Enzymatic/chemical synthesis and biological evaluation of seven-membered iminocyclitols

Article abstract of DOI:10.1021/ja960975c

Several polyhydroxyperhydroazepines have been obtained either by chemoenzymatic or chemical synthesis. Condensation of (±)-3-azido-2-hydroxypropanaldehyde and dihydroxyacetone phosphate (DHAP) in the presence of a DHAP dependent aldolase followed by treat

Full text of DOI:10.1021/ja960975c

VOLUME 118, NUMBER 33
AUGUST 21, 1996
© Copyright 1996 by the
American Chemical Society
Enzymatic/Chemical Synthesis and Biological Evaluation of
Seven-Membered Iminocyclitols
Francisco Mor´ıs-Varas, Xin-Hua Qian, and Chi-Huey Wong*
Contribution from the Department of Chemistry, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed March 25, 1996X
Abstract: Several polyhydroxyperhydroazepines have been obtained either by chemoenzymatic or chemical synthesis.
Condensation of (()-3-azido-2-hydroxypropanaldehyde and dihydroxyacetone phosphate (DHAP) in the presence
of a DHAP dependent aldolase followed by treatment with acid phosphatase and an isomerase gave a 6-azido-6-
deoxyaldopyranose, which upon reductive amination afforded the title compound. The iminocyclitols can also be
obtained by chemical manipulations of aldopyranoses, protected as benzyl glycosides or diisopropylidene ethers.
Thus, D-galactose leads to a meso-3,4,5,6-tetrahydroxyperhydroazepine, D-mannose to a derivative with a C2 symmetry
axis, and N-acetylglucosamine to a 6-acetamidoiminocyclitol. Asymmetrization of the meso azasugar was carried
out by chemical means, to yield a 3-methoxy-4,5,6-trihydroxyazepane. An attempted enzymatic synthesis of the
methoxy derivatives of these azasugars was unsuccessful, leading, however, to both enantiomers of 1-deoxy-2-O-
methylmannojirimycin. Some of these compounds display significant activity as glycosidase inhibitors, with Ki
values from moderate to low micromolar range. Though all these iminocyclitols do not inhibit the mechanistically
related HIV protease, the 3,6-dibenzyl derivative 30 showed moderate inhibition. The X-ray structure of 7 indicates
a pseudochair conformation.
Glycosidases and glycosyltransferases are targets of inhibition
as these enzymes are involved in the processing and synthesis
of complex carbohydrates which are essential for various
biological recognition processes.1,2 Much work has been
devoted to the study of five- and six-membered azasugar
families, as they are thought to mimic the transition state of
these enzymatic reactions.3,4
We describe here new efficient methods for the synthesis of
3,4,5,6-tetrahydroxyperhydroazepines 1-8 and evaluation of this
class of seven-membered iminocyclitols as inhibitors of gly-
cosidases. These seven-membered ring species are regarded
to be conformationally more flexible than the five- and six-
membered counterparts and hence may adopt a quasi-flattened
conformation with minimum energetic demand, which could
lead to a favorable binding in the enzyme active site. These
heterocycles have been described in the literature,5,6 but little
X Abstract published in AdVance ACS Abstracts, August 1, 1996.
(1) (a) Look, G. C.; Fotsch, C. H.; Wong, C.-H. Acc. Chem. Res. 1993,
26, 182. (b) Winchester, B.; Fleet, G. W. J. Glycobiology 1992, 2, 199. (c)
Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 521
(2) (a) Legler, G. AdV. Carbohydr. Chem. Biochem. 1990, 48, 319. (b)
Sinnott, M. L. Chem. ReV. 1990, 90, 1171. (c) Kirby, A. J. AdV. Phys. Org.
Chem. 1994, 29, 87. (d) Jacob, G. S. Curr. Opin. Struct. Biol. 1995, 5,
605.
(3) (a) McCarter, J. D.; Withers, S. G. Curr. Opin. Struct. Biol. 1994, 4,
885. (b) Ganem, B.; Papandreou, G. J. Am. Chem. Soc. 1991, 113, 8984;
(c) Papandreou, G.; Tong, M. K.; Ganem, B. J. Am. Chem. Soc. 1993, 115,
11682.
(4) (a) Fotsch, C.; Wong, C.-H. Tetrahedron Lett. 1994, 35, 3481. (b)
Jeong, J.-H.; Murray, B. W.; Takayama, S.; Wong, C.-H. J. Am. Chem.
Soc., 1996, 118, 4227.
(5) (a) Paulsen, H.; Todt, K. Chem. Ber 1967, 100, 512. (b) Poitout, L.;
Merrer, Y. L.; Depezay, J-C. Tetrahedron Lett. 1994, 35, 3293. (c) Lohray,
B. B.; Jayamma, Y.; Chatterjee, M. J. Org. Chem. 1995, 60, 5958.
(6) Farr, R. A.; Holland, A. K.; Huber, E. W.; Peet, N. P.; Weintraub,
P. M. Tetrahedron 1994, 50, 1033.
(7) Bernotas, R. C.; Pezzone, M. A.; Ganem, B. Carbohydr. Res. 1987,
167, 305.
S0002-7863(96)00975-4 CCC: $12.00 © 1996 American Chemical Society

7648 J. Am. Chem. Soc., Vol. 118, No. 33, 1996
Mor´ıs-Varas et al.
Table 1. Inhibition of Glycosidases with Various Seven-Membered Iminocyclitolsa
R- mannosidase R-galactosidase â-galactosidase
â-glucosidase
from sweet
almonds
R-fucosidase
compd [I],
from jack
beans
from green
from
R-glucosidase
from yeast
â-N-acetylglucosaminidase from bovine
no.
µM
coffee beans Aspergillus niger
from jack beans
kidney
1
2
3
4
120 31 (187 ( 26) 87 (5.4 ( 0.56)
9
13
16
NI
35
95 (4.6 ( 0.7)
160 18
240 NI
25 (216 ( 19)
49 (50 ( 4)
NI
21
15
NI
14
NI
NI
13
57
NI
NI
15
NI
94 (4.6 ( 0.4)
22
16
240 30 (364 ( 49) NI
224 14 NI
89 (10.6 ( 0.7)
42
44
88 (23.4 ( 3.8)
5
5
41 (269 ( 20)
6b
200 81 (25.7 ( 1.3) 65 (67 ( 4.5)
95 (6.5 ( 1.2) 78 (29.4 ( 2.2) 92 (12.8 ( 0.7)
NI
3
89 (22.7 ( 2.6)
7b,c 160 11
8b,c 200 48
NI
31
6
NI
14
6
NI
78 (30.5 ( 2.5)
a % inhibition at specified [I]; all show competitive inhibition, and Ki values (µM) are given in parentheses. NI stands for no inhibition. b Prepared
through epoxide opening according to the procedure described previously.2 c See refs 10 and 11 for spectral and physical data.
Scheme 1a
is known regarding their biological activities, with the exception
that one related compound was reported to have no inhibition
activity against R-mannosidase.6 We have found, however, that
many of these iminocyclitols are potent inhibitors of glycosi-
dases and some even exhibit higher inhibition potencies than
the five- and six-membered counterparts (Table 1).
As shown in Table 1, for each of the seven glycosidases
investigated, there is at least one seven-membered iminocyclitol
which exhibits potent inhibition of the enzyme with Ki in the
low micromolar range. Interestingly, compound 3 (Ki ) 4.6
µM) is better than 1-deoxy-N-acetylglucojirimycin (Ki ) 9.8
µM)1c as an inhibitor of â-N-acetylglucosaminidase, 6 (Ki )
6.5 µM) is better than 1-deoxygalactojirimycin (Ki > 1 mM)7
as an inhibitor of â-galactosidase, and 6 (Ki ) 25.7 µM) is better
than 1-deoxymannojirimycin (Ki ) 150 µM)8 as an inhibitor
of R-mannosidase. Compound 1 (Ki ) 9.4 µM) is, however,
weaker than 1-deoxygalactojirimycin (Ki ) 1.5 nM) as an
a Conditions: (a) pH 6.7, DHAP, FDPA; (b) pH 4.5, Pase; (c) pH
7.2, TAKASWEET (Glcl), 26% for 10; (d) H2 (50 psi), Pd/C, H2O, 2
d, 91-54%; (e) pH 6.7, DHAP, RhaA; 30% for 11; (f) pH 7.2, RhaI;
(g) pH 6.7, DHAP, FucA; (h) pH 7.2, Fucl 21% for 12.
inhibitor of R-galactosidase7 and also weaker (Ki ) 4.6 µM)
than 1-deoxyfucojirimycin (Ki ) 5 nM)9 as an inhibitor of
R-fucosidase. A similar situation was observed for five-
membered iminocyclitols.1c It is also interesting that, of the
iminocyclitols examined, one of them with a C2 symmetry (6)
inhibits all glycosidases, including R-mannosidase. Benzylation
of the nitrogen group of 3 and 6 (resulting in compounds 7 and
8) does not improve inhibition activity except in the case of
R-fucosidase.
products 6-azido-6-deoxy-D-glucopyranose (10) and 6-azido-
6-deoxy-L-galactopyranose (12) obtained were subjected to Pd-
mediated reductive amination15 in aqueous solution in the
presence of H2 (50 psi) to give 3(R),4(R),5(R),6(S)-tetrahydrox-
yperhydroazepine (1) and the meso-iminocyclitol 3(S),4(R),5-
(S),6(R)-tetrahydroxyperhydroazepine (2), respectively.
The chemical synthesis of iminocyclitols starts with 6-azido-
6-deoxysugars prepared from readily available protected monosac-
charides (diisopropylidene sugars or benzyl pyranosides), the
key step being again the reductive amination of a 6-azido-6-
deoxyaldohexopyranose. Thus, 6-azido-6-deoxy-D-galactopy-
ranose (14) (enantiomer of compound 12) undergoes ring
expansion Via Pd-mediated reductive amination in water to give
The enzymatic syntheses of iminocyclitols involve the
combined use of aldolases and isomerases (Scheme 1).12-14
Condensation of (()-3-azido-2-hydroxypropanaldehyde (9) and
dihydroxyacetone phosphate (DHAP) in the presence of an
aldolase followed by treatment with acid phosphatase (Pase)
and an isomerase gave 6-azido-6-deoxyaldopyranoses, which
upon reductive amination15 afforded the corresponding 3,4,5,6-
tetrahydroxyazepane. Isomerization of the ketose to aldose was
performed with the use of glucose and fucose isomerases (GlcI
and FucI), giving the equilibrium favoring aldose, but rhamnose
isomerase (RhaI) was unable to isomerize azidoketose 11. The
(13) FucI prefers (2S,3R)- and RhaI prefers (2R,3R)-aldose as substrate.
Glucose isomerase prefers (2R,3S) aldose as substrate. For synthethic work
using isomerases see: (a) Fessner, W.-D.; Bad´ıa, J.; Eyrisch, O.; Schneider,
A.; Sinerius, G. Tetrahedron Lett. 1992, 33, 5231. (b) Alajar´ın, R.; Garc´ıa-
Junceda, E.; Wong, C.-H. J. Org. Chem. 1995, 60, 4294. (c) Wong, C.-H.;
Alajar´ın, R.; Mor´ıs-Varas, F.; Blanco, O.; Wong, C.-H. J. Org. Chem. 1995,
60, 7360. (d) Page, P.; Blonski, C.; Pe´rie´ J. Tetrahedron, 1996, 52, 1557.
(14) de Raadt, A.; Stu¨tz, A. E. Tetrahedron Lett. 1992, 33, 189.
(15) (a) Paulsen, H.; Sangster, I.; Heyns, K. Chem. Ber. 1967, 100, 802.
In an attempt to prepare seven-membered iminocyclitols from an azido
ketose catalyzed by Pd on C in the presence of H2 (50 psi) the only product
obtained was the aminohexopyranose: (b) Liu, K. C.; Pederson, R. L.;
Wong, C. H. J. Chem. Soc., Perkin Trans 1 1991, 1991, 2669.
(8) Tulsiani, D. R. P.; Harris, T. M.; Touster, O. J. Biol. Chem. 1982,
257, 7936.
(9) Fleet, G. W. J.; Shaw, A. N.; Evans, S. V.; Fellows, L. E. J. Chem.
Soc., Chem. Commun. 1985, 841.
(10) Qian, X.; Mor´ıs-Varas, F.; Wong, C.-H. Bioorg. Med. Chem. Lett.
1996, 6, 117.
(11) Qian, X.; Mor´ıs-Varas, F.; Fitzgerald, M. C.; Wong, C.-H. Bioorg.
Med. Chem., in press.
(12) Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic
Chemistry; Pergamon: Oxford, 1994.

SeVen-Membered Iminocyclitols
J. Am. Chem. Soc., Vol. 118, No. 33, 1996 7649
Scheme 2a
Scheme 4a
a Conditions: (a) BnOH, 80 °C, Et2O‚BF3, 75%; (b) 2,2-dimethox-
ypropane, p-TSA, DMF, rt, 95%; (c) MeI, NaH, THF, rt, 93%; (d)
80% AcOH, 80 °C, 95%; (e) H2 (50 psi), Pd/C, H2O, 2 d, 90%.
Scheme 5a
a Conditions: (a) (i) Ph3P, DEAD, THF, 0 °C; (ii) (EtO)2P(O)N3,
80%; (b) 80% AcOH, 70 °C, 95%; (c) H2 (50 psi), Pd/C, H2O, 2 d,
90%.
Scheme 3a
a Conditions: (a) TsCl, Py, 0 °C, 12 h, 61-68%; (b) NaN3 (5 equiv),
NH4Cl, (5 equiv), EtOH/H2O, 9:1, reflux, 12 h, 70%; (c) H2 (1 atm),
Pd/C, H2O, 12 h, 85%; (d) BnOH, HCl, 80 °C, 30%.
a Conditions: (a) (i) pH 6.7, DHAP, FDPA; (ii) pH 4.5, acid Pase;
(b) (i) pH 6.7, DHAP, RhaA; (ii) pH 4.5, acid Pase; (c) H2, 50 psi, rt,
MeOH, 3 h, 90-95%.
2 (Scheme 2). During the hydrogenation, compounds 2 and
the intermediate 17 were observed by NMR in 12 h. After 2
days, 17 disappeared and only 2 was obtained. It appears that
17 is formed via intramolecular dehydration of the intermediate
16.
Benzyl pyranosides are easily prepared from aldopyranoses
and represent a convenient entry point to these seven-membered
ring heterocycles. We illustrate this approach by using benzyl
mannopyranoside and benzyl N-acetylglucosamine as starting
materials (Scheme 3). This approach is much more convenient
than the bisepoxide opening described previously, since mixtures
of six- and seven-membered rings are completely avoided.
Tosylation of the primary hydroxyl group of 18 and benzyl
pyranoside of 20 followed by azide displacement affords the
azides 19 and 21. Hydrogenolysis of both compounds afforded
the desired iminocyclitols 3 and 4.
The meso-iminocyclitol 2 was asymmetrized to (3S,4R,5S,6R)-
3-methoxy-4,5,6-trihydroxyazepine (5) from 6-azido-6-deoxy-
D-galactopyranoside (14) Via benzyl glycosilation, isopropy-
lidene protection, methylation, and reductive amination of the
2-O-methyl glycoside 23, as shown in Scheme 4.
In an attempt to prepare 5 enzymatically, (()-3-azido-2-
hydroxy-propanaldehyde dimethyl acetal was acetylated and
kinetically resolved by lipase PS800 mediated hydrolysis.16 The
products from this enzymatic reaction were methylated (MeI,
NaH, THF) to give both enantiomers of 3-azido-2-O-methyl-
propanaldehyde diethyl acetal (24 and 25), which were hydro-
lyzed and reacted with DHAP in the presence of fructose 1,6-
diphosphate aldolase (FDPA) and rhamnulose 1-phosphate
aldolase (RhaA), respectively. In both cases the methylated
hydroxy aldehydes were substrates for the aldolases and
(3S,4R,5R)-6-azido-5-methoxy-1,3,4-trihydroxyhexan-2-one (26)
and (3R,4S,5S)-6-azido-5-methoxy-1,3,4-trihydroxyhexan-2-one
(27) were obtained. These ketoses were, however, not accepted
as substrates for GlcI and RhaI, respectively. These observa-
tions are in agreement with the conclusion made by our group17
and others18 that fructose analogues modified at carbon 5
(inversion, deoxygenation, or blocked hydroxyl) are not isomer-
ized by GlcI. Similarly, (3R,4R,5S)-6-azido-5-methoxy-1,3,4-
trihydroxyhexan-2-one was not accepted by FucI. The ketoses
26 and 27 were then converted to (2R,3R,4S,5S)-2-hydroxym-
ethyl-3,4-dihydroxy-5-methoxypiperidine (2-O-methyl-1-deoxy-
mannojirimycin) (28) and the enantiomer 29, respectively, under
the reductive amination condition at 50 psi for 3 h in MeOH.
None of these two azasugars exhibited significant inhibition
when tested for the glycosidases mentioned above.
At this stage it is not clear how these seven-membered
iminocyclitols act as inhibitors of glycosidases. The X-ray
crystal structure of a representative compound (7) was deter-
mined11 and shown to adopt a pseudochair conformation (Figure
1 and Table 2).
(17) Durrwachter, J. R.; Drueckhammer, D. G.; Nozaki, K.; Sweers, H.
M.; Wong, C. H. J. Am. Chem. Soc. 1986, 108, 7812.
(18) Berger, A.; de Raadt, A.; Gradnig, G.; Grasser, M.; Lo¨w, H.; Stu¨tz,
A. E. Tetrahedron Lett. 1992, 33, 7125.
(16) von der Osten, C. H.; Barbas, C. F.; Wong, C. H.; Pederson, R. L.;
Sinskey, A. J.; Wang, Y. F. J. Am. Chem. Soc. 1989, 111, 3924.

7650 J. Am. Chem. Soc., Vol. 118, No. 33, 1996
Mor´ıs-Varas et al.
noside was purchased from Toronto Research Chemicals Inc. and
diphenylphosphoryl azide from Fluka, and the rest of the chemicals
and solvents were purchased from Aldrich and used without further
treatment. Dowex 50W-X8 (Biorad, 200-400 mesh, H+ form) was
+
converted to NH4 form by passing 2 N ammonium hydroxyde and
thoroughly washed with purified water prior to use. Aldol condensation
was monitored enzymatically by DHAP consumption.21 The phos-
phatase-catalyzed hydrolysis was monitored by TLC (silica gel 60 from
Merck). Isomerization was monitored by 13C-NMR (100 MHz) analysis
of the anomeric center in the cyclized form. Nuclear magnetic
resonance (1H, 400 MHz; 13C, 100 MHz) spectra were obtained using
1
D2O (δ ) 4.65 ppm) or CD3OD (δ ) 3.5 ppm for H and 49.9 for
13C). Flash chromatography was carried out with silica gel 60 (230-
400 mesh). Inhibitory analysis were performed on a Beckman DU-70
spectrophotometer at 400 nm.
Figure 1. X-ray crystal structure of 7.
3(R),4(R),5(R),6(S)-Tetrahydroxyazepane (1). 3-Azido-2-hydrox-
ypropanaldehyde (9) was formed in situ as follows: the diethyl acetal
of 9 (1.512 g, 8 mmol) was dissolved in water (3 mL), and Dowex
50W-X8 (H+ form, 200-400 mesh) was added until pH < 2. The
mixture was heated at 50 °C for 8 h, and then the resin was filtered off
and washed with water. A solution of DHAP (250 mM, 15 mL, 3.8
mmol) was added, and the mixture was adjusted to pH 6.8 with 6 N
NaOH. Rabbit muscle aldolase, RAMA (840 µL, 300 units), was
added, the mixture was stirred gently at room temperature until DHAP
analysis indicated > 90% conversion, and the pH was adjusted to 4.7
with HCl. Acid phosphatase (780 µL, 300 units) was added and the
mixture heated at 37 °C until the organic phosphate was hydrolyzed
completely as indicated by TLC analysis (EtOH/NH4OH, 1:1). 6-Azido-
6-deoxyfructose was purified by silica gel chromatography (CH2Cl2/
MeOH, 6:1) to yield 465 mg (61%) of a product with data in accordance
with those reported previously.22 A 70 mg portion of this product was
dissolved in 2 mL of Tris buffer (50 mM, 2 mM Mn2+, pH 7.7), and
200 mg of immobilized glucose isomerase (TAKASWEET, Miles Labs)
was added. The mixture was shaken at 37 °C for 24 h. 1H- and 13C-
NMR analysis indicated the presence of a mixture of aldose 10 and
ketose (∼65:35). The enzyme was filtered off, solvent evaporated under
reduced pressure, and the residue chromatographed carefully on silica
gel (CHCl3/MeOH, 6:1) to yield 30 mg (42%, 26% overall) of 6-azido-
6-deoxy-D-glucopyranose (10). Spectral data are in agreement with
those reported previously.23 Azidoaldose 10 was hydrogenated at 50
psi in water using Pd/C as catalyst. The reaction was monitored by
NMR and was complete after 48 h. The catalyst was removed by
filtration and the solvent evaporated under reduced pressure. 3(R),4-
(R),5(R),6(S)-tetrahydroxyazepane (1) was obtained in 94% yield (24%
overall): 1H-NMR (D2O, δ, ppm) 3.85 (1H, ddd, J ) 1.7, 3.6, 5.4 Hz,
H3 or H6); 3.59 (2H, m, overlapped H4 + H5); 3.48 (1H, q, J ) 5.5
Hz, H3 or H6); 2.7-2.8 (4H, dd, J ) 14.5, 5.4 Hz, and d, J ) 5.4 Hz,
overlapped 2H2 + 2H7); 13C-NMR (D2O, δ, ppm), 75.18, 74.62, 73.28,
71.21, 69.30, 49.46, 49.20; MS (FAB+) (M + H) expected 164.0923,
observed 164.0920.
Table 2. Bond Lengths and Angles for Compound 7
Bond Lengths (Å) (Standard Deviation ( 10-3 Å)
O(1)-C(9)
O(3)-C(11)
N(1)-C(7)
N(1)-C(8)
C(1)-C(2)
C(3)-C(4)
C(5)-C(6)
C(8)-C(9)
C(10)-C(11)
C(12)-C(13)
1.432(4)
1.423(5)
1.474(5)
1.484(5)
1.386(8)
1.358(10)
1.379(6)
1.534(6)
1.536(5)
1.531(6)
O(2)-C(10)
O(4)-C(12)
N(1)-C(13)
C(1)-C(6)
C(2)-C(3)
C(4)-C(5)
C(6)-C(7)
C(9)-C(10)
C(11)-C(12)
1.438(4)
1.441(4)
1.479(5)
1.377(7)
1.385(9)
1.388(7)
1.511(6)
1.516(6)
1.507(6)
Bond Angles (deg) (Standard Deviation ( 10-1 deg)
C(7)-N(1)-C(13)
108.0(3) C(7)-N(1)-C(8)
114.1(3) C(6)-C(1)-C(2)
119.4(6) C(4)-C(3)-C(2)
119.9(5) C(6)-C(5)-C(4)
118.7(4) C(1)-C(6)-C(7)
120.4(5) N(1)-C(7)-C(6)
117.7(3) O(1)-C(9)-C(10)
109.4(3) C(10)-C(9)-C(8)
108.0(3) O(2)-C(10)-C(11)
114.5(3) O(3)-C(11)-C(12)
112.9(3)
120.8(5)
120.4(5)
120.8(5)
120.9(4)
113.1(3)
109.8(3)
112.6(3)
109.7(3)
108.0(3)
C(13)-N(1)-C(8)
C(3)-C(2)-C(1)
C(3)-C(4)-C(5)
C(1)-C(6)-C(5)
C(5)-C(6)-C(7)
N(1)-C(8)-C(9)
O(1)-C(9)-C(8)
O(2)-C(10)-C(9)
C(9)-C(10)-C(11)
O(3)-C(11)-C(10)
O(4)-C(12)-C(11)
109.6(3) C(12)-C(11)-C(10) 115.9(3)
109.9(3) O(4)-C(12)-C(13)
109.3(3)
114.1(3)
C(11)-C(12)-C(13) 114.6(3) N(1)-C(13)-C(12)
These iminocyclitols were also tested as inhibitors of a
mechanistically related enzyme, i.e., the HIV protease, but no
inhibition was observed. The 3,6-dibenzyl derivatives, espe-
cially 30, are, however, moderate inhibitor of the HIV protease
6-Azido-6-deoxyrhamnulose (11). 3-Azido-2-hydroxypropanalde-
hyde (9) generated as before (diethyl acetal; 284 mg, 1.5 mmol) was
mixed with a solution of DHAP (230 mM, 4.34 mL, 1 mmol), and the
mixture was adjusted to pH 6.8 with 6 N NaOH, followed by addition
of rhamnulose 1-phosphate aldolase (270 µL, 10 units). After 24 h
DHAP analysis indicated 95% conversion, and the pH was adjusted to
4.7 with HCl. Acid phosphatase (200 µL, 100 units) was added and
the mixture heated at 37 °C until the organic phosphate was hydrolyzed
completely as indicated by TLC analysis (EtOH/NH4OH, 1:1). 6-Azido-
6-deoxyrhamnulose (11) was purified by silica gel chromatography
(CHCl3/MeOH, 6:1) to yield 60 mg (30%). Spectral data are the same
as for the enantiomeric fructose analog22 with opposite sign for the
with Ki around 350 µM.10 Interestingly these benzyl derivatives
are not inhibitors of the glycosidases in this study.
In summary, we have demonstrated new and effective
syntheses of various seven-membered iminocyclitols which have
been shown to be a new class of glycosidase inhibitors and may
be useful as new templates for the development of HIV protease
inhibitors.
optical rotation, [R]D ) -8.9° (c ) 1.95, MeOH). Several attemps
25
to isomerize this product in 2 mL of Tris buffer (50 mM, 2 mM Mn2+
,
pH 7.7) and in the presence of variable amounts of rhamnose isomerase
(19) Henderson, I.; Garc´ıa-Junceda, E.; Liu, K. K.-C.; Chen, Y.-L.; Shen,
G.-J.; Wong, C.-H. Bioorg. Med. Chem. 1994, 2, 837.
Experimental Section
(20) Garc´ıa-Junceda, E.; Shen, G.-J.; Alajar´ın, R.; Wong, C.-H. Bioorg.
Med. Chem. 1995, 3, 1349.
(21) Bergmeyer, H. U. Methods of Enzymatic Analysis, 3rd ed.; Verlag
Chemie: Deerfield, FL, 1984; Vol. 2, p 146.
(22) Straub, A.; Effenberger, F.; Fischer, P. J. Org.Chem. 1990, 55, 3926.
(23) Durrwachter, J. R.; Wong, C. H. J. Org. Chem. 1988, 53, 4175.
Materials and Methods. Rabbit muscle aldolase (E.C. 4.1.2.13)
and acid phosphatase (E.C. 3.1.3.2) were purchased from Sigma. The
enzymes fuculose-1-phosphate aldolase, rhamnulose 1-phosphate al-
dolase, fucose isomerase, and rhamnose isomerase were prepared in
our laboratory as described previously.19,20 Benzyl R-D-mannopyra-

SeVen-Membered Iminocyclitols
J. Am. Chem. Soc., Vol. 118, No. 33, 1996 7651
were unsuccesful. The mixture was shaken at 37 °C and the reaction
as eluent to yield 531 mg (68%) of benzyl 6-tosyl-6-deoxy-D-
mannopyranoside. To a solution of this compound (120 mg, 0.29
mmol) in EtOH/H2O, 9:1, were added 5 equiv of NaN3 and 5 equiv of
NH4Cl, and this mixture was refluxed overnight, the solvent removed
in vacuo, and the residue chromatographed in SiO2 using EtOAc as
eluent to yield benzyl 6-azido-6-deoxy-D-mannopyranoside (19) (60
mg, 70%): 13C-NMR (CDCl3, δ, ppm) 136.61, 128.43, 128.10, 127.99,
69.27 (benzyl group), 98.82, 71.53 (double intensity), 70.65, 68.14,
51.25. A solution of this product (26 mg, 0.09 mmol) in water (3 mL)
was added to 20 mg of Pd/C and hydrogenated at 50 psi for 24 h. The
catalyst was removed by filtration and the filtrate purified by ion-
exchange chromatography (Dowex-50W, NH4+ form, 1 × 20 cm) eluted
first with water and then with a NH4OH gradient, 0 f 1 N. The
fractions containing the product were pooled, and HCl was added to
form the hydrochloric acid salt of 3(R),4(R),5(R),6(R)-tetrahy-
droxyazepane (3) (16 mg, 85%);5b 1H-NMR (D2O, δ, ppm) 4.15 (2H,
m, H4 + H5); 3.70 (2H, s, H3 + H6); 3.25 (4H, dd, J ) 14.4, 6.4 Hz,
2H2 + 2H7); 13C-NMR (D2O, δ, ppm) 73.26, 67.03, 45.13.
1
monitored by H- and 13C-NMR; no aldose peaks were detected even
after 3 days.
3(S),4(R),5(S),6(R)-Tetrahydroxyazepane (2). Enzymatic Syn-
thesis. 3-azido-2-hydroxypropanaldehyde (9) prepared as described
previously (diethyl acetal; 756 mg, 4 mmol) was mixed with a solution
of DHAP (262 mM, 7.6 mL, 2 mmol) at pH 6.5. Fuculose 1-phosphate
aldolase was added (640 µL, 10 units), and the mixture was stirred at
room temperature. After 18 h DHAP analysis indicated 91% conver-
sion, and the pH was adjusted to 4.7 with HCl. After deprotection of
the phosphate, 6-azido-6-deoxyfuculose was isolated by Dowex-50W
(Ba2+ form, 3 × 20 cm) chromatography15b using water as eluent to
yield 99 mg (25%): 1H-NMR (D2O, δ, ppm, major anomer only) 4.15
(1H, t, J ) 4.5 Hz, H4); 4.05 (1H, d, J ) 4.5 Hz, H3); 4.00 (1H, m,
J ) 2, 4.5, 8.5 Hz, H5); 3.3-3.5 (4H, m, overlapped signals, 2H1 and
2H6); 13C-NMR (D2O, δ, ppm) 102.78, 78.43, 70.96, 70.43, 62.45,
50.99 (major), 105.16, 77.65, 76.88, 71.29, 62.22, 50.32 (minor); MS
(FAB+) (M+) expected 205.0699, observed 205.0691. This product
was dissolved in 2 mL of Tris buffer (50 mM, 2 mM Mn2+, pH 7.7),
and 1200 units (2 mL) of fucose isomerase were added. The mixture
was stirred at room temperature for 24 h. 1H- and 13C-NMR analysis
indicated the consumption of the ketose and the appearence of a new
compound, aldose 12 (∼95:5). The enzyme was precipitated with
acetone and solvent evaporated under reduced pressure, and the residue
was chromatographed on Dowex-50W (Ba2+ form, 3 × 20 cm) using
EtOH/H2O, 1:1, as eluent to yield 82 mg (82%, 21% overall) of 6-azido-
6-deoxy-L-fucopyranose (12). The NMR data were the same as those
reported previously.13c To a solution (H2O/THF, 3:1, 4 mL) of 12 (60
mg) was added a catalytic amount of Pd/C, and the mixture was
hydrogenated at 50 psi. After 20 h of reaction azasugar 2 was obtained
as the major product, but NMR analysis also showed another minor
compound, characterized as 8-oxa-2-aza-4,6,7-trihydroxybicyclo[3.2.1]-
heptane (17): 1H-NMR (CD3OD, δ, ppm) 4.92 (1H, d, J ) 6 Hz);
4.36 (1H, d, J ) 2.5 Hz); 4.32 (1H, ddd, J ) 1, 2.5, 6 Hz); 4.09 (1H,
d, J ) 4.3 Hz); 3.91 (1H, m); 3.18 (1H, ddd, J ) 1, 8.8, 13.5 Hz);
2.87 (1H, dd, J ) 13.5, 10.8 Hz); 13C-NMR (CD3OD, δ, ppm) 89.03,
87.18, 82.43, 79.05, 66.64, 46.67. After an additional period of time
(∼2 d) the bicyclic compound disappeared completely and 3(S),4(R),5-
(S),6(R)-tetrahydroxyazepane (2) was the only product detectable (55
mg, 91%, 19% overall): 1H-NMR (D2O, δ, ppm) 3.82 (2H, d, J ) 6.5
Hz, H4 + H5); 3.66 (2H, dd, J ) 4.5, 6.1 Hz, H3 + H6); 2.77-2.67
(4H, dd, J ) 14.8, 4.5 Hz, 2H2 + 2H7); 13C-NMR (D2O, δ, ppm)
74.28, 70.90, 51.43; MS (FAB+), (M + H) expected 164.0923, observed
164.0918.
3(S),4(R),5(S),6(R)-Tetrahydroxyazepane (2). Chemical Synthe-
sis. To a solution of 1,2:3,4-diisopropylidene-D-galactose (13) (3.67
g, 14.1 mmol) in THF (30 mL) at 0 °C were added DEAD (2.26 mL,
14.1 mmol) and PPh3 (3.79 g, 14.1 mmol), and the mixture was stirred
for 15 min. Then, diphenylphosphoryl azide (3.04 mL, 14.1 mmol)
was added dropwise and the reaction mixture stirred overnight. The
solvent was evaporated under reduced pressure, and the product was
purified by silica gel chromatography (CH2Cl2) to yield 3.25 g (80%)
of 6-azido-6-deoxy-1,2:3,4-diisopropylidene-D-galactose: 1H-NMR
(CDCl3, δ, ppm) 5.51 (d, J ) 5 Hz, H1); 4.60 (dd, J ) 1.5, 8 Hz, H3);
4.30 (dd, J ) 1.5, 5 Hz, H2); 4.15 (dd, J ) 2, 8 Hz, H4); 3.90 (ddd,
J ) 2, 5, 7.5 Hz, H5); 3.50 (dd, J ) 12.5, 7 Hz, H6); 3.35 (dd, J )
12.5, 5 Hz, H6′); 13C-NMR (CDCl3, δ, ppm): 110.23, 109.43, 26.13,
26.04, 24.98, 24.51 (isopropylidene), 96.88, 71.53, 71.16, 70.74, 67.36,
50.91 (sugar). This compound was treated with 80% AcOH at 70 °C
for 3 h (TLC showed completion, CH2Cl2/MeOH, 4:1). The solvent
was removed in vacuo to yield 6-azido-6-deoxy-D-galactose (14) (2.15
g, 95%); the spectral data are the same as those reported for the
enantiomer.13c This product (60 mg) was hydrogenated at 50 psi in
H2O/THF, 3:1, for 2 d to give 3(S),4(R),5(S),6(R)-tetrahydroxyazepane
(2) (50 mg, 90%).
6(R)-Acetamido-3(S),4(R),5(S)-trihydroxyazepane (4). To 11 g
of N-acetylglucopyranose 20 was added 68 mL of benzyl alcohol, and
HCl gas was passed for 3 min. The mixture was allowed to react for
3 h, and the precipitate was collected and washed with cold water,
cold ether, and hexane (yield 30%). This benzyl glucopyranoside (500
mg, 1.67 mmol) was subjected to the same sequence as that of
compound 18 above, to yield benzyl 2-acetamido 6-azido-2,6-dideoxy-
glucopyranoside (241 mg, 43% overall yield) (21): 13C-NMR (CD3-
OD, δ, ppm, R-anomer only) 175.68, 23.68 (acetamide); 140.61, 131.27,
131.15, 130.78, 71.89 (benzyl group); 99.09, 74.64 (double intensity),
73.89, 56.62, 54.03 (sugar moiety). A sample of this compound (34
mg, 0.1 mmol) was hydrogenated over Pd/C in H2O/THF, 4:1, overnight
and the azasugar 4 purified by ion-exchange chromatography (Dowex-
+
50W, NH4 form, 1 × 20 cm) eluted first with water and then with a
NH4OH gradient, 0 f 1 N. The fractions containing the product were
pooled, and HCl was added to form the hydrochloric acid salt of 6(R)-
acetamido-3(S),4(R),5(S)-trihydroxyazepane (4) (14 mg, 60%): 1H-
NMR (D2O, δ, ppm) 4.15 (1H, dt, J ) 2, 6.5 Hz, H3); 3.87 (1H, dt, J
) 3, 8 Hz, H6); 3.68 (1H, t, J ) 8 Hz, H5); 3.59 (dd, J ) 2, 8 Hz,
H5); 3.32-3.12 (4H, m, 2H2 + 2H7); 13C-NMR (D2O, δ, ppm) 75.23,
72.05, 66.83, 49.28, 45.73, 45.18, 21.86.
3(S),4(R),5(S),6(R)-3-Methoxy-4,5,6-trihydroxyazepane (5).
A
suspension of 6-azido-6-deoxy-D-galactose (14) (1.63 g, 7.9 mmol)
obtained as described above in benzyl alcohol (5 mL) was heated to
80 °C, and BF3‚OEt2 (984 µL, 8 mmol) was added dropwise. After
20 min the suspension became transparent. The solution was allowed
to cool. The reaction mixture was passed through a SiO2 column.
Benzyl alcohol came out first using EtOAc/hexane, 1:5, and then benzyl
6-azido-6-deoxy-D-galactopyranoside was separated from the furanose
byproduct with EtOAc as eluent (1.21 g, 75%, R/â, 60:40): 1H-NMR
(CD3OD, δ, ppm, R-anomer only) 7.4-7.6 (5H, benzyl group); 5.3
(H1, overlapped by HDO signal); 4.98 and 4.78 (2H, AB system, J )
12 Hz, CH2-OBn); 4.18 (1H, dd, J ) 4.2, 8.7 Hz, H5); 4.0 (3H,
overlapped, H2, H3, H4); 3.75 (1H, dd, J ) 13, 8.7 Hz, H6); 3.49
(1H, dd, J ) 13, 4.2 Hz, H6′); 13C-NMR (CD3OD, δ, ppm, R-anomer
only) 139.76, 131.64, 130.31, 100.28, 72.54, 72.36, 72.10, 71.36, 70.89,
53.62. To a solution of this product (400 mg, 1.35 mmol) in DMF (4
mL) were added 2,2-dimethoxypropane (1 mL, large excess) and a
catalytic amount of p-tosylic acid. The reaction was driven overnight
under argon at room temperature and then extracted with Et2O. Further
purification by flash chromatography (EtOAc/hexanes, 1:1) yielded 429
mg (95%) of benzyl 6-azido-6-deoxy-3,4-isopropylidene-D-galactopy-
ranoside (22): 13C-NMR (CDCl3, δ, ppm, R-anomer only) 136.81,
128.43, 128.32, 128.11, 69.68 (benzyl group); 109.73, 27.33, 25.62
(isopropylidene); 96.15, 75.49, 73.06 (doublet), 68.65, 68.12, 51.13
(sugar). To a solution of this latter product (204 mg, 0.6 mmol) in dry
THF (5 mL) were added MeI (38.6 µL, 0.62 mmol) and NaH (16 mg,
0.66 mmol), and the mixture was reacted at room temperature for 2 h
and then was extracted with CH2Cl2 and the resulting syrup treated
with 80% AcOH at 70 °C for 3 h. Evaporation of the solvent with
added water (three times) yielded 163 mg (88% from 22) of benzyl
6-azido-6-deoxy-2-O-methyl-D-galactopyranoside (23): 1H-NMR (CD3-
OD, δ, ppm, R-anomer only) 7.4-7.6 (5H, benzyl group); 5.28 (1H,
d, J ) 3.7 Hz, H1); 4.95 and 4.75 (2H, AB system, J ) 12 Hz, CH2-
3(R),4(R),5(R),6(R)-Tetrahydroxyazepane (3). To a solution of
benzyl mannopyranoside (18) (500 mg, 1.9 mmol) in dry pyridine (5
mL) at 0 °C was added tosyl chloride (381 mg, 2 mmol) dissolved in
CH2Cl2, and the reaction was monitored by TLC (EtOAc). After 4 h
the starting material was consumed completely. The reaction mixture
was extracted with CH2Cl2 and washed with 1 N HCl, saturated
NaHCO3, and brine, and purified by flash chromatography using EtOAc

7652 J. Am. Chem. Soc., Vol. 118, No. 33, 1996
Mor´ıs-Varas et al.
OBn); 4.15 (1H, dd, J ) 4, 8.8 Hz, H5); 4.05 (1H, dd, J ) 3.4, 10 Hz,
H3); 3.85 (1H, d, J ) 3.4 Hz, Hr); 3.75 (1H, dd, J ) 13, 8.7 Hz, H6);
3.45 (1H, dd, J ) 13, 4 Hz, H6′); 3.68 (1H, dd, J ) 10, 3.7 Hz, H2);
3.60 (3H, s, MeO-); 13C-NMR (CD3OD, δ, ppm, R-anomer only)
140.51, 131.22, 130.73, 97.87, 80.66, 72.87, 72.78, 71.67, 71.59, 59.69,
53.98. A sample of this product (105 mg, 0.34 mmol) was hydroge-
nated at 50 psi over Pd/C in water for 2 d to yield (3S,4R,5S,6R)-3-
methoxy-4,5,6-trihydroxyazepane (5) (54 mg, 90%): 1H-NMR (D2O,
δ, ppm) 4.29 (1H, dd, J ) 1.3, 6.3 Hz, H4 or H5); 4.09 (1H, dd, J )
1.3, 7.1 Hz, H5 or H4); 3.97 (1H, dt, J ) 4.5, 7.1 Hz, H3 or H6); 3.5
(4H, Me and H6 or H3 overlapped by Me); 3.27 (1H, dd, J ) 14.3,
4.5 Hz, H2 or H7); 3.27 and 2.03 (1H each, dd, J ) 14.3, 4.4 Hz, 2
H2 or 2H7); 3.7 and 4.1 (1H each, dd, J ) 14.3, 4.4 Hz, 2H2′ or 2H7′);
13C-NMR (D2O, δ, ppm) 83.99, 77.04, 75.33, 73.66, 58.67(OMe), 54.78,
50.85; MS (FAB+), (M + H) expected 178.1079, observed 178.1072.
3(S),4(R),5(S)-6-Azido-5-methoxy-1,3,4-trihydroxyhexan-2-one (26).
A solution of 3-azido-2(R)-O-methylpropanal diethyl acetal (24) (934
mg, 4.6 mmol) prepared by methylation of 3-azido-2(R)-hydroxypro-
panal diethyl acetal obtained enzymatically)16 was dissolved in water
(5 mL), and Dowex 50W-X8 (H+ form, 200-400 mesh) was added
until pH < 2. The mixture was heated at 50 °C for 8 h, and then the
resin was filtered off and washed with a minimum amount of water. A
solution of DHAP (262 mM, 7.6 mL, 2 mmol) was added, and the
mixture was adjusted to pH 6.5 with 6 N NaOH. Fructose 1,6-
diphosphate aldolase was added (1.4 mL, 500 units), and the mixture
was stirred at room temperature. After 4 h the pH was adjusted to 4.7
with HCl. Acid phosphatase (500 µL, 400 units) was added and the
mixture heated at 37 °C. The enzyme was precipitated by adding
acetone and the residue evaporated and chromatographed on SiO2 (CH2-
Cl2/MeOH, 6:1), to give 400 mg (40%) of 3(S),4(R),5(S)-6-azido-5-
methoxy-1,3,4-trihydroxyhexan-2-one (26): 1H-NMR (D2O, δ, ppm)
4.48 and 4.35 (1H each, J ) 19.8 Hz, H1 and H1′); 4.38 (1H, d, J )
3 Hz, H3); 3.85 (1H, dd, J ) 9.2, 1.9 Hz, H4), 3.7 (1H, dd, J ) 13.6,
2.8 Hz, H6); 3.45, (1H, ddd, J ) 9.2, 2.8, 3.7 Hz, H5); 3.33 (3H, s,
Me); 3.30 (1H, dd, J ) 13.5, 3.7 Hz, H6′); 13C-NMR (D2O, δ, ppm)
213.22, 78.55, 74.85, 70.26, 65.98, 57.36, 48.97.
S-enantiomer was treated in the same manner as described for the
R-enantiomer. In this case rhamnulose 1-phosphate aldolase was added
(0.5 mL, 80 units), and after usual monitoring and workup, 380 mg
(38%) of (3R,4S,5S)-6-azido-5-methoxy-1,3,4-trihydroxyhexan-2-one
(27) was obtained. The NMR data were identical to those of 26.
2-O-Methyl-1-deoxy-D-mannojirimycin (28). A sample of 26 (39
mg, 0.17 mmol) was hydrogenated in MeOH (2mL) over Pd/C at 50
psi for 3 h. After the catalyst was filtered off and the solvent
evaporated, 29 mg (95%) of 2-O-methyl-1-deoxy-D-mannojirimycin
(28) was obtained: 1H-NMR (CD3OD, δ, ppm) 3.91 (1H, dd, J ) 4.5,
11.1 Hz, H6); 3.96 (1H, J ) 11.1, 3 Hz, H6′); 3.75 (1H, t, J ) 9.6 Hz,
H4); 3.65 (1H, ddd, J ) 2.7, 2.8 Hz, 1.2 Hz, H2); 3.60 (4H, Me and
H3 overlapped by Me); 3.40 (1H, dd, J ) 14, 2.7 Hz, H1); 2.75 (1H,
dd, J ) 14, 1.2 Hz, H1′); 2.55 (1H, ddd, J ) 9.7, 4.4 Hz, 3.2 Hz, H5);
13C-NMR (CD3OD, δ, ppm) 178.58, 74.50, 68.08, 60.52, 59.88, 55.44,
43.71.
2-O-Methyl-1-deoxy-L-mannojirimycin (29). By using the same
procedure as above for the D-enantiomer, 120 mg of 27 gave 88 mg
(90%) of 2-O-methyl-1-deoxy-L-mannojirimycin (29), with NMR data
identical to those of 28.
Inhibition Analysis. Inhibition analyses were performed at 37 °C
in 0.1 M HEPES buffer, pH 6.8, except for R-fucosidase (assayed at
37 °C in 50 mM sodium acetate buffer, pH 6.0). The amount of enzyme
added in each assay was 0.05 unit (0.1 unit for â-galactosidase and
R-fucosidase). For each inhibitor, four inhibitor concentrations, ranging
from 0 to 3 times Km, were used to obtain a set of data. p-(Nitrophenyl)
glycosides were used as substrates, and the release of p-nitrophenol
was monitored at 400 nm for 2 min. Data were collected and fitted to
a Michaelis-Menten curve by using the program HyperCleland for
enzyme kinetics analysis.24
Acknowledgment. We thank the NIH for financial support
(Grant NIH GM44154). F.M.-V. thanks the Ministerio de
Educacio´n y Ciencia (Spain) for a postdoctoral fellowship and
Dr. Brion W. Murray for help with biological assays.
3(R),4(S),5(S)-6-Azido-5-methoxy-1,3,4-trihydroxyhexan-2-one (27).
A solution of 3-azido-2(S)-O-methylpropanal diethyl acetal (25) (832
mg, 4.1 mmol) prepared in the same way as above from the
JA960975C
(24) Cleland, W. W. Methods Enzymol. 1979, 63, 103.