Cyclic Amidine Sugars as Transition-State Analogue Inhibitors of Glycosidases: Potent Competitive Inhibitors of Mannosidases

Article abstract of DOI:10.1021/ja037822r

A series of monocyclic glycoamidines bearing different exocyclic amine, alcohol, or alkyl-functionalities and bicyclic amidines derived from D-glucose and D-mannose were synthesized and tested as inhibitors of various glycosidases. All the prepared compou

Full text of DOI:10.1021/ja037822r

Published on Web 01/31/2004
Cyclic Amidine Sugars as Transition-State Analogue
Inhibitors of Glycosidases: Potent Competitive Inhibitors of
Mannosidases
Marie-Pierre Heck,*,†, Ste´phane P. Vincent,§,‡ Brion W. Murray,§,#
Franc¸ois Bellamy,| Chi-Huey Wong,§ and Charles Mioskowski*,†,
Contribution from the CEA, CE-Saclay, SerVice de Marquage Mole´culaire et de Chimie
Bioorganique, Baˆt 547, De´partement de Biologie Joliot-Curie, 91191 Gif Sur YVette, France;
UniVersite´ Louis Pasteur, Laboratoire de Synthe`se Bio-Organique associe´ au CNRS, Faculte´ de
Pharmacie, 74 route du Rhin BP 24, 67401 Illkirch, France; The Scripps Research Institute,
Department of Chemistry, 10550 North Torrey Pines Road, La Jolla, California 92037;
Laboratoires Fournier, 50 rue de Dijon, 21121 Daix, France
Received August 8, 2003; E-mail: heck@dsvidf.cea.fr; mioskow@bioorga.u-strasbg.fr
Abstract: A series of monocyclic glycoamidines bearing different exocyclic amine, alcohol, or alkyl
functionalities and bicyclic amidines derived from D-glucose and D-mannose were synthesized and tested
as inhibitors of various glycosidases. All the prepared compounds demonstrated good to excellent inhibition
toward glycosidases. In particular, the biscationic D-mannoamidine 9b bearing an exocyclic ethylamine
moiety proved to be a selective competitive inhibitor of R- and â-mannosidases (Ki ) 6 nM) making it the
most potent inhibitor of these glycosidases reported to date. A favorable B2,5 boat conformation might explain
the selectivity of mannosidase inhibition compared to other glycosidases.
Introduction
classes of inhibitors have been discovered, some of them giving
interesting insights into the mechanism of enzymatic glycoside
hydrolysis.
Glycosciences are emerging as a key research field at the
frontiers of biology, synthetic and supramolecular chemistry,
and enzymology.1 Among the carbohydrate processing enzymes,
glycosidases have been identified as an important class of
therapeutic targets with applications in the treatment of influenza
infection,2 cancer,3 AIDS,4 and diabetes.5 Thus, numerous
Two general classes of glycosidase inhibitors can be de-
fined: (i) natural products and synthetic analogues whose design
has been inspired by the inhibitory activity of the natural
inhibitors6 and (ii) inhibitors whose design has been rationally
conceived from the mechanism of the enzymatic reaction. The
latter class of inhibitors comprises transition-state analogues of
the glycoside cleavage process,7 mechanism-based inactivators,8
and conformationally locked molecules.9
The transition state of this enzymatic hydrolysis may strongly
vary as a function of the type of glycosidase: for instance,
glycosidases proceeding through retention or inversion of
configuration at the anomeric center do not share the same
mechanism.8c,d Furthermore, stereoelectronic factors may play
a key role in the pathway through which a glycoside is
enzymatically hydrolyzed.10 Thus, the transition-state or the
† CEA, CE-Saclay.
Universite´ Louis Pasteur.
§ The Scripps Research Institute.
‡ Current address: Ecole Normale Supe´rieure, De´partement de Chimie,
24 rue Lhomond, 75231 Paris, France.
# Current address: Pfizer La Jolla Laboratories, 10628 Science Center
Drive, San Diego, CA 92121, USA.
| Laboratoires Fournier.
(1) (a) Sears, P.; Wong, C.-H. Science 2001, 291, 2344-2350. (b) Bertozzi,
C. R.; Kiessling, L. Science 2001, 291, 2357-2364.
(2) (a) von Itzstein, M.; Wu, W.-Y.; Kok, G. B.; Pegg, M. S.; Dyason, J. C.;
Jin, B.; Phan, T. V.; Smythe, M. L.; White, H. F.; Oliver, S. W.; Colman,
P. M.; Varghese, J. N.; Ryan, D. M.; Woods, J. M.; Bethell, R. C.; Hotman,
V. J.; Cameron, J. M.; Penn, C. R. Nature 1993, 363, 418-423. (b) Kim,
C. U.; Lew, W.; Williams, M. A.; Liu, H.; Zhang, L.; Swaminathan, S.;
Bischofberger, N.; Chen, M. S.; Mendel, D. B.; Tai, C. Y.; Laver, W. G.;
Stevens, R. C. J. Am. Chem. Soc. 1997, 119, 681-690. (c) Chand, P.;
Kotian, P. L.; Dehghani, A.; El-Kattan, Y.; Lin, T.-H.; Hutchison, T. L.;
Sudhakar Babu, Y.; Bantia, S.; Elliott, A. J.; Montgomery, J. A. J. Med.
Chem. 2001, 44, 4379-4392.
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Weinheim, 1999. (b) Berecibar, A.; Grandjean, C.; Siriwardena, A. Chem.
ReV. 1999, 99, 779-844. (c) Winchester, B.; Fleet, G. W. J. Glycobiology
1992, 2, 199-210.
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Vasella, A. T. Angew. Chem., Int. Ed. 1999, 38, 750-770. (c) Bols, M.
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9
10.1021/ja037822r CCC: $27.50 © 2004 American Chemical Society
J. AM. CHEM. SOC. 2004, 126, 1971-1979
1971

A R T I C L E S
Heck et al.
high-energy intermediates (such as oxycarbenium species) may
adopt very different conformations depending on the structure
of the sugar-substrate and the R/â-nature of the glycosidase.11
The analysis of glycosidases sequences has allowed their
classification into families and superfamilies on the basis of
their sequence similarities and mechanism of action. To date,
over 80 families have been defined,12 which means that
numerous distinct mechanistic pathways are likely occurring
among the whole glycosidase family. Because of this multiplicity
of mechanisms, it is difficult to define whether a given inhibitor
is a transition-state analogue or an opportunistic binder of the
catalytic pocket. Hundreds of natural or synthetic inhibitors have
already been described,6,13 among them some rationally designed
mechanism-based inhibitors. However, only few molecules
display inhibitory activity in the nanomolar range. In this study,
we describe the synthesis of a new family of glycoamidines
which display a low nanomolar and selective inhibition pattern
for mannosidases.
Figure 1. Expected transition state in a glycosidase reaction (left) and
designed biscationic mannoamidines as transition-state analogues (right).
Design of the Inhibitors: Targeting an Additional Ionic
Interaction at the Vicinity of the Anomeric Center. Cationic
glycosides can be viewed as early transition-state analogues (if
they mimic the initial protonation of the exocyclic oxygen) or
late transition-state analogues (if they mimic high-energy
intermediates such as oxycarbenium species, after disruption
of the glycosidic bond).7d Thus, glycoside analogues adopting
Figure 2. Designed inhibitors.
decided to explore this design by adding a supplementary
electrostatic interaction to the glycoamidine core structure of
the inhibitor. Indeed, most of the glycosidases have two
carboxylic residues acting as general base and general acid at
the vicinity of the substrate’s anomeric center (or, in the case
of retaining glycosidases, one carboxylate acts as a nucleophile
to give a covalent intermediate) (Figure 1, left). Once the
inhibitor is protonated, the amidine functionality is a monocation
that can ionically interact with only one of the two carboxylates.
Thus, the addition of a supplementary amino group through a
short spacer attached to the exocyclic nitrogen might provide a
new ionic interaction with the second carboxylate (Figure 1,
right).
a
4H3 half-chair conformation tend to mimic the glycosyl
oxycarbenium species often considered close to the transition
state. The first inhibitors designed in this way were the
glycoamidines, glycoamidrazones, and glycoamidoxime devel-
oped by Ganem et al.14 Then, substituted amidines,15 amidine
pseudodisaccharides,16 mannohydroximolactone,15c and fuco-
amidrazone17 were reported as well and showed good inhibition
levels for glycosidases. All these inhibitors were designed to
mimic both the half-chair conformation and the charge devel-
oped around the anomeric center.
The glycoamidines reported by Ganem14 displayed very good
inhibition properties especially in the mannosidase series. We
In principle, adding a properly placed electrostatic interaction
should dramatically increase the binding strength of a given
inhibitor. We then designed a set of monocyclic amidines in
the gluco and the manno series, bearing an ethyl-, a propyl-, or
a butylamino group linked to the exocyclic nitrogen (7b, 9b,
7c, 9c, 7d, 9d; Figure 2). In an attempt to demonstrate the role
of this additional amine, analogues bearing an alkyl chain (7a,
9a), a hydroxyl function (7e, 9e), and a six-membered ring cyclic
amidine (12, 13) were synthesized (Figure 2).
(10) (a) Sinnott, M. L. Chem. ReV. 1990, 90, 1171-1202. (b) Franck, R. W.
Bioorg. Chem. 1992, 20, 77-88.
(11) (a) Vasella, A.; Davies, G. J.; Bo¨hm, M. Curr. Opin. Chem. Biol. 2002, 6,
619-629. (b) Sidhu, G.; Withers, S. G.; Nguyen, N. T.; McIntosh, L. P.;
Ziser, L.; Brayer, G. D. Biochemistry 1999, 38, 5346-5354. (c) Sabini,
E.; Sulzenbacher, G.; Dauter, M.; Dauter, Z.; Jorgensen, P. L.; P.; Schulein,
M.; Dupont, C.; Davies, G. J.; Wilson, K. S. Chem. Biol. 1999, 6, 483-
492. (d) Vocadlo, D. J.; Davies, G. J.; Laine, R.; Withers, S. G. Nature
2001, 412, 835-838.
(12) (a) Henrissat, H.; Bairoch, A. Biochem. J. 1996, 316, 695-696. (b)
Henrissat, B.; Davies, G. Curr. Opin. Struct. Biol. 1997, 7, 637-644.
(13) (a) Asano, N.; Nash, R. J.; Molyneux, R. J.; Fleet, G. W. J. Tetrahedron:
Asymmetry 2000, 11, 1645-1680. (b) Jacob, G. S. Curr. Opin. Struct. Biol.
1995, 5, 605-611. (c) Legler, G. In Carbohydrate Mimics; Chapleur, Y.,
Ed.; Wiley-VCH: Weinheim, 1998; pp 463-490. (d) Winchester, B.
Biochem. Soc. Trans. 1992, 20, 699-705.
(14) (a) Tong, M. K.; Papandreou, G.; Ganem, B. J. Am. Chem. Soc. 1990,
112, 6137-6139. (b) Ganem, B.; Papandreou, G. J. Am. Chem. Soc. 1991,
113, 8984-8985. (c) Papandreou, G.; Tong, M. K.; Ganem, B. J. Am. Chem.
Soc. 1993, 115, 11682-11690. (d) Pan, Y. T.; Kaushal, G. P.; Papandreaou,
G.; Ganem, B.; Elbein, A. J. Biol. Chem. 1992, 267, 8313-8318.
(15) (a) Ble´riot, Y.; Genre-Grandpierre, A.; Tellier, C. Tetrahedron Lett. 1994,
35, 1867-1870. (b) Ble´riot, Y.; Dintinger, T.; Genre-Grandpierre, A.;
Pradines, M.; Tellier, C. Bioorg. Med. Chem. Lett. 1995, 5, 2655-2660.
(c) Therisol, H.; Letourneux, Y.; Therisod, M. Bioorg. Med. Chem. Lett.
1998, 8, 371-372. (d) Ble´riot, Y.; Dintinger, T.; Guillo, N.; Tellier C.
Tetrahedron Lett. 1995, 36, 5175-5176. (e) Guo, W.; Hiratake, J.; Ogawa,
K.; Yamamoto, M.; Ma, S.-J.; Sakata, K. Bioorg. Med. Chem. Lett. 2001,
11, 467-470.
Synthesis of the Glycoamidines. The general synthetic route
to protected glucoamidines 4a-e and mannoamidines 5a-e was
carried out as summarized in Scheme 1, following the procedure
reported by Ganem.14c A slight modification was applied using
benzyl ethers as protective groups to improve compound stability
and to facilitate purifications.
The sequence started with 2,3,4,6 tetra-O-benzyl-D-glucono-
lactam 1 which was prepared in four steps (42% overall yield)
from commercially available 2,3,4,6-tetra-O-benzyl-D-gluco-
pyranose.18 Lactam 1 was then reacted with Lawesson’s
reagent19 (C6H6, reflux, 8 h, 84% yield) to afford the corre-
sponding glucothionolactam 2 as previously reported.20 Treat-
(16) (a) Knapp, S.; Purandare, A.; Rupitz, K.; Withers S. G. J. Am. Chem. Soc
1994, 116, 7461-7462. (b) Knapp, S.; Choe, Y. H.; Reilly, E. Tetrahedron
Lett. 1993, 34, 4443-4446.
(17) Schedler, D. J. A.; Bowen, B. R.; Ganem, B. Tetrahedron Lett. 1994, 35,
3845-3848.
(18) (a) Overkleeft, H. S.; van Wiltenburg, J.; Pandit, U. K. Tetrahedron Lett.
1993, 34, 2527-2528. (b) Overkleeft, H. S.; van Wiltenburg, J.; Pandit,
U. K. Tetrahedron 1994, 50, 4215-4224.
(19) Pedersen, B. S.; Scheibye, S.; Nilsson, N. H.; Lawesson, S.-O. Bull. Soc.
Chim. Belg. 1978, 87, 293-297.
9
1972 J. AM. CHEM. SOC. VOL. 126, NO. 7, 2004

Cyclic Amidine-Sugars as Glycosidase Inhibitors
A R T I C L E S
Scheme 1. Preparation of Glycoamidines from Gluconolactama
Scheme 2. Preparation of Glycoamidines from Mannonolactama
a Reagents and conditions: (a) Lawesson’s reagent, C6H6, reflux, 8 h;
(b) Meerwein’s salt, CH2Cl2, 0 °C, 1.5 h, 95% yield; (c) PrNH2, CH2Cl2,
0 °C to rt, 12 h.
a Reagents and conditions: (a) Lawesson’s reagent, C6H6, reflux, 8 h;
(b) Meerwein’s salt, CH2Cl2, 0 °C, 1.5 h; (c) R1NH2, CH2Cl2, 0 °C to rt,
12 h (for details see Table 1).
5c, 4d, 5d; entries 4-6) were prepared in good yields (88-
93%) in a ratio of 3:7 in favor of the mannoepimer. Similarly
when 3-(tert-butyldimethylsiloxy)-1-propylamine25 was reacted
with glucoiminoether 3, protected propanol glucoamidine 4e and
mannoamidine 5e were isolated in 16% and 67% yields,
respectively (entry 7).
To confirm and characterize the two epimers formed during
the condensation of amine with D-glucoiminothioether 3, we
further studied the products obtained starting from D-mannoimi-
nothioethyl ether 16 with propylamine. Iminothioether 16 was
synthesized in two steps from tetrabenzylmannonolactam 14 as
reported in Scheme 2.
Table 1. Obtention of Glucoamidines 4 and Mannoamidines 5
1
entry
R NH
equiv
4 (%)
5 (%)
2
1
2
3
4
5
6
7
CH3(CH2)2NH2
CH3(CH2)2NH2
CH3(CH2)2NH2
BocNH(CH2)2NH2
BocNH(CH2)3NH2
BocNH(CH2)4NH2
TBDMSO(CH2)3NH2
1
1.8
10
1.8
1.8
1.8
1.8
4a (69)
4a (29)
4a (25)
4b (23)
4c (26)
4d (22)
4e (16)
5a (23)
5a (68)
5a (70)
5b (70)
5c (66)
5d (66)
5e (67)
Mannonolactam 1426 was treated with Lawesson’s reagent
to afford mannothionolactam 15 (89% yield), which was then
reacted with Meerwein’s salt to generate mannoiminothioether
16 (95% yield). Subsequent treatment of 16 with 1.8 equiv of
propylamine gave a separable 9:1 mixture of two products which
were identified as mannoamidine 5a (90% yield) and glucoa-
midine 4a (10% yield). This experiment demonstrated that under
these conditions mannoiminothioether 16 is less epimerized.
Removal of the protective groups to provide the final inhibitors
was then performed as summarized in Scheme 3.
Hydrogenolysis of 4a and 5a with 30% palladium on activated
carbon gave propylglucoamidine 7a (93% yield) and propyl-
mannoamidine 9a (81% yield), respectively. Deprotection of
the benzylglucoamidine carbamates 4b, 4c, 4d and benzylm-
annoamidine carbamates 5b, 5c, 5d with trifluoroacetic acid
afforded the benzylglucoamidine amines 6b, 6c, 6d (89%, 86%,
83% yields, respectively) and benzylmannoamidine amines 8b,
8c, 8d (91%, 92%, 88% yields, respectively). Debenzylation
reaction of benzylglucoamidines alkylamine 6b, 6c, 6d gave
glucoamidines alkylamine 7b, 7c, 7d (95%, 89%, 91% yields,
respectively), and benzylmannoamidines alkylamine 8b, 8c, 8d
afforded the mannoamidine alkylamines 9b, 9c, 9d (89%, 88%,
91% yields, respectively).
ment of 2 with Meerwein’s salt yielded glucoiminothioethyl
ether 3 (95% yield). Iminothioether 3 was then treated with a
series of amines R1NH2 and afforded a separable mixture of
21
glucoamidine 4a-e22 and mannoamidine 5a-e.23 The results
are summarized in Table 1.
We first studied the ratio of amine to use in the coupling
reaction (Table 1, entries 1-3). Glucoiminothioethyl ether 3
reacted with 1 equiv of propylamine affording a 3:1 mixture of
propylglucoamidine 4a and propylmannoamidine 5a (69% and
23% yields, respectively, entry 1) which were separated by
chromatography on silica gel.22 To our surprise, when glu-
coiminothioethyl ether 3 was treated with a slight excess of
propylamine (1.8 equiv, entry 2), propylmannoamidine 5a was
isolated as a major product (68% yield) compared to propyl-
glucoamidine 4a (29% yield, entry 2). The same 3:7 ratio in
manno/glucoamidine and the global yield were conserved when
3 was treated with an excess of propylamine (10 equiv, entry
3). The formation of D-mannoamidine 5a starting from D-
glucoiminothioether 3 is probably the result of an epimerization
at C2 during the reaction with propylamine. Ganem et al.14c
have previously observed such an epimerization when reacting
D-mannothiolactam with a large excess of ammonia and have
isolated glucoamidine as the sole product in 70% yield.
For the following experiments, glucoiminothioether 3 was
reacted with 1.8 equiv of amines to obtain in one step the two
epimers D-glucoamidine 4a-e and D-mannoamidine 5a-e
(Table 1). Gluco- and mannoamidines bearing a Boc-protected
aminoethyl, aminopropyl, and aminobutyl groups24 (4b, 5b, 4c,
Removal of TBDMS group in benzylglucoamidine 4e and
benzylmannoamidine 5e using pyridinium-p-toluenesulfonate in
EtOH gave benzylglucoamidine propyl alcohol 6e (61% yield)
and benzylmannoamidine propyl alcohol 8e (80% yield).
Hydrogenolysis of benzylglucoamidine 6e and benzylmano-
amidine 8e yielded, respectively, glucoamidine propyl alcohol
7e (92% yield) and mannoamidine propyl alcohol 9e (86%
yield).
(20) Hoos, R.; Naughton, A. B.; Thiel, W.; Vasella, A.; Weber, W.; Rupitz, K.;
Withers, S. G. HelV. Chim. Acta 1993, 76, 2666-2686. As reported,
thiolactam 2 gave an inseparable crystalline 9:1 mixture of D-glucothiolac-
tam and D-mannothiolactam.
(21) The amines were commercially available or synthesized; see refs 24 and
25.
(22) The less polar isomer (minor) was identified as D-glucoamidine, and the
more polar isomer was assigned having a D-manno configuration.
(23) Theoretical calculations were not employed to predict the endo or exo
constitution of the CdN bond. Based on previous studies reported by
Vasella (ref 20), we postulate the presence of an exocyclic CdN bond.
(24) For the preparation of N-tert-butoxycarbonyl alkanediamines, see: Krapcho,
A. P.; Kuell, C. S. Synth. Commun. 1990, 20, 2559-2564. Guo, H.; Naser,
S. A.; Ghobrial, G.; Phanstiel, O., IV. J. Med. Chem. 2002, 45, 2056-
2063.
(25) For the synthesis of 3-(tert-butyldimethylsiloxy)-1-propylamine, see: Grillot,
A.-L.; Hart, D. J. Tetrahedron 1995, 51, 11377-11392.
(26) Tetrabenzylmannolactam 14 was prepared following the procedure reported
by Pandit (ref 18b).
9
J. AM. CHEM. SOC. VOL. 126, NO. 7, 2004 1973

A R T I C L E S
Heck et al.
Scheme 3. Preparation of Final Glycoamidinesa
Table 2. Inhibitory Potencies of Synthesized Glycoamidines (Ki
Values in µM)27
glycosidasesa
entry inhibitor R-Man â-Man R-Glc â-Glc R-Gal â-Gal R-Fuc â-NAcGl
1
2
3
4
5
7a 2.3
7b 0.06
7c 0.09
7d 0.12
7e 1.7
3.3
13
32
14
39
31
3.6
34
71
50
184
192
44
31 295
53 96
44 91
6 221
374
339
252
136
4500
900
99
0.13
0.41
0.71
0.87
0.19
451
20 28 000 1000 470
6
7
9a 0.11
9b 0.006 0.009
125 111
81 6600
4700 2200 34
218
82
6
8
9c 0.019 0.15 2870
22
1400 119
8.5
85
9
9d 0.012 0.06 155 315
1800 156 10
1200 235 79
2000 1400 75
7400 6300 930
112
1300
1100
750
10
11
12
9e 0.08
12 8.8
13 6.0
0.15
nd
nd
38
1100
1800
44
nd
nd
a Commercially available glycosidases: R-Man ) R-Mannosidase (jack
beans), â-Man ) â-Mannosidase (snail acetone powder), R-Glc ) R-Glu-
cosidase (yeast type III), â-Glc ) â-Glucosidase (almond), R-Gal )
R-Galactosidase (green coffee beans), â-Gal ) â-Galactosidase (Aspergillus
orizae), R-Fuc ) R-Fucosidase (bovine kidney), â-N-AcGl ) N-Acetyl-
â-glucosaminidase (jack beans), nd ) not determined.
good agreement with earlier studies.14 Indeed, the glycoamidine
core structure, even in the gluco series with an inverted
configuration at C2, reaches a low nanomolar inhibition level
with mannosidases, whereas all the other glycosidases were, at
best, inhibited in the micromolar range.27
a Reagents and conditions: (a) H2, 30% Pd/C, EtOH, 12 h; (b) TFA,
CH2Cl2, 0 °C to rt, 1.5 h; (c) PPTS, EtOH, 65 °C, 7 h.
r-/â-Glycosidase Selectivity. Glucoamidines 7a-7e (entries
1-5) and mannoamidines 9a-d (entries 6-9) bearing alkyl-
amino groups were almost always better competitive inhibitors
of R-glycosidases than â-glycosidases. Nevertheless, this se-
lectivity is moderate: depending on the length of the alkyl chain,
Ki values for â-mannosidases are 1.5 to 7 times higher than
values for R-mannosidases. This difference is not significant
enough to draw any conclusion regarding a difference in the
mechanistic pathways between R- and â-mannosidases. For
instance, isofagomine derivatives,7d,28 N-iminosugars,29 as well
as mannosyl derivatives locked in a 1,4B conformation9 display
a greater R/â-glycosidase selectivity leading information in the
difference of conformations or charge localization at the
transition state of R- or â-glycosidases. The mannoamidines
9a-d inhibiting R- and â-mannosidases in the nanomolar range,
we can reasonably conclude that they should mimic a “late”
high-energy intermediate common to R- and â-mannoside
hydrolysis.
Scheme 4 . Preparation of Bicyclic Amidinesa
a Reagents and conditions: (a) (i) Br(CH2)3NH2, EtOH, 0 °C to rt, 8 h,
(ii) K2CO3, 0 °C to rt, 6 h; (b) H2, Pd/C, EtOH, 12 h.
Bicyclic amidines 12 and 13 were synthesized as illustrated
in Scheme 4. Iminothioether 3 was treated with 3-bromo-
propylamine and K2CO3 in CH2Cl2 to yield a 1:7 mixture of
protected bicyclic glucoamidine 10 (10% yield) and bicyclic
mannoamidine 11 (69% yield) which were separated by
chromatography on silica gel. Hydrogenolysis of 10 and 11 with
30% palladium on activated carbon gave D-gluco-1,5-diazabicyclo-
[4,4,0]decene 12 (95% yield) and D-manno-1,5-diazabicyclo-
[4,4,0] decene 13 (90% yield).
All the synthesized amidines (7a-e, 9a-e, 12, 13) were
shown to be stable for months at -40 °C.
Effects of the Additional Amino Group and Spacer
Length. The glycoamidines 7b-7d and 9b-d (entries 2-4,
6-8) were designed to engender two tight ionic interactions
between the two catalytic carboxylates of the enzyme (the
general protonator and the base/nucleophile), and both ami-
Discussion of the Inhibitory Activity. Glucoamidines 7a-e
and 12 and mannoamidines 9a-e and 13 were then evaluated
against a wide variety of commercially available glycosidases:
R-mannosidase (jack beans), â-mannosidase (snail acetone
powder), R-glucosidase (yeast type III), â-glucosidase (almond),
R-galactosidase (green coffee beans), â-galactosidase (Aspergil-
lus Orizae), R-fucosidase (bovine kidney), N-acetyl-â-glu-
cosaminidase (jack beans). The Ki values obtained are summa-
rized in Table 2. Lineweaver-Burk plots were drawn, thus
showing the competitive character of the inhibitions described
in Table 2.
Glycoamidines Selectively Inhibit Mannosidases in the
Low Nanomolar Range. Mannoamidines 9a-e (entries 6-9)
display a much better inhibitory activity toward R- and
â-mannosidases than the corresponding glucoamidines 7a-7e
(entries 1-5) toward R- and â-glucosidases. This result is in
(27) Ki values were measured at pH 6.8 (pH 6 for the fucosidase). The pH
dependency of glycoamidine inhibition against R-mannosidase and â-glu-
cosidase has been previously reported. Ganem et al. have showed that,
between pH 4.5 and 7, the inhibition of â-glucosidase is not pH dependent;
see ref 14a-c. Ble´riot et al. have reported that the inhibition of R-man-
nosidase by N-benzylmannoamidine slightly increased between pH 5
(optimum mannosidase activity) and pH 6.8; see ref 15b. However the
comparison of Ki values determined at different pH should be done with
care. For instance, Davies et al. have recently reported that the pH
dependency of the catalytic efficiency and the inhibition constant might
not correspond: Varrot; A.; Tarling, C. A.; Macdonald, J. M.; Stick, R.
V.; Zechel, D. L.; Withers, S. G.; Davies, G. J. J. Am. Chem. Soc. 2003,
125, 7496-7497.
(28) Jespersen, T. M.; Dong, W.; Sierks, M. R.; Skrydstrup, T.; Lundt, I.; Bols,
M. Angew. Chem., Int. Ed. Engl. 1994, 33, 1778-1779.
(29) Ichikawa, Y.; Igarashi, Y.; Ichikawa, M.; Suhara, Y. J. Am. Chem. Soc.
1998, 120, 3007-3018.
9
1974 J. AM. CHEM. SOC. VOL. 126, NO. 7, 2004

Cyclic Amidine-Sugars as Glycosidase Inhibitors
A R T I C L E S
dinium and tethered amino group. The aminomannoamidines
9b-d (entries 7-9) were potent inhibitors of R- and â-man-
nosidases in the low nanomolar range. The shortest length amine
9b (entry 7), bearing an ethylene linker between the amidine
and amine functions, was very potent toward R- and â-man-
nosidases (Ki ) 6 nM and 9 nM, respectively), but 9b was a
moderate inhibitor against the other enzymes tested. These
results make 9b the most potent and selective inhibitor reported
to date. Both propylamine mannoamidine 9c (entry 8) and the
butylamine mannoamidine 9d (entry 9) also showed good
potency and selectivity toward mannosidases with Ki in the range
of 10-20 nM. For R-mannosidase, the Ki values of propyl
derivative 9a (entry 6) and alcohol 9e (entry 10) are very similar
compared to the corresponding propylamino derivative 9c (entry
8): the presence of the amino group is very significant and has
to be explained by an ionic interaction, otherwise the alcohol
9e would be a more potent inhibitor than 9c. As it was expected,
the results showed that the amidine 9b bearing the shortest
spacer displayed the best inhibitory activity (entry 7). The effect
of the tether length is particularly significant for the inhibition
of the â-mannosidase. Nevertheless, the fact that butylaminoa-
midine 9d (entry 9) was more potent than propylaminoamidine
9c (entry 8) is still unclear. Although our results support a new
beneficial interaction with one of the two catalytic carboxylates,
we cannot exclude the possibility that the amino functionality
interact with another properly placed residue. Only a cocrys-
tallization of one of these inhibitors with the mannosidases
would unambiguously answer this question at the molecular
level. Derivatives of aminomethylpyrrolidine diol were previ-
ously reported as dicationic mimics of a transition or intermedi-
ate structure of an R-mannosidase-catalyzed hydrolysis and
displayed a competitive and good selectivity toward R-man-
nosidase from jack bean (Ki ) 7.4 µM).30
Figure 3. Conformational analysis.
4H3 half-chair conformation (b, Figure 3). However, it does not
imply that a molecule adopting this conformation will give rise
to the tightest or the most selective binding to a given
glycosidase. For instance, Vasella et al.9a have shown that
polyhydroxylated isoquinuclidines locked in a 1,4B boat con-
formation display strong and selective binding to â-mannosidase
(c, Figure 3, Ki ) 0.17 µM, IC50 ) 0.68 µM for snail
â-mannosidase, IC50 ) 9.6 mM for jack bean R-mannosidase).
Since mannoamidines 9a-d do not display significant
selective inhibition between R- and â-mannosidases, they
probably do not bind these glycosidases in a 1,4B boat conforma-
Effect of Glycobicyclic Amidine 12 and 13. Bicyclic
glucoamidine 12 (entry 11) and mannoamidine 13 (entry 12)
showed good inhibition properties for R-mannosidase (Ki ) 9
µM and 6 µM respectively), but the inhibitions were much
weaker for the other enzymes. The introduction of a fused six-
membered ring on the glycoamidine core structure did not
improved the inhibition level.
4
tion. Indeed a stabilized H3 transition state was reported for
inverting R-mannosidase,31 and other glycosides have been
shown to adopt a B2,5 boat conformation as mannonolactam in
solution or solid state32 (Figure 3, d) and mannonolactone.33
Moreover, crystallographic studies of â-mannanase 26A from
P. cellulosa bound to a fluorinated trisaccharide inactivator
showed a distortion of the carbohydrate suggesting a B2,5 boat
conformation for the transition state, and the family 11-xylanases
displayed a covalent xylobiosyl-enzyme intermediate in the same
B2,5 conformation.34
Effect of the Inversion of the Stereochemistry at the
2-Position. Inversion of the stereochemistry on C2 from manno
to gluco configuration gives rise to an expected loss of inhibitory
activity on the mannosidase inhibitions with a factor of 5 to 15
(Table 2: glucoamidines 7a-d (entries 1-4) vs mannoamidines
9a-d (entries 6-10)). The same effect was also observed for
glucosidases: glucoamidines 7a-d were 3 to 20 times more
potent on glucosidases than the corresponding mannoamidines
9a-d. The intriguing point was that glucoamidines only inhibit
R- and â-glucosidases in the low micromolar range (4-70 µM),
suggesting a major difference in the relative mechanism of
glucosidases and mannosidases which might be explained by
stereoelectronic and conformational factors.
Since the double bond of the amidine functionality was
proved to be exocyclic,20 mannoamidines, 9a-d should possess
close structural features to mannonolactam d. Therefore,
amidines 9a-d might also bind mannosidases in a B2,5 boat
conformation (f, Figure 3) preferentially to a 4H3 conformation
(e, Figure 3). As reported for mannonolactam d, a significant
NOE effect between H2 and H5 was observed for amidine 9a
in D2O, showing that its conformation in aqueous solution is
(31) (a) Valle´e F.; Karaveg, K.; Herscovics, A.; Moremen, K. W.; Howell, P.
L. J. Biol. Chem. 2000, 29, 41287-41298. (b) Davies, G. J.; Ducros, V.
M.-A.; Zechel, D. L. Biochem. Soc. Trans. 2003, 31, 523-527.
(32) Nishimura, Y.; Adachi, H.; Satoh, T.; Shitara, E.; Nakamura, H.; Kojima,
F.; Takeuchi, T. J. Org. Chem. 2000, 65, 4871-4882.
(33) Walaszek, Z.; Horton, D.; Ekiel, I. Carbohydr. Res. 1982, 106, 193-201.
(34) (a) Ducros, V. M.-A.; Zechel, D. L.; Murshudov, G. N.; Gilbert, H. J.;
Szabo, L.; Stoll, D.; Withers, S. G.; Davies, G. J. Angew. Chem., Int. Ed.
2002, 41, 2824-2827. (b) Sabini, E.; Sulzenbacher, G.; Dauter, M.; Dauter,
Z.; Jorgensen, P. L.; Schu¨lein, M.; Dupont, C.; Davies, G. J.; Wilson, K.
S. Chem. Biol. 1999, 6, 483-492. (c) Numao, S.; Kuntz, D. A.; Withers,
S. G.; Rose, D. R. J. Biol. Chem 2003, in press.
Conformational Analysis. Due to the double bond between
the anomeric carbon and the endocyclic oxygen, the oxycar-
benium species (a, b; Figure 3), likely a high-energy intermedi-
ate of the enzymatic hydrolysis, is most often represented in its
(30) Popowycz, F.; Guerber-Lemaire, S.; Demange, R.; Rodriguez-Garcia, E.;
Carmona Asenjo, A. T.; Robina, I.; Vogel, P. Bioorg. Med. Chem. Lett.
2001, 11, 2489-2493.
9
J. AM. CHEM. SOC. VOL. 126, NO. 7, 2004 1975

A R T I C L E S
Heck et al.
Table 3. Inhibitory Activities of Newly Synthesized and Known
Mannosidase inhibitors toward Jack Bean R-Mannosidasea
A favorable B2,5 boat conformation might explain the selectivity
of the mannosidase inhibition compared to other glycosidases.
To further improve the selectivity and the binding strength of
these amidines, it can be envisaged attaching either an hydro-
phobic moiety or a second carbohydrate part to the terminal
amino group.
Experimental Section
General Methods. All reagents were commercial grade and were
used as received without further purification. All reactions were
performed under inert atmosphere, and anhydrous solvents were dried
and distilled over appropriate desiccant prior to use. Thin-layer
chromatogry (TLC) and flash chromatography separations were,
respectively, performed on precoated silica gel 60 F254 plates (Merck,
0.25 mm) and on Merck silica gel 60 (230-400 mesh). 1H NMR spectra
were recorded at 300 MHz, and 13C NMR spectra were obtained at 75
MHz in the specified solvent. Mass spectra were recorded at 70 eV on
a Finnigan-Mat 4600 using chemical ionization mode (CI-NH3 or CI-
CH4). ESITOF (electrospray) mass spectra were mesured on a Mariner
Perspective Biosystem apparatus. High-resolution mass spectra HRMS/
LSIMS+ were performed by the Centre Regional de Mesures Physiques
de l’Ouest on a Zabspec TOF Micromass (matrix: 3-nitrobenzyl
alcohol). IR spectra were recorded on NaCl plates as thin film on an
FTIR instrument. Optical rotations were recorded on a Perkin-Elmer
241 MC polarimeter. Elemental analyses were measured at the Service
de Microanalyze de Gif sur Yvette (ICSN).
a 1, Amidine 9b; 2, Ganem’s amidine; 3, D-Mannoamidrazone; 4,
Mannostatin A; 5, (-)-Swainsonine; 6, Methylaminopentacyclitol.
4
closer to a B2,5 boat (f, Figure 3) than to a H3 half-chair (e,
Figure 3). This conformational property should be responsible
for the inhibitory potency and the selectivity of both manno-
and glucoamidines against mannosidases and should also explain
why the glucoamidines do not bind glucosidases in a low
nanomolar range. In agreement with Davies,34 we can assume
that all glycosidases do not react through a 4H3 half-chair
transition state.
Inhibitory Activities of Mannoamidine 9b Compared to
Known Inhibitors. Comparison of the inhibitory potency of
our best inhibitor 9b with known inhibitors of mannosidases is
reported in Table 3.35 Amidine 9b inhibited jack bean R-man-
nosidase in the nanomolar range (entry 1). Ganem’s amidine14a
(entry 2) and amidrazone14c (entry 3) were proved to be good
inhibitors of R-mannosidase with a Ki in the low micromolar
range. Mannostatin A36 (entry 4), swainsonine37 (entry 5), and
methylaminopentacyclitol38 (entry 6) were the most potent
inhibitors of R-mannosidase reported thus far with Ki values
comprised between 50 and 200 nM. To our knowledge, these
results prove that mannoamidine 9b is the first inhibitor
displaying a Ki value below 10 nM against jack bean R-man-
nosidase.
2,3,4,6-Tetra-O-benzyl-D-gluconoiminothioether 3. To a solution
of gluconothiolactam 2 (300 mg, 0.54 mmol) in dry CH2Cl2 (13 mL)
at 0 °C was added triethyloxonium tetrafluoroborate (Meerwein’s salt)
(112 mg, 0.59 mmol). The mixture was stirred at 0 °C for 1 h 30 min
and was used directly in the next step. The crude was concentratred
under vacuum to give 3 (298 mg, 95% yield): 1H NMR (CDCl3) δ
7.40-7.21 (m, 20H), 4.80-4.31 (m, 10H), 4.08 (dd, J ) 4.4, 6.9 Hz,
1H), 3.99 (dd, J ) 2.7, 10.6 Hz, 1H), 3.87 (dd, J ) 6.9, 9.0 Hz, 1H),
3.50 (dd, J ) 2.2, 10.7 Hz, 1H), 3.18 (q, J ) 7.5 Hz, 2H), 1.42 (t, J
) 7.5 Hz, 3H); 13C NMR (CDCl3) δ 192.7, 136.9, 136.6, 135.5, 128.9-
128.0, 81.4, 78.6, 76.3, 74.9, 73.6, 73.4, 66.7, 63.6, 25.7, 11.8; IR (neat,
cm-1) 3565, 2930, 2874, 1674, 1604, 1455, 1072, 744, 700; MS (DCI/
NH3) m/z (M + H)+ ) 582.
General Procedure for the Preparation of Amidines 4a, 5a, 4b,
5b, 4c, 5c, 4d, 5d, 4e, 5e. To a stirred solution of freshly prepared
glucoiminothioether 3 (0.17 mmol, 1 equiv) in 4 mL of dry CH2Cl2 at
0 °C was added a solution of amine (0.3 mmol, 1.8 equiv) in 2 mL of
dry CH2Cl2. The reaction mixture was allowed to warm to room
temperature and was stirred for 8 h. The solution was concentrated
under vacuum and purified by flash column chromatography on silica
gel eluting with CH2Cl2/MeOH (98:2) to give glucoamidine (less polar
isomer) and mannoamidine (more polar isomer).
N,N-Propyl-2,3,4,6-tetra-O-benzyl-D-glucoamidine 4a and N,N-
Propyl-2,3,4,6-tetra-O-benzyl-D-mannoamidine 5a. Glucoiminothio-
ether 3 (65 mg, 0.11 mmol) was reacted with propylamine (16 µL, 0.2
mmol) to give glucoamidine 4a (19 mg, 29%) and mannoamidine 5a
(44 mg, 68%) following the general procedure. 4a: 1H NMR (CDCl3)
δ 7.40-7.16 (m, 20H), 4.93 (d, J ) 11.6 Hz, 1H), 4.79 (s, 2H), 4.63-
4.36 (m, 7H), 4.01-3.96 (m, 2H), 3.88-3.79 (m, 2H), 3.51 (dd, J )
2.8, 10.3 Hz, 1H), 3.22 (t, J ) 6.9 Hz, 2H), 1.50 (q, J ) 7.2 Hz, 2H),
0.87 (t, J ) 7.3 Hz, 3H); 13C NMR (CDCl3) δ 162.4, 137.4, 137.2,
136.9, 136.0, 129.1-128.1, 81.4, 77.8, 75.9, 74.8, 74.4, 73.5, 73.0,
80.0, 57.6, 43.7, 21.1, 10.8; IR (neat, cm-1) 3301, 2877, 1680, 1455,
1094, 743, 699; MS (ESITOF) m/z (M)+ ) 578; [R]25D ) -20.6 (c )
1.95, CH2Cl2). 5a: 1H NMR (CDCl3) δ 7.37-7.08 (m, 20H), 4.59-
4.25 (m, 10H), 3.91 (t, J ) 2.8 Hz, 1H), 3.78-3.61 (m, 4H), 3.38-
3.36 (m, 2H), 1.62 (q, J ) 7.2 Hz, 2H), 0.92 (t, J ) 7.3 Hz, 3H); 13C
NMR (CDCl3) δ 162.1, 137.7, 136.8, 135.6, 128.9-127.8, 73.5, 73.2,
Conclusion
In conclusion, we have designed and synthesized a series of
biscationic glycoamidines in the gluco and manno series which
displayed excellent inhibition properties against R- and â-man-
nosidases. Moreover, we have demonstrated the enhancement
of binding interactions triggered by an additional amino
functionality anchored to the amidine unit through a short spacer.
(35) Except for amidines, inhibitors showing Ki < 0.2 µM were only reported
in Table 2.
(36) Tropea, J. E.; Kaushal, G. J.; Pastushak, I.; Mitchell, M.; Aoyagi, T.;
Molyneux, R. M.; Elbein, A. D. Biochemistry 1990, 29, 10062-10069.
(37) (a) Cenci di Bello, I.; Fleet, G. W. J.; Namgoong, S. K.; Tadano, K.-I.;
Winchester, B. Biochem. J. 1989, 259, 855-861. (b) Hembre, E. J.; Pearson,
W. H. Tetrahedron 1997, 53, 11021-11032. (c) Elbein, A. D. Annu. ReV.
Biochem. 1987, 56, 497-534.
(38) Farr, R. A.; Peet, N. P.; Kang, M. S. Tetrahedron Lett. 1990, 31, 7109-
7112.
73.1, 72.5, 72.4, 71.9, 71.6, 68.2, 55.6, 43.6, 21.1, 10.8; IR (neat, cm-1
)
9
1976 J. AM. CHEM. SOC. VOL. 126, NO. 7, 2004

Cyclic Amidine-Sugars as Glycosidase Inhibitors
A R T I C L E S
3307, 2942, 2362, 1681, 1574, 1455, 1361, 1092, 819; MS (DCI/NH3)
3364, 2366, 1681, 1455, 1076, 711; MS (DCI/NH3) m/z (M + H)+
)
m/z (M)+ ) 579; [R]24 ) +6.1 (c ) 2.77, CH2Cl2).
595; [R]27 ) +7.9 (c ) 1.15, CH2Cl2).
D
D
N,N-(3-Hydroxypropyl)-D-glucoamidine 7e. Amidine 6e (11 mg,
0.02 mmol) was hydrogenated in a manner similar to the preparation
of 7a to give deprotected glucoamidine 7e (4 mg, 92% yield): 1H NMR
(CD3OD) δ 4.19 (d, J ) 9.7 Hz, 1H), 3.79-3.71 (m, 3H), 3.65 (t, J )
5.8 Hz, 2H); 3.57-3.31 (m, 4H), 1.78-1.68 (m, 2H); 13C NMR (CD3-
OD) δ; 166.3, 73.5, 69.2, 67.1, 62.1, 60.8, 59.9, 40.4, 31.2, 18.2; IR
(neat, cm-1) 3331, 2466, 1667, 1064, 521; HRMS calcd C9H19N2O5
(M + H)+ 235.1294, found 235.1290. [R]28D ) +1.3 (c ) 1.08, CH3-
OH).
N,N-Propyl-D-glucoamidine 7a. To a stirred solution of amidine
4a (17 mg, 0.03 mmol) in EtOH (2.5 mL) was added 30% Pd on
activated carbon. The mixture was stirred under hydrogen (1 atm) for
12 h and was filtered through a pad of Celite and washed with EtOH.
The solvent was concentrated under vacuum to afford the amidine 7a
(5.9 mg, 93% yield): 1H NMR (CD3OD) δ 4.17 (d, J ) 9.6 Hz, 1H),
3.82-3.75 (m, 4H), 3.62-3.55 (m, 3H), 3.33-3.30 (m, 1H), 1.68 (q,
J ) 7.2 Hz, 2H), 0.98 (t, J ) 7.3 Hz, 3H); 13C NMR (CD3OD) δ
165.9, 74.2, 69.9, 62.4, 61.5, 60.9, 44.5, 22.2, 11.3; HRMS calcd for
C9H19N2O4 (M + H)+ 219.1345, found 219.1337; [R]28D ) +1.2 (c )
1.69, CH3OH).
N,N-Propyl-D-mannoamidine 9a. Amidine 5a (35 mg, 0.05 mmol)
was hydrogenated in a manner similar to the preparation of 7a to give
deprotected amidine 9a (10.5 mg, 81% yield): 1H NMR (CD3OD) δ
4.62 (d, J ) 3.1 Hz, 1H), 3.98 (t, J ) 3.1 Hz, 1H), 3.90 (dd, J ) 3.5,
4.0 Hz, 1H), 3.84 (dd, J ) 4.9, 11.3 Hz, 1H), 3.74 (dd, J ) 5.7, 11.4
Hz, 1H), 3.38 (q, J ) 4.9 Hz, 1H), 3.36-3.25 (m, 2H), 1.63 (q, J )
7.3 Hz, 2H), 0.94 (t, J ) 7.3 Hz, 3H); 13C NMR (CD3OD) δ 166.0,
73.3, 69.6, 67.2, 62.4, 60.9, 44.3, 22.2, 11.2; IR (neat cm-1) 3326,
2970, 2466, 1681, 1455, 1316, 1035; HRMS calcd for C9H19N2O4 (M
+ H)+ 219.1345, found 219.1347; [R]27D ) -9.7 (c ) 1.32, CH3OH).
N,N-[3-(tert-Butyldimethylsiloxy)propyl]-2,3,4,6-tetra-O-benzyl-
D-glucoamidine 4e and N,N-[3-(tert-Butyldimethylsiloxy)propyl]-
2,3,4,6-tetra-O-benzyl-D-mannoamidine 5e. Glucoiminothioether 3
(200 mg, 0.34 mmol) was reacted with 3-(tert-butyldimethylsiloxy)-
1-propylamine25 (117 mg, 0.6 mmol) to give glucoamidine 4e (39 mg,
16%) and mannoamidine 5e (163 mg, 67%) following the general
procedure. 4e: 1H NMR (CDCl3) δ 7.39-7.09 (m, 20H), 4.90 (d, J )
11.8 Hz, 1H), 4.77 (s, 2H), 4.62-4.28 (m, 6H), 3.94-3.85 (m, 3H),
3.79-3.67 (m, 4H), 3.58-3.49 (m, 1H), 3.40-3.36 (m, 2H), 1.74-
1.69 (m, 2H), 0.89 (s, 9H), 0.06 (s, 6H); 13C NMR (CDCl3) δ 162.2,
137.8, 136.8, 135.4, 129.1-127.4, 73.4, 73.1, 73.0, 72.4, 72.2, 72.1,
71.7, 68.3, 62.3, 55.7, 41.4, 29.8, 26.2, 18.7, 0.3; IR (neat, cm-1) 3296,
N,N-(3-Hydroxypropyl)-D-mannoamidine 9e. Amidine 8e (58 mg,
0.097 mmol) was hydrogenated in a manner similar to the preparation
of 7a to give deprotected mannoamidine 9e (19 mg, 86% yield): 1H
NMR (CD3OD) δ 4.65 (d, J ) 3.0 Hz, 1H), 4.01 (t, J ) 3.1 Hz, 1H),
3.89 (dd, J ) 3.2, 4.0 Hz, 1H), 3.83 (dd, J ) 4.8, 11.4 Hz, 1H), 3.75
(dd, J ) 5.3, 11.4 Hz, 1H), 3.65 (t, J ) 5.8 Hz, 2H), 3.55-3.41 (m,
3H), 1.34-1.26 (m, 2H); 13C NMR (CD3OD) δ 166.5, 73.5, 70.1, 67.2,
62.7, 61.0, 59.7, 40.1, 31.4; IR (neat, cm-1) 3364, 2366, 1681, 1455,
1076, 711; HRMS cald for C9H19N2O5 (M + H)+ 235.1294, found
235.1284; [R]27 ) -5.8 (c ) 1.29, CH3OH).
D
N,N-[2-(tert-Butoxycarbonylamino)ethyl]-2,3,4,6-tetra-O-benzyl-
D-glucoamidine 4b and N,N-[2-(tert-Butoxycarbonylamino)ethyl]-
2,3,4,6-tetra-O-benzyl-D-mannoamidine 5b. Glucoiminothioether 3
(100 mg, 0.17 mmol) was reacted with 2-(tert-butoxycarbonylamino)-
1-ethylamine (52 mg, 0.31 mmol) to give glucoamidine 4b (28 mg,
23%) and mannoamidine 5b (83 mg, 70%) following the general
procedure. 4b: 1H NMR (CDCl3) δ 8.71-8.60 (br s, 1H), 7.40-7.19
(m, 20H), 5.35-5.32 (br s, 1H), 4.81 (q, J ) 11.2 Hz, 2H), 4.73 (s,
2H), 4.57-4.47 (m, 4H), 4.36 (d, J ) 11.8 Hz, 1H), 3.99-3.68 (m,
3H), 3.76 (dd, J ) 2.9, 10.4 Hz, 1H), 3.53 (dd, J ) 2.6, 10.2 Hz, 1H),
3.51-3.48 (m, 2H), 3.38-3.34 (m, 2H), 1.40 (s, 9H); 13C NMR (CDCl3)
δ 163.2, 158.2, 137.4, 137.2, 137.0, 135.9, 128.7, 127.8, 81.1, 80.5,
77.4, 74.2, 74.1, 73.3, 73.1, 68.9, 57.4, 43.9, 38.3, 28.3; IR (neat, cm-1
)
2929, 1685, 1515, 1081, 738; MS (ESITOF) m/z (M)+ 679, (M + H)+
680; [R]27D ) -4.7 (c ) 1.95, CH2Cl2). 5b: 1H NMR (CDCl3) δ 7.36-
7.06 (m, 20H), 5.49-5.46 (br s, 1H), 4.78 (d, J ) 11.8 Hz, 1H), 4.59-
4.49 (m, 4H), 4.41-4.23 (m, 4H), 3.90 (dd, J ) 2.6, 2.7 Hz, 1H),
3.78-3.72 (m, 3H), 3.68-3.41 (m, 6H), 1.41 (s, 9H); 13C NMR (CDCl3)
δ 162.9, 158.5, 137.8, 137.0, 136.9, 135.9, 129.0-127.9, 80.5, 73.6,
2929, 1682, 1455, 1091, 741, 699; MS (DCI/NH3) m/z (M + H)+
)
1
709. 5e: H NMR (CDCl3) δ 8.03-7.95 (br s, 1H), 7.39-7.07 (m,
20H), 4.61-4.23 (m, 9H), 3.87 (d, J ) 2.8 Hz, 1H), 3.81-3.56 (m,
8H), 1.92-1.86 (m, 2H), 0.9 (s, 9H), 0.06 (s, 6H); 13C NMR (CDCl3)
δ 162.2, 137.8, 136.9, 135.4, 129.1, 127.4, 73.4, 73.1, 73.0, 72.2, 72.1,
73.4, 73.2, 72.6, 72.3, 71.6, 68.5, 55.9, 44.0, 38.5, 28.4; IR (neat, cm-1
)
71.8, 68.3, 62.3, 55.7, 41.4, 29.8, 26.2, 18.7, -4.9; IR (neat cm-1
)
3292, 2929, 1685, 1515, 1456, 1081, 739; MS (ESITOF) m/z (M)+
3297, 2928, 1681, 1454, 1256, 1093, 837, 736; MS (DCI/NH3) m/z
(M + H)+ 709. Anal. Calcd for C43H56N2O5Si : C, 72.88; H, 7.99.
Found: C, 72.85; H, 7.89.
679; [R]27 ) -8.7 (c ) 1.82, CH2Cl2).
D
N,N-(2-Aminoethyl)-2,3,4,6-tetra-O-benzyl-D-glucoamidine 6b. To
a solution of glucoamidine 4b (22 mg, 0.03 mmol) in CH2Cl2 (1.5 mL)
was added trifluoroacetic acid (50 µL, 1.57 mmol) at 0 °C, and the
reaction was stirred 6 h at room temperature. The mixture was
concentrated to yield 6b (16 mg, 89% yield) which was pure enough
to be used in the next step without further purification. 1H NMR (CD3-
OD) δ 7.33-7.21 (m, 20H), 4.90-4.67 (m, 2H), 4.64-4.36 (m, 7H),
4.04-3.97 (m, 2H), 3.94-3.91 (m, 1H), 3.71-3.57 (m, 4H), 3.18 (t,
J ) 6.3 Hz, 2H); 13C NMR (CD3OD) δ 165.7, 139.0, 138.8, 137.9,
129.7, 129.2, 81.9, 77.5, 75.9, 74.6, 74.4, 73.7, 69.4, 58.5, 40.9, 38.4;
N,N-(3-Hydroxypropyl)-2,3,4,6-tetra-O-benzyl-D-glucoamidine 6e.
To a stirred solution of glucoamidine 4e (30 mg, 0.04 mmol) in EtOH
(3 mL) was added pyridinium p-toluenesulfonate (6 mg, 0.02 mmol).
The reaction was heated at 60 °C for 6 h. The solvent was concentrated,
and the crude product was purified by column chromatography CH2-
Cl2/MeOH (95:5) to yield compound 6e (15.2 mg, 61% yield): 1H NMR
(CDCl3) δ 7.60-7.56 (br s, 1H), 7.42-7.14 (m, 20H), 4.90 (d, J )
11.3 Hz, 1H), 4.89 (s, 1H), 4.65-4.32 (m, 7H), 3.98-3.90 (m, 2H),
3.75 (dd, J ) 2.4, 9.8 Hz, 1H), 3.64 (t, J ) 5.0 Hz, 2H), 3.51 (dd, J
) 2.8, 10.2 Hz, 1H), 3.48-3.44 (m, 1H), 3.18-3.05 (br s, 1H), 1.72-
1.55 (m, 4H); 13C NMR (CDCl3) δ 162.4, 137.5, 137.1, 136.5, 129.0-
128.0, 81.9, 78.2, 75.6, 75.1, 74.3, 74.2, 73.6, 70.4, 62.4, 55.2, 40.1,
26.4; IR (neat cm-1) 3252, 2925, 1681, 1455, 1075, 738, 698; MS (DCI/
NH3) m/z (M + H)+ 595.
N,N-(3-Hydroxypropyl)-2,3,4,6-tetra-O-benzyl-D-mannoami-
dine 8e. In a manner similar to the preparation of hydroxyamidine 6e,
amidine 5e (144 mg, 0.2 mmol) was converted to 8e (97 mg, 80%
yield). 1H NMR (CDCl3) δ 8.11-7.92 (br s, 1H), 7.36-7.10 (m, 20H),
4.72 (d, J ) 11.8 Hz, 1H), 4.62-4.30 (m, 9H), 3.78 (dd, J ) 2.6, 3.1
Hz, 1H), 3.74-3.54 (m, 8H), 1.85-1.79 (m, 2H); 13C NMR (CDCl3)
δ 162.6, 137.7, 137.0, 136.9, 135.9, 128.9-128.0, 73.7, 73.5, 73.3,
73.2, 72.9, 72.6, 71.7, 68.7, 60.1, 55.6, 40.4, 29.7; IR (neat cm-1):
MS (ESITOF) m/z (M + Na)+ 602; [R]27 ) +5.9 (c ) 1.19, CH3-
D
OH).
N,N-(2-Aminoethyl)-2,3,4,6-tetra-O-benzyl-D-mannoamidine 8b.
From 5b (82 mg, 0.12 mmol), compound 8b was obtained (63 mg,
92% yield) following the preparation reported for 6b from 4b. 1H NMR
(CD3OD) δ 7.39-7.12 (m, 20H), 4.82 (s, 1H), 4.68-4.64 (m, 2H),
4.52-4.24 (m, 6H), 3.87-3.85 (m, 2H), 3.69 (t, J ) 9.9 Hz, 2H), 3.62-
3.59 (m, 3H), 3.20-3.17 (m, 2H); 13C NMR (CD3OD) δ 165.7, 138.9,
137.7, 130.1-128.9, 75.5, 74.6, 74.5, 74.1, 73.9, 73.7, 72.6, 68.8, 57.2,
40.4, 38.5; MS (ESITOF) m/z (M + Na)+ 602; [R]27 ) -2.5 (c )
D
1.81, CH3OH).
N,N-(2-Aminoethyl)-D-glucoamidine 7b. Glucoamidine 6b (15 mg,
0.025 mmol) was hydrogenated in a manner similar to the preparation
9
J. AM. CHEM. SOC. VOL. 126, NO. 7, 2004 1977

A R T I C L E S
Heck et al.
calcd for C9H20N3O4 (M + H)+ 234.1454, found 234.1455; [R]27
+3.3 (c ) 1.57, MeOH).
N,N-(3-Aminopropyl)-D-mannoamidine 9c. Mannoamidine 8c (96
of 7a to give glucoamidine 7b (5.2 mg, 95% yield): 1H NMR (CD3-
OD) δ 4.22 (d, J ) 9.7 Hz, 1H), 3.91-3.61 (m, 6H), 3.50-3.47 (m,
1H), 3.29-3.25 (m, 2H); 13C NMR (CD3OD) δ 167.4, 74.0, 70.3, 69.1,
62.6, 61.0, 40.4, 38.5; HRMS calcd for C8H18N3O4 (M + H)+ 220.1297,
)
D
mg, 0.135 mmol) was hydrogenated in a manner similar to the
found 220.1294; [R]27 ) +3.9 (c ) 1.11, CH3OH).
1
preparation of 7a to give mannoamidine 9c (28.2 mg, 88% yield). H
D
NMR (CD3OD) δ 4.64 (d, J ) 3.0 Hz, 1H), 3.99 (t, J ) 2.9 Hz, 1H),
3.90-3.73 (m, 3H), 3.57-3.40 (m, 3H), 2.98 (t, J ) 7.8 Hz, 2H), 2.01-
1.92 (m, 2H); 13C NMR (CD3OD) δ 166.9, 73.7, 69.8, 67.3, 62.1, 61.0,
39.7, 38.0, 26.9; IR (neat, cm-1) 2980, 1681, 1434, 1061; HRMS calcd
N,N-(2-Aminoethyl)-D-mannoamidine 9b. Mannoamidine 8b (80
mg, 0.13 mmol) was hydrogenated in a manner similar to the
preparation of 7a to give deprotected mannoamidine 9b (27 mg, 89%
yield): 1H NMR (CD3OD) δ 4.71 (d, J ) 3.2 Hz, 1H), 4.05 (t, J ) 3.0
Hz, 1H), 3.94-3.91 (m, 2H), 3.89-3.74 (m, 3H), 3.46-3.42 (m, 1H),
3.26 (t, J ) 6.5 Hz, 2H); 13C NMR (CD3OD) δ 167.7, 73.8, 69.8, 67.5,
62.0, 61.2, 40.3, 38.7; HRMS calcd for C8H18N3O4 (M + H)+ 220.1297,
for C9H19N3O4 (M + H)+ 234.1454, found 234.1456. [R]26 ) +0.7
D
(c ) 1.34, CH3OH).
N,N-[4-(tert-Butoxycarbonylamino)butyl]-2,3,4,6-tetra-O-benzyl-
D-glucoamidine 4d and N,N-[4-(tert-Butoxycarbonylamino)butyl]-
2,3,4,6-tetra-O-benzyl-D-mannoamidine 5d. Glucoiminothioether 3
(200 mg, 0.34 mmol) was reacted with 4-(tert-butoxycarbonylamino)-
1-butylamine (116 mg, 0.62 mmol) to give glucoamidine 4d (53 mg,
22%) and mannoamidine 5d (160 mg, 66%) following the general
procedure. 4d: 1H NMR (CDCl3) δ 7.35-7.22 (m, 21H), 4.89 (d, J )
11.5 Hz, 1H), 4.82-4.36 (m, 9H), 3.96-3.78 (m, 4H), 3.53 (dd, J )
2.4, 10.6 Hz, 1H), 3.31 (t, J ) 6.7 Hz, 2H), 3.07-3.05 (m, 2H), 1.57-
1.46 (m, 4H), 1.43 (s, 9H); 13C NMR (CDCl3) δ 162.7, 156.7, 137.5,
137.2, 137.1, 136.1, 129.1, 127.9, 81.1, 77.6, 75.6, 74.7, 74.1, 73.5,
73.1, 69.2, 57.2, 42.1, 39.5, 28.5, 27.2, 24.5; IR (neat, cm-1) 2933,
1683, 1454, 1074; MS (ESITOF) m/z (M + H)+ 707; [R]26D ) -17.1
(c ) 1.3, CH2Cl2). 5d: 1H NMR (CDCl3) δ 7.62-7.51 (br s, 1H),
7.38-7.09 (m, 20H), 4.87-4.85 (br s, 1H), 4.74 (d, J ) 11.8 Hz, 1H),
4.64-4.27 (m, 8H), 3.91 (t, J ) 2.9 Hz, 1H), 3.76-3.67 (m, 4H), 3.44
(q, J ) 6.9 Hz, 2H), 3.09-3.05 (m, 2H), 1.69-1.64 (m, 2H), 1.56-
1.49 (m, 2H), 1.43 (s, 9H); 13C NMR (CDCl3) δ 162.35, 156.8, 137.8,
136.9, 135.9, 129.0-127.9, 79.5, 73.8, 73.5, 73.3, 72.6, 72.3, 71.8,
61.4, 55.9, 41.9, 39.4, 28.5, 27.2, 24.6; IR (neat, cm-1) 3302, 2933,
found 220.1298; [R]27 ) -4.7 (c ) 1.22, CH3OH).
D
N,N-[3-(tert-Butoxycarbonylamino)propyl]-2,3,4,6-tetra-O-ben-
zyl-D-glucoamidine 4c and N,N-[3-(tert-Butoxycarbonylamino)-
propyl]-2,3,4,6-tetra-O-benzyl-D-mannoamidine 5c. Glucoiminothio-
ether 3 (200 mg, 0.34 mmol) was reacted with 3-(tert-butoxycarbonyl-
amino)-1-propylamine (108 mg, 0.61 mmol) to give glucoamidine 4c
(62 mg, 26%) and mannoamidine 5c (157 mg, 66%) following the
general procedure. 4c: 1H NMR (CDCl3) δ 7.36-7.23 (m, 20H), 5.08-
4.95 (br s, 1H), 4.88 (d, J ) 11.2 Hz, 1H), 4.77 (s, 2H), 4.76-4.49
(m, 5H), 4.35 (d, J ) 11.2 Hz, 1H), 4.01-3.89 (m, 2H), 3.87 (dd, J )
6.1, 9.5 Hz, 1H), 3.83 (dd, J ) 2.7, 10.1 Hz, 1H), 3.52 (dd, J ) 2.8,
9.9 Hz, 1H), 3.42-3.38 (br s, 2H), 3.11-3.07 (m, 2H), 1.74-1.70 (m,
2H), 1.63-1.60 (br s, 1H), 1.44 (s, 9H); 13C NMR (CDCl3) δ 162.7,
157.8, 137.6, 137.4, 137.2, 136.2, 130.0-127.8, 81.6, 79.9, 75.5, 74.5,
74.3, 73.5, 72.6, 69.3, 57.5, 39.2, 36.9, 29.1, 28.5; IR (neat, cm-1
)
3297, 2933, 1682, 1519, 1455, 1074, 738, 699; MS (DCI/NH3) m/z
(M + H)+ 694; [R]26 ) -14.6 (c ) 1.44, CH2Cl2). 5c: 1H NMR
D
(CDCl3) δ 8.62-8.55 (br s, 1H), 7.43-7.12 (m, 20H), 5.34 (t, J ) 3.5
Hz, 1H), 5.67 (d, J ) 11.8 Hz, 1H), 4.77-4.30 (m, 8H), 3.98 (s, 1H),
3.85-3.70 (m, 4H), 3.61-3.43 (m, 2H), 3.18-3.07 (m, 2H), 1.61 (br
s, 2H), 1.47 (s, 9H); 13C NMR (CDCl3) δ 162.5, 157.8, 137.9, 137.1,
136.0, 128.9-127.9, 79.8, 73.6, 73.3, 73.4, 72.6, 71.8 68.3, 55.8, 39.2,
37.0, 29.0, 28.5; IR (neat, cm-1) 3295, 2977, 1695, 1517, 1453, 1367,
1014, 738, 700; MS (DCI/NH3) m/z (M + H)+ 694; [R]26D ) +1.1 (c
) 2.55, CH2Cl2).
N,N-(3-Aminopropyl)-2,3,4,6-tetra-O-benzyl-D-glucoamidine 6c.
From 4c (24 mg, 0.03 mmol), compound 6c was obtained (18 mg,
86% yield) following the same procedure reported for 6b from 4b. 1H
NMR (CD3OD) δ 7.37-7.21 (m, 20H), 4.85-4.34 (m, 10H), 4.01-
3.94 (m, 2H), 3.91-3.88 (m, 1H), 3.66 (dd, J ) 3.6, 10.3 Hz, 1H),
3.58 (dd, J ) 3.8, 10.2 Hz, 1H), 3.39 (t, J ) 6.7 Hz, 2H), 2.94 (t, J )
7.6 Hz, 2H), 1.96-1.91 (m, 2H); 13C NMR (CD3OD) δ 164.8, 138.9,
138.7, 137.8, 130.1-129.1, 81.5, 77.6, 75.5, 75.4, 74.2, 73.4, 73.7,
69.3, 57.9, 40.4, 38.0, 26.7; IR (NaCl, cm-1) 3261, 3032, 1678, 1455,
1074, 744, 699; MS (DCI/NH3) m/z (M + H)+ 594; [R]23D ) +0.9 (c
) 1.38, MeOH).
N,N-(3-Aminopropyl)-2,3,4,6-tetra-O-benzyl-D-mannoamidine 8c.
From 5c (146 mg, 0.21 mmol), mannoamidine 8c was obtained (115
mg, 92% yield) following the preparation reported for 6b from 4b. 1H
NMR (CD3OD) δ 7.38-7.09 (m, 20H), 4.81 (d, J ) 11.9 Hz, 1H),
4.64 (d, J ) 12.0 Hz, 2H), 4.50-4.21 (m, 6H), 3.89-3.81 (m, 2H),
3.66-3.64 (m, 1H), 3.61-3.54 (m, 2H), 3.51-3.46 (m, 2H), 2.93 (t,
J ) 7.1 Hz, 2H), 1.99-1.89 (m, 2H); 13C NMR (CD3OD) δ 164.9,
138.9, 138.7, 138.5, 137.8, 130.1-128, 96, 75.6, 74.4, 74.1, 73.7, 72.6,
68.8, 57.1, 39.9, 37.9, 26.8; IR (neat, cm-1) 3298, 2875, 2101, 1681,
1455, 1092; MS (DCI/NH3) m/z (M + H)+ 594.
N,N-(3-Aminopropyl)-D-glucoamidine 7c. Glucoamidine 6c (15
mg, 0.025 mmol) was hydrogenated in a manner similar to the
preparation of 7a to give glucoamidine 7c (5.3 mg, 89% yield): 1H
NMR (CD3OD) δ 4.20 (d, J ) 6.5 Hz, 1H), 3.88-3.77 (m, 3H), 3.66-
3.55 (m, 2H), 3.49-3.45 (m, 2H), 2.98 (t, J ) 10.2 Hz, 2H), 2.06-
1.95 (m, 2H); 13C NMR (CD3OD) δ 166.6, 74.2, 70.1, 69.3, 62.5, 61.2,
39.7, 38.1, 26.8; IR (neat, cm-1) 3330, 2528, 1681, 1434, 1061; HRMS
1683, 1454, 1074, 743, 700; MS (ESITOF) m/z (M)+ 707; [R]26
-8.9 (c ) 1.94, CH2Cl2).
)
D
N,N-(4-Aminobutyl)-2,3,4,6-tetra-O-benzyl-D-glucoamidine 6d.
From 4d (50 mg, 0.07 mmol) compound, 6d was obtained (36 mg,
83% yield) following the same procedure reported for the synthesis of
1
6b from 4b. H NMR (CD3OD) δ 7.37-7.22 (m, 20H), 4.95-4.35
(m, 12H), 4.00-3.94 (m, 2H), 3.89-3.87 (m, 2H), 3.66 (dd, J ) 3.6,
10.3 Hz, 1H), 3.57 (dd, J ) 3.8, 10.2 Hz, 1H), 2.88-2.84 (m, 2H),
1.65-1.62 (m, 2H); 13C NMR (CD3OD) δ 164.4, 138.9, 138.7, 137.9,
129.7-129.1, 81.5, 77.6, 75.6, 75.4, 74.2, 74.1, 73.3, 69.3, 57.7, 42.7,
40.1, 25.6, 25.5; IR (neat, cm-1) 3032, 1678, 1455, 1074. MS (DCI/
NH3) m/z (M + H)+ 608; [R]26 ) +1.9 (c ) 1.42, CH3OH).
D
N,N-(4-Aminobutyl)-2,3,4,6-tetra-O-benzyl-D-mannoamidine 8d.
From 5d (134 mg, 0.19 mmol) compound, 8d was obtained (101 mg,
88% yield) following the preparation reported for 6b from 4b. 1H NMR
(CD3OD) δ 7.39-7.12 (m, 20H), 4.82 (d, J ) 12.0 Hz, 1H), 4.68 (s,
1H), 4.64-4.62 (m, 3H), 4.52-4.25 (m, 6H), 3.88-3.85 (m, 2H), 3.68-
3.65 (m, 1H), 3.61-3.54 (m, 2H), 3.42-3.39 (m, 2H), 2.86-2.84 (m,
2H), 1.58-1.53 (m, 2H); 13C NMR (CD3OD) δ 164.6, 139.1, 138.8,
138.5, 137.7, 130.0-128.9, 75.7, 74.4, 74.1, 73.7, 72.7, 68.9, 57.1,
42.2, 40.2, 25.7, 25.6; IR (neat, cm-1) 2929, 1680, 1455, 1074, 1028;
MS (DCI/NH3) m/z (M + H)+ 608; [R]27 ) +5.2 (c ) 2.21, CH3-
D
OH).
N,N-(4-Aminobutyl)-D-glucoamidine 7d. Glucoamidine 6d (30 mg,
0.049 mmol) was hydrogenated in a manner similar to the preparation
of 7a to give glucoamidine 7d (11.1 mg, 91% yield): 1H NMR (CD3-
OD) δ 4.19 (d, J ) 9.7 Hz, 1H), 3.88-3.78 (m, 4H), 3.58 (t, J ) 9.3
Hz, 1H), 3.46-3.43 (m, 4H), 2.99-2.96 (m, 2H), 1.74-1.72 (m, 2H);
13C NMR (CD3OD) δ 166.2, 74.2, 70.1, 69.4, 62.3, 61.3, 42.3, 40.3,
25.7, 25.6; IR (neat, cm-1) 3326, 2970, 1681, 1455, 1035; HRMS calcd
for C10H22N3O4 (M + H)+ 248.1610, found 248.1610; [R]27 ) -0.8
D
(c ) 1.62, CH3OH).
N,N-(4-Aminobutyl)-D-mannoamidine 9d. Mannoamidine 8d (96
mg, 0.158 mmol) was hydrogenated in a manner similar to the
preparation of 7a to give mannoamidine 9d (28.7 mg, 89% yield): 1H
9
1978 J. AM. CHEM. SOC. VOL. 126, NO. 7, 2004

Cyclic Amidine-Sugars as Glycosidase Inhibitors
A R T I C L E S
NMR (CD3OD) δ 4.67 (d, J ) 3.2 Hz, 1H), 4.02 (t, J ) 3.1 Hz, 1H),
3.94-3.85 (m, 2H), 3.78 (dd, J ) 5.9, 11.3 Hz, 1H), 3.46-3.40 (m,
3H), 2.99-2.94 (m, 2H), 1.73-1.71 (m, 4H); 13C NMR (CD3OD) δ
166.5, 73.6, 69.7, 67.2, 62.4, 61.1, 42.0, 40.2, 25.7, 25.6; IR (neat,
cm-1) 2970, 1681, 1455, 1030; HRMS calcd for C10H22N3O4 (M +
3.52 (m, 2H), 3.42 (d, J ) 9.0, 9.4 Hz, 1H); 13C NMR (CDCl3) δ
199.4, 137.8, 137.5, 137.3, 128.5-127.7, 79.6, 78.3, 73.9, 73.6, 73.2,
72.9, 72.3, 70.5, 59.6; IR (neat cm-1) 3194, 2921, 2865, 1529, 1454,
1098, 739, 698; MS (DCI/NH3) m/z (M + H)+ 554, (M + NH4)+ 571.
Anal. Calcd for C34H35NO4S: C, 73.75; H, 6.37. Found: C, 73.75; H,
H)+ 248.1610, found 248.1616; [R]27 ) -2.1 (c ) 1.0, CH3OH).
6.35. [R]24 ) -31.3 (c ) 1.67, CH2Cl2).
D
D
2,3,4,6-Tetra-O-benzyl-D-gluco-1,5-diazabicyclo[4,4,0]decene 10
and 2,3,4,6-Tetra-O-benzyl-D-manno-1,5-diazabicyclo[4,4,0]decene
11. To a stirred solution of 3-bromopropylamine hydrobromide (68
mg, 0.31 mmol) and triethylamine (43 µL, 0.31 mmol) in CH2Cl2 (1.5
mL) at 0 °C was added dropwise a solution of glucoiminothioether 3
(100 mg, 0.17 mmol) in CH2Cl2 (5 mL). The mixture was stirred at
room temperature for 6 h. Powdered K2CO3 (24 mg 0.17 mmol) was
added at 0 °C, and the reaction was stirred at room temperature for 6
h. The solution was concentrated and submitted to column chroma-
tography (CH2Cl2/MeOH, 98:2) to yield glucoamidine 10 (10 mg, 10%
yield) and mannoamidine 11 (68.3 mg, 69% yield). 10: 1H NMR
(CDCl3) δ 7.36-7.15 (m, 20H), 4.86 (d, J ) 2.8 Hz, 1H), 4.70-4.34
(m, 8H), 3.86-3.82 (m, 2H), 3.70-3.58 (m, 5H), 3.44-3.22 (m, 2H),
2.31-2.15 (m, 2H); 13C NMR (CDCl3) δ 160.0, 137.0, 136.9, 136.2,
136.0, 129.0-127.8, 73.9, 73.8, 73.4, 73.2, 73.0, 72.6, 72.5, 72.2, 71.1,
67.9, 63.1, 45.5, 38.8, 18.3; IR (neat, cm-1) 3337, 1662, 1454, 1070,
748, 700; MS (ESITOF) m/z (M)+ 576; [R]24D ) -10 (c ) 1.8, CHCl3).
11: 1H NMR (CDCl3) δ 7.40-7.12 (m, 20H), 4.89-4.31 (m, 9H),
3.84 (dd, J ) 3.1, 4.3 Hz, 1H), 3.71-3.58 (m, 6H), 3.45-3.39 (m,
2H), 2.03-1.94 (m, 2H); 13C NMR (CDCl3) δ 160.5, 136.9-135.3,
129.1-128.0, 80.9, 75.7, 74.5, 74.1, 73.6, 73.4, 72.5, 67.3, 65.2, 45.5,
38.9, 18.5; IR (neat, cm-1) 2876, 1660, 1455, 1361, 1067, 745, 700;
2,3,4,6-Tetra-O-benzyl-D-mannoimino Thioether 16. To a solution
of 15 (125 mg, 0.25 mmol) in dry CH2Cl2 (7 mL) at 0 °C was added
Meerwein’s salt (52 mg, 0.275 mmol). The mixture was stirred at 0
°C for 1 h 30 min and was used directly in the next step. To be analyzed,
the crude was concentratred under vacuum to give 16 (125 mg, 95%
1
yield); H NMR (CDCl3) δ 7.4-7.07 (m, 20H), 4.76-4.27 (m, 10H),
4.10-4.04 (m, 2H), 3.92 (dd, J ) 2.7, 5.7 Hz, 1H), 3.82-3.77 (m,
1H), 3.33-3.17 (m, 2H), 1.42 (t, J ) 7.2 Hz, 3H); 13C NMR (CDCl3)
δ 192.7, 137.4, 136.5, 136.4, 128.7-127.8, 81.3, 78.6, 76.6, 74.0, 73.5,
73.2, 72.7, 71.6, 68.5, 60.9, 25.1, 11.5; IR (neat, cm-1) 3568, 2874,
1603, 1454, 1070, 1026, 749, 699, 600; MS (DCI/NH3) m/z (M + H)+
582.
Inhibition Analysis. R-Mannosidase (jack beans), â-mannosidase
(snail acetone powder), R-glucosidase (yeast type III), â-glucosidase
(almond), R-galactosidase (green coffee beans), â-galactosidase (As-
pergillus orizae), R-fucosidase (bovine kidney), and N-acetyl-â-
glucosaminidase (jack beans) were purchased from Sigma Chemical
Company and used without further purification. The substrates for the
glycosidases were the appropriate p-nitrophenylglycosides and were
purchased from Sigma Chemical Company. The 1 mL enzymatic assays
typical contained 0.05 units of enzyme. The assay buffer for all the
enzymatic reactions except for R-L-fucosidase was 50 mM N-[2-
hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES, pH 6.8).
An R-L-fucosidase assay was conducted in a 50 nM sodium acetate
buffer, pH 6.0. Enzyme activity was determined by monitoring the
production of the p-nitrophenylate anion at 400 nm on a Beckman DU70
spectrophotometer. Km values for all the substrates were determined
before Ki measurements. Substrate concentrations for the inhibition
studies were chosen to be in the neighborhood of the determined Km.
Ki values were determined from five inhibitor concentrations. Double
reciprocal analysis was used to establish that the inhibitors were
competitive. The precise Ki values were derived from nonlinear least-
squares fits of the data to the kinetic equation for competitive inhibition.
MS (ESITOF) m/z (M)+ 576; [R]24 ) +7.2 (c ) 0.4, CH3OH).
D
D-Gluco-1,5-diazabicyclo[4,4,0]decene 12. Glucoamidine 10 (10
mg, 0.017 mmol) was hydrogenated in a manner similar to the
preparation of 7a to give mannoamidine 12 (3.5 mg, 95% yield): 1H
NMR (CD3OD) δ 4.21 (d, J ) 9.8 Hz, 1H), 4.09-4.01 (m, 3H), 3.98-
3.79 (m, 4H), 2.15-2.00 (m, 2H), 0.91 (t, J ) 5.4 Hz, 2H); 13C NMR
(CD3OD) δ 163.2, 74.1, 70.1, 69.4, 62.3, 59.5, 46.7, 39.1, 19.4; IR
(neat, cm-1) 3343, 2894, 2484, 1658, 1323, 1061; HRMS calcd for
C9H17N2O4 (M + H)+ 217.1188, found 217.2000; [R]23D) -3.7 (c )
1.9, MeOH).
D-Manno-1,5-diazabicyclo[4,4,0]decene 13. Mannoamidine 11 (60
mg, 0.104 mmol) was hydrogenated in a manner similar to the
preparation of 7a to give mannoamidine 13 (20.2 mg, 90% yield): 1H
NMR (CD3OD) δ 4.66 (d, J ) 3.2 Hz, 1H), 3.99 (t, J ) 2.9 Hz, 1H),
3.92-3.71 (m, 3H), 3.58-3.45 (m, 3H), 2.15-2.09 (m, 2H), 0.89 (t,
J ) 6.4 Hz, 2H); 13C NMR (CD3OD) δ 163.4, 73.7, 69.5, 66.7, 61.1,
59.5, 46.8, 39.1, 19.7. IR (neat, cm-1) 3348, 2951, 2478, 1659, 1324,
1059; HRMS cald for C9H17N2O4 (M + H)+ 217.1188, found 217.1180;
Acknowledgment. This work is dedicated by M.-P.H. with
best wishes to Prof. A. Valleix on the occasion of his retirement,
and he is gratefully acknowledged for carrying out mass spectra
analyses. We thank Prof. B. Winchester (Institute of Child
Health, London) for human glycosidase inhibitions essays, Dr.
A.-M. Aubertin (Faculte´ de Me´decine, Strasbourg, France) for
biological studies, and the Laboratoires Fournier (Daix, France)
for fellowships and discussions.
[R]23 ) +0.95 (c ) 1.15, MeOH).
D
2,3,4,6-Tetra-O-benzyl-D-mannothiolactam 15. To a solution of
mannolactam 14 (144 mg, 0.26 mmol) in dry benzene (5 mL) was
added Lawesson’s reagent (65 mg, 0.16 mmol), and the resulting
solution was refluxed for 2 h. The mixture was concentrated under
vacuum. Flash chromatography of the crude product (hexanes/EtOAc
8:2) gave the thiolactam 15 (134 mg, 89% yield): 1H NMR (CDCl3)
δ 8.15 (br s, 1H), 7.48-7.18 (m, 20H), 5.06 (d, J ) 12 Hz, 1H), 4.85-
4.44 (m, 7H), 4.40 (d, J ) 2.4 Hz, 1H), 3.91-3.79 (m, 2H), 3.59-
Supporting Information Available: 1H NMR and 13C NMR
spectra for compounds 7a, 9a, 7b, 9b, 7c, 9c, 7d, 9d, 7e, 9e,
12, and 13. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA037822R
9
J. AM. CHEM. SOC. VOL. 126, NO. 7, 2004 1979