N-alkylated derivatives of 1,5-dideoxy-1,5-iminoxylitol as β-xylosidase and β-glucosidase inhibitors

Article abstract of DOI:10.1007/s007060200028

A range of lipophilic derivatives of the D-xylosidase inhibitor 1,5-dideoxy-l,5-iminoxylitol was synthesized by N-alkylation of the parent compound with different alkyl halides. Inhibitory activities of the products obtained were measured with β-glucosida

Full text of DOI:10.1007/s007060200028

Monatshefte fuÈr Chemie 133, 555±560 ꢀ2002)
N-Alkylated Derivatives of 1,5-Dideoxy-
1,5-iminoxylitol as b-Xylosidase
and b-Glucosidase Inhibitorsa
1
2
1;Ã


Herwig Hausler , Karen Rupitz , Arnold E. Stutz
,
and Stephen G. Withers2;Ã
1


Glycogroup, Institut fur Organische Chemie der Technischen Universitat Graz,
A-8010 Graz, Austria
2
Department of Chemistry, University of British Columbia, Vancouver, B.C.,
V6T 1Z1 Canada
Summary. A range of lipophilic derivatives of the D-xylosidase inhibitor 1,5-dideoxy-1,5-imino-
xylitol was synthesized by N-alkylation of the parent compound with different alkyl halides. Inhibi-
tory activities of the products obtained were measured with ꢀ-glucosidase from Agrobacterium sp.,
a family 1 enzyme, as well as with ꢀ-xylosidase from Thermoanaerobacterium saccharolyticum
belonging to family 39 of the glycohydrolases. Whereas the former enzyme, which has low
ꢀ-xylosidase activity, was only moderately inhibited, with Ki values in the millimolar range, good
inhibition was observed with the latter one. Inhibitors with terminally functionalized N-alkyl chains
open up opportunities for novel applications as af®nity ligands as well as for various types of tagging
for diagnostic purposes.
Keywords. 1,5-Dideoxy-1,5-iminoxylitol; ꢀ-Glucosidase; ꢀ-Xylosidase; Glycosidase inhibitor.
Introduction
D-Xylans are polysaccharides consisting of ꢀ-1,4-linked D-xylopyranosyl moieties
and can be found in the cell walls of all land plants and most plant parts [1].
Their biodegradation relies on ꢀ-1,4-xylanases ꢀEC 3.2.1.8) and ꢀ-xylosidases
ꢀEC 3.2.1.37). Xylanases are endo-enzymes attacking internal glycosidic linkages,
whereas xylosidases release xylose by attack of the terminal glycosidic bond at
the non-reducing end of the oligomer. These enzymes are produced by bio-
degradative microorganisms such as fungi and bacteria that are important for
carbon turnover in nature. For example, 35% of birch wood ꢀdry weight) consists
of xylans. In pulp and paper technology, these enzymes are employed in a pre-
bleach treatment to remove residual xylans, thereby reducing the need for
expensive and environmentally questionable chemicals in the process. The food
a
Dedicated to Prof. Gunter Legler on the occasion of his 75th birthday
Corresponding authors. E-mail: stuetz@orgc.tu-graz.ac.at, withers@chem.ubc.ca
Ã


H. Hausler et al.
556
and feed industry has also been identi®ed as a large potential area for application
of xylanases.
Low molecular weight inhibitors of xylanases and xylosidases are not only
useful tools for enzyme characterization and puri®cation but might also serve
other, more `applied' purposes. For example, powerful and inexpensive inhibitors
could be employed in crop protection against xylan degrading plant pathogens or
as components in wood preservation coatings and paints. Iminosugars including
iminoalditols have been found to be powerful inhibitors of many glycosidases with
Ki values in the micromolar and, in quite a few cases, even nanomolar range [2].
Some of these compounds, due to their interference with biochemical pathways,
exert interesting biological activities [2]. Many have become valuable diagnostic
tools for studies of enzyme active sites and catalytic mechanisms. Despite the
abundance of xylosidases and xylanases and the fact that they play important roles
in the degradation of hemicelluloses, corresponding inhibitors such as 1,5-dideoxy-
1,5-iminoxylitol ꢀ1) have not yet gained as much attention as the related hexo-
pyranoside mimetics.
1,5-Dideoxy-1,5-iminoxylitol ꢀ1) is the paradigmatic example of a reversible,
xyloside mimicking inhibitor. Its ®rst synthesis has been reported by Paulsen in the
mid-nineteen sixties ꢀits precursor, 5-amino-5-deoxyxylose, was independently
reported by both Paulsen and Hanessian) [3]. Subsequently, this compound was
synthesized by several other groups [4]. Examples of its biochemical applications
have remained rare and largely con®ned to studies of ꢀ-glucosidases, despite the
fact that this compound could be a useful probe for xylanase=ꢀ-xylosidase research.
Based on recent ®ndings with related glycosidase inhibitors, in particular deriv-
atives of the powerful ꢀ-glucosidase inhibitor 2,5-dideoxy-2,5-imino-D-mannitol,
which indicated that lipophilic chain extensions can improve the inhibitory activity
of such derivatives [5], we prepared a small exploratory set of simple N-alkylated
derivatives of compound 1 featuring simple hydrocarbon substituents as well as
terminally functionalized chains suitable for tagging or immobilization studies.
Results and Discussion
Parent compound 1 was readily accessible from D-xylose as mentioned previously
according to Scheme 1. Controlled conversion of D-xylose gave the corresponding
furanoid 1,2-O-isopropylidene protected derivative 2 which was regioselectively
O-tosylated at the primary position in dichloromethane=pyridine employing the
standard procedure. Conventional reaction of 1,2-O-isopropylidene-5-O-tosyl-ꢁ-
D-xylofuranose ꢀ3) with sodium azide in N,N-dimethylformamide furnished the
corresponding 5-azidodeoxysugar 4 [6]. Deprotection employing acidic ion
‡
exchange resin Amberlite IR 120 [H ] gave the free sugar 5 which was cyclized
by conventional catalytic hydrogenation and concomitant intramolecular reductive
amination to yield the desired key intermediate 1.
Its reaction with bromohexane, 3-phenyl-1-bromopropane, methyl 6-bromo-
hexanoate, and 6-bromohexanoic nitrile led smoothly to the corresponding
N-alkyl derivatives 6±9. From 8, the 6-aminohexyl substituted compound 10
was formed by catalytic hydrogenation over palladium hydroxide on charcoal.
Compounds 8±10 can be modi®ed at their terminal functional groups e.g. by

ꢀ-Xylosidase and ꢀ-Glucosidase Inhibitors
557
Scheme 1
Scheme 2
Table 1. Inhibition constants ꢀKi values, ꢂM) for substituted iminoxylitols with ꢀ-glucosidase
from Agrobacterium sp. at pH 7.0 as well as with ꢀ-xylosidase from Thermoanaerobacterium
saccharolyticum at pH 5.5
1
6
7
8
9
10
ꢀ-glucosidase
ꢀ-xylosidase
50
2600
88
1600
135
3600
25
2000
35
2500
118
206
attachment onto a polymer support for af®nity chromatography or by other types of
transformations.
Preliminary screening of inhibitory activities were conducted with ꢀ-gluco-
sidase from Agrobacterium sp. as well as with ꢀ-xylosidase from Thermoanaero-
bacterium saccharolyticum ꢀTable 1). All N-alkylated compounds were found to be
moderate inhibitors of the ꢀ-glucosidase employed with activities lower than that
of parent compound 1.
Conversely, with the ꢀ-xylosidase employed in this study, N-alkylation
improved activities by a factor of 2 to 3 ꢀcompounds 6, 7, and 10) as compared


H. Hausler et al.
558
to the parent compound. Gratifyingly, in the case of nitrile 8, an increase of
inhibitory potency of nearly one order of magnitude was observed. These pre-
liminary results suggest that ꢀ-xylosidase inhibitors of the iminoxylitol type with
interesting properties should become available by ®ne-tuning the nature as well as
the length of the N-alkyl substituent.
Experimental

Melting points were recorded on a Tottoli apparatus ꢀBuchi) and are uncorrected. Optical rotations
were measured on a JASCO Digital Polarimeter or with a Perkin Elmer 341 instrument with a path
length of 10 cm. NMR spectra were recorded at 200 ꢀ1H) and at 50.29 MHz ꢀ13C) on a Varian Gemini
spectrometer. Chemical shifts are given in ꢃ relative to the solvent resonances. The signals of the
protecting groups were found in the expected regions and are not listed explicitly. Thin-layer
chromatography was performed on precoated aluminum sheets ꢀMerck 5554). Plates were stained
with a mixture of 10% ammonium molybdate ꢀw=v) in 10% H2SO4 containing 0.8% CeꢀSO4)2
ꢀw=v). For column chromatography, Silica Gel 60 ꢀE. Merck) was used employing CHCl3:MeOH:
concd. aq. ammonia ˆ 100:100:1 as the solvent system. Elemental analyses agreed favourably with
the calculated values.
General method for N-alkylation reactions employing bromoalkyl reagents
To a 5% solution of compound 1 in MeOH, 3 equivalents of the respective bromoalkane and 3
equivalents of pyridine were added, and the mixture was kept at ambient temperature for 48 h.
Removal of solvents under reduced pressure followed by chromatography of the residue furnished
compounds 6±8.
N-Hexyl-1,5-dideoxy-1,5-iminoxylitol ꢀ6; C11H23NO3)
Following the general procedure, 170 mg 1 ꢀ1.28 mmol) were reacted with 530 mm3 1-bromohexane
ꢀ3.78 mmol). Compound 6 ꢀ215 mg) was obtained in 78% yield.
M.p.: 79±82ꢀC; 1H NMR ꢀCD3OD, ꢃ, 200 MHz): 3.78 ꢀ2H, m, H-2, H-4), 3.46 ꢀ1H, broad, H-3),
3.33 ꢀ2H, m, H-1a, H-5a), 2.98 ꢀ2H, m, H-1e, H-5e), 2.80 ꢀ2H, broad, H-10, H-100), 1.71 ꢀ2H, m, H-20,
H-200), 1.37 ꢀ6H, m, H-30, H-300, H-40, H-400, H-50, H-500), 0.92 ꢀ3H, t, H-60, H-600, H-6000) ppm; the
poor resolution of signals is due to inherent conformational ¯exibility of the molecule; 13C NMR
ꢀCD3OD, ꢃ, 50 MHz): 68.1 ꢀC-2, C-3, C-4), 57.4 ꢀC-1, C-5), 55.4 ꢀC-10), 31.3, 26.4, 24.5, 22.4, 13.1
ꢀC-20, C-30, C-40, C-50, C-60) ppm.
N-%3-Phenyl)-propyl-1,5-dideoxy-1,5-iminoxylitol ꢀ7; C14H21NO3)
Employing the general procedure, 142 mg 1 ꢀ107 mmol) and 48 mm3 3-phenyl-1-bromopropane
ꢀ3.15 mmol) furnished 160 mg of 7 ꢀ59%) as a wax.
1H NMR ꢀCD3OD, ꢃ, 200 MHz): 3.67 ꢀ2H, ddd, J1a,2 ˆ J4,5a ˆ 7.5 Hz, J2,3 ˆ J3,4 ˆ 8.4 Hz,
J1e,2 ˆ J4,5e ˆ 4.4 Hz, H-2, H-4), 3.15 ꢀ1H, H-3), 3.01 ꢀ2H, dd, J1a,1e ˆ J5a,5e ˆ 11.4 Hz, H-1a, H-5a),
2.70±2.12 ꢀ6H, m, H-1b, H-5b, H-10, H-100, H-30, H-300), 1.76 ꢀ2H, m, H-20, H-200) ppm; 13C NMR
ꢀCD3OD, ꢃ, 50 MHz): 145.1, 132.3, 132.2, 129.9 ꢀphenyl), 79.8 ꢀC-3), 72.9 ꢀC-2, C-4), 60.8 ꢀC-1,
C-5), 60.4 ꢀC-10), 36.7, 31.1 ꢀC-20, C-30) ppm.
N-%5-Cyano)-pentyl-1,5-dideoxy-1,5-iminoxylitol ꢀ8; C11H20N2O3)
Iminoalditol 1 ꢀ170 mg, 1.28 mmol) was reacted with 250 mm3 6-bromohexanoic nitrile ꢀ1.92 mmol)
to give 134 mg ꢀ46%) 8.

ꢀ-Xylosidase and ꢀ-Glucosidase Inhibitors
559
1
M.p.: 103±107ꢀC; H NMR ꢀCD3OD, ꢃ, 200 MHz): 3.55 ꢀ2H, ddd, J1a,2 ˆ J4,5a ˆ J2,3 ˆ J3,4
ˆ
9.2 Hz, J1e,2 ˆ J4,5e ˆ 4.4 Hz, H-2, H-4), 3.18 ꢀ1H, H-3), 3.06 ꢀ2H, dd, J1a,1e ˆ J5a,5e ˆ 11.0 Hz, H-1a,
H-5a), 2.52 ꢀ4H, m, H-10, H-100, H-50, H-500), 2.14 ꢀ2H, m, H-1b, H-5b), 1.82±1.38 ꢀ6H, m, H-20,
H-200, H-30, H-300, H-40, H-400) ppm; 13C NMR ꢀCD3OD, ꢃ, 50 MHz): 119.7 ꢀCN), 79.0 ꢀC-3), 69.7
ꢀC-2, C-4), 57.6 ꢀC-1, C-5), 57.2 ꢀC-10), 26.2, 25.3, 25.1, 16.1 ꢀC-20, C-30, C-40, C-50) ppm.
N-Methoxycarbonylpentyl-1,5-dideoxy-1,5-iminoxylitol ꢀ9; C12H23NO5)
To a 5% solution of 240 mg of the starting material 1 ꢀ1.80 mmol), 294 mm3 adipic acid methyl ester
hemialdehyde ꢀ2.7 equivalents) and 120 mg PdꢀOH)2=C ꢀ20%) were added, and the mixture was
stirred under H2 for 5 h. After removal of the catalyst, the ®ltrate was concentrated under reduced
pressure. Chromatography of the residue gave 394 mg 9 ꢀ89%).
1
M.p.: 118±119ꢀC; H NMR ꢀCD3OD, ꢃ, 200 MHz): 3.66 ꢀ3H, s, OMe), 3.49 ꢀ2H, ddd, J1a,2
ˆ
J4,5a ˆ 10.5 Hz, J1e,2 ˆ J4,5e ˆ 4.8 Hz, J2,3 ˆ J3,4 ˆ 9.2 Hz, H-2, H-4), 3.09 ꢀ1H, H-3), 3.00 ꢀ2H, dd,
J1a,1b ˆ J5a,5b ˆ 11.0 Hz, H-1a, H-5a), 2.37 ꢀ4H, m, H-10, H-100, H-50, H-500), 1.92 ꢀ2H, H-1b, H5b),
1.76±1.24 ꢀ6H, m, H-20, H-200, H-30, H-300, H-40, H-400) ppm; 13C NMR ꢀCD3OD, ꢃ, 50 MHz): 174.7
ꢀC-60), 79.2 ꢀC-3), 70.2 ꢀC-2, C-4), 58.3 ꢀC-1, C-5), 57.6 ꢀC-10), 50.8 ꢀOCH3), 33.5 ꢀC-50), 26.8, 26.2,
24.7 ꢀC-20, C-30, C-40) ppm.
N-%6-Amino)-hexyl-1,5-dideoxy-1,5-iminoxylitol ꢀ10; C11H24N2O3)
To a 5% solution of 112 mg 8 ꢀ0.49 mmol), 105 mg PdꢀOH)2=C ꢀ20%) were added, and the mixture
was stirred under H2 for 5 days. After removal of the catalyst, the ®ltrate was concentrated under
reduced pressure. Chromatography of the residue gave 52 mg of syrupy 10 ꢀ46%).
1H NMR ꢀD2O, ꢃ, 200 MHz): 3.45 ꢀ2H, ddd, J1e,2 ˆ J4,5e ˆ 4.8 Hz, J2,3 ˆ J3,4 ˆ 10.1 Hz, H-2,
H-4), 3.15 ꢀ1H, H-3), 3.05 ꢀ2H, dd, J1a,1b ˆ J5a,5b ˆ 11.0 Hz, H-1a, H-5a), 2.81 ꢀ2H, t, J ˆ 7.3 Hz,
H-10, H-100), 2.49 ꢀ2H, m, H-60, H-600), 2.13 ꢀ2H, dd, H-1b, H-5b), 1.60±1.04 ꢀ8H, m, H-20, H-200,
H-30, H-300, H-40, H-400, H-50, H-500) ppm; 13C NMR ꢀD2O, ꢃ, 50 MHz): 77.4 ꢀC-3), 68.8 ꢀC-2, C-4),
57.0 ꢀC-1, C-5), 56.1 ꢀC-10), 39.5 ꢀC-60), 26.7, 26.0, 25.4, 24.8 ꢀC-20±C-50) ppm.
Inhibition constants ꢀKi values, ꢂM) for substituted iminoxylitols with Agrobacterium sp.
ꢀ-glucosidase ꢀ8.5 Â 10À5 mg Á cmÀ3) were determined at 37ꢀC in pH 7.0 Na3PO4 buffer ꢀ45 mM)
containing 0.1% bovine serum albumin using a ®xed concentration of substrate, 4-nitrophenyl-
ꢀ-D-glucopyranoside ꢀ0.11 mM ˆ 1.5  Km). Inhibition constants ꢀKi values, ꢂM) for substituted
iminoxylitols with Thermoanaerobacterium saccharolyticum ꢀ-xylosidase ꢀ0.10 mg Á cmÀ3) were
determined at 37ꢀC in pH 5.5 sodium citrate ± phosphate buffer containing 0.01% bovine serum
albumin using a ®xed concentration of substrate, phenyl-ꢀ-D-xylopyranoside ꢀ1.7 mM ˆ 1.0  Km).
Acknowledgements
Financial support by the Austrian Fonds zur Forderung der Wissenschaftlichen Forschung ꢀFWF),
Vienna, Project P 13593 CHE, and by the Protein Engineering Network of Centers of Excellence of
Canada are appreciated.
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H. Hausler et al.: ꢀ-Xylosidase and ꢀ-Glucosidase Inhibitors
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Received October 25, 2001. Accepted November 19, 2001