Fluorous iminoalditols: A new family of glycosidase inhibitors and pharmacological chaperones

Article abstract of DOI:10.1002/cbic.201000192

A collection of new reversible glycosidase inhibitors of the iminoalditol type featuring N-substituents containing perfluorinated regions has been prepared for evaluation of physicochemical, biochemical and diagnostic properties. The vast variety of feasi

Full text of DOI:10.1002/cbic.201000192

DOI: 10.1002/cbic.201000192
Fluorous Iminoalditols: A New Family of Glycosidase
Inhibitors and Pharmacological Chaperones
Georg Schitter,[a] Andreas J. Steiner,[a] Gerit Pototschnig,[a] Elisabeth Scheucher,[a]
Martin Thonhofer,[a] Chris A. Tarling,[b] Stephen G. Withers,[b] Katrin Fantur,[c]
Eduard Paschke,[c] Don J. Mahuran,[d] Brigitte A. Rigat,[d] Michael B. Tropak,[d]
Carina Illaszewicz,[a] Robert Saf,[e] Arnold E. Stꢀtz,*[a] and Tanja M. Wrodnigg[a]
A collection of new reversible glycosidase inhibitors of the imi-
noalditol type featuring N-substituents containing perfluorinat-
ed regions has been prepared for evaluation of physicochemi-
cal, biochemical and diagnostic properties. The vast variety of
feasible oligofluoro moieties allows for modular approaches to
customised structures according to the intended applications,
which are influenced by the fluorine content as well as the dis-
tance of the fluorous moiety from the ring nitrogen. The first
examples, in particular in the d-galacto series, exhibited excel-
lent inhibitory activities. A preliminary screen with two human
cell lines showed that, at subinhibitory concentrations, they
are powerful pharmacological chaperones enhancing the activ-
ities of the catalytically handicapped lysosomal d-galactosidase
mutants associated with GM1 gangliosidosis and Morquio B
disease.
Introduction
Organic products containing more than one fluorine atom, in
particular oligofluoroalkyl compounds with three or (prefera-
bly) more fluorine atoms have recently attracted pronounced
interest as so-called “fluorous” substances. Their unique prop-
erties can be exploited by applying “fluorous technologies”.
Perfluorocarbons such as perfluorinated butyltetrahydrofuran
were initially developed as respiratory gas carriers and blood
substitutes; this was most impressively exemplified by the
“submerged mouse” breathing a perfluoroalkyl solution of
oxygen.[1] The term “fluorous chemistry”, that is, the chemistry
of such oligofluoro compounds, was introduced in a seminal
paper by Horvath and Rabai[2] in 1994 in context with the bi-
phasic separation techniques employing a third phase, the “flu-
orous phase”, in addition to the common aqueous and organic
media usually utilised. Pioneered by groups such as Curran’s,[3]
fluorous chemistry has developed prolifically, and oligofluoro
substituents in reagents or substrates and starting materials
have been exploited for the easy and convenient separation of
intermediates or the removal of excess reagents and undesired
side-products from complex reaction mixtures in a vast range
of fluorous-compound-based isolation and purification tech-
niques.[4] Noteworthy, the noncovalent attachment of small
molecules to surfaces by fluorous interactions to form micro-
arrays for the discovery of histone deacetylase inhibitors was
recently reported by Schreiber and his group.[5] Such fluorous
microarrays for function-selective protein “fishing” have since
attracted considerable interest. For example, Spring and co-
workers attached fluorous-tagged biotin to a fluorous-surface-
based microarray and could demonstrate the biotin–avidine in-
teraction with Cy3- and Cy5-labelled avidine.[6] By the same
token, Pohl and her group have shown the interactions of
sugars that had been attached to a fluorous surface with con-
canavalin A (conA) exploiting fluorescein-tagged conA[7] and
have nicely extended the technology since then.[8] Trifluorome-
thionine was recently employed to produce a highly active flu-
orous DNA polymerase as a 19F NMR spectroscopic probe.[9]
Likewise, engineering protein properties with other fluorous
amino acids provided more stable analogues due to potent
packing effects.[10]
One of the truly fascinating fields of glycochemistry and bio-
chemistry are the iminosugar-based natural and synthetic gly-
cosidase inhibitors[11] resulting from the formal replacement of
the ring oxygen by a basic nitrogen atom, which is able to
[a] G. Schitter, A. J. Steiner, G. Pototschnig, E. Scheucher, M. Thonhofer,
C. Illaszewicz, Prof. A. E. Stꢀtz, Prof. T. M. Wrodnigg
Glycogroup, Deparment of Organic Chemistry
Graz University of Technology
Stremayrgasse 16, 8010 Graz (Austria)
Fax: (+43)316-873-8740
E-mail: Stuetz@tugraz.at
[b] C. A. Tarling, Prof. S. G. Withers
Department of Chemistry, University of British Columbia
2036 Main Mall, Vancouver, B.C., V6T 1Z1 (Canada)
[c] K. Fantur, Prof. E. Paschke
Department of Pediatrics, Medical University Graz
Auenbruggerplatz 30, 8010 Graz (Austria)
[d] Prof. D. J. Mahuran, B. A. Rigat, Prof. M. B. Tropak
Department of Laboratory Medicine and Pathobiology
Sick Kids Hospital, University of Toronto
555 University Avenue, Ontario M5G 1X8 (Canada)
[e] Prof. R. Saf
Institute for Chemistry and Technology of Materials
Graz University of Technology
Stremayrgasse 16, 8010 Graz (Austria)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000192.
2026
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Fluorous Iminoalditols
form powerful ionic bonds with carboxyl groups in the en-
zymes’ active sites. Due to their notable biological activities
and pharmacological properties of selected derivatives, imi-
noalditols such as compounds 1–3 have enjoyed great interest
For the exploratory studies described, due to our current
main interest in galactosidases, emphasis was laid on the d-
galacto series represented by compounds 5–8 and 12. As an
internal standard for probing the selectivity of these galactosi-
dase inhibitors, N-acetylhexosaminidase inhibitor 10 was pre-
pared. Glucosidase inhibitor 15 was chosen for comparison
with previously prepared unsubstituted or nonfluorous ana-
logues.
For easy access to the desired structures, a set of fluorous
reagents A–D was prepared by exploiting the unique proper-
ties of oligofluoro compounds,[16] in particular, the strong acidi-
ty (1,1,1,3,3,3-hexafluoropropan-2-ol, pKa =9.3) of fluorous alco-
hols.[17] For the synthesis of compound 5, fluorous hexyl ethers
6 and 8, as well as acetal 7, reductive amination of easily avail-
able, partially protected l-lyxo-hexos-5-ulose 4 was the key
step. Reaction of compound 4 with the amine generated from
fluorous azide A (available by reaction of the commercial iodo
compound with sodium azide under phase-transfer condi-
tions[16]) under hydrogenation conditions gave desired imino-
galactitol derivative 5 (Scheme 1).
over the past three decades. Surprisingly, in the light of the
above, fluorous iminoalditols featuring alkyl groups containing
more than three fluorine atoms have not been investigated
systematically as biological probes for glycosidases to date.
We are interested in investigating the opportunities coming
with this new class of “fluorous iminoalditols” as glycosidase
probes. Not only could the “fluorous properties” as mentioned
in the introduction, be interesting for analytical and likely also
diagnostic purposes, but the oligofluoro moieties would also
provide extreme hydrophobicity and possibly lead to improved
activities or selectivities as compared to the already quite
potent “simple” lipophilic N-alkyl derivatives internationally in-
vestigated and exploited thus far.[12] Indeed, lipophilic glycosi-
dase inhibitors have recently been used as pharmacological
chaperones to enhance lysosomal enzyme activities that are
deficient in one of the more than 40 different lysosomal stor-
age disorders.[13] In many of these diseases, mutant lysosomal
enzymes that cannot obtain and/or retain their functional con-
formation are recognised as misfolded by the quality control
machinery in the endoplasmatic reticulum (ER) and are eventu-
ally targeted for degradation; this results in decreased intracel-
lular activity of the enzyme. Some inhibitors can act as phar-
macological chaperones in cells and bind to and stabilise the
functional conformation of the mutant enzymes, enabling their
exit from the ER (their site of synthesis, folding and assembly)
and subsequent transport to the lysosome.[14] Although suc-
cessful, only a limited number of b-galactosidase inhibitors
have been shown to enhance the activity of mutant lysosomal
b-galactosidase in fibroblast cell lines from patients with the
lysosomal storage disorders GM1 gangliosidosis and Mor-
quio B.[15] Here we show that some of the “fluorous iminoaldi-
tols” have the potential to act as efficient pharmacological
chaperones.
Scheme 1. Synthesis of iminogalactitol derivative 5. a) H2, Pd/C, MeOH;
b) H3O+.
Fluorous amines B–D were made available by Mitsunobu re-
action of the commercial N-benzyloxycarbonyl-6-aminohexanol
with the respective oligofluoro alcohols providing the corre-
sponding ethers in good yields (Scheme 2). Interestingly, in the
Scheme 2. Synthesis of fluorous amines B–D.
case of 1,1,1,3,3,3-hexafluoropropan-2-ol, under the reaction
conditions employed, the initially formed corresponding ether
reacted with another molecule of the fluorous alcohol to give
stable acetal C, which bears four trifluoromethyl moieties.
Reaction of ulosose 4 with B, C and D, respectively, provided
galactosidase inhibitors 6–8 in fair yields (Scheme 3).
Analogously, treatment of GlcNAc-derived ulososide 9[18]
with fluorous amine C gave N-acetylhexosaminidase inhibitor
10 (Scheme 4).
Results and Discussion
Syntheses and structural variability
To acquire an initial picture on the viability of the new class of
fluorous iminoalditols, a set of quite different structures (5–8,
10, 12, 15) was selected to cover a range of configurations,
polarities, steric demands, as well as linker moieties such as
ethers, amides and carbamates.
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A. E. Stꢀtz et al.
inhibitors devoid of the fluorous substructures under consider-
ation. N-Acetyl-b-d-hexosaminidase inhibitor 10 was screened
with human lysosomal hexosaminidase A and exhibited the
same IC50 of 6 mm as the N-unsubstituted parent compound, 2-
acetamino-1,2,5-trideoxy-1,5-imino-d-glucitol.[21] Glucosidase in-
hibitor 15 turned out slightly better (by a factor of two) than
its parent 13 (Table 1).
Gratifyingly, in the d-galacto series all new compounds
except 5 exhibited pronouncedly improved activities with the
b-galactosidases probed and exhibited Ki values of
one up to two orders of magnitude smaller than
parent compound 3. Inhibitors 5–8 showed good sol-
ubility and suitable activities for the intended pur-
pose and were screened with human lysosomal a-
and b-galactosidases from placenta as well as human
lysosomal b-galactosidase from skin fibroblasts. Inhib-
itory activities with b-galactosidase were better than
or ranging around the value of parent compound 3
with very good selectivities when compared to the
values with the corresponding a-specific enzyme
(Table 1). Compounds 6–8 were thus selected for preliminary
pharmacological chaperoning experiments.
Scheme 3. Synthesis of galactosidase inhibitors 6–8. a) H2, Pd/C, MeOH;
b) H3O+.
Scheme 4. Synthesis of N-acetylhexosaminidase inhibitor 10. a) H2, Pd/C, MeOH.
The reaction of N-(6-Amino)hexyl-1-deoxy-d-galactonojirimy-
cin[19] (11) with commercial reagent E yielded the correspond-
ing fluorous urethane 12
(Scheme 5).
In the l-ido series, compound
13[20] with methyl 6-oxocaproate
gave intermediate 14 under
standard hydrogenation condi-
tions. Saponification of the ester
14 followed by reaction of the
free carboxylate 14a with freshly
prepared nonafluorohexyl amine
A furnished fluorous amide 15
(Scheme 6).
Table 1. Ki values [mm] of compounds.
Compounds
Enzymes[a]
ABG
E. c.
GCB
HLbG[b]
HLbG[c]
HLaG[c]
HexA
3
5
6
7
100
450
0.7
12
2.1
n.d.
5
13
3.5
0.013
3.2
0.36
2.9
1.4
n.d.
11
0.6
15
5.1
0.02
16
28
15
33
n.d.
n.d.
n.d.
n.d.
n.i.
n.i.
n.i.
n.i.
n.i.
6
n.i.
n.d
n.d.
0.36
0.10
0.39
0.12
n.i.
n.i.
n.d.
n.d.
0.37
0.39
1.1
n.d.
0.60
n.d.
n.d.
0.8
3.5
0.8
n.i.
n.i.
n.d.
n.d.
8
10
12
13
15
26
11
n.d.
n.d.
Final products 5–8, 10, 12 and
15 were screened by employing
two standard d-galactosidases
(b-galactosidase from E. coli and
[a] ABG=b-glucosidase/b-galactosidase Agrobacterium sp. (pH 7); E.c.=b-galactosidase E. coli (pH 7); GCB=a-
galactosidase green coffee beans (pH 6.5); HLbG=human lysosomal b-galactosidase (pH 4.5); HLaG=human
lysosomal a-galactosidase (pH 4.5). For N-acetylhexosaminidase A (HexA), IC50 is given. [b] From skin fibroblasts;
[c] From placenta (Km =0.2 mm).[28] n.d.=not determined, n.i.=no inhibition.
a-galactosidase
from
green
coffee beans) as well as the b-
glucosidase/b-galactosidase from
Agrobacterium sp. and compared with the structural parent
compounds 3 (for 5–12) and 13 (for compound 15). All com-
pounds were nicely soluble under the respective conditions
employed and did not show significant differences to related
Previously, Tominaga and co-workers[15a] showed that com-
pound 3 acted as a pharmacological chaperone in GM1 gan-
gliosidosis patient fibroblasts, increasing the enzyme activity of
mutant b-galactosidase bearing mutations I51T, R201H or
Scheme 5. Synthesis of fluorous urethane 12. c) NEt3, DMF.
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Fluorous Iminoalditols
Scheme 6. Synthesis of fluorous amide 15. a) H2, Pd/C, MeOH; d) NaOH; e) HBTU, NEt3, DMF.
Conclusions
R457Q, by 2.2, 2.6 and 6.1-fold, respectively. It is important to
bear in mind that a maximal response was achieved only after
using 500 mm of compound 3. Inhibitors 6 and 8 were also
evaluated as potential pharmacological chaperones in GM1 pa-
tient fibroblasts heterozygous for the mutation R148S/D332N
in b-galactosidase. Although both compounds exhibited similar
strength of binding to human b-galactosidase, only compound
8 significantly increased enzyme activity (>1.6-fold) relative to
DMSO-treated cells. The differences in enzyme enhancement
efficacy might be due to differences in intracellular bioavaila-
bility and/or metabolism, as has been postulated for some b-
glucocerebrosidase pharmacological chaperones.[14c] Whereas 3
gave maximal enzyme enhancement response in patient cells
at a concentration of 500 mm, compound 8 gave a maximal re-
sponse at a more than tenfold-lower concentration of 30 mm
(Figure 1).
From this first set of structural variations and configurations it
may be expected that fluorous iminoalditol derivatives will be
powerful glycosidase inhibitors with a variety of options as
provided by the nature and the lengths of the spacer arms
and by the number and distribution of fluorine atoms in the
fluorous substituents, both of which are expected to alter and
possibly improve the interactions with lipophilic pockets adja-
cent to the enzymes’ active sites.[12c,d,f] Interesting selectivities
such as observed in the d-galacto series (HLbG/HLaG) might
also be found with other configurations. Thus, some of these
derivatives might offer worthwhile chemical and biological
properties for further exploitation.
Experimental Section
General methods: Optical rotations were measured on a Perkin–
Elmer 341 polarimeter at the wavelength of 589 nm and a path
length of 10 cm at 208C. NMR spectra were recorded on a Varian
INOVA 500 operating at 599.82 MHz (1 H), and at 125.894 MHz (13C)
or on a Bruker Ultrashield spectrometer at 300.36 and 75.53 MHz,
respectively. CDCl3 was employed for protected compounds and
[D4]MeOH or D2O for unprotected inhibitors. Chemical shifts are
listed in ppm by employing residual, nondeuterated solvent as the
internal standard. The signals of the protecting groups were found
in the expected regions and are not listed explicitly. Mass spectra
were recorded on an Agilent Systems Quadrupole LC–MS in the
positive mode. Electron impact (EI, 70 eV) HRMS spectra were re-
corded on Waters GCT Premier equipped with direct insertion (DI).
MALDI-TOF mass spectrometry was performed on a Micromass Tof-
Spec 2E time-of-flight mass spectrometer. Analytical TLC was per-
formed on precoated aluminium plates silica gel 60 F254 (E. Merck
5554), detected with UV light (254 nm), 10% vanillin/sulfuric acid
as well as ceric ammonium molybdate (100 g ammonium molyb-
date/8 g ceric sulfate in 1 L 10% H2SO4) and heated on a hotplate.
Preparative TLC was performed on precoated glass plates silica
gel 60 F254, 0.5 mm (E. Merck 5744). For column chromatography
silica gel 60 (230–400 mesh, E. Merck 9385) was used.
Figure 1. Compound 6 increases b-Gal activity in a GM1 Gangliosidosis pa-
tient fibroblast. b-Gal activity (hydrolysis of 4-methylumbelliferyl b-d-galacto-
pyranoside fluorogenic substrate) in lysates from 6 or 8; treated cells were
expressed relative to DMSO-treated cells. Cells were grown for five days in
the presence of increasing concentration of 6 or 8. Error bars correspond to
an average reading error of 7.4%.
Furthermore, compounds 6–8 were screened in a juvenile
GM1 gangliosidosis mutation cell line (p.R201C), which is
known to be chaperone-sensitive.[15a,22] For this particular cell
line, compounds 6–8 exhibited very similar efficacy between 5
and 50 mm. A fivefold increase of residual b-galactosidase activ-
ity representing 35% of normal cells was obtained at 5 and
20 mm with compound 6. Compound 7 led to a fourfold in-
crease (ca. 40% of normal activity) at 20 and 50 mm. With in-
hibitor 8, the observed increase at 20 mm was also fourfold
(37% of normal cells). Compound 3 did not exhibit any appre-
ciable chaperone activity across the entire concentration range
screened but acted as an inhibitor.
Kinetic studies
Agrobacterium sp. b-galactosidase/-glucosidase: was purified and
assayed as described.[23] Kinetic studies were performed at 378C in
pH 7.0 sodium phosphate buffer (50 mm) containing 0.1% bovine
serum albumin by using 7.2ꢂ10À5 mgmLÀ1 enzyme. Approximate
values of Ki were determined by using a fixed concentration of
substrate, 4-nitrophenyl b-d-glucopyranoside (0.11 mm=1.5Km)
and inhibitor concentrations ranging from 0.2 to 5-times the Ki
value that was ultimately determined. A horizontal line drawn
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A. E. Stꢀtz et al.
through 1/Vmax in a Dixon plot of this data (1/V vs [I]) intersects the
experimental line at an inhibitor concentration equal to ÀKi. Full Ki
determinations when required, were performed by using the same
range of inhibitor concentrations while also varying substrate (4-ni-
trophenyl glucoside) concentrations from approximately 0.015 mm
to 0.6 mm. Data were analysed by direct fit to the Michaelis–
Menten equation describing reaction in the presence of inhibitors
by using the program GraFit.
d-galactopyranoside (2.5 mm) as the substrate (Km =0.2 mm). Reac-
tions were performed at 378C in pH 4.5 citrate phosphate buffer
(100 mm) as described previously.[25] IC50 values (mm) were extract-
ed from the enzyme activity curves in presence of increasing con-
centrations of the inhibitor using nonlinear regression as imple-
mented within Graphpad Prism 5.
IC50 of hexosaminidase inhibitor 10 was determined with purified
human placental N-acetyl-b-d-hexosaminidase A (hexA; final
0.3 mgmLÀ1) and methylumbelliferyl N-acetyl-b-d-glucosaminide
(final 1.6 mm) in citrate phosphate buffer (100 mm) at pH 4.5 in the
presence of 0.025% human serum albumin at 378C for 30 min.
E. coli b-galactosidase: (Sigma) kinetic studies were performed at
378C in pH 7.0 sodium phosphate buffer (50 mm), using 1 nm
enzyme. Approximate values of Ki were determined by using a
fixed concentration of substrate, 2-nitrophenyl b-d-galactopyrano-
side (0.15 mm=1.5Km) and inhibitor concentrations ranging from
0.2 times to five times the Ki value ultimately determined. A hori-
zontal line drawn through 1/Vmax in a Dixon plot of this data (1/V
vs [I]) intersects the experimental line at an inhibitor concentration
equal to ÀKi.
Intracellular b-galactosidase activity: Infantile GM1 gangliosidosis
(R148S/D332N) fibroblasts[26] were treated with escalating doses of
compounds 6 and 8 (dissolved in DMSO) for 5 d. Intracellular b-gal-
actosidase activity was measured by using the fluorogenic sub-
strate 4-methylumbelliferyl b-d-galactopyranoside as previously de-
scribed.[25]
Green coffee bean a-galactosidase: (Sigma) kinetic studies were
performed at 378C in pH 6.5 sodium phosphate buffer (50 mm) by
using 2 nm enzyme. Approximate values of Ki were determined by
using a fixed concentration of substrate, 4-nitrophenyl b-d-galacto-
pyranoside (0.7 mm=1.5Km) and inhibitor concentrations ranging
from 0.2 to 5-times the Ki value that was ultimately determined. A
horizontal line drawn through 1/Vmax in a Dixon plot of this data
(1/V vs [I]) intersects the experimental line at an inhibitor concen-
tration equal to ÀKi.
Juvenile GM1 gangliosidosis (p.R201C) human skin fibroblasts were
grown until semiconfluency in 6-well plates. Compounds were
added at concentrations from 0–500 mm and incubated for four
more days at 378C. Intracellular b-galactosidase activity was deter-
mined as described.[22]
General procedure for intramolecular reductive amination: One
equivalent of azide A or the respective N-benzyloxycarbonyl com-
ponent B, C or D was added to a 0.03m solution of 4 (or 9) in
MeOH/H2O (15:1, v/v), Pd(OH)2/C (20%, 0.1 equiv) and the hetero-
geneous reaction mixture was stirred under an atmosphere of H2
at ambient pressure and RT until TLC indicated completed conver-
sion of the starting material. After filtration and removal of the sol-
vent under reduced pressure, the residue was dissolved in MeOH/
H2O 1:1 (v/v) and the pH value was adjusted to pH 1 with aq conc
HCl to remove the isopropylidene group. The solution was concen-
trated under reduced pressure and the remaining residue was sub-
jected to fluorous-phase extraction and subsequently purified by
column chromatography on silica gel (CHCl3/MeOH/concd NH4OH,
500:100:6; v/v/v), yielding the target compound as a colourless
syrup. The formation of l-altro-configured products from d-galac-
tose derived ulosose 4 and l-ido configuration from ulososide 9,
respectively, was not observed.
Human ß-galactosidase: Human skin fibroblasts were grown in
minimal essential medium (MEM) with Earle’s Salts (PAA, Pasching,
Austria) containing 10% foetal bovine serum, 400 mm l-glutamine
and 50 mgmLÀ1 gentamicin at 378C and 5% CO2. All cells used in
this study were between the third and nineteenth passages. Cells
were harvested by trypsinisation or scraping as described earlier
and cell homogenates were prepared in Eppendorf tubes in a
0.9% NaCl solution containing 0.01% Triton. Modified b-gal assays
were used to estimate the IC50 values of putative b-gal inhibitors.
For triplicate assays, confluent fibroblast cells from healthy patients
(3ꢂ75 mL flasks) were harvested by trypsinisation, resuspended in
0.9% NaCl (1.5 mL) containing 0.01% Triton, sonicated and centri-
fuged. For inhibition assays, cell homogenate (20 mL; 40 mL total
protein) was mixed with prewarmed (90 mL; 378C) b-gal substrate
solution and inhibitor solutions (10 mL) in final concentrations rang-
ing from 0–100 mm. Substrate blanks contained 0.9% NaCl (20 mL)
instead of cell homogenate, whereas enzyme blanks were made
with substrate buffer without b-gal substrate. The samples were in-
cubated for 30 min at 378C in a water bath, and the reaction was
stopped by addition of stop buffer (2.5 mL). The amount of hydro-
lysed 4-methylumbelliferone was determined with a Luminescence
Spectrometer (LS50B, Perkin–Elmer) with an excitation wavelength
of 360 nm and emission at 450 nm. Data analysis was performed
with Microcal Origin v6.0 by using the IC50 module based on
sigmoid curve fitting. For enzyme kinetic experiments cells from
healthy controls were prepared as described for IC50 determination.
To determine Ki values, fixed concentrations of each inhibitor were
used while varying the b-gal substrate concentration from 100–
450 mm. Data analysis was performed with Microcal Origin v6.0 by
using a nonlinear curve-fitting module based on the Michaelis–
Menten equation for competitive inhibitors.
General procedure for Mitsunobu reactions on N-benzyloxycar-
bonyl-6-aminohexanol; Reagents B, C, and D: Triphenylphos-
phane (3.15 g, 12 mmol, 3 equiv) was added to a 2% solution of
commercial N-benzyloxycarbonyl-6-aminohexanol (1.0 g, 4.0 mmol)
in dry THF. The mixture was cooled to 08C and diethylazodicarbox-
ylate (DEAD, 1.84 mL, 12 mmol, 3 equiv) was added dropwise. The
reaction mixture was brought to RT, and the respective fluorous al-
cohol (3 equiv) was added. When TLC indicated completed conver-
sion of the starting material, the solvent was removed under re-
duced pressure and the crude residue was partly purified by fluo-
rous solid-phase extraction by employing MeOH/H2O for loading
and washing followed by THF as the eluent. Subsequent purifica-
tion on silica gel provided compounds B, C, and D in yields of
1
91%, 82%, and 80%, respectively. B: H NMR (CDCl3): d=5.19 (d,
J=11.2 Hz, 2H), 4.74 (brs; NH), 3.98 (t, J=5.9 Hz, 2H), 3.18 (dd, J=
13.2 Hz, J=6.9 Hz, 2H), 1.67 (m, 2H), 1.51 (m, 2H), 1.45–1.30 ppm
(m, 4H); 13C NMR (CDCl3): d=156.6, 136.9, 130.3, 128.8 (2C), 128.6,
128.2, 122.7 (q, JC,F =291 Hz), 82.8 (hep, JC,F =28.1 Hz), 66.7, 66.4,
41.1, 30.0, 29.8, 26.6, 25.5 ppm; MS: m/z calcd for C23H25NO3F6Na:
500.1636 [M+Na]+, found: 500.1612.
The inhibitory activity of compounds 3 and 5–8 against human ly-
sosomal a- as well as b-galactosidase was also evaluated by using
a lysosomal-enzyme-enriched conA fraction from human placenta
as the enzyme source.[24] For the b-galactosidase, 4-nitrophenyl b-
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Fluorous Iminoalditols
1
1
C: H NMR (CDCl3): d=5.12 (s, 2H), 4.92 (q, JH,CF3 5.2 Hz, 1H), 4.47
foam. [a]2D0 =À15.2 (c=0.5 in MeOH); H NMR ([D4]MeOH): d=3.98
(brs, 1H; NH), 4.02 (t, J=6.2 Hz, 2H), 3.19 (q, J=6.7 Hz, 2H), 1.68
(m, 2H), 1.52 (m, 2H), 1.44–1.30 ppm (m, 4H); 13C NMR (CDCl3): d=
124.2–116.6 (m, JC,F =293 Hz, 4C; CF3), 95.2 (p, JC,F =32.8 Hz), 69.7,
69.4 (m, JC,F =34.6 Hz; CHCF3), 66.7, 40.9, 29.9, 29.4, 26.3, 25.1 ppm;
MS: m/z calcd for C20H21NO4F12Na: 590.1177 [M+Na]+, found:
590.1153.
(m, 1H; H4), 3.84–2.76 (m, 3H; H2, H6a, H6b), 3.57 (m, 2H; H6’a,
H6’b), 3.21 (dd, J2,3 =9.7 Hz, J3,4 =3.3 Hz, 1H; H3), 2.98 (dd, J1eq,2
=
4.7 Hz, J1eq,1ax =11.2 Hz, 1H; H1eq), 2.68 (m, 1H; H1’ax), 2.51 (m,
1H; H1’b), 2.37 (m, 1H; H5), 2.12 (dd, J1ax,2 =10.8 Hz, 1H; H1eq),
1.73 (m, 2H; H5’), 1.58–1.49 (m, 2H; H-2’), 1.49–1.42 (m, 2H), 1.36–
1.27 ppm (m, 2H); 13C NMR ([D4]MeOH): d=131.6, 129.9, 129.3,
129.2, 124.0 (q, 2 C, JC,F =290 Hz, C2’’), 84.1 (m, JC,F =29 Hz, C1’’),
77.3 (C3), 72.3 (C4), 69.0 (C2), 67.6, 65.2, 62.4 (C5, C6, C6’), 58.1,
54.0 (C1, C1’), 30.7, 28.3, 26.8, 24.9 ppm (C2’, C3’, C4’, C5’); MS: m/z
calcd for C21H29NO5F6H: 490.4674 [M+H]+, found: 490.50.
1
D: H NMR (CDCl3): d=5.19 (d, J=11.2 Hz, 2H), 4.76 (brs, 1H; NH),
3.55 (t, J=6.8 Hz, 2H), 3.18 (m, 2H), 1.70 (m, 2H), 1.53 (m, 2H),
1.43 (m, 2H), 1.35 (m, 2H); 13C NMR (CDCl3): d=120.7 (q, JC,F
=
293 Hz, 3C), 80.0 (m, JC,F =29.6 Hz), 69.9, 66.9, 41.2, 30.1, 29.8, 26.5,
25.3; MS: m/z calcd for C18H20NO3F9Na: 492.1197 [M+Na]+, found:
492.1172.
N-[(1,1,1,3,3,3-Hexafluoropropyl-2-oxy)-1,1,1,3,3,3-hexafluoro-
propyl-2-oxy]hexyl-1,5-dideoxy-1,5-imino-d-galactitol (7): By fol-
lowing the general procedure for Mitsunobu reactions, the reaction
of ulosose 4 (415 mg, 1.76 mmol) and reagent C gave fluorous
inhibitor 7 (306 mg, 30%) as a white foam. [a]2D0 =À11.5 (c=0.8 in
MeOH); 1H NMR ([D4]MeOH): d=5.76 (m, J1’’F =5.3 Hz, 1H; H1’’),
4.09 (m, 2H; H6’), 3.98 (m, 1H; H4), 3.79 (m, 3H; H2, H6a, H6b),
3,4-O-Isopropylidene-l-arabino-hexos-5-ulose (4): A 20% solution
of
1,2:3,4-di-O-isopropylidene-5-deoxy-hex-5-eno-l-arabino-pyra-
nose[27] (10.0 g, 41.3 mmol) in CH2Cl2 was added to a 10% solution
of 3-chloroperbenzoic acid (13.8 g, 61.6 mmol, 77%) in CH2Cl2, and
the reaction was stirred at RT for 4 h. After completed conversion
of the starting material, the mixture was cooled to À188C, and a
white precipitate formed, which was removed by filtration. The fil-
trate was washed consecutively with sat. aq NaHCO3 and H2O until
the pH value of the aqueous layer was neutral. The organic phase
was separated, dried (Na2SO4) filtered and concentrated under re-
duced pressure. The crude residue was taken up in dry MeOH and
the solution was cooled to À308C. NaOMe (1m in MeOH) was
added carefully to adjust the pH value to 10. After completed con-
version of the intermediate, the reaction mixture was neutralised
with acidic ion exchange resin Amberlite IR 120 [H+]. Following
removal of the resin by filtration, the solution was concentrated
under reduced pressure. Chromatography of the residue provided
the title compound (mixture of several pyranoid and furanoid tau-
tomers, 6.54 g, 67%) as off-white foam, which was immediately
used in the next step.
3.20 (m, J2,3 =9.3 Hz, 1H; H3), 2.98 (dd, J1eq,2 =2.8 Hz, J1ax,1eq
=
10.3 Hz, 1H; H1eq), 2.72 (m, 1H; H1’a), 2.37 (m, 1H; H1’b), 2.11 (dd,
J
1ax,2 =10.9 Hz, 1H; H1ax), 1.80–1.69 (m, 2H), 1.60–1.48 (m, 2H),
1.48–1.40 (m, 2H), 1.37–1.25 ppm (m, 2H); 13C NMR ([D4]MeOH):
d=125.7–117.9 (m, JC,F =286 Hz, 2C), 120.2 (m, JC,F =293 Hz, 2C),
96.5 (m, JC,F =32.7 Hz), 77.3 (C3), 72.2 (C4), 71.2 (C2), 70.2 (m, JC,F
=
34.1 Hz), 69.0 (C5), 65.2 (C6’), 62.4 (C6), 58.0 (C1’), 53.9 (C1), 30.3,
28.1, 26.3, 25.0 ppm (C2’, C3’, C4’, C5’); MS: m/z calcd for
C18H25NO6F12H: 580.3918 [M+H]+, found: 580.3956.
N-(Nonafluoro-tert-butyloxy)hexyl-1,5-dideoxy-1,5-imino-d-ga-
lactitol (8): By following the general procedure for Mitsunobu reac-
tions, the reaction of ulosose 4 (100 mg, 0.42 mmol) with reagent
D gave fluorous inhibitor 8 (158 mg, 78%) as a white foam. [a]D20
=
À14.7 (c=1.8 in MeOH); 1H NMR ([D4]MeOH): d=4.08 (dd, J=
5.3 Hz, J=5.9 Hz, 2H; H6’), 3.98 (m, 1H; H4), 3.80 (m, 3H; H2, H6a,
H6b), 3.21 (dd, J2,3 =9.3 Hz, J3,4 =2.8 Hz, 1H; H3), 2.98 (dd, J1ax,1eq
=
N-(3,3,4,4,5,5,6,6,6-Nonafluoro)hexyl-1,5-dideoxy-1,5-imino-d-ga-
11.2 Hz, J1eq,2 4.4 Hz, 1H; H1eq), 2.72 (m, 1H; H1’a), 2.51 (m, 1H;
H1’b), 2.40 (m, 1H; H5), 2.11 (dd, J1ax,2 10.1 Hz, 1H; H1ax), 1.70 (m,
2H; H2’), 1.51 (m, 2H; H5’), 1.43 (m, 2H), 1.32 ppm (m, 2H);
13C NMR ([D4]MeOH): d=121.9 (m, 3 C, JC,F =299 Hz, 3 C2’’), 81.1
(m, JC,F 29.5 Hz, C1’’), 77.2 (C3), 72.2 (C4), 71.5 (C2), 69.0 (C6’), 65.2
(C5), 62.4 (C6), 58.0 (C1), 53.9 (C1’), 30.8, 28.1, 26.4, 24.9 ppm (C2’,
C3’, C4’, C5’); MS: m/z calcd for C16H24NO5F9H: 482.1589 [M+H]+,
found: 482.1638.
lactitol (5): 3,3,4,4,5,5,6,6,6-Nonafluorohexyl azide
A (290 mg,
1.00 mmol) and Pd/C (10%, 50 mg) were added a to a 1% metha-
nolic solution of 3,4-O-isopropylidene-l-arabino-hexos-5-ulose (4,
406 mg, 1.72 mmol), and the mixture was stirred under an atmos-
phere of H2 at ambient pressure for 36 h. The catalyst was re-
moved by filtration, and the filtrate was concentrated under re-
duced pressure. The crude residue was taken up in a mixture of
1:1 MeOH/H2O (15 mL), and CHCl3 (6 mL) was added to provide a
clear solution. The pH value was adjusted to 1 by addition of conc.
HCl. After completed removal of the isopropylidene group as indi-
cated by TLC, the reaction mixture was concentrated under re-
duced pressure. Chromatography (CHCl3/MeOH/NH4OH 600:100:7
v/v/v) gave free inhibitor 5 (163 mg, 23% from 4) as a white
powder. [a]2D0 =À6.9 (c=1.9 in MeOH); 1H NMR ([D4]MeOH): d=
3.93 (m, 1H; H4), 3.81 (m, 3H; H2, H6a, H6b), 3.24 (dd, J2,3 =8.8 Hz,
N-[(1,1,1,3,3,3-Hexafluoropropyl-2-oxy)-1,1,1,3,3,3-hexafluoro-
propyl-2-oxy]hexyl-2-acetamino-1,2,5-trideoxy-1,5-imino-d-gluci-
tol (10): H2O (1 mL), Pd/C (10%, 50 mg) and compound
C
(0.750 mg, 7.87 mmol) were added to a 2% MeOH solution of ulo-
soside 9[18] (530 mg, 1.55 mmol), and the mixture was stirred under
an atmosphere of H2 at ambient pressure for 24 h, when TLC indi-
cated completed conversion of the starting material 9. The catalyst
was removed by filtration, and the filtrate was concentrated under
reduced pressure. Chromatography (CHCl3/MeOH/NH4OH 80:10:1,
v/v/v) furnished 10 (376 mg, 39%) as a colourless syrup. [a]2D0 =7.1
(c=1.4 in H2O); 1H NMR ([D4]MeOH): d=5.81 (hept, J1’’,F =5.3 Hz,
J
3,4 =2.5 Hz, 1H; H3), 3.10 (m, 1H; H1’a), 3.03 (m, 1H; H1’b), 2.92
(dd, J1ax,1eq =11.3 Hz, J1eq,2 =2.7 Hz, 1H; H1eq), 2.60–2.28 (m, JC,F
17.3 Hz, 2H; H2’), 2.18 ppm (dd, J1ax,2 =10.1 Hz, 1H; H1ax); 13C NMR
([D4]MeOH): d=122.5–107.4 (m, 4 C, C3’, C4’, C5’, C6’), 77.0 (C3),
72.6 (C4), 68.9 (C2), 64.8 (C5), 63.2 (C6), 57.8 (C1), 45.2 (t, JC,F =4 Hz,
H1’’), 4.09 (t, J5’,6’ =5.9 Hz, 2H; H6’), 3.88 (dd, J5,6a =1.5 Hz, J6a,6b
11.5 Hz, 1H; H6a), 3.84–3.77 (m, 2H; H2, H6b), 3.38 (dd, J3,4
9.2 Hz, 1H; H4), 3.20 (dd, J2,3 =10.0 Hz, 1H; H3), 3.00 (dd, J1ax,1eq
=
=
=
C1’), 26.9 ppm (t,
JC,F =21.4 Hz, C2’); MS m/z calcd for
C12H16N2O4F9H: 410.1014 [M+H]+, found: 410.1026, 432.0850
[M+Na]+.
11.3 Hz, J1eq,2 =4.2 Hz, 1H; H1eq), 2.80 (m, 1H; H1’a), 2.55 (m, 1H;
H1’b), 2.13 (m, 1H; H5), 2.08 (dd, J1ax,2 =11.0 Hz, 1H; H1ax), 1.95 (s,
3H; NAc), 1.78–1.70 (m, 2H), 1.53–1.39 (m, 4H), 1.36–1.26 ppm (m,
N-(a,a-Di-trifluoromethyl)benzyloxyhexyl-1,5-dideoxy-1,5-imino-
d-galactitol (6): By following the general procedure for the Mitsu-
nobu reactions, the reaction of ulosose 4 (100 mg, 0.42 mmol) and
reagent B gave fluorous inhibitor 6 (147 mg, 71%) as a white
2H); 13C NMR ([D4]MeOH): d=173.6 (NHAc), 125.8–117.9 (m, JC,F
=
293 Hz, 4CF3), 96.5 (m, JC,F =32.9 Hz), 77.7 (C3), 72.8 (C4), 71.2 (C6’),
70.2 (m, JC,F =34.1 Hz), 67.5 (C5), 59.8 (C6), 55.6, 53.4 , 51.9 (C1, C1’,
ChemBioChem 2010, 11, 2026 – 2033
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
2031

A. E. Stꢀtz et al.
C2), 30.3 (C5’), 28.1, 26.3, 25.5 (C2’, C3’, C4’), 22.7 ppm (NHAc); MS:
m/z calcd for C20H28N2O6F12Na: 643.1653 [M+Na]+, found:
643.1652.
H6b), 3.91–3.84 (m, 1H; H4), 3.78 (dd, J1ax,2 =4.5 Hz, 1H; H2), 3.53
(t, 2H; H1’’), 3.46 (m, 1H; H5), 3.38 (m, 1H; H1ax), 3.28–3.18 (m,
3H; H1eq, H1’), 2.54–2.34 (m, 2H; H2’’), 2.26 (t, 2H; H5’), 1.88–1.63
(m, 4H; H2’, H4’), 1.49–1.36 (m, 2H; H3’); 13C NMR ([D4]MeOH): d=
176.1 (CONH), 122.5–107.6 (m, 4C, C4F9), 72.2 (C3), 70.7 (C2), 69.6
(C4), 63.9 (C5), 59.6 (C6), 54.9 (C1), 53.7 (C1’), 36.6 (C5’), 32.6 (t,
N-[4-(3,3,4,4,5,5,6,6,6-Nonafluorohexyl)benzyloxycarbonyl-ami-
no]hexyl-1,5-dideoxy-1,5-imino-d-galactitol (12): Commercially
available 2,5-dioxopyrrolidin-1-yl 4-(3,3,4,4,5,5,6,6,6-nonafluorohex-
yl)benzyl carbonate E (204 mg, 0.42 mmol) was added to a solution
of N-(6-amino)hexyl-1-deoxy-d-galactonojirimycin 11[19] (108 mg,
0.41 mmol) and Et3N (86 mg, 0.82 mmol) in dry DMF (15 mL), and
the reaction mixture was stirred at RT for 90 min. After completed
conversion, MeOH (5 mL) was added, and the solvents were re-
moved under reduced pressure. Chromatography of the residue
J
C,F =4.9 Hz, C1’’), 31.2 (t, JC,F =21.1 Hz, C2’’), 27.3, 26.3 (3 C, C2’, C3’
C4’); MS: m/z calcd for C18H27N2O5F9H: 523.1854 [M+H]+, found:
523.1802.
Acknowledgements
1
provided 12 (128 mg, 49%). [a]2D0 =4.5 (c=0.7 in MeOH); H NMR
Financial support by the Austrian Ministry of Science and Re-
search (Project GZ 200.156/2-VI/1a/2006), the Austrian Research
Society for Mucopolysaccharidoses and Related Diseases as well
as by the Canadian Institutes of Health Research (CIHR Team
Grant CTP-82944) is gratefully acknowledged.
([D4]MeOH): d=7.40–7.26 (m, 4H), 5.08 (s, 2H; CH2Ph), 4.03 (dd,
J
3,4 =3.0 Hz, J4,5 =1.9 Hz, 1H; H4), 3.86 (ddd, J1ax,2 =10.5 Hz, J1eq,2 =
4.7 Hz, J2,3 =9.2 Hz, 1H; H2), 3.86–3.81 (m, 2H; H6a, H6b), 3.27 (dd,
1H; H3), 3.14 (t, J=6.9 Hz, 2H; H6’), 3.05 (dd, J1ax,1eq =11.2 Hz, 1H;
H1eq), 3.00–2.92 (m, 2H), 2.78 (m, 1H; H1’a), 2.59 (m, 1H; H1’b),
2.54–2.38 (m, 3H; H5, 2 H2’’), 2.21 (dd, 1H; H1ax), 1.65–1.47 (m,
4H), 1.46–1.26 ppm (m, 4H); 13C NMR ([D4]MeOH): d=158.9, 140.3,
136.9, 129.5, 129.3, 122.1–108.0 (m, C4F9), 77.1 (C3), 72.1 (C4), 68.8
(C2), 67.0 (CH2Ph), 65.4 (C5), 62.2 (C6), 57.9 (C1), 54.0 (C1’), 41.7
Keywords: chaperones · enzymes · fluorine · glycosidases ·
inhibitors
(C6’), 33.6 (t, JC,F =22 Hz, C2’’), 30.9, 28.2, 27.7, 27.0 ppm (t, JC,F
5 Hz, C1’’), 25.0; MS: m/z calcd for C26H35N2O6F9H: 643.5722
[M+H]+, found: 643.5703.
=
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N-Methoxycarbonylpentyl-1,5-dideoxy-1,5-imino-l-iditol
(14):
Adipic acid hemialdehyde methylester (1.1 mL, 7.9 mmol) was
added to a 10% methanolic solution of 1,5-dideoxy-1,5-imino-l-
iditol[20] 13 (1.26 g, 7.22 mmol), and the mixture was stirred with
Pd/C (10%, 260 mg) under an atmosphere of H2 at ambient pres-
sure until TLC indicated completed conversion of the starting imi-
nosugar. The catalyst was removed by filtration, the filtrate was
concentrated under reduced pressure, and the remaining residue
was chromatographed to give 14 as a colourless syrup (1.10 g,
1
52%). [a]2D0 =À0.1 (c=6.5 in MeOH); H NMR ([D4]MeOH): d=3.86–
3.79 (m, 2H; H6a, H6b), 3.71–3.69 (m, 1H; H4), 3.65 (s, 3H; OCH3),
3.58–3.50 (m, 1H; H2), 3.39 (dd, J2,3 =J3,4 =8.3 Hz, 1H; H3), 3.05 (m,
1H; H5), 2.82–2.74 (m, 2H; H1eq, H1’a), 2.68–2.63 (m, 1H; H1’b),
2.59 (dd, J1ax,1eq =11.2 Hz, J1ax,2 =10.8 Hz, 1H; H1ax), 2.33 (t, 2H;
H5’), 1.63, 1.53, 1.33 ppm (3 m, 2H each, H2’, H3’, H4’); 13C NMR
([D4]MeOH): d=174.8 (C6’), 74.6 (C3), 71.5, 70.0 (C2, C4), 63.2 (C5),
56.5 (C6), 54.2 (C1), 51.7 (C1’), 51.0 (OMe), 35.6 (C5’), 27.0, 26.6,
24.7 ppm (C2’, C3’, C4’); MS: m/z calcd for C13H25NO6H: 292.3553
[M+H]+, found: 292.3600 [M+H]+.
N-Carboxypentyl-1,5-dideoxy-1,5-imino-l-iditol (14a) and N-
[(3,3,4,4,5,5,6,6,6-Nonafluoro)hexylaminocarbonyl]pentyl-1,5-di-
deoxy-1,5-imino-l-iditol (15): NaOH (0.5m, 2.0 mL) was added to
an ice-cold 1% solution of 14 (237 mg, 0.81 mmol) in dioxane/H2O
(1:1, v/v), and the mixture was stirred until TLC (CHCl3/MeOH/
NH4OH, 500:100:6) indicated completed saponification. The mix-
ture was brought to pH 6 by addition of ion-exchange resin IR 120
[H+]. After removal of the resin by filtration, the filtrate was con-
centrated under reduced pressure to give carboxylic acid 14a,
which was immediately used in the next step. Et3N (120 mL,
0.9 mmol), and HBTU (320 mg, 0.9 mmol) were added to a 1%
DMF solution of crude 14a (230 mg, 0.83 mmol), compound A
(3 mmol, 3.3 equiv), and the reaction mixture was stirred at RT
until TLC indicated quantitative consumption of the iminoalditol.
The solvent was removed under reduced pressure and the remain-
ing residue was chromatographed (CHCl3/MeOH/NH4OH 700:100:8)
to provide 15 (200 mg, 47%) as a colourless syrup. [a]2D0 =4.4 (c=
1
1.1 in MeOH); H NMR ([D4]MeOH): d=4.01–3.92 (m, 3H; H3, H6a,
2032
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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 2026 – 2033

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Received: March 25, 2010
Published online on August 16, 2010
ChemBioChem 2010, 11, 2026 – 2033
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