N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols: A new family of activity promotors for a GM1-gangliosidosis related human lysosomal β-galactosidase mutant

Article abstract of DOI:10.1016/j.carres.2017.03.009

From 1,2;3,4-di-O-isopropylidene-α-D-galactopyranose, a series of highly functionalized (hydroxymethyl)cyclopentanes was easily available. In line with reports by Reymond and J?ger on similar structures, these amine containing basic carbasugars are potent inhibitors of β-D-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for G_M1-gangliosidosis-associated lysosomal acid β-galactosidase mutant R201C, thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.

Full text of DOI:10.1016/j.carres.2017.03.009

Accepted Manuscript
N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols: A new family of activity
promotors for a G -gangliosidosis related human lysosomal β-galactosidase mutant
M1
Michael Schalli, Christina Tysoe, Roland Fischer, Bettina M. Pabst, Martin Thonhofer,
Eduard Paschke, Tanja Rappitsch, Arnold E. Stütz, Marion Tschernutter, Werner
Windischhofer, Stephen G. Withers
PII:
S0008-6215(17)30089-7
10.1016/j.carres.2017.03.009
CAR 7338
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 2 February 2017
Revised Date: 2 March 2017
Accepted Date: 10 March 2017
Please cite this article as: M. Schalli, C. Tysoe, R. Fischer, B.M. Pabst, M. Thonhofer, E. Paschke,
T. Rappitsch, A.E. Stütz, M. Tschernutter, W. Windischhofer, S.G. Withers, N-Substituted 5-amino-1-
hydroxymethyl-cyclopentanetriols: A new family of activity promotors for a G -gangliosidosis
M1
related human lysosomal β-galactosidase mutant, Carbohydrate Research (2017), doi: 10.1016/
j.carres.2017.03.009.
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ACCEPTED MANUSCRIPT

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Correspondence to
Arnold E. Stütz
Glycogroup, Institut für Organische Chemie
Technische Universität Graz
Stremayrgasse 9
A-8010 Graz
Austria
Phone 0043 316 873 32430
Fax: 0043 316 873 32402
email:stuetz@tugraz.at
N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols: A new
family of activity promotors for a GM1-gangliosidosis related
human lysosomal β-galactosidase mutant
Michael Schallia, Christina Tysoeb, Roland Fischerc, Bettina M. Pabstd, Martin Thonhofera,
Eduard Paschked, Tanja Rappitscha, Arnold E. Stütza,*, Marion Tschernutterd,
Werner Windischhoferd, Stephen G. Withersb
aGlycogroup, Institute of Organic Chemistry, Graz University of Technology,
Stremayrgasse 9, A-8010 Graz, Austria
bChemistry Department, University of British Columbia,
2036 Main Mall, Vancouver, BC, Canada V6T 1Z1
cInstitute of Inorganic Chemistry Graz University of Technology,
Stremayrgasse 9, A-8010 Graz, Austria
dLaboratory of Metabolic Diseases, Department of Pediatrics, MedUni Graz,
Auenbruggerplatz 30, A-8036 Graz,Austria

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ABSTRACT
From 1,2;3,4-di-O-isopropylidene-α-
(hydroxymethyl)cyclopentanes was easily available. In line with reports by Reymond and Jäger
on similar structures, these amine containing basic carbasugars are potent inhibitors of β-
D
-galactopyranose, a series of highly functionalized
D
-
galactosidases and, for the first time, could be shown to act as pharmacological chaperones for
GM1-gangliosidosis-associated lysosomal acid β-galactosidase mutant R201C, thus representing
a new structural type of pharmacological chaperones for this lysosomal storage disease.
Keywords:
Aminocyclopentane, Carbafuranose, Galactosidase Inhibitor, Pharmacological
Chaperone, GM1-Gangliosidosis.
1. Introduction
Due to the vital roles of glycoside hydrolases, specific inhibitors may exert notable biological
activities against various pathological processes, including certain forms of hereditary lysosomal
disorders.1,2,3,4 This latter group of about 50 metabolic diseases arises from mutations in specific
genes that lead to deficiencies in enzymes involved in the lysosomal degradation of glycolipids
and glycans. Considerable efforts have been made to provide novel therapeutic compounds that
may relieve the various symptoms arising from the inability of lysosomal glycosidases to
degrade their respective substrates. The latter consequently accumulate in the cells and lead to
irreversible damage of nerve tissue, bones as well as various organs. For some of these
lysosomal disorders, hematopoietic stem cell transplant,5 gene therapy6,7 as well as combination
therapies8 by additional administration of small molecules are envisaged to become tools of
profound intervention.
Efforts of many groups have shown that imino sugar- as well as carba sugar-based
glycomimetics may provide suitable therapeutic agents that function either by inhibiting
upstream enzymes, thereby reducing the production of metabolites that cannot be degraded

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rapidly enough by the respective mutant enzyme (substrate reduction therapy, SRT) or by
assisting the folding and transport of mutant enzymes to the lysosome (chaperone mediated
therapy, CMT), for which it was suggested that sub-inhibitory concentrations of active site
specific molecules (pharmacological chaperones) could be exploited.9,10,11,12,13,14,15,16,17 Mutant
proteins that cannot obtain/retain their functional conformation are recognized as misfolded by
the quality control machinery in the endoplasmic reticulum and are eventually targeted for
degradation. These carba or imino sugars can bind to and stabilize mutant forms of enzymes
such as the lysosomal β-glucosidase, β-galactosidase or β-N-acetylhexosaminidase in their
functional folded conformations thus facilitating their exit from the endoplasmic reticulum and
their subsequent transport to the lysosome.
Guiding contributions by various leaders in the field and recent reviews18,19 have shown that
non-polar N-substituted iminoalditols and similarly functionalized structures bind better than
their more polar parent compounds through stronger interactions with the aglycon binding site or
with lipophilic pockets around the enzymes’ active sites. In this context, N-butyl-1-
deoxynojirimycin is one of the best studied iminosugars, thus far.4 Other researchers20,21,22,23
have observed that large, lipophilic substituents, for example, adamantyl capped spacer arms,
greatly improve the interaction between iminoalditol derivatives and the lysosomal β-
glucosidase.
Compounds 1-3 have been found in nature. The so-called isoimino sugars invented by Lundt24
and Bols25 are not known natural products. The first such isoimino sugar, isofagomine (4), was
found to be a potent β-glucosidase inhibitor. C-5a-Chain extended derivatives such as C-nonyl
analog 5, introduced by Fan and co-workers26 turned out to be even more powerful. Derivatives
6 and 7 were synthesized and investigated by Withers and collaborators.27
GM1-Gangliosidosis and Morquio B disease are two manifestations of mutations on the GLB1
gene which encodes the vital lysosomal β-galactosidase. This enzyme is responsible for removal
of a β-galactosyl residue from the non-reducing end of its substrate, converting gangliosides GM1

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and GA1 into GM2 and GA2, respectively. Depending on the location of the mutation, the resulting
lysosomal storage disorder is neurodegenerative (GM1-gangliosidosis) or bone-destructive
(Morquio B).28,29
Figure 1: Glucosidase and galactosidase inhibitors as potential therapeutic principles for
lysosomal deficiencies of D-glucosidase and D-galactosidase, respectively.
Only three distinct types of β-D-galactosidase inhibitors have been investigated as potential
pharmacological chaperones for these two disorders. The first compound reported, carbasugar
NOEV (N-octyl-epi-valienamine, 8) is a powerful inhibitor30 and reportedly even at sub-
micromolar concentrations a highly potent pharmacological chaperone for a wide range of
mutants of lysosomal β-galactosidase.31 More recently, selected unbranched analogs lacking the
hydroxymethyl group have been presented and their activities with patients’ skin fibroblasts
carrying the R201C mutation were evaluated.32 Other potential chaperones were based on the
structure of compound 2 which, by various structural modifications, could be transformed into a
relatively small number of derivative classes (10-16, in the case of several N-substituent-

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variations in the same publication, only the most active is depicted) with interesting properties as
experimental pharmacological chaperones.33,34,35,36,37,38,39,40,41
C-5a-substituted derivatives of the isoiminosugar 4-epi-isofagomine42 (17), such as compounds
18 and 19 have recently been reported and were found to be potent β-galactosidase inhibitors and
superior pharmacological chaperones for selected GM1-gangliosidosis and Morquio B disease cell
lines.43,44
Amongst other types of potentially suitable sugar analogs in this context, carbasugars are a
noteworthy and biologically powerful family, second only to the much larger group of imino and
isoimino sugars. Comprehensive reviews have covered this important field recently.45,46,47 Both
pyranoid and furanoid-like carbasugars have been found in nature, with 5a-carba-α-D-
galactopyranose having been the first such product discovered.48 Examples of natural products
containing cyclopentane-derived carbasugars include trehazolines,49,50 allosamidine,51 and the
nucleoside analogs neplanocin52 and aristeromycin.53 Based on these and related structures,
various non-natural derivatives have been prepared for several purposes. Included in these are
synthetic
D-galacto-related derivatives of valienamine and validamine, which have been
investigated as pharmacological chaperones for a range of lysosomal storage disorders, including
GM1-gangliosidosis.54
We have now become interested in another substance class, the aminocyclopentane-based
carbasugars (hydroxymethyl branched di- and trihydroxycyclopentylamines). There are only
three leading references presenting such sugar analogs as proven
D
-galactosidase inhibitors.55
Even more surprisingly, this family of compounds reportedly harbours some of the most
powerful inhibitors of β-galactosidases known to date featuring inhibition constants in the sub-
nanomolar range.55 In particular, the β-
D-galacto configured cyclopentyl amine bearing a (4-
bromo)benzyl moiety at the nitrogen (20, figure 2), has been found to excel in terms of inhibitory
activity.55b,c

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Figure 2: Inhibitor 20
With a view to extending the selection of chaperone structures for GM1-gangliosidosis and
Morquio B, we wanted to explore whether such furanoid
lead compounds.
D-galactosidase inhibitors were suitable
2. Results and discussion
2.1. Synthesis
Our initial approach was based on Jäger’s55b route: Starting from 1,2;4,6-di-O-isopropylidene-α-
-galactose (21), employing Garegg’s conditions, known 6-deoxyiodosugar 22 was prepared.
D
Taking advantage of the classical Vasella approach,56 its reaction with Zn-dust under slightly
acidic conditions provided open-chain intermediate 23. Previously reported57 vitamin B12
catalysis in this fragmentation reaction was found not to be necessary for satisfactory yields. By
subsequent reaction with N-benzylhydroxylamine the initially formed oxime was smoothly
converted by intramolecular 2+3 cycloaddition57 with excellent stereoselectivity into desired
known bicyclic cyclopentane derivative 24 the structure of which was unambiguously confirmed
by X-ray crystallography (fig. 3). Nonetheless, despite all efforts made, we were not able to
exceed an isolated yield of 40% in this cyclization step. Unexpectedly, formation of the second
possible isomer could not be detected under the conditions employed. Catalytic hydrogenolysis
under acidic conditions of the oxime ether bond with concomitant de-N-benzylation and removal
of the isopropylidene moiety provided core structure 25 in only six steps from
(26.3% overall yield from 22)
D-galactose.

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OH
I
O
O
a
b
O
O
O
O
O
O
O
O
21
22
HO
6
5
6
O
HO
NBn
c
d
HO
5
NH2
O
4
1
4
1
O
O
3
2
3
2
O
O
O
OH
HO
OH
23
24
25
e
h
HO
6
5
O
OH
N
NH2
f
g
N
O
4
1
O
O
3
2
O
O
O
OH
OH
OH
26
27
28
Scheme 1: a) PPh3, I2, imidazole, toluene; b) Zn dust, NH4Cl, MeOH, 81%; c) BnNHOH.HCl,
NaHCO3, NaOMe, MeOH, 41%; d) H2, Pd(OH)2/C (20%), MeOH/HCl, 78%; e) H2NOH
(50wt% in H2O), MeOH, 91% over 2 steps from 22; f) NaOCl (10-15% active chlorine), silica
gel, 2-propanol, 81%; g) LiAlH4, THF, 78%; h) MeOH/H2O 4:1, HCl conc., 87%.
Aiming to improve the yields overall and, in particular, in the carbacyclization step, we turned to
a modified route based on exploiting unsubstituted hydroxylamine and subsequent oxidative
conditions in the ring closing reaction58 but employing 2-propanol in homogenous solution as
solvent in the cyclization step, thus reducing the reaction times from several hours to around 60
min and improving yields by a factor of more than 2. Thus, open-chain unsaturated sugar 23 was
reacted with hydroxylamine to give oxime 26, which, in turn, smoothly reacted in the presence
of NaOCl providing isoxazoline 27 in good yields. X-Ray crystallography unambiguously
established the correct configuration at the branching point also nicely showing the bowl-like
structure of 27 which only allows for hydride access from the exo-face. LiAlH4-mediated
reduction gave partially protected carbasugar 28 which was deprotected under acidic conditions
to furnish free central intermediate 25 in 49.6 % overall yield from 22.

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Figure 3. Carbasugar precursors 24 (CCDC 1530133; left) and 27 (CCDC 1530134).
With compound 25 readily at hand, subsequent conventional chemoselective N-alkylation
furnished, in one single step each, a range of N-modified final products featuring “D-galacto”
configuration (Scheme 2).
Scheme 2: a) RBr, NaHCO3, DMF, (20: 80%; 29: 83%; 30: 87%; 32: 72%); b) H2, Pd(OH)2/C,
acetone, MeOH, 90%.
Thus, individual reaction of compound 25 with 1-bromohexane, as well as (3-phenyl)-1-
bromopropane gave new products 29 and 30, respectively. Conventional catalytic reductive
amination with acetone led to new N-isopropyl derivative 31. For internal biochemical
comparison, known compounds 32 and 20 (the latter - as mentioned - one of the most powerful
β-galactosidase inhibitors reported thus far)55 were also prepared by reaction with benzyl
bromide and (4-bromo)benzyl bromide, respectively.

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2.2.Biological evaluation
Inhibitory activities of free aminocyclopentane tetraols 20, as well as 29 - 32 in comparison with
unsubstituted parent compound 25 and bench mark molecule 8 are summarized in Table 1. In all
cases where inhibition was observed the pattern of inhibition was competitive. It is noteworthy
that none of the compounds inhibited either of the α-glycosidases tested. Likewise, inhibition
was reasonably galactosidase-specific relative to glucosidase. Nonetheless, undesired high
activities with recombinant lysosomal glucocerebrosidase were noted in 75% of the compounds
probed in this study and suggest structural re-iteration attempts to reduce this feature. Of the
inhibitors tested, compounds 20, 29 and 30 showed the best performance against the mammalian
β-galactosidases, thus these were taken forward for cell-based testing.
Evaluation of inhibitors 20, 29 and 30 as potential chaperones for the R201C mutant enzyme
(Fig. 2) employing patients’ skin fibroblasts revealed an effective concentration range of two
orders of magnitude with an enhancement of 3.5- to 4-fold β-galactosidase activity at a
concentration of 2-2.5 µM, 5-fold (compounds 20 and 29) to 6-fold activity (compound 30) at 10
µM and a maximum chaperone effect of more than 8-fold activity at 50 µM in case of compound
30. For comparison, in-house data for compound 8 are also depicted (Fig. 4, black graph).
Figure 4: Activity enhancements with compounds 20, 29 and 30 (in comparison with pyranoid
carbasugar 8) with human lysosomal β-galactosidase mutant R201C

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10
Table 1. Ki-values [µM] of compounds with ABG = β-glucosidase/β-galactosidase from Agrobacterium sp.;
E. coli = lac Z β-galactosidase from E. coli; Bovine Liver = β-galactosidase from bovine liver; Fabrazyme = commercial recombinant
lysosomal α-galactosidase; S. cer. = α-glucosidase from S. cerevisiae; GCase = recombinant human lysosomal β-D-glucocerebrosidase;
β-Gal. human lys. = human lysosomal acid β-galactosidase; IC50 [µM] with β-Gal (human lysosomal);
N.I. = no inhibition, with Ki > 2 mM; n.d., not determined.
Compounds
Enzyme
20
25
29
30
31
32
8
0.0010
0.095
0.0046
0.0062
3.8
0.013
7.7
Abg (β-Glc/Gal)
E. coli (β-Gal)
0.0039
0.0030
N.I.
33.3
54.3
N.I.
N.I.
0.102
0.049
N.I.
0.061
0.085
N.I.
5.0
13.0
N.I.
N.I.
0.065
0.14
N.I.
2.8
0.8759
N.I.
Bovine liver (β-Gal)
Fabrazyme (α-Gal)
S. cer (α-Glc)
N.I.
N.I.
N.I.
N.I.
N.I.
GCase (β-Glc)
0.067
0.47
78.1
n.d.
0.23
1.44
0.21
1.44
N.I.
n.d.
0.38
n.d.
83
β-Gal hum. lys (IC50)
0.51

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3. Conclusions
Following a simple protocol previously outlined by Vasella’s and Jäger’s groups, a series of
novel derivatives of 25, bearing chain extensions at N-1 have been made available and were
screened with a range of
D-galactosidases including human lysosomal β-galactosidase. New
inhibitors, compounds 29 and 30, turned out to be highly potent and exhibited encouraging dose-
dependent chaperoning profiles with enzyme mutant R201C. Results obviously merit screening
for potential chaperone activity with other frequent mutants of this enzyme.
4. Experimental
4.1. General methods
Optical rotations were measured at 20° C on a Perkin Elmer 341 polarimeter at a wavelength of
589 nm and a path length of 10 cm. NMR spectra were recorded on a Varian INOVA 500
operating at 499.82 MHz (1H), 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 methanol-d4 or D2O for unprotected inhibitors. Carbon and hydrogen numbering
in NMR spectra was conducted in analogy to carbohydrate nomenclature and clockwise, starting
with the amino bearing carbon as C-1. Chemical shifts are listed in delta employing residual,
non-deuterated solvent as the internal standard. Signals were assigned unambiguously by COSY,
HSQC as well as APT analysis. The signals of the protecting groups as well as of the N-
substituents were found in the expected regions and are only listed explicitly when overlapping
with important spectral features of the respective compound. For crucial intermediates, structures
were confirmed by XRD structural analysis: Suitable single crystals of compounds were
immersed in silicone oil, mounted using a glass fiber and frozen in the cold nitrogen stream
(100K). X-ray diffraction data were collected at low temperature on a Bruker Kappa APEX II
diffractometer using Mo Ka radiation (l = 0.71073 Å) generated by an INCOATEC micro-focus
source. The data reduction and absorption correction was performed with the Bruker SMART60

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and Bruker SADABS,61 respectively. The structures were solved with SHELXT62 by direct
methods and refined with SHELXL62 by least-square minimization against F2 using first
isotropic and later anisotropic thermal parameters for all non-hydrogen atoms. Hydrogen atoms
were added to the structure models on calculated positions using the riding model. The space
group assignments and structural solutions were evaluated using PLATON.63,64
MALDI-TOF Mass Spectrometry was performed on a Micromass TofSpec 2E Time-of-Flight
Mass Spectrometer. Analytical TLC was performed on precoated aluminum plates silica gel 60
F254 (E. Merck 5554) and detected with UV light (254 nm). For staining, a solution of vanillin
(9 g) in a mixture of H2O (950 mL)/ EtOH (750 mL)/ H2SO4 (120 mL) or ceric ammonium
molybdate (100 g ammonium molybdate/8 g ceric sulfate in 1 L 10% H2SO4) were employed
followed by heating on a hotplate. For column chromatography, silica gel 60 (230-400 mesh, E.
Merck 9385) or silica gel 60 (Acros Organics, AC 24036) were used.
4.2. Kinetic Studies
Kinetic studies were performed at 37°C in an appropriate buffer using a known concentration of
enzyme (specific conditions depicted below). Ki determinations were performed using the
corresponding 4-nitrophenyl α- or β-
D
-galactopyranoside as substrate, except for Fabrazyme and
-glucosylpyranoside65 were
GCase, where 2,4-dinitrophenyl α-
D
-galactopyranoside and β-D
employed. In a typical assay, the enzyme was incubated with different inhibitor concentrations
for up to 5 minutes before initiating the reaction by the addition of substrate. The initial reaction
rate was measured by monitoring the increase in absorbance at 400 nm for up to ten minutes. Ki
determinations were performed using at least two different substrate concentrations. For each
inhibitor, a range of four to six inhibitor concentrations bracketing the Ki value ultimately
determined was used for each substrate concentration. Dixon plots (1/v vs [I]) were constructed

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to validate the use of competitive inhibition model. The data were then fit using non-linear
regression analysis with Grafit 7.0.
N.I. stands for no inhibition or weak inhibition with an estimated Ki value higher than 2 mM.
Specific assay conditions for each enzyme:
Agrobacterium sp. β-glucosidase:66,67 50 mM sodium phosphate buffer (pH 7) using 1.85x10-4
mg/mL of enzyme (Km = 4.1 mM).
E.coli lac z β-galactosidase: 50 mM sodium phosphate, 1.0 mM MgCl2 (pH 7) using 6.4x10-4
mg/mL of enzyme (Km = 60 µM).
Bovine liver β-galactosidase: 50 mM sodium phosphate buffer (pH 7) using 4.9x10-2 mg/mL of
enzyme (Km = 0.65 mM).
Fabrazyme (Acid α-galactosidase): 20 mM sodium citrate, 50 mM sodium phosphate, 1.0 mM
tetrasodium EDTA, 0.25% v/v Triton X-100® and 0.25% w/v taurocholic acid buffer (pH5.5)
using 5x10-5 mg/mL of enzyme (Km = 0.65 mM).
GCase (β-glucocerebrosidase): 20 mM sodium citrate, 50 mM sodium phosphate, 1.0 mM
tetrasodium EDTA, 0.25% v/v Triton X-100® and 0.25% w/v taurocholic acid buffer (pH 7)
using 4x10-5 mg/mL of enzyme (Km = 2.7 mM).
Human β-galactosidase and β-hexosaminidase activity measurements were performed in
duplicate assays, unless otherwise stated. Fibroblast cells were harvested by trypsinization in
0.9% NaCl containing 0.01% Triton, homogenized by sonication (3 x 10 sec, Sonifier Bandelin
Sonopuls) and centrifuged at 13,000 rpm for 2 min in a table top centrifuge (Biofuge Pico,
Heraeus). Protein amounts were determined according to the method of Lowry.68
For assessment of β-Gal activity, 20 µl of cell homogenate were mixed with 100 µl of 0.5 mM 4-
methylumbelliferyl-β-D-galactopyranoside (Sigma-Aldrich), in 100 mM citrate buffer (pH 4.0)
containing 100 mM NaCl and 0.02% NaN3. After incubation at 37°C for 30 min, the reaction
was stopped by adding 2.5 ml 400 mM glycine/NaOH (pH 10.4).

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β-Hexosaminidase activity was measured by adding 10 µl homogenate to 90 µl 0.9% NaCl and
100 µl of 1 mM 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich) in 100 mM
citrate buffer (pH 4.6) containing 0.2% BSA and 0.04% NaN3. The reaction was stopped after 10
min at 37°C by addition of 2.5 ml of 400 mM glycine/NaOH (pH 10.4). The amount of
hydrolyzed 4-methylumbelliferone was determined with a fluorescence spectrometer (Hitachi
F7000).
Modified β-Gal assays were used to estimate the half maximal inhibitory concentration (IC50) of
the particular chaperone. For IC50 determination, 0.001 to 100 µM of chaperone was added to the
assay mixture. Activity was measured in normal fibroblasts. Data analysis was performed with
Microcal™ Origin® v6.0 using a non-linear curve fitting module based on sigmoid curve fitting.
4.2.1. Patients and cell lines
Cell lines from one GM1-Gangliosidosis-patient (R201C) and one WT, were exposed to
compounds 20, 29, and 30, for evaluation of their chaperone effects.
Human skin fibroblasts were grown in minimal essential medium (MEM) with Earle’s Salts
(SIGMA) containing 10% fetal bovine serum, 400 µM L-glutamine, and 50 µg/ml gentamycin at
37°C and 5% CO2. All cells used in this study were between the third and nineteenth passages.
Potential chaperones were dissolved in DMSO and diluted in 10 mM phosphate buffer (pH 7.0)
containing 100 mM NaCl, 0.01% NaN3, and 0.01% Triton for the IC50-measurements and in
MEM for the in-vivo tests. The total DMSO concentration in the media was less than 1% and had
no effect on β-Gal activity or cell viability.
4.2.2. Treatment of cultured fibroblasts
Fibroblasts were grown to semi-confluency in 6-well plates. Particular chaperone was added to
the culture medium at following concentrations: 100µmol-0,02µmol. Cells were incubated for

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four additional days at 37°C. Cells were harvested by scraping and prepared for β-galactosidase
and β-hexosaminidase assays as described above.
4.3. General procedures
General procedure for N-alkylation: To a 10% solution of free aminocyclopentane tetraol 25 in
DMF, solid NaHCO3 (4 equiv.) and the respective alkyl bromide (2 equiv.) were added and the
mixture was strirred at 60 °C until TLC indicated completed conversion of the starting material.
The reaction was quenched with MeOH, the resulting suspension was concentrated under
reduced pressure, and the remaining residue was passed over a short column of silica gel.
4.4. (3aR,3bS,6aR,7S,7aR)-Hexahydro-5,5-dimethyl-1-(phenylmethyl)-1H-
[1,3]Dioxolo[3,4]cyclopent[1,2-c]isoxazol-7-ol (24)
A suspension of Zn dust (1.4 g, 21.9 mmol) and NH4Cl (1.2 g, 21.9 mmol) in MeOH (15 mL)
was stirred at ambient temp for 30 min. A solution of deoxyiodo sugar 22 (0.54 g, 1.46 mmol) in
MeOH (6 mL) was added and the mixture was stirred or 60 min. After filtration over a short plug
of silica gel, the filtrate was concentrated under reduced pressure, the residue was taken up in
ethyl acetate and the organic layer was washed with water, dried (Na2SO4) and removed under
reduced pressure providing compound 23 (0.29 g, 1.19 mmol, 81.4%) as a pale yellow oil. The
resulting crude material was directly taken into the next step.
To a stirred solution of N-benzylhydroxylamine.HCl (279 mg, 1.75 mmol) in MeOH (6 mL), a
methanolic solution of NaOMe (6 mL, 1 M) and solid NaHCO3 (490 mg, 5.83 mmol) were
added. After 15 min, a solution of crude enosugar 23 in MeOH (6 mL) was added and the
mixture was stirred at 50 °C for 6 h. The resulting reaction mixture was diluted with ethyl acetate
and washed with water. The organic layer was dried (Na2SO4), filtered and concentrated under
reduced pressure. The remaining residue was purified on silica gel to provide desired N-

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benzylisoxazolidine 24 (141 mg, 0.48 mmol, 40.8%). Crystallization from MeOH/ethyl acetate
provided pale yellow crystals which were suitable for X-ray structure analysis.
1
[ꢀ]ꢂꢁꢃ= +62.9 (c 1.70, CHCl3); m.p.: 108-110°C; H NMR (300 MHz, CDCl3): δ =4.51 (dd, 1H,
J4,5 7.1 Hz, H-4), 4.30 (dd, 1H, J3,4 6.3 Hz, H-3), 4.16 (dd, 1H, J5,6a 4.2 Hz, J6a,6b 8.8 Hz, H-6a),
3.98-3.82 (m, 3H, H-2, H-6b, CH2Ar), 3.62 (d, 1H, CH2Ar), 3.48 (dd, 1H, J1,2 8.0 Hz, J1,5 7.1
Hz, H-1), 3.18 (m, 1H, H-5), 1.42, 1.22 (2s, 3H each, C(CH3)2); 13C NMR (75.5 MHz,
CDCl3) δ = 136.7 (ipso Ar), 129.1, 128.5, 127.5 (Ar), 112.7 (C(CH3)2), 86.4 (C-3), 79.7 (C-2),
77.5 (C-1), 77.1 (C-4), 64.9 (C-6), 59.7 (CH2Ar), 45.6 (C-5), 27.3, 25.5 (C(CH3)2); MS: Calcd
for [C16H21NO4H]: m/z 292.1549 [M+H]+; found [M+H]+ 292.1613.
4.5. (1S,2S,3S,4R,5R)-4-Amino-5-hydroxymethyl-1,2,3-cyclopentanetriol (25)
Condition A:
To a solution of intermediate 24 (83,0 mg, 0,29 mmol) in MeOH (4 mL), HCl (conc., 100 µL)
and Pd(OH)2/C (20%, 50 mg) were added and the mixture was stirred under an atmosphere of H2
at ambient pressure for 23 h. The catalyst was removed by filtration and washed with MeOH.
Combined solvents were removed under reduced pressure and the remaining residue was
subjected to silica gel chromatography to give free aminocyclopentane 25 (36.2 mg, 0.22 mmol,
77.9%) as a pale yellow syrup.
Condition B:
To a solution of compound 28 (60.0 mg, 0.30 mmol) in 5 ml MeOH/H2O 4:1, HCl conc.(50 µl)
was added and the mixture was stirred for 60 minutes. After complete conversion indicated by
TLC, the reaction mixture was evaporated to dryness. Purification on silica gel gave 25 (41.7
mg, 0.26 mmol, 86.6%) as a pale yellow syrup.
1
[ꢀ]ꢂꢁꢃ= +24.6 (c 0.97, H2O), H NMR (300 MHz, D2O) δ= 4.14 (dd, 1H, J4,5 3.5 Hz, H-4), 4.11
(dd, 1H, J1,2 4.7 Hz, J2,3 7.4 Hz, H-2), 3.89 (dd, 1H, J5,6a 4.0 Hz, J6a,6b 11.5 Hz, H-6a), 3.88 (m,
1H, H-3), 3.82 (dd, 1H, J5,6b 8.4 Hz, H-6b), 3.47 (dd, 1H, J1,2 4.7 Hz, J1,5 8.4 Hz, H-1); 13C NMR

17
ACCEPTED MANUSCRIPT
(75.5 MHz, D2O) δ= 81.0 (C-2), 77.9 (C-3), 72.3 (C-4), 57.0 (C-6), 56.7 (C-1), 41.9 (C-5); MS:
Calcd for [C6H13NO4H]: m/z 164.0923 [M+H]+; found [M+H]+ 164.0929.
4.6. (E/Z)-5,6-Dideoxy-5-eno-3,4-O-isopropylidene-L-arabinohexose oxime (26)
A suspension of Zn dust (3.7 g, 55.9 mmol) and NH4Cl (3.0 g, 55.9 mmol) in 40mL MeOH is
stirred for 30 minutes at ambient temperature. A solution of 22 (1.38 g, 3.73 mmol) in 5mL
MeOH is added, stirred for additional 60 minutes filtrated over silica gel concentrated under
reduced pressure diluted with ethyl acetate and washed with H2O. The combined organic layers
were dried over Na2SO4 and evaporated. The resulting residue was diluted in 20ml MeOH and
H2NOH (50 %wt in H2O, 685 µl, 11.2 mmol) was added and stirred for additional 60 minutes at
ambient temperature. After full conversion of the starting material the reaction was evaporated to
dryness. 26 (680.1 mg, 3.38 mmol, 90.7 % over 2 steps) was observed as an inseparable mixture
of E/Z as a colourless syrup.
Major isomer: 13C NMR (75.5 MHz CDCl3) δ = 150.1 (C-1), 133.5 (C-5), 119.9 (C-6), 109.3
(C(CH3)2), 79.1 (C-3), 78.9 (C-4), 68.2 (C-2), 27.2, 25.0 (C(CH3)2);
Minor isomer: 13C NMR (75.5 MHz CDCl3) δ =152.5 (C-1), 133.5 (C-5), 120.1 (C-6), 109.2
(C(CH3)2), 79.1 (C-3), 78.2 (C-4), 64.5 (C-2), 26.8, 24.7 (C(CH3)2).
4.7. (3aS,4S,5S,6S)-6-Hydroxy-4,5-isopropylidenedioxy-3a,4,5,6-tetrahydro-3H-
cyclopent[c]isoxazole (27)
To a stirred solution of 26 (590.7 mg, 2.94 mmol) in 20 ml 2-propanol was added silica gel (8 g)
and 4.5 ml NaOCl (10-15% active chlorine) and stirred for 60 minutes at ambient temperature.
After full conversion of the starting material, the reaction mixture was filtered and diluted with
CH2Cl2 and consecutively washed with H2O, HCl (2 N) and saturated NaHCO3. The combined
organic layers were dried (Na2SO4) and concentrated under reduced pressure. Purification on

18
ACCEPTED MANUSCRIPT
silica gel afforded 27 (473.3 mg, 2.38 mmol, 80.9%) as a colourless syrup. Recrystallisation with
ethyl acetate/cyclohexane gave colourless crystals for XRD analysis.
[ꢀ]ꢂꢁꢃ = -140.2 (c 1.62, CHCl3); m.p.: 113-116°C 1H NMR (300 MHz) CDCl3) δ = 4.70 (dd, 1H,
;
J3,4 5.2 Hz, H-3), 4.61 (dd, 1H, J4,5 5.6 Hz, H-4), 4.47 (s, 1H, H-2), 4.41 (dd, 1H, J5,6a 8.2 Hz,
J6a,6b 11.7 Hz, H-6a), 4.31 (dd,1H, J5,6b 8.4 Hz, H-6b), 4.04 (m, 1H, H-5), 3.39 (bs, 1H, 2-OH),
1.34, 1.24 (2s, 3H each, C(CH3)2); 13C NMR (75.5 MHz, CDCl3) δ = 165.3 (C-1), 111.6
(C(CH3)2), 90.1 (C-3), 75.1 (C-4), 70.0 (C-6), 68.9 (C-2), 54.4 (C-5), 26.4, 24.7 (C(CH3)2).
MS: Calcd for [C9H13NO4H]: m/z 200.0923 [M+H]+; Found [M+H]+ 200.0936.
4.8. (1S,2S,3S,4R,5R)-1,2-O-Isopropylidene-4-amino-5-hydroxymethyl-1,2,3-
cyclopentanetriol (28)
To a stirred suspension of LiAlH4 (902 mg 24 mmol) in THF (40 ml) a solution of compound 27
(470.2 mg, 2.4 mmol) in 15ml THF was added dropwise at 0°C. After full conversion of the
starting material, H2O and NaOH (3M) was added dropwise until the reaction mixture turned
milky white. The resulting suspension was filtered and evaporated to dryness. Purification on
silica gel gave 28 (374.0 mg, 1.84 mmol; 78.0%) as a colourless oil.
[ꢀ]ꢂꢁꢃ = +1.2 (c 1.35, MeOH) 1H NMR (300 MHz, MeOH-d4) δ = 4.80 (dd, 1H, J4,5 5.2 Hz, H-
;
4), 4.47 (d, 1H, J3,4 5.7 Hz, H-3), 4.13 (s, 1H, H-2), 3.94 (dd, 1H, J5,6a 7.2 Hz, J6a,6b 11.1 Hz, H-
6a), 3.85 (dd, 1H, J5,6b 8.2 Hz, H-6b), 3.35 (m, 1H, H-1), 2.59 (m, 1H, H-5), 1.47, 1.31 (2s, 3H
each, C(CH3)2); 13C NMR (75.5 MHz, MeOH-d4) δ= 112.1 (C(CH3)2) 87.4 (C-3), 81.7 (C-4),
79.9 (C-2), 60.7 (C-1), 58.2 (C-6), 47.0 (C-5), 26.2, 23.0 (C(CH3)2).
MS: Calcd for [C9H17NO4H]: m/z 204.1236 [M+H]+; Found [M+H]+ 204.1221.
4.9. (1S,2S,3S,4R,5R)-N-Hexyl-4-amino-5-hydroxymethyl-1,2,3-cyclopentanetriol (29)
Following the general procedure, compound 25 (20.1 mg, 0.12 mmol) was treated with 1-
bromohexane (19 µL, 0.14 mmol) in the presence of solid NaHCO3 (40 mg, 0.48 mmol) for 90 h.

19
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Purification on silica gel afforded N-hexyl derivative 29 (25.4 mg, 0.10 mmol, 83.4%) as a white
wax.
1
[ꢀ]ꢂꢁꢃ= +24.0 (c 0.78, MeOH), H NMR (300 MHz, MeOH-d4) δ= 4.22 (dd, 1H, H-2), 4.04 (dd,
1H, J3,4 4.2 Hz, H-4), 3.92 (m, 2H, H-6a, H-6b), 3.80 (dd, 1H, J2,3 6.6 Hz, H-3), 3.48 (dd, 1H,
J1,2 8.7 Hz, J1,5 8.7 Hz, H-1), 3.28-3.03 (m, 2H, H-1’), 2.60 (m, 1H, H-5), 1.86-1.64 (m, 2H, H-
2’), 1.47-1.28 (m, 6H, H-3’, H-4’, H-5’), 0.93 (t, 3H, H-6’); 13C NMR (75.5 MHz, MeOH-d4) δ=
80.2 (C-2), 79.8 (C-3), 73.3 (C-4), 64.9 (C-1), 58.6 (C-6), 48.5 (C-1‘) 43.7 (C-5), 32.4, 27.2,
26.7, 23.5 (C-2‘, C-3‘, C-4‘, C-5‘), 14.3 (C-6‘).
MS: Calcd for [C12H25NO4H]: m/z 248.1862 [M+H]+; Found [M+H]+ 248.1864.
4.10. (1S,2S,3S,4R,5R)-N-(3-Phenyl)propyl-4-amino-5-hydroxymethyl-1,2,3-cyclopentanetriol
(30)
Following the general procedure, compound 25 (19.5 mg, 0.12 mmol) was treated with 3-phenyl-
1-bromopropane (20.5 µL, 0.13 mmol) in the presence of solid NaHCO3 (40 mg, 0.48 mmol) for
96 h. Purification on silica gel gave (3-phenyl)propyl derivative 30 (29.4 mg, 0.10 mmol, 87.4%)
as a colorless syrup.
1
[ꢀ]ꢂꢁꢃ= +26.8 (c 1.14, MeOH), H NMR (300 MHz, MeOH-d4) δ= 4.14 (dd, 1H, J1,2 5.4 Hz, H-
2), 3.97 (dd, 1H, J3,4 4.2 Hz, H-4), 3.85 (m, 2H, H-6a, H-6b), 3.73 (dd, 1H, J2,3 6.7 Hz, H-3),
3.41 (dd, 1H, J1,2 5.4 Hz, J1,5 8.9 Hz, H-1), 3.23-2.99 (m, 2H, H-1’); 2.71-2.61 (t, 2H, H-3’), 2.53
(m, 1H, H-5), 2.13-1.91 (m, 2H, H-2’); 13C NMR (75.5 MHz, MeOH-d4) δ=141.6 (ipso Ar),
129.6, 129.4, 127.4 (Ar), 80.2 (C-2), 79.8 (C-3), 73.3 (C-4), 65.0 (C-1), 58.6 (C-6), 43.7 (C-1’),
33.6 (C-3’), 28.5 (C-2’).
MS: Calcd for [C15H23NO4H]: m/z 282.1705 [M+H]+; Found [M+H]+ 282.1709.
4.11. (1S,2S,3S,4R,5R)-N-isopropyl-4-amino-5-hydroxymethyl-1,2,3-cyclopentanetriol (31)

20
ACCEPTED MANUSCRIPT
To a stirred solution of 25 (42.8 mg, 0.26 mmol) in MeOH (3 ml) and HCl conc. (10 µL),
acetone (29 µl, 0.39 mmol) was added and the mixture was stirred under an atmosphere of
hydrogen at ambient pressure in prescence of Pd(OH)2/C (20 %; 40 mg) for 24 hours. The
catalyst was removed by filtration and washed with MeOH. The solvent was removed under
reduced pressure and the remaining residue was chromatographed on silica gel to give compound
31 (48.7 mg, 0.24 mmol, 90.5%) as a colourless wax.
[ꢀ]ꢂꢁꢃ = +19.8 (c 1.31, H2O) 1H NMR (300 MHz, D2O) δ= 4.25 (dd, 1H, J2,3 6.9 Hz, H-2), 4.11
;
(dd, 1H, J3,4 4.1 Hz, H-4), 3.93-3.79 (m, 3H, H-3, H-6a, H-6b), 3.69 (dd, 1H, J1,2 6.0 Hz, J1,5 9.1
Hz, H-1), 3.63 (m, 1H, CH(CH3)2), 2.65 (m, 1H, H-5), 1.32 (m, 6H, CH(CH3)2); 13C NMR (75.5
MHz, D2O) δ= 78.6 (C-2), 77.4 (C-3), 71.4 (C-4),59.9 (C-1), 57.1 (C-6), 50.6 (CH(CH3)2), 41.5
(C-5), 19.1, 17.5 (CH(CH3)2).
MS: Calcd for [C9H19NO4H]: m/z 206.1392 [M+H]+; Found [M+H]+ 206.1381.
4.12. (1S,2S,3S,4R,5R)-N-benzyl-4-amino-5-hydroxymethyl-1,2,3-cyclopentanetriol (32)
Following the general procedure, compound 25 (19.8 mg, 0.12 mmol) was treated with benzyl
bromide (16 µL, 0.13 mmol) in the presence of solid NaHCO3 (40 mg, 0.48 mmol). Purification
on silica gel yielded N-benzyl derivative 32 (22.0 mg, 0.09 mmol, 71.6%) as a colorless wax.
[ꢀ]ꢂꢁꢃ= +39.9 (c 1.35, MeOH); 1H NMR (300 MHz, D2O) δ= 4.50 (d, 1H, CH2Ar), 4.38 (dd, 1H,
J1,2 5.8 Hz, J2,3 7.4 Hz, H-2), 4.27 (d, 1H, CH2Ar), 4.06 (dd, 1H, J3,4 3.8 Hz, H-4), 3.94-3.74 (m,
13
3H, H-3, H-6a, H-6b), 3.51 (dd, 1H, J1,5 9.0 Hz, H-1), 2.61 (m, 1H, H-5); C NMR (75.5 MHz,
D2O) δ= 130.1, 129.8, 129.8, 129.3 (Ar), 78.5 (C-2), 77.7 (C-3), 71.4 (C-4), 61.3 (C-1), 56.9 (C-
6), 50.1 (CH2Ar), 41.4 (C-5).
MS: Calcd for [C13H19NO4Na]: m/z 276.1212 [M+Na]+; Found [M+Na]+ 276.1205.
4.13. (1S,2S,3S,4R,5R)-N-(4-bromo)benzyl-4-amino-5-hydroxymethyl-1,2,3-cyclopentanetriol
(20)

21
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Following the general procedure, compound 25 (19.3 mg, 0.12 mmol) was treated with (4-
bromo)benzyl bromide (32.5 mg, 0.13 mmol) in the presence of solid NaHCO3 (40 mg, 0.48
mmol). Purification on silica gel gave N-(4-bromo)benzyl derivative 20 (31.5 mg, 0.10 mmol,
80.2%) as a slightly yellow syrup.
[ꢀ]ꢂꢁꢃ= +17.6 (c 0.48, H2O); 1H NMR (300 MHz, D2O) δ= 4.16-4.06 (m, 2H, H-2, H-4), 3.98 (d,
1H, CH2Ar), 3.88-3.73 (m, 4H, H-3, H-6a, H-6b, CH2Ar), 3.11 (dd, 1H, J1,2 5.0 Hz, J1,5 8.8 Hz,
H-1), 2.46 (m, 1H, H-5); 13C NMR (75.5 MHz, D2O) δ= 137.0 (ipso Ar), 131.6, 130.5 (Ar),
120.8 (ArBr), 82.1 (C-2), 78.3 (C-3), 72.0 (C-4), 61.7 (C-1), 57.4 (C-6), 50.1 (CH2Ar), 43.1 (C-
5).
MS: Calcd for [C13H18BrNO4H]: m/z 332.0497 [M+H]+; Found [M+H]+ 332.0863.
Acknowledgment
Financial support by the Austrian Fonds zur Förderung der Wissenschaftlichen Forschung
(FWF), Vienna, (Project P 24815-B21) is gratefully acknowledged. SGW thanks GlycoNet, the
Canadian Network of Centres of Excellence in glycoscience, for financial support.
References and notes
1.
2.
3.
4.
Compain, P.; Martin, O. R. In Iminosugars – From synthesis to therapeutic applications,
Compain, P.; Martin, O. R. Eds.,Wiley: Chichester, 2007, pp 1-6.
Wennekes, T.; van den Berg, R. J. B. H. N.; Boot, R. G.; van der Marel, G. A.;
Overkleeft, H. S.; Aerts, J. M. F. G. Angew. Chem. Int. Ed. 2009, 48, 8848-8869.
Fan, J.-Q. In Iminosugars – From synthesis to therapeutic applications, Compain, P.;
Martin, O. R. Eds.,Wiley: Chichester, 2007, pp 225-247.
Butters, T. In Iminosugars – From synthesis to therapeutic applications, Compain, P.;
Martin, O. R. Eds.,Wiley: Chichester, 2007, pp. 249-268.

22
ACCEPTED MANUSCRIPT
5.
Lund, T. C. Ped. Endocrinol. Rev. 2013, 11, 91-98.
6.
Biffi, A. Hum. Mol. Genet. 2016, 25, R65-R75.
7.
Yew, N. S.; Cheng, S. H. Ped. Endocrinol. Rev. 2013, 11, 99-109.
Macauley, S. L. Pediatr. Endocrinol. Rev. Suppl. 1, 2016, 13, 639-648.
Fan, J.-Q.; Ishii, S.; Asano, N.; Suzuki, Y. Nat. Med. 1999, 5, 112-115.
Morello, J.-P.; Petäjä-Repo, U. E.; Bichet, D. G.; Bouvier, M. Trends Pharm. Sci. 2000,
21, 466-469.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Suzuki, Y.; Ogawa, S.; Sakakibara, Y. Persp. Med. Chem. 2009, 3, 7-19.
Parenti, G.; Pignata, C.; Vajro, P.; Salerno, M. Int. J. Mol. Med. 2013, 31, 11-20.
Suzuki, Y. Proc. Jap. Acad., Ser B: Phys. Biol. Sci. 2014, 90, 145-162.
Parenti, G.; Moracci, M.; Fecarotta, S.; Andria, G. Fut. Med. Chem. 2014, 6, 1031-1045.
Small, S. A. Proc. Natl. Acad. Sci. USA 2014, 111, 12274-12275.
Covertino, M.; Das, J.; Dokholyan, N. V. Chem. Biol. 2016, 11, 1471-1489.
Sanchez-Fernandez, E. M.; Garcia Fernandez, J. M.; Ortiz Mellet, C. Chem. Commun.
2016, 5497-5515.
18.
Boyd, R. E.; Lee, G.; Rybczynski, P.; Benjamin, E. R.; Khanna, R.; Wustman, B. A.;
Valenzano, K. J. J. Med. Chem. 2013, 56, 2705-2725.
19.
20.
Wrodnigg, T. M.; Stütz, A. E. Curr. Enzyme Inhib. 2012, 8, 47-99
Sawkar, A. R.; Adamski-Werner, S. L.; Cheng, W-C.; Wong, C.-H.; Beutler, E.; Zimmer
K-P.; Kelly, J. W. Chem. Biol. 2005, 12, 1235-1244.
21.
22.
Wennekes, T.; van den Berg, R. J. B. H. N.; Donker, W.; van der Marel, G. A.; Strijland,
A.; Aerts, M. F. G.; Overkleeft, H. S. J. Org. Chem. 2007, 72, 1088-1097.
Alfonso, P.; Andreu, V.; Pino-Angeles, A.; Moya-Garcia, A. A.; Garcia-Moreno, M. I.;
Rodriguez-Rey, J. C.; Sanchez-Jimenez, F.; Pocovi, M.; Ortiz Mellet, C.; Garcia
Fernandez, J. M.; Giraldo, P. ChemBioChem. 2013, 14, 943-949.

23
ACCEPTED MANUSCRIPT
23.
24.
25.
26.
27.
28.
Li, Z.; Li, T.; Dai, S.; Xie, X.; Ma, X.; Zhao, W.; Zhang, W.; Li, J.; Wang, P. G.
ChemBioChem. 2013, 14, 1239-1247.
Jespersen, T. M.; Dong, W.; Sierks, M. R.; Skrydstrup, T.; Lundt, I.; Bols, M. Angew.
Chem. Int. Ed. Engl. 1994, 33, 1778-1779.
Jespersen, T. M.; Bols, M.; Sierks, M. R.; Skrydstrup, T. Tetrahedron, 1994, 50, 13449-
13460.
Zhu, X. X.; Sheth, K. A.; Li, S.; Chang, H. H.; Fan, J. Q. Angew. Chem. Int. Ed. 2005, 44,
7450-7453.
Hill, T.; Tropak, M. B.; Mahuran, D.; Withers, S. G. ChemBioChem 2011, 12, 2151-
2154.
Suzuki, Y.; Oshima, A.; Nanba, E. In The Metabolic and Molecular Bases of Inherited
Disease, C. R. Shriver, A. L. Beaudet, W. S. Sly, and D. Valle, Ed. McGraw-Hill: New
York, 2001; pp. 3775-3809.
29.
30.
Brunetti-Pierri, N.; Scaglia, F. Mol. Genet. Metabol. 2008, 94, 391-396.
Matsuda, J.; Suzuki, O.; Oshima, A.; Yamamoto, Y.; Noguchi, A.; Takimoto, K.; Itoh,
M.; Matsuzaki, Y.; Yasuda, Y.; Sakata, Y.; Nanba, E.; Higaki, K.; Ogawa, Y.; Tominaga,
L.; Ohno, K.; Iwasaki, H.; Watanabe, H.; Brady, R. O.; Suzuki Y. Proc. Natl. Acad. Sci.
USA 2003, 100, 15912-15917.
31.
32.
Higaki, K.; Li, L.; Okuzawa, S.; Takamuram, A.; Yamamoto, K.; Adachi, K.; Paraguison,
R. C.; Takai, T.; Ikehata, H.; Tominaga, L.; Hisatome, I.; Iida, M.; Ogawa, S.; Matsuda,
J.; Ninomiya, H.; Sakakibara, Y.; Ohno, K.; Suzuki, Y.; Nanba E. Hum. Mutat. 2011, 32,
843-852.
Kuno, S.; Higaki, K.; Takahashi, A.; Nanba, E.; Ogawa, S. Med. Chem. Commun. 2015,
6, 306-310.

24
ACCEPTED MANUSCRIPT
33.
34.
Sanchez-Fernandez, E. M.; Garcia Fernandez, J. M.; Ortiz Mellet, C. Chem. Commun.
2016, 52, 5497-5515
Takai, T.; Higaki, K.; Aguilar-Moncayo, M.; Mena-Barragan, T.; Hirano, Y.; Yura, K.;
Yu, L.; Ninomiya, H.; Garcia-Moreno, M. I.; Sakakibara, Y.; Ohno, K.; Nanba, E.; Ortiz
Mellet, C.; Garcia Fernandez, J. M.; Suzuki Y. Mol. Ther. 2013, 21, 526-532.
Siriwardena, A.; Sonawane, D. P.; Bande, O. P.; Markad, P. R.; Yonekawa, S.; Tropak,
M. B.; Ghosh, S.; Chopade, B. A.; Mahuran, D. J.; Dhavale D. D. J. Org. Chem. 2014,
79, 4398-4404.
35.
36.
37.
38.
39.
Kasperzyk, J. L.; El-Abbadi, M. M.; Hauser, E. C.; d’Azzo, A.; Platt, F. M.; Seyfried, T.
N. J. Neurochemistry 2004, 89, 645-653
Rigat, B. A.; Tropak, M. B.; Buttner, J.; Crushell, E.; Benedict, D.; Callahan, J. W.;
Martin, D. R.; Mahuran D. J. Mol. Genet. Metab. 2012, 107, 203-212.
Fantur, K.; Hofer, D.; Schitter, G.; Steiner, A. J.; Pabst, B. M.; Wrodnigg, T. M., Stütz,
A. E.; Paschke E. Mol. Genet. Metab. 2010, 100, 262-268.
Schitter, G.; Steiner, A. J.; Pototschnig, G.; Scheucher, E.; Thonhofer, M.; Tarling, C. A.;
Withers, S. G.; Fantur, K.; Paschke, E.; Mahuran, D. J.; Rigat, B. A.; Tropak, M. B.;
Illaszewicz, C.; Saf, R.; Stütz, A. E.; Wrodnigg T. M. ChemBioChem 2010, 11, 2026-
2033.
40.
Suzuki, H.; Otho, U.; Higaki, K.; Mena-Barragan, T.; Aguilar-Moncayo, M.; Ortiz
Mellet, C.; Nanba, E.; Garcia Fernandez, J. M.; Suzuki, Y.; Shimizu, T. J. Biol. Chem.
2014, 289, 14560-14568.
41.
42.
Front, S.; Gallienne, E.; Charollais-Thoenig, J.; Demotz, S.; Martin, O. ChemMedChem
2016, 11, 133-141.
Ichikawa, Y.; Igarashi, Y.; Ichikawa, M.; Suhara, Y. J. Am. Chem. Soc. 1998, 120, 3007-
3018.

25
ACCEPTED MANUSCRIPT
43.
44.
45.
Thonhofer, M.; Weber, P.; Gonzalez Santana, A.; Tysoe, C.; Fischer, R.; Pabst, B. M.;
Paschke, E.; Schalli, M.; Stütz, A. E.; Tschernutter, M.; Windischhofer, W.; Withers, S.
G. Carbohydr. Res. 2016, 429, 71-80.
Front, S.; Biela-Banas, A.; Burda, P.; Ballhausen, D.; Higaki, K.; Caciotti, A.; Morrone,
A.; Charollais-Thoenig, J.; Galliene, E.; Demotz, S.; Martin, O. R. Eur. J. Med. Chem.
2017, 126, 160-170.
Roscales, S.; Plumet, J. Int. J. Carbohydr. Chem. 2016, ID 4760548,
http://dx.doi.org/10.1155/2016/4760548
46.
47.
48.
Lahiri, R.; Ansari, A. A.; Vankar, Y. D. Chem. Soc. Rev. 2013, 42, 5102-5118.
Arjona, O.; Lopez, J. C.; Plumet, Chem. Rev. 2007, 107, 1919-2038.
Miller, T. W.; Arison, B. H.; Albers Schonberg, G. Biotechn. Bioeng. 1973, 15, 1075-
1080.
49.
Nakayama, T.; Amachi, T.; Murao, S.; Sakai, T.; Shin, T.; Kenny, P. T. M.; Iwashita, T.;
Zagorski, M.; Komura, H.; Nomoto, K. Chem. Commun. 1991, 14, 919-921.
Kobayashi, Y. Carbohydr. Res. 1999, 315, 3-15.
50.
51.
Sakuda, S.; Isogai, A.; Matsumoto, S.; Suzuki, A.; Koseki, K. Tetrahedron Lett. 1986, 27,
2475-2478.
52.
53.
Hayashi, M.; Yainuma, S.; Yoshioka, H, Nakatsu, K. J. Antibiot. 1981, 34, 675-680.
Kishi, T.; Muroi, M.; Kusaka, T.; Nishikawa, M.; Kamiya, K.; Mizuno, K. Chem. Pharm.
Bull. 1972, 20, 940-946.
54.
Kuno, S.; Higaki, K.; Takahashi, A.; Nanba, E.; Ogawa, S. Med. Chem. Commun. 2015,
6, 306-310.
55. a) Leroy, E.; Reymond, J.-L. Org. Lett. 1999, 1, 775-777; b) Greul, J. N.; Kleban, M.;
Schneider, B.; Picasso, S.; Jäger, V. ChemBioChem 2001, 2, 368-370; c) Gartenmann
Dickson, L.; Leroy, E.; Reymond, J.-L. Org. Biomol. Chem. 2004, 2, 1217-1226.

26
ACCEPTED MANUSCRIPT
56. Bernet, B.; Vasella, A. Helv. Chim. Acta 1979, 62, 1990-2016.
57. Kleban, M.; Kautz, U.; Greul, J.; Hilgers, P.; Kugler, R.; Dong, H.-Q.; Jäger, V. Synthesis
2000, 1027-1033.
58. Gallos, J. K.; Koumbis, A. E.; Xiraphaki, V. P.; Dellios, C. C.; Coutouli-Argyropoulou
Tetrahedron 1999, 55, 15167-15180.
59
Suzuki, Y.; Ogawa, S.; Sakakibara, Y. Perspect. Med. Chem. 2009, 3, 7-19.
Bruker (2007). SMART. Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2001). SADABS. Bruker AXS Inc., Madison, Wisconsin, USA.
Sheldrick, G. M. Acta Crystallogr. 2015, A71, 3–8.
60.
61.
62.
63.
64.
65.
66.
67.
68.
Spek, A. L. J. Appl. Crystallogr. 2003, 36, 7-13.
Spek, A. L. Acta Crystallogr. Sect. D 2009, 65, 148-155.
Chen, H.-M.; Withers, S.G. ChemBioChem 2007, 8, 719-722.
Prade, H.; Mackenzie, L. F.; Withers, S. G. Carbohydr. Res. 1998, 305, 371-381.
Kempton, J. B.; Withers, S. G. Biochem. 1992, 31, 9961-9969.
Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193,
265-275.

ACCEPTED MANUSCRIPT
A simple, short and high yielding route enables access to amino(hydroxymethyl)pentanetriols.
Additional modifications are available by simple 1-N-alkylation.
Final products are potent inhibitors of a panel of β-galactosidases.
New compounds act as efficient experimental pharmacological chaperones for GM1-gangliosidosis-
related R201C mutant of human lysosomal β-galactosidase.