Ϫ100 (c ϭ 0.10, MeOH). Ϫ 1H NMR (300 MHz, CDCl3): δ ϭ
0.98 (d, J5,Me ϭ 6.8 Hz, 3 H, CMe), 1.36 (dd, J6gem ϭ 13.9 Hz,
J5,6b ϭ 4.5 Hz, 6b-H), 1.45 (dd, J5,6a ϭ 13.9 Hz, 1 H, 6a-H),
1.95Ϫ2.00 (m, 1 H, 5-H), 2.58 (d, J7gem ϭ 13.2 Hz, 7a-H), 2.79 (d,
Y. Kameda, K. Kawashima, M. Takeuchi, K. Ikeda, N. Asano,
K. Matsui, Carbohydr. Res. 1997, 300, 259Ϫ264.
S. Ogawa, M. Oya, T. Toyokuni, N. Chida, T. Suami, Bull.
Chem. Soc. Jpn. 1983, 56, 1441Ϫ1445; S. Ogawa, M. Suzuki,
T. Tonegawa, Bull. Chem. Soc. Jpn. 1988, 61, 1824Ϫ1826;
A part of this work has been previously reported: S. Ogawa, R.
Sekura, A. Maruyama, T. Odagiri, H. Yuasa, H. Hashimoto,
Carbohydr. Lett. 2000, 4, 13Ϫ20. A. Maruyama, R. Sekura, T.
Odagiri, H. Yuasa, H. Hashimoto, S. Ogawa, reported on the
78th Annual Meeting of Chemical Society of Japan, Chiba,
Japan, March, 2000, Abstract 4 E5 02, p. 864.
The racemic form of 10 was first synthesized in a similar man-
ner: S. Ogawa, T. Toyokuni, M. Omata, N. Chida, T. Suami,
Bull. Chem. Soc. Jpn. 1980, 53, 455Ϫ457; the -enantiomer was
also prepared[16] later.
S. Ogawa, Y. Iwasawa, T. Nose, T. Suami, S. Ohba, M. Ito, Y.
Saito, J. Chem. Soc., Perkin Trans. 1 1985, 903Ϫ906.
B. Helferich, E. Himmen, Ber. Dtsch. Chem. Ges. 1928, 61,
1825Ϫ1835.
Treatment of 16 with sodium cyanide in DMF (4 d at 110 °C)
resulted in a cleavage of the epoxide ring to give a single nitrile,
isolated as the 2-mesylate in 72% overall yield.
Under the influences of either DBU/toluene (ca. 40 °C), 0.2
 MeONa/MeOH (reflux), or NaH/DMF (40 °C Ǟ reflux),
compound 19 did not undergo elimination reaction. Addition
of a trace of MeOH to the reaction mixture under NaH/DMF
conditions resulted in the formation of the single elimination
product 21. Some isomerized alkenes have been shown to be
formed when increasing the amount of MeOH.
1 H, 7b-H), 3.55 (d, J2,3 ϭ 9.8 Hz, 1 H, 2-H), 3.62 (dd, J3,4
ϭ
3.2 Hz, 1 H, 3-H), 3.73 (br. s, 1 H, 4-H). Ϫ HRMS [C8H18NO4,
[14]
Mϩ ϩ H]: calcd. 192.1236; found 192.1222.
(1R,2S,3S,4R,6R)-1-(Aminomethyl)-5-methyl-1,2,3,4-cyclohexane-
tetrol (8): Compound 32 (5 mg, 14 µmol) was deprotected and re-
duced as in the preparation of 5, and the product was purified as
in the preparation of 7 to give 8 (2 mg, ca. 100%) as a syrup;
[15]
1
[α]2D0 ϭ Ϫ36 (c ϭ 0.10, MeOH). Ϫ H NMR (300 MHz, CDCl3):
δ ϭ 0.99 (d, J5,Me ϭ 6.1 Hz, 3 H, CMe), 1.19Ϫ1.62 (m, 3 H, 5-H,
6,6-H), 2.62 (d, J7gem ϭ 12.6 Hz, 1 H, 7a-H), 3.08 (d, 1 H, 7b-H),
3.39 (dd, J2,3 ϭ 10.3, J3,4 ϭ 3.0 Hz, 1 H, 3-H), 3.70 (br. s, 1 H, 4-
H), 3.74 (d, 1 H, 2-H). Ϫ HRMS [C8H18NO4, Mϩ ϩ H]: calcd.
192.1236; found 192.1210.
[16]
[17]
[18]
Biological Assay: α-Fucosidase from bovine kidney, bovine serum
albumin (BSA), and p-nitrophenyl α--fucopyranoside (pNP-Fuc)
were purchased from Sigma. Hydrolysis of pNP-Fuc by α-fucosid-
ase was carried out in wells of microplates precoated with BSA,
and the liberated p-nitrophenol was quantified by measuring ab-
sorbance at 400 nm with a microplate reader (BioRad 550) under
alkaline conditions. The mixture of pNP-Fuc (0.54Ϫ1.37 µ), α-
fucosidase (1.3 ng), BSA (38 µg), and an appropriate amount of
inhibitor in 17 µ citrate buffer (pH ϭ 6.0, 45 µL) was incubated
at 25 °C for 20 min, and was then treated with 50 m glycine buffer
(pH ϭ 10.1, 90 µL). Ki values listed in Table 1 were estimated from
LineweaverϪBurk plots.
[19]
[20]
Both addition of a 5-crown-5 ether and use of aqueous DMF
as a solvent resulted in an increased yield of the elimination
product.
M. Carpintero, C. Jaramillo, A. Fernadez-Mayoralas, Eur. J.
Org. Chem. 2000, 1285Ϫ1296.
S. Ogawa, Y. Shibata, K. Miyazawa, T. Toyokuni, T. Iida, T.
Suami, Carbohydr. Res. 1987, 163, 53Ϫ62.
[21]
[22]
[23]
Acknowledgments
The authors thank Miss L. Zhao for elementary analyses and Ya-
makawa Chemical Industry Co. Ltd. (Tokyo) for provision of op-
tical resolution reagents.
R. L. Whistler, M. S. Feather, D. L. Ingles, J. Am. Chem. Soc.
1962, 84, 122Ϫ127; Y. Ding, O. Hindsgaul, Bioorg. Chem. Lett.
1998, 8, 1215Ϫ1220 and references therein.
Received July 27, 2000
[O00395]
974
Eur. J. Org. Chem. 2001, 967Ϫ974