Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: Notable β-galactosidase inhibitors and activity promotors of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C

Article abstract of DOI:10.1016/j.carres.2016.03.020

From an easily available partially protected analog of 1-deoxy-L-gulo-nojirimycin, by chain-branching at C-4 and suitable modification, lipophilic analogs of the powerful β-D-galactosidase inhibitor 4-epi-isofagomine have been prepared. New compounds exhibit considerably improved inhibitory activities when compared with the unsubstituted parent compound and may serve as leads toward new pharmacological chaperones for G_M1-gangliosidosis and Morquio B disease.

Full text of DOI:10.1016/j.carres.2016.03.020

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Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine:
notable β-galactosidase inhibitors and activity promotors of
GM1-gangliosidosis related human lysosomal β-galactosidase
mutant R201C
a
a
b
b
Martin Thonhofer , Patrick Weber , Andres Gonzalez Santana , Christina Tysoe ,
c
d
d
a
a,
Roland Fischer , Bettina M. Pabst , Eduard Paschke , Michael Schalli , Arnold E. Stütz *,
d
d
b
Marion Tschernutter , Werner Windischhofer , Stephen G. Withers
a
Glycogroup, Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria
Chemistry Department, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1
Institute of Inorganic Chemistry, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria
Department of Pediatrics, Laboratory of Metabolic Diseases, MedUni Graz, Auenbruggerplatz 30, A-8036 Graz, Austria
b
c
d
A R T I C L E
I N F O
A B S T R A C T
Article history:
From an easily available partially protected analog of 1-deoxy-L-gulo-nojirimycin, by chain-branching
at C-4 and suitable modification, lipophilic analogs of the powerful β-D-galactosidase inhibitor 4-epi-
isofagomine have been prepared. New compounds exhibit considerably improved inhibitory activities
when compared with the unsubstituted parent compound and may serve as leads toward new pharma-
cological chaperones for GM1-gangliosidosis and Morquio B disease.
Received 8 February 2016
Received in revised form 21 March 2016
Accepted 22 March 2016
Available online
©
2016 Elsevier Ltd. All rights reserved.
Keywords:
Iminoalditol
4
-Epi-isofagomine
Galactosidase inhibitor
Pharmacological chaperone
GM1-gangliosidosis
1
. Introduction
These glycosides consequently accumulate in the cells and lead to
irreversible damage of nerve tissue, bones as well as various organs
resulting in painful long-term suffering of patients and their fami-
lies without the hope for a cure.
Imino sugars in general and, in particular, iminoalditols, such as
1
-deoxynojirimycin (1) and its epimer at C-4 (2) as well as bicy-
clic analog castanospermine (3) are typical examples of powerful
Efforts of many groups have shown that imino sugar- as well as
carba sugar-based glycomimetics may provide suitable therapeu-
tic agents that function either by inhibiting upstream enzymes and,
thus, by reducing the production of metabolites that cannot be de-
graded 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 concentra-
tions of active site specific molecules (pharmacological chaperones)
inhibitors of glycoside hydrolases, and as such have proved to be
1
–5
valuable tools for enzymatic investigation and characterization.
Due to the vital roles of these enzymes, specific inhibitors may exert
notable biological activities against various pathological pro-
6–9
cesses, including certain forms of hereditary lysosomal disorders.
This latter group of about 50 metabolic diseases arises from mu-
tations in specific genes that lead to deficiencies in enzymes involved
in the lysosomal degradation of glycolipids and glycans. Consider-
able 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.
6
could be exploited. Whereas 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
coordinate and stabilize such mutant enzymes including lyso-
somal β-glucosidase, β-galactosidase or β-N-acetylhexosaminidase
in their functional folded conformations thus supporting their exit
from the endoplasmic reticulum and their subsequent transport to
the lysosome.
*
Corresponding author. Glycogroup, Institut für Organische Chemie, Technische
Universität Graz, Stremayrgasse 9, A-8010 Graz, Austria. Tel.: +0043 316 873 32430;
fax: +0043 316 873 32402.
E-mail address: stuetz@tugraz.at (A.E. Stütz).
http://dx.doi.org/10.1016/j.carres.2016.03.020
0008-6215/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Martin Thonhofer, et al., Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable β-galactosidase inhibitors and activity promotors
of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C, Carbohydrate Research (2016), doi: 10.1016/j.carres.2016.03.020

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M. Thonhofer et al./Carbohydrate Research ■■ (2016) ■■–■■
Guiding contributions by various leaders in the field and recent
inhibitors have been investigated as potential pharmacological
10,11
reviews
have shown that N-substituted iminoalditols and sim-
chaperones thus far. The first compound, carba sugar NOEV (N-
octyl-epi-valienamine, 8), turned out a powerful inhibitor18 and
even at sub-micromolar concentrations a highly potent pharma-
cological chaperone for a wide range of mutants of lysosomal
ilarly 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
1
9
β-galactosidase. More recently, selected unbranched analogs
lacking the hydroxymethyl group have been presented and their
activities with patients’ skin fibroblasts carrying the R201C muta-
9
studied iminosugars, thus far. Other researchers such as Wong and
his group as well as Aerts and Overkleeft and their co-workers13
have observed that large, lipophilic N-alkyl substituents, in partic-
ular adamantyl capped spacer arms, greatly improve the interaction
between iminoalditol derivatives and the lysosomal β-glucosidase
12
20
tion were reported. Other potential chaperones were structurally
based on compound 2 which by various types of structural modi-
fications could be transformed into a relatively small number
of typical derivatives (10–16, in case of several N-substituent-
variations in the same publication, only the most active is depicted)
with interesting properties as experimental pharmacological
(
Fig. 1).
Whereas compounds 1–3 have been found in nature, the so-
called isoimino sugars invented by Lundt14 and Bols and their
groups in 1994 are not natural products. They were designed on the
hypothesis that sugar related compounds that can stabilize posi-
tive charge at the (formal) anomeric position may be interesting
glycosidase inhibitors. Indeed, 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, intro-
duced by Fan and co-workers,16 turned out even more powerful.
Other chain-extended derivatives 6 and 7 were synthesized and in-
vestigated by Withers and collaborators.17
15
2
1–28
chaperones.
Potentially interesting derivatives of the isoiminosugar
2
9
4-epi-isofagomine (17), whose chaperoning activity was only re-
28
cently reported, for the same purpose have been lacking. To date,
only 5a-C-methyl analog 18 has been prepared (in different context)
3
0
by a de-novo approach.
Motivated by the early encouraging results with our 1-deoxy-
2
5,26,31
D-galacto-nojirimycin derivatives
we became interested in
extending our collection of experimental chaperone candidates for
For lysosomal β-galactosidase associated with GM1-gangliosidosis
and Morquio B disease, only two distinct types of β-D-galactosidase
G
M1-gangliosidosis and have recently targeted C-5a-modifications
3
2
of parent compound 17.
HO
R3
HO
R3
R2
R1
HO
R1
R2
R4
N
N
HO
NH
HO
OH
HO
OH
HO
1
2
3
1
2
3
4
1
2
R = H
R = H
R = OH
3
4 R = OH R = H
R = H
R = H
1
2
3
1
2
3
4
R = H
R = OH
R = H
5 R = OH R = H
R = H
R = C H
9
19
1
2
3
1
2
3
4
1
1
2 R = C H
R = OH
R = H
6 R = OH R = H
R = H
R = (CH ) CH OH
4
9
2 2
2
1
2
3
1
2
3
4
3 R = C H
R = OH
R = H
7 R = OH R = H
R = C H R = C H
9
19
3
7
3
7
1
2
3
4
1
1
7 R = H
R = OH R = H
R = H
1
2
3
4
8 R = H
R = OH R = CH R = H
3
O
HO
HO
2
R = OH
MeO
NH
4
3
R = H
1
1
4 R =
NHoctyl
HO
NH
O
3
NHdansyl
HO
OH
HO
OH
2
R = OH
R = H
3
8
9
1
O
1
1
5 R =
R2
C(CF3)3
S
6
Nbutyl
2
N
R1
HO
NH
R = OH
1
2
1
1
1
R = phenyl R = OH
S
3
R = H
HO
1
2
1a R = C H
R = H
4
9
1
6 R =
NHbutyl
HO
OH
HO
OH
1
0
Fig. 1. Glucosidase and galactosidase inhibitors as potential therapeutic principles for lysosomal deficiencies of d-glucosidase and d-galactosidase, respectively.
Please cite this article in press as: Martin Thonhofer, et al., Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable β-galactosidase inhibitors and activity promotors
of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C, Carbohydrate Research (2016), doi: 10.1016/j.carres.2016.03.020

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2
. Results and discussion
To gain regioselective access to C-5a-modifications, the primary
hydroxyl group 26 was protected as methoxymethylene acetal 32
followed by fluoride catalyzed cleavage of the silyl ether to provide
alcohol 33. X-Ray crystallography unambiguously revealed the iden-
tity of this key intermediate (CCDC 1465279) (Picture 1).
2.1. Synthesis
Starting from known 5-aminodeoxy septanuronic ester 19 (easily
available from D-mannose in six steps), by reduction of the ester
moiety (20) and subsequent catalytic hydrogenation/concomitant
intramolecular reductive amination, partially protected 6-homo de-
rivative 21 of 1-deoxy-L-gulo-nojirimycin was prepared and
N-protected (22). Regioselective O-silylation at C-7 (23) followed
by oxidation of C-4 provided ulose 24. Its reaction with methy-
lene triphenyl phosphorane or other one-carbon Wittig reagents such
as methoxymethylene triphenyl phosphorane invariantly was met
with failure to provide the desired branched chain compound due
to pronounced enolization tendency of the carbonyl group. The same
was true for attempted Peterson olefination approaches with
By oxidation to aldehyde 34 and subsequent chain extension, C
as well as C chains featuring terminal nitrile groups (compounds
4
6
35 and 36, respectively) were formed. From these, the correspond-
ing primary amines 37 and 38, respectively, were liberated by
catalytic hydrogenation over Raney-Ni en route to the desired
N-dansyl derivatives 39 and 40 which upon acidic deprotection pro-
2
5,34
vided “glycogroup-typical”
free inhibitors 41 and 42.
2.2. D-Galactosidase inhibition
Inhibitory activities of free iminosugars 28, 29, 31, 41 as well as
42 in comparison with unsubstituted parent compound 17 as well
as bench mark molecule 8 are summarized in Table 1. In all cases
of confirmed activity, the inhibition was competitive.
(
trimethylsilyl)methyl magnesium chloride under all conditions
probed. Cerium chloride modification of the (trimethylsilyl)methyl
3
3
Grignard reagent following a particular protocol by Li and Yang
and subsequent spontaneous elimination of trimethyl silanol during
attempted chromatography of the reaction mixture finally gave
desired exo-methylene compound 25, but only in inacceptable yields
Interestingly, branched iminotetraol 29 was found to be a good
inhibitor of β-D-galactosidases. Epimer 28 was practically devoid
of inhibitory activity against the enzymes probed. 6-Deoxygenation
(derivative 31) mainly reduced the activity against E. coli
β-galactosidase, whereas inhibitory power with the other two
β-galactosidases probed was practically retained when compared
to parent 29. α-Galactosidase Fabrazyme was not inhibited by any
of these new compounds.
(23%) (Scheme 1).
Despite all other efforts made, in our hands, only the Tebbe re-
action allowed convenient access to the desired crucial intermediate
5 in fair yield. Stereoselective hydroboration led to the 4-epi
2
isofagomine derivative 26 in 51% yield. Due to steric interference
of the neighboring silyloxyethyl moiety with the approach of the
borane, diastereomeric side product 27 was also formed in this re-
action (21%). For unambiguous structural assessment, compound
Gratifyingly, dansylaminoalkyl substituted 4-epi-isofagomines 41
and 42 are powerful β-galactosidase inhibitors with practically no
affinity to the α-galactosidases employed in this study.
2
7 was deprotected to free iminoalditol 28 which could be crys-
With human lysosomal β-galactosidase, polyol 29 was an, albeit
mediocre, inhibitor (IC50 = 40 μM). Compound 41 had IC50 = 1.5 μM,
homolog 42 showed IC50 = 0.38 μM.
tallized for X-ray crystallography (CCDC 1465280). Deprotection of
2
6 gave free iminosugar 29.
By catalytic hydrogenation (Pd/C 5%), 6-deoxy derivative 30
and the corresponding free compound 31 were also prepared from
compound 25 (Scheme 2).
Evaluation of inhibitor 42 as a potential chaperone for the R201C
mutant enzyme employing patients’ skin fibroblasts revealed an ef-
fective concentration range of three orders of magnitude with an
6
7
NHBn
NHBn
HO
O
O
HO
O
O
OR2
OBn
OBn
a
b
4
2
5
1
NR1
3
EtO
O
O
O
O
O
1
2
1
9
20
21 R = H
2
2
R = H
c
d
1
2
2 R = Boc
R = H
1
2
3 R = Boc
R = TBS
6
R2
1´
2´
R2
X
R1
R1
g
h
OTBS
26
27
OR3
OH
NH
e
5
3
5a
1
i
NBoc
4
NBoc
HO
O
O
3
0
O
O
HO
1
2
3
1
2
2
2
4 X = O
5 X = CH
26 R = CH OH R = H
R = TBS
29 R = CH OH R = H
2
2
f
1
2
3
1
2
2
27 R = H
R = CH2OH R = TBS
28 R = H
R = CH2OH
1
2
3
1
2
3
0 R = CH
R = H
R = H
31 R = CH
R = H
3
3
Scheme 1. (a) LAH, THF, 94%; (b) H2, Pd(OH)2/C (20%), MeOH, 86%; (c) (Boc)2O, Et3N, MeOH, 90%; (d) TBSCl, imidazole, DMF, 83%; (e) Dess–Martin oxidation, CH2Cl2, 97%;
.
(
f) Tebbe’s reagent, THF, 94%; (g) BH3 THF, THF, 72%; (h) H2, Pd(OH)2/C (20%), MeOH, 88%; (i) HCl, MeOH, 85–92%.
Please cite this article in press as: Martin Thonhofer, et al., Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable β-galactosidase inhibitors and activity promotors
of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C, Carbohydrate Research (2016), doi: 10.1016/j.carres.2016.03.020

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1
HO
O
R O
MOMO
O
d
e
OTBS
OR2
O
3
5
6
a
c
NBoc
NBoc
O
NBoc
3
O
O
O
2
6
1
2
34
3
3
2 R = MOM R = TBS
b
1
2
3 R = MOM R = H
MOMO
O
MOMO
O
HO
HO
X
NHR
NHdansyl
f
h
n
n
NBoc
NBoc
NH
O
O
HO
3
3
5
6
X = CN
X = (CH ) CN
37 R = H
n = 2
41 n = 2
42 n = 4
g
g
39 R = dansyl n = 2
2
2
3
4
8
0
R = H
R = dansyl n = 4
n = 4
.
Scheme 2. (a) MOMCl, DIPEA, CH2Cl2, 87%; (b) TBAF 3H2O, THF/H2O, 92%; (c) Dess–Martin oxidation, 95%; (d) (EtO)2POCH2CN, t-BuOK, THF, 73%; (e) Ph3P(CH2)3CNBr, LDA,
THF, 60%; (f) H2, Raney-Ni, MeOH, 75–84%; (g) dansyl chloride, Na2CO3, MeOH; (h) HCl, MeOH, 49–59% (over 2 steps).
enhancement of 3.5-fold β-galactosidase activity at a concentra-
tion as low as 0.02 μM, 6-fold activity at 0.1 μM and a maximum
chaperone effect of 10-fold activity at 2.5 μM (Compound 8: 5.2-
fold at 2 μM; parent compound 17: 2.7-fold at 10 μM). At higher
concentrations, dose-dependent inhibition gradually reduced the
desired enhancement effect to a level of 9-fold at 12.5 μM. Com-
pound 41 was considerably less powerful but showing good activity
enhancement of around 9-fold between 20 and 100 μM (Fig. 2).
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
1
13
599.82 MHz ( H), and at 125.894 MHz ( C) or on a Bruker Ultrashield
spectrometer at 300.36 and 75.53 MHz, respectively. CDCl was em-
ployed for protected compounds and methanol-d or D O for
3
3
. Conclusions
4
2
unprotected inhibitors. Chemical shifts are listed in delta employ-
ing residual, non-deuterated solvent as the internal standard. Signals
were assigned unambiguously by COSY, HSQC as well as APT anal-
ysis. 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
iminoalditol. For crucial intermediates, structures were confirmed
by XRD structural analysis. MALDI-TOF Mass Spectrometry was
performed on a Micromass TofSpec 2E Time-of-Flight Mass Spec-
trometer. Analytical TLC was performed on precoated aluminum
plates silica gel 60 F254 (E. Merck 5554) and detected with UV light
Following a simple protocol, a series of novel derivatives of 4-epi-
isofagomine, bearing chain extensions at C-5a, have been made
available and were screened with a range of D-galactosidases in-
cluding human lysosomal β-galactosidase. Two new inhibitors,
compounds 41 and 42, turned out highly potent and exhibited in-
teresting dose dependent chaperoning profiles at low concentrations
with enzyme mutant R201H. Results obviously merit screening for
potential chaperone activity with other frequent mutants of this
enzyme. Screening with selected Morquio B cell lines is currently
in progress.
.
Picture 1. Crystal structures of compounds 28 HCl (CCDC 1465280) and 33 (CCDC 1465279).
Please cite this article in press as: Martin Thonhofer, et al., Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable β-galactosidase inhibitors and activity promotors
of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C, Carbohydrate Research (2016), doi: 10.1016/j.carres.2016.03.020

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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; GCB = α-galactosidase from green coffee beans
Compounds
Enzyme
H
N
OH
OH
OH
NHdansyl
2
NHdansyl
4
NHoctyl
OH
H
N
H
N
H
N
OH
H
N
H
N
OH
OH
OH
OH
OH H C
OH
3
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
17
28
29
31
41
42
8
ABG
E. coli
Bovine Liver
GCB
Fabrazyme
β-Gal human lys (IC50)
0.29
0.031
10
140
N.I.
927
147.5
N.I.
n.d.
N.I.
2.9
3.6
0.049
0.0037
0.151
848
N.I.
2.67
0.0175
0.0021
0.0014
n.d.
N.I.
0.38
—
—
0.106
4.89
n.d.
N.I.
305
6.57
n.d.
N.I.
n.d.
0.87 (ref. 35)
3.1 (ref. 35)
—
99.7 (ref. 36)
n.d.
30
0.125 (ref. 37)
Fabrazyme = commercial recombinant lysosomal α-galactosidase; IC50 [μM] with β-Gal (human lysosomal); N.I. = no inhibition, with Ki > 2 mM; n.d., not determined.
(
H
254 nm). For staining, a solution of vanillin (9 g) in a mixture of
O (950 mL)/EtOH (750 mL)/H SO (120 mL) or ceric ammonium
molybdate (100 g ammonium molybdate/8 g ceric sulfate in
L 10% H SO ) was employed followed by heating on a hotplate.
minutes before initiating the reaction by the addition of sub-
strate. The initial reaction rate was measured by monitoring the
increase in absorbance at 400 nm for up to ten minutes. K deter-
i
minations were performed using at least two different substrate
concentrations. For each inhibitor, a range of four to six inhibitor
i
concentrations bracketing the K value ultimately determined was
used for each substrate concentration. Dixon plots (1/v vs [I]) were
constructed to validate the use of competitive inhibition model. The
data were then fit using non-linear regression analysis with Grafit
2
2
4
1
2
4
For column chromatography, silica gel 60 (230–400 mesh,
E. Merck 9385) or silica gel 60 (Acros Organics, AC 24036) was
used.
4
.2. Kinetic studies
7
.0.
N.I. stands for no inhibition or weak inhibition with an esti-
mated K value higher than 2 mM.
Kinetic studies were performed at 37 °C in an appropriate buffer
using a known concentration of enzyme (specific conditions de-
picted below). K determinations were performed using the
i
i
Specific assay conditions for each enzyme:
corresponding 4-nitrophenyl α- or β-D-galactopyranoside as
substrate, except for Fabrazyme, where 2,4-dinitrophenyl
α-galactopyranoside was employed. In a typical assay, the enzyme
was incubated with different inhibitor concentrations for up to 5
Agrobacterium sp. β-glucosidase:39,40 50 mM sodium phos-
phate buffer (pH 7) using 1.85 × 10 mg/mL of enzyme
(K = 4.1 mM).
m
38
−4
Fig. 2. Chaperoning activities of inhibitors 41 (black) and 42 (red) in comparison with values of NOEV (8, ref. 18) and parent 4-epi-isofagomine (17, ref. 36) as activity en-
hancement at given concentrations.
Please cite this article in press as: Martin Thonhofer, et al., Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable β-galactosidase inhibitors and activity promotors
of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C, Carbohydrate Research (2016), doi: 10.1016/j.carres.2016.03.020

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E. coli lac z β-galactosidase: 50 mM sodium phosphate, 1.0 mM
MgCl (pH 7) using 6.4 × 10 mg/mL of enzyme (K = 60 μM).
2 m
4.3. General procedures
−
4
Bovine liver β-galactosidase: 50 mM sodium phosphate buffer
4.3.1. General procedure A
pH 7) using 4.9 × 10− mg/mL of enzyme (K
2
= 0.65 mM).
The respective starting material was dissolved in MeOH and HCl
concd was added dropwise to adjust to pH 1. After completed
removal of all protecting groups, the solvents were removed under
reduced pressure. Subsequent purification on silica gel provided the
respective desired title compound.
(
m
Green Coffee Bean α-galactosidase: 50 mM sodium citrate buffer
−
3
(
pH 6.5) using 4.6 × 10 mg/mL of enzyme (K
m
= 0.56 mM).
Fabrazyme (Acid α-galactosidase): 20 mM sodium citrate,
5
0 mM sodium phosphate, 1.0 mM tetrasodium EDTA, 0.25% v/v
®
Triton X-100 and 0.25% w/v taurocholic acid buffer (pH 5.5) using
5
× 10− mg/mL of enzyme (K
5
= 0.65 mM).
4.3.2. General procedure B
m
Human β-galactosidase and β-hexosaminidase activity mea-
surements 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
2 2
To a 10% solution of the respective alcohol in CH Cl , Dess–
Martin periodinane (1.1–1.2 equiv) was added. After completed
conversion of the starting material, the reaction was carefully
3
quenched with saturated aq NaHCO . The organic layer was dried
(
1
2 × 10 sec, Branson Sonifier II, W-250) and centrifuged at
3,000 rpm for 2 min in a table top centrifuge (Biofuge Pico,
Heraeus). Protein amounts were determined according to the
(Na SO ), filtered and concentrated under reduced pressure. Puri-
fication of the remaining residue by column chromatography on silica
gel afforded the respective oxidation product.
2
4
4
1
method of Lowry.
4.3.3. General procedure C
For assessment of β-Gal activity, 20 μL of cell homogenate were
A 10% solution of the respective nitrile in MeOH was stirred with
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% NaN . After incubation at 37 °C
3
for 30 min, the reaction was stopped by adding 2.5 ml 400 mM
glycine/NaOH (pH 10.4).
2 2
Raney-Ni (moist in H O) under an atmosphere of H at ambient pres-
sure for 30–60 min. After complete conversion of the starting
material, the catalyst was filtered off. Removal of the solvent under
reduced pressure and subsequent chromatography on silica gel af-
forded the respective desired amine.
β-Hexosaminidase activity was measured by adding
1
4
0 μL homogenate to 90 μL 0.9% NaCl and 100 μL of 1 mM
-methylumbelliferyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich)
4.3.4. General procedure D
2 3
To a suspension of the respective amine and Na CO (2.5–3.0
in 100 mM citrate buffer (pH 4.6) containing 0.2% BSA and 0.04%
NaN . 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 hy-
drolyzed 4-methylumbelliferone was determined with a fluorescence
spectrometer (Hitachi F7000).
equiv) in MeOH, dansyl chloride (1.2 equiv) was added. After com-
pleted conversion of the starting material, the reaction mixture was
evaporated to dryness. The remaining residue was quickly passed
through silica gel to provide the crude product which was imme-
diately taken to the next step.
3
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.
4.4. Benzyl N-benzyl-5-amino-5,6-dideoxy-2,3-O-isopropylidene-α-
L-gulo-heptofuranoside (20)
4
To a 10% suspension of LiAlH (0.68 g, 18.00 mmol) in dry THF,
a 3% solution of ester 19 (5.3 g, 12.00 mmol) in dry THF was added
dropwise at −0 °C. After completed conversion of the starting ma-
2
terial (5–15 min), the reaction was quenched with H O (3 mL). By
addition of 10 mL NaOH (3 N) and stirring for further 60 min at
ambient temperature aluminum salts were precipitated and could
4.2.1. Patients and cell lines
Cell lines from one GM1-Gangliosidosis-patient (R201C) and one
2 4
be removed by filtration. The filtrate was dried (Na SO ), filtered and
WT were exposed to compounds 29, 41 as well as 42 for evalua-
tion of their chaperone effects.
concentrated under reduced pressure. The resulting syrup was pu-
rified by flash column chromatography (C/EE 1:2 v/v) to give alcohol
20
1
Human skin fibroblasts were grown in minimal essential medium
20 (4.5 g, 11.26 mmol, 93.8%).
(300 MHz, MeOH-d
2,3 6.0 Hz, J3,4 3.6 Hz, H-3), 4.49 (dd, 1H, H-2), 3.63 (m, 2H, 2× H-7),
3.53 (dd, 1H, J4,5 8.8 Hz, H-4), 3.11 (ddd, 1H, J5,6a 4 Hz, J5,6b 7.5 Hz,
[
α
]
=−20.3 (c 2.02, MeOH); H NMR
D
(MEM) with Earle’s Salts (SIGMA) containing 10% fetal bovine serum,
4
): δ = 4.72 (d, 1H, J1,2 4.2 Hz, H-1), 4.60 (dd, 1H,
4
00 μM L-glutamine, and 50 μg/mL gentamycin at 37 °C and 5% CO
2
.
J
All cells used in this study were between the third and nineteenth
passages.
1
3
H-5), 1.80–1.53 (bm, 2H, H-6a, H-6b); C NMR (75.5 MHz, MeOH-
): δ = 140.6, 139.1 (ipso NBn, ipso OBn), 129.7–128.3 (aromatic OBn,
NBn), 114.4 [C(CH ], 103.4 (C-1), 81.1, 81.0, 80.1 (C-2, C-3, C-4), 73.0
(OCH Ph), 61.2 (C-7), 56.6 (C-5), 52.1 (NCH
[(CH
Potential chaperones were dissolved in DMSO and diluted in
d
4
10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl, 0.01%
3 2
)
NaN , and 0.01% Triton for the IC50-measurements and in MEM for
3
2
2
Ph), 32.8 (C-6), 26.2, 25.6
H]: m/z 414.2281[M + H] ; Found
+
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.
3
)
2
C]. MS: Calcd for [C20
H
31NO
5
+
[M + H] 414.2273; MS: Calcd for [C20
[M + Na] ; Found [M + Na] 436.2107.
H
31NO
5
Na]: m/z 436.2100
+
+
4.5. 1,5,6-Trideoxy-1,5-imino-2,3-O-isopropylidene-L-gulo-heptitol
4.2.2. In-vivo treatment of cultured fibroblasts
(21)
Fibroblasts were grown to semi-confluency in 6-well plates. Par-
ticular chaperone was added to the culture medium at following
concentrations: 41: 100 μmol to 5 μmol; 42: 12.5 μmol to 0.02 μmol.
Cells were incubated for four additional days at 37 °C. Cells were
harvested by scraping and prepared for β-galactosidase and
β-hexosaminidase assays as described above.
A solution of Compound 20 (4.5 g, 11.26 mmol) in MeOH (15 mL)
was stirred with Pd(OH) /C under an atmosphere of H at ambient
pressure overnight. After completed conversion of the starting ma-
terial (indicated by TLC), the catalyst was filtered off and the solvent
was removed under reduced pressure. Chromatography on silica gel
2
2
Please cite this article in press as: Martin Thonhofer, et al., Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable β-galactosidase inhibitors and activity promotors
of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C, Carbohydrate Research (2016), doi: 10.1016/j.carres.2016.03.020

ARTICLE IN PRESS
M. Thonhofer et al./Carbohydrate Research ■■ (2016) ■■–■■
7
(
CHCl
3
/MeOH/concd NH
4
OH 8:4:0.01 v/v/v) afforded compound 21
2× H-6); 13C NMR (75.5 MHz, CDCl
Boc), 111.7 [C(CH ], 80.7 [OC(CH
7), 59.1 (C-2), 42.2 (C-1), 32.8 (C-6), 28.5 [OC(CH
[(CH C], 26.1, 18.43, −5.2, −5.3 (TBS); MS: Calcd for [C21
m/z 452.2444 [M + Na] ; Found [M + Na] 452.2544.
3
): δ = 204.8 (C-4), 155.2 (C
=
O,
20
(2.1 g, 9.66 mmol, 85.8%) as a colorless syrup.
[
α
]
=−28.4 (c 1.12,
)
3 2
3
)
3
], 76.7, 76.6 (C-3, C-2), 59.7 (C-
], 26.6, 25.0
SiNa]:
D
1
MeOH); H NMR (300 MHz, MeOH-d
4
): δ = 4.30 (ddd, 1H, J1,2ax 6.3 Hz,
3 3
)
J1,2eq 4.8 Hz, J2,3 6 Hz, H-2), 4.18 (dd, 1H, J3,4 3 Hz, H-3), 3.80 (dd, 1H,
3
)
2
H39NO
6
+
+
J
4,5 3 Hz, H-4), 3.68 (m, 2H, 2× H-7), 3.25–3.13 (m, 2H, H-1eq, H-5),
1
3
2
.88 (dd, 1H, J1ax,1eq 13.5 Hz, H-1ax), 1.77 (m, 2H, 2× H-6); C NMR
): δ = 110.3 [C(CH ], 76.9 (C-3), 71.4 (C-2), 68.5
C-4), 59.98 (C-7), 52.6 (C-5), 45.4 (C-1), 33.8 (C-6), 27.9, 25.7
(
(
[
75.5 MHz, MeOH-d
4
3 2
)
4.9. N-tert-Butyloxycarbonyl-7-O-tert-butyldimethylsilyl-1,5,6-
trideoxy-1,5-imino-2,3-O-isopropylidene-4-C-methylene-L-ribo-
heptitol (25)
+
(CH
3
)
2
C]; MS: Calcd for [C10
H
19NO
4
Na]: m/z 240.1212 [M + Na] ;
+
Found [M + Na] 240.1225.
To a solution of compound 24 (964 mg, 2.24 mmol) in dry THF
45 mL), Tebbe’s reagent (0.5 M in toluene, 4.9 mL, 2.45 mmol) was
added under an atmosphere of nitrogen at −25 °C. After 120 min
at −20 °C, the reaction was stirred at ambient temp for another 12 h.
2
The reaction mixture was diluted with Et O (80 mL), and NaOH (1.5
N, 1.94 mL, 2.91 mmol) was added dropwise. After vigorous stir-
ring for further 12 h, the mixture was filtered. The filtrate was
2 4
washed with water and the organic layer was dried (Na SO ). The
4
.6. N-tert-Butyloxycarbonyl-1,5,6-trideoxy-1,5-imino-2,3-O-
(
isopropylidene-L-gulo-heptitol (22)
To a suspension of compound 21 (2.1 g, 9.66 mmol) in MeOH
(
8 mL) and Et
was added and the mixture was stirred for 8 h. When TLC (CHCl
MeOH/concd NH OH 3:1:0.01 v/v/v) indicated full conversion of the
starting material, the suspension was concentrated and the remain-
ing residue was diluted with CH Cl and consecutively washed with
HCl (6%) and saturated aq NaHCO . The combined organic layers were
dried (Na SO ), filtered and evaporated to dryness. Chromatograph-
ic purification on silica gel (C/EE 1:1 v/v) gave compound 22 (2.75 g,
3 2
N (4.0 mL, 28.99 mmol), (Boc) O (2.5 g, 11.59 mmol)
3
/
4
solvent was removed under reduced pressure and the residue was
purified by flash column chromatography (silica gel, C/EE 8:1 v/v)
2
2
3
2
D
0
to give 25 (897 mg, 2.09 mmol, 93.5%).
[
α
]
= +9.0 (c 4.33, CHCl
3
);
2
4
1
3
H NMR (300 MHz, CDCl ): δ = 5.30, 5.19 (2s, 2H, 2× exo-methylene),
4.72 (bs, 1H, H-5), 4.64 (d, 1H, J2,3 7.1 Hz, H-3), 4.25 (bs, 1H, H-2),
4.05 (m, 1H, H-1eq), 3.75-3.57 (2H, 2× H-7), 2.82 (m, 1H, H-1ax),
2
D
0
8
CHCl
4
3
.66 mmol, 89.7%) as a pale yellow syrup.
[
α
]
= +25.3 (c 4.75,
1
3
); H NMR (300 MHz, MeOH-d
4
): δ = 4.18 (m, 2H, H-2, H-3),
1
3
2
(
.09–1.76 (m, 2H, 2× H-6); C NMR (75.5 MHz, CDCl
O, Boc), 143.0 (C-4), 116.0 (exo-methylene), 109.5 [C(CH
79.9, 79.6 [OC(CH ], 76.9, 75.3 (C-2, C-3), 60.7, 60.4 (C-7), 52.5, 51.7
C-5), 43.4, 42.4 (C-1), 35.4, 35.2 (C-6), 28.5 [OC(CH ], 26.1 (TBS),
6.7, 24.9 [(CH C], 18.4, −5.2 (TBS); MS: Calcd for [C22 SiNa]:
m/z 450.2652 [M + Na] ; Found [M + Na] 450.2767.
3
): δ = 155.6
.00 (dd, 1H, J1ax/1eq 14.Hz, J1eq,2 2.8 Hz, H-1eq), 3.95 (m, 2H, H-5),
.88-3.83 (m, 1H, H-4), 3.58-3.40 (m, 2H, 2× H-7), 3.28–3.08 (m, 1H,
C
=
3 2
) ],
1
3
3 3
)
H-1ax), 2.00–1.60 (m, 2H, 2× H-6); C NMR (75.5 MHz, MeOH-
): δ = 158.0, (C O, Boc), 110.3 [C(CH ], 81.2, 81.1 [OC(CH ], 76.6,
6.2, 77.5, 77.4 (C-2, C-3), 68.2 (C-4), 59.8 (C-7), 53.0, 51.9 (C-5), 52.6,
(
2
3 3
)
d
4
7
4
=
3
)
2
3 3
)
)
3 2
H41NO
5
+
+
1.5 (C-1), 31.8, 30.7 (C-6), 28.7 [OC(CH
3
)
3
], 27.3, 24.9 [(CH
3
)
2
C]; MS:
+
+
Calcd for [C15
H
27NO
6
Na]: m/z 340.1736 [M + Na] ; Found [M + Na]
3
40.1731.
4.10. (5aS)-N-tert-Butyloxycarbonyl-3,4-O-isopropylidene-5a-C-
(
tert-butyldimethylsilyloxy)ethyl-4-epi-isofagomine (26) and (5aS)-
4
.7. N-tert-Butyloxycarbonyl-7-O-tert.butyldimethylsilyl-1,5,6-
N-tert-Butyloxycarbonyl-3,4-O-isopropylidene-5a-C-(tert-
butyldimethylsilyloxy)ethyl-4,5-di-epi-isofagomine (27)
trideoxy-1,5-imino-2,3-O-isopropylidene-L-gulo-heptitol (23)
To a 20% solution of compound 22 (2.75 g, 8.66 mmol) in dry DMF,
imidazole (1.77 g, 25.99 mmol) and TBSCl (1.40g, 9.53 mmol) were
added at ambient temperature. After full conversion of the start-
ing material (indicated by TLC) the reaction was quenched with
MeOH (15 mL) and the solvent was removed under reduced pres-
To a stirred solution of compound 25 (453 mg, 1.06 mmol) in dry
.
THF (4 mL), BH
3
THF (1 M, 5.3 mL) was added dropwise at 0 °C under
an atmosphere of nitrogen. After stirring for 12 h, the reaction was
allowed to reach ambient temperature. To the ice cooled reaction
mixture, H
added and the mixture was stirred for additional 8 h. The organic
layer was separated and dried (Na SO ) and was filtered, and the
2 2 2
O (5 mL), NaOH (3 M, 8 mL) and H O (33%, 8 mL) were
sure. The residue was diluted with CH
with HCl (6%) and saturated NaHCO . The combined organic layers
were dried (Na SO ), filtered and concentrated. Chromatography on
silica gel (C/EE 10:1 v/v) provided compound 23 (3.1 g, 7.18 mmol,
2 2
Cl and consecutively washed
3
2
4
2
4
solvent was evaporated to dryness. Purification of the residue on
silica gel (C/EE 15:1 v/v) afforded alcohols 26 (241 mg, 0.54 mmol,
51.0%) and 27 (101 mg, 0.23 mmol, 21.4%). Compound 26:
2
D
0
1
82.9%) as a pale colorless syrup.
[
α
]
= +53.5 (c 0.94, CHCl
3
); H NMR
(
2
300 MHz, CDCl
3
): δ = 4.48–4.35 (m, 2H, H-2, H-3), 4.35-3.89 (m, 2H,
20
D
1
[
α
(
]
= +52.6 (c 5.32, CHCl
3
); H NMR (300 MHz, CDCl
3
): δ = 4.49
1
3
× H-7), 3.40–3.19 (m, 1H, H-1ax), 2.08–1.88 (m, 2H, 2× H-6);
C
m, 1H, H-4), 4.32–3.94 (m, 3H, H-2eq, H-3, H-5a), 3.82 (bs, 2H, 2×
H-6), 3.69 (bs, 2H, 2× H-2′), 2.94–2.48 (m, 2H, H-2ax, 6-OH), 2.05–
NMR (75.5 MHz, CDCl
3
): δ = 155.7 (C
], 73.8 (C-3), 73.1, 72.9 (C-4), 66.7, 66.6 (C-2), 60.6, 60.4
C-7), 53.3, 52.8 (C-5), 41.9, 40.9 (C-1), 32.4, 31.4 (C-6), 28.6
OC(CH ], 26.5, 24.4 [(CH C], 25.94, 18.25, −5.6 (TBS); MS: Calcd
SiNa]: m/z 454.2601 [M + Na] ; Found [M + Na]
3 2
=O, Boc), 109.3 [C(CH ) ], 79.5
[
(
[
OC(CH
3
)
3
13
1
.60 (m, 3H, H-5, 2× H1′); C NMR (75.5 MHz, CDCl
3
): δ = 155.8
], 74.6, 74.5 (C-
), 74.0, 73.8 (C-3), 63.2, 63.0 (C-6), 60.7, 60.6 (C-2′), 47.5, 47.0 (C-
a), 42.9, 41.9 (C-2), 41.1, 40.8 (C-5), 36.0, 35.7 (C-1′), 28.6 [OC(CH ],
6.2, 24.3 [(CH C], 26.0, 18.3, −5.3 (TBS). MS: Calcd for
SiNa]: m/z 468.2757 [M + Na] ; Found [M + Na] 468.3375.
3 2 3 3
(C =O, Boc), 108.9 [C(CH ) ], 79.8, 79.4 [OC(CH )
3
)
3
3 2
)
4
5
2
+
+
for [C21H41NO
6
3 3
)
4
54.2617.
3 2
)
+
+
[C
22
H
43NO
6
2
D
0
1
4.8. N-tert-Butyloxycarbonyl-7-O-tert-butyldimethylsilyl-1,5,6-
Compound 27:
CDCl ): δ = 4.45-4.17 (bs, 2H, H-2eq, H-5a), 4.16–4.02 (m, 2H, H-3,
H-4), 3.89–3.49 (m, 4H, 2× H-6, 2× H-2′), 3.24 (bs, 1H, H-2ax), 2.09
[ ]
a =−24.1 (c 1.75, CHCl
3
); H NMR (300 MHz,
trideoxy-1,5-imino-2,3-O-isopropylidene-L-ribo-hept-4-ulose (24)
3
13
Following general procedure B, alcohol 23 (1.39 g, 3.22 mmol)
was treated with Dess–Martin periodinane (1.64 g, 3.86 mmol). Pu-
rification on silica gel (C/EE 7:1 v/v) afforded 24 (1.34g, 3.12 mmol,
(m, 1H, H-5), 1.76–1.51 (m, 2H, 2× H-1′); C NMR (75.5 MHz, CDCl
δ = 155.5 (C O, Boc), 108.9 [C(CH ], 80.1 [OC(CH ], 74.4, 72.0
(C-3, C-4), 63.3 (C-6), 61.0 (C-2′), 49.1 (C-5a), 43.9 (C-5), 40.4, 39.4
(C-2), 29.7 (C-1′), 28.4 [OC(CH ], 28.3, 26.4 [(CH C], 26.0, 18.4,
−5.3, −5.4 (TBS); MS: Calcd for [C22 SiNa]: m/z 468.2757
3
):
=
3
)
2
3 3
)
2
D
0
1
9
(
6.9%) as a colorless syrup.
300 MHz, CDCl ): δ = 4.80 (bs, 1H, H-5), 4.60–4.19 (m, 3H, H-1eq,
H-2, H-3), 3.66 (m, 2H, 2× H-7), 3.08 (m, 1H, H-1ax), 1.99 (m, 2H,
[
α
]
= +24.5 (c 2.17, CHCl
3
); H NMR
3
)
3
3 2
)
3
H43NO
6
+
+
[M + Na] ;Found [M + Na] 468.2781.
Please cite this article in press as: Martin Thonhofer, et al., Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable β-galactosidase inhibitors and activity promotors
of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C, Carbohydrate Research (2016), doi: 10.1016/j.carres.2016.03.020

ARTICLE IN PRESS
8
M. Thonhofer et al./Carbohydrate Research ■■ (2016) ■■–■■
4.11. (5aS)-5a-C-(Hydroxy)ethyl-4,5-di-epi-isofagomine (28)
4.15. (5aS)-N-tert-Butyloxycarbonyl-5a-C-(tert-butyldimethylsilyloxy)
ethyl-3,4-O-isopropylidene-6-O-methoxymethylene-4-epi-
Following general procedure A, compound 27 (79 mg, 0.18 mmol)
isofagomine (32)
was converted into title compound 28, which was an amorphous
solid. Recrystallization in MeOH and Et
drops of aq HCl afforded the corresponding hydrochloride 28 HCl
2
O in the presence of a few
To a solution of compound 17 (162 mg, 0.36 mmol) and
diisopropyl ethyl amine (172 μL, 1.02 mmol) in CH Cl (5 mL),
2 2
chloromethyl methyl ether (MOMCl, 36 μL, 0.47 mmol) was added
dropwise. After completed conversion, the reaction mixture was con-
.
2
D
0
(
H
36 mg, 0.16 mmol, 89.2%) as colorless crystals.
[
α
]
=−13.6 (c 0.91,
1
2
O) (hydrochloride); H NMR (300 MHz, D
2
O, DCl): δ = 4.22 (ddd,
1
H, J2ax,3 7.9 Hz, J2eq,3 3.8 Hz, J3,4 6.8 Hz, H-3), 4.04 (dd, 1H, J4,5 3.1 Hz,
secutively washed with HCl (6%) and saturated NaHCO
(Na SO ) combined organic layers were evaporated to dryness. Chro-
matographic purification of the remaining residue on silica gel (C/
3
. The dried
H-4), 3.96 (dd, 1H, J5,6 5.4 Hz, J6,6 11.7 Hz, 1× H-6), 3.88–3.68 (m, 4H,
2
4
H-5a, 1× H-6, 2× H-2′), 3.35 (dd, 1H, J2ax,2eq 12.9 Hz, H-2eq), 3.23 (dd,
H, H-2ax), 2.35 (m, 1H, H-5), 2.05–1.93 (m, 2H, 2× H-1′); 1 C NMR
3
EE 8:1 v/v) gave 32 (155 mg, 0.32 mmol, 87.1%).
20
= +86.5 (c 1.10,
1
[
α
]
D
1
(75.5 MHz, D
2
O, DCl): δ = 67.9 (C-4), 64.1 (C-3), 59.0 (C-2′), 58.9 (C-
CDCl
3
); H NMR (300 MHz, CDCl
3
): δ = 4.48 (m, 1H, H-4), 4.20–
6
), 52.5 (C-5a), 43.0 (C-2), 40.8 (C-5), 28.0 (C-1′); MS: Calcd for
3.87 (m, 2H, H-2eq, H-3), 3.82–3.54 (m, 5H, H-5a, 2× H-6, 2× H-2′),
2.88–2.67 (m, 1H, H-2ax), 1.94–1.54 (m, 3H, H-5, 2× H-1′); C NMR
+
+
13
[
C
8
H
17NO
4
H]: m/z 192.1236 [M + H] ; Found [M + H] 192.1229.
(
75.5 MHz, CDCl
(MOM), 79.6, 79.2 [OC(CH
C-6), 60.6, 60.4 (C-2′), 55.2 (MOM), 48.3, 47.5 (C-5a), 52.9, 41.9 (C-
2), 39.9, 39.4 (C-5), 36.7, 36.3 (C-1′), 28.4 [OC(CH ], 26.2, 24.2
[(CH C], 26.0, 18.3, −5.3 (TBS); MS: Calcd for [C24 SiNa]: m/z
512.3019 [M + Na] ; Found [M + Na] 512.3071.
3
): δ = 155.4 (C
3 2
= O, Boc), 108.2 [C(CH ) ], 96.5
4.12. (5aS)-5a-C-(2-Hydroxy)ethyl-4-epi-isofagomine (29)
3 3
) ], 73.3, 73.2 (C-3), 71.3, 71.2 (C-4), 66.9
(
Following general procedure A, compound 26 (61 mg, 0.14 mmol)
3 3
)
was converted into 29. Purification on silica gel (CHCl
concd NH OH 8:4:1 v/v/v) afforded 29 (24 mg, 0.13 mmol, 91.7%)
as amorphous solid.
NMR (300 MHz, D O, DCl): δ = 4.22 (dd, 1H, J3,4 2.7 Hz, J4,5 2.5 Hz, H-4),
.94 (ddd, 1H, J2ax,3 11.7 Hz, J2eq,3 5 Hz, H-3), 3.87–3.73 (m, 3H, J5,6
3
/MeOH/
)
3 2
H47NO
7
+
+
4
20
1
[ ]
α
2
=−18.1 (c 0.73, H O) (hydrochloride); H
D
2
4
.16. (5aS)-N-tert-Butyloxycarbonyl-5a-C-(2-hydroxyl)ethyl-3,4-O-
3
5
3
1
isopropylidene-6-O-methoxymethylene-4-epi-isofagomine (33)
Hz, 1× H-6, 2× H-2′), 3.70 (dd, 1H, J5,6 7.3 Hz, J6,6 11.5 Hz, 1× H-6),
.45 (ddd, 1H, J5,5a 11.5 Hz, J5,1′ 3.2 Hz, J5a, 1′ 8.4 Hz, H-5a), 3.25 (dd,
H, J2ax,2eq 12 Hz, H-2eq), 3.12 (dd, 1H, H-2ax), 2.15–2.05 (m, 1H, 1×
.
Silyl ether 23 (98 mg, 0.20 mmol) and TBAF3H
2
O (200 mg) were
2
stirred in THF/H O (100:1 v/v) at 50 °C. When TLC indicated com-
13
H-1′), 2.03–1.92 (m, 1H, H-5), 1.86–1.71 (m, 1H, 1× H-1′); C NMR
75.5 MHz, D O, DCl): δ = 66.9 (C-4), 65.6 (C-3), 59.6 (C-6), 58.2 (C-
′), 51.7 (C-5a), 42.5 (C-5), 42.2 (C-2), 31.0 (C-1′); MS: Calcd for
pleted cleavage of the protecting group, the solvents were removed
under reduced pressure. Purification of the remaining slurry on silica
gel (C/EE 1:1 v/v) afforded the free alcohol 33 (69 mg, 0.18 mmol,
(
2
2
+
+
[
C
8
H
17NO
4
H]: m/z 192.1236 [M + H] ; Found [M + H] 192.1240.
20
D
1
9
1.7%).
[ ]
α
= +37.1 (c 1.93, CHCl ); H NMR (300 MHz, CDCl ):
3
3
δ = 4.52 (dd, 1H, J3,4 7.7 Hz, J4,5 2.4 Hz, H-4), 4.22–4.05 (m, 1H, H-3),
4
.13. (5aS)-N-tert-Butyloxycarbonyl-6-deoxy-3,4-O-isopropylidene-
3.99–3.86 (m, 2H, H-2eq, H-5a), 3.75–3.60 (m, 2H, 2× H-6), 3.58–
.40 (m, 2H, 2× H-2′), 2.73 (dd, 1H, J2ax,2eq 14.7 Hz, J2ax,3 2 Hz, H-2ax),
5a-C-(tert-butyldimethylsilyloxy)ethyl-4-epi-isofagomine (30)
3
1
.99–1.87 (m, 1H, 1× H-1′), 1.73–1.62 (m, 1H, H-5), 1.40–1.29 (m,
A solution of compound 25 (165 mg, 0.39 mmol) in MeOH
13
1
H, 1× H1′); C NMR (75.5 MHz, CDCl
3
): δ = 157.2 (C
], 73.15 (C-3), 71.4 (C-4), 67.3
C-6), 58.1 (C-2′), 55.4 (MOM), 46.5 (C-5a), 42.4 (C-2), 41.1 (C-5), 36.8
C-1′), 28.4 [OC(CH ], 26.3, 24.2 [(CH C]; MS: Calcd for
Na]: m/z 398.2155 [M + Na] ; Found [M + Na]+
=O, Boc), 108.5
(
10 mL) was stirred with Pd(OH)
2
/C (20%, ca. 50 mg), under an at-
[
(
(
[
3 2 3 3
C(CH ) ], 96.6 (MOM), 80.5 [OC(CH )
mosphere of H
2
at ambient pressure for 16 h. After completed
conversion of the starting material (indicated by TLC), the catalyst
was filtered off and the solvent was removed under reduced pres-
3
)
3
3 2
)
+
18 7
C H33NO
sure. Chromatography on silica gel (C/EE 3:1 v/v) afforded compound
3
98.2517.
1
3
4
0 (107 mg, 0.34 mmol, 87.9%). H NMR (300 MHz, CDCl
3
): δ = 4.39–
< 1 Hz,
.09 (m, 2H, H-3, H-4), 3.94 (dd, 1H, J2ax,2eq 14.8 Hz, J2eq,
3
4.17. (5aS)-N-tert-Butyloxycarbonyl-5a-C-(1-oxo)ethyl-3,4-O-
H-2eq), 3.87 (m, 1H, H-5a), 3.60–3.38 (m, 2H, 2× H-2′), 2.71 (dd, 1H,
2ax,3 < 1 Hz, H-2ax), 1.92 (m, 1H, 1× H-1′), 1.46 (m, 1H, H-5a), 1.22
m, 1H, 1× H-1′), 1.16 (d, 3H, 3× H-6); 13C NMR (75.5 MHz, CDCl
):
δ = 157.4 (C O, Boc), 108.4 [C(CH ], 80.3 [OC(CH ], 76.0, 73.9
C-3, C-4), 58.2 (C-2′), 50.2 (C-5a), 42.0 (C-2), 36.4 (C-1′), 35.5 (C-
), 28.5 [OC(CH ], 26.33, 24.2 [(CH C], 16.7 (C-6); MS: Calcd for
Na]: m/z 338.1943 [M + Na] ; Found [M + Na] 338.1945.
isopropylidene-6-O-methoxymethylene-4-epi-isofagomine (34)
J
(
3
Following general procedure B, alcohol 24 (110 mg, 0.29 mmol)
was treated with Dess–Martin periodinane (149 mg, 0.35 mmol).
Purification on silica gel (C/EE 1:1 v/v) afforded 34 (104 mg,
=
3
)
2
3 3
)
(
5
3
)
3
3 2
)
2
D
0
+
+
0.27 mmol, 94.8%) as a colorless syrup.
[
α
]
= +56.2 (c 3.43, CHCl
3
);
[C
16
H
29NO
5
1
3
H NMR (300 MHz, CDCl ): δ = 9.96 (s, 1H, H-2′), 4.43 (m, 1H, H-4),
4
2
.28–3.87 (m, 3H, H-2eq, H-3, H-5a), 3.74–3.48 (m, 2H, 2× H-6), 2.99–
.78 (m, 1H, H-2ax), 2.77–2.37 (m, 2H, 2× H-1′), 1.99–1.81 (m, 1H,
4
.14. (5aS)-6-Deoxy-5a-C-hydroxyethyl-4-epi-isofagomine (31)
13
H-5); C NMR (75.5 MHz, CDCl
3
): δ = 199.9, 199.4 (C-2′), 154.5, 153.9
], 95.6 (MOM), 79.7, 79.0 [OC(CH ], 72.7
C-4), 70.9 (C-3), 66.7 (C-6), 54.43 (MOM), 47.0, 46.3 (C-5a), 46.6,
], 25.2,
23.3 [(CH ) C]; 34 hydrate: MS: Calcd for [C18H33NO Na]: m/z
Following general procedure A, compound 30 (37 mg, 0.12 mmol)
(C
=
O, Boc), 107.7 [C(CH
)
3 2
3 3
)
was converted into title compound 31, which was an amorphous
solid. Recrystallization in MeOH and Et
drops of aq HCl afforded the corresponding hydrochloride 31 HCl
(
2
O in the presence of a few
.
3 3
46.3 (C-1′), 41.7, 40.7 (C-2), 39.3, 38.3 (C-5), 27.4 [OC(CH )
2
D
0
3
2
8
(21 mg, 0.10 mmol, 84.6%) as white crystals.
[
α
]
=−10.7 (c 0.48,
+
+
1
414.2104 [M + Na] ; Found [M + Na] 414.0195.
H
2
O) (hydrochloride); H NMR (300 MHz, D
2
O, DCl): δ = 3.97 (m, 1H,
H-3), 3.92 (m, 1H, H-4), 3.78 (m, 2H, 2× H-2′), 3.30–3.17 (m, 2H,
H-2eq, H-5a), 3.08 (dd, 1H, J2ax,2eq = J2ax,3 11.5 Hz, H-2ax), 2.13–2.00
4.18. 4-[(5aS)-(N-tert-Butyloxycarbonyl-3,4-O-isopropylidene-6-O-
methoxymethylene-4-epi-isofagomin-5a-yl)]-but-2-enoic nitrile
(35)
(
m, 1H, 1× H-1′), 1.92 (ddq, 1H, J4,5 2 Hz, J5,5a 13.7 Hz, J5,6 6.9 Hz,
1
3
H-5), 1.82–1.67 (m, 1H, 1× H-1′), 1.06 (d, 3H, 3× H-6); C NMR
75.5 MHz, D O, DCl): δ = 70.4 (C-4), 65.6 (C-3), 58.2 (C-2′), 54.2 (C-
a), 42.35 (C-2), 35.83 (C-5), 30.9 (C-1′), 13.9 (C-6); MS: Calcd for
(
5
2
To a suspension of t-BuOK (45 mg, 0.40 mmol) in dry THF (3 mL),
diethyl cyanomethyl phosphonate (48 μL, 0.45 mmol) was added
+
+
[
C
8
H
17NO
3
H]: m/z 176.1287 [M + H] ; Found [M + H] 176.178.
Please cite this article in press as: Martin Thonhofer, et al., Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable β-galactosidase inhibitors and activity promotors
of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C, Carbohydrate Research (2016), doi: 10.1016/j.carres.2016.03.020

ARTICLE IN PRESS
M. Thonhofer et al./Carbohydrate Research ■■ (2016) ■■–■■
9
dropwise at 0° C. The mixture was stirred for 10 min before allow-
ing the reaction to warm up to ambient temperature. After additional
0 min, a 5% solution of compound 34 (83 mg, 0.22 mmol) in dry
4.21. (5aS)-N-tert-Butyloxycarbonyl-5a-C-(6-amino)hexyl-3,4-O-
isopropylidene-6-O-methoxymethylene-4-epi-isofagomine (38)
4
THF was added dropwise. The reaction mixture was stirred until full
conversion of the starting material was detected (TLC). The mixture
was consecutively extracted with HCl (6%) and with saturated aq
Nitrile 36 (75 mg, 0.17 mmol) was converted into amine 38 by
general procedure C. Purification on silica gel (CHCl /MeOH/concd
3
NH
4
0
OH 8:4:0.01 v/v/v) provided 38 (64 mg, 0.15 mmol, 84.1%).
2
D
1
3
NaHCO , dried (Na
2
SO
4
) and filtered. The filtrate was concentrated
[
α
]
= +27.4 (c 1.05, MeOH); H NMR (300 MHz, CHCl
3
): δ = 4.57
under reduced pressure and the remaining crude product was pu-
(dd, 1H, J3,4 7.9 Hz, J4,5 2.4 Hz, H-4), 4.25 (m, 1H, H-3), 3.99 (m, 1H,
H-2eq), 3.84–3.55 (m, 3H, H-5a, 2× H-6), 2.96–2.77 (m, 3H, H-2ax,
2× H-6′), 1.88 (m, 1H, H-5), 1.76–1.32 (m, 10H, 2× H-1′, 2× H-2′, 2×
rified on silica gel (C/EE 4:1 v/v) to provide 35 (mixture of the E/Z-
isomers, 64 mg, 0.16 mmol, 72.6%). 1H NMR (300 MHz, CHCl
):
δ = 6.83–6.45 (m, 1H, H-2′), 5.34 (m, 1H, H-3′), 4.49 (m, 1H, H-4),
3
13
H-3′, 2× H-4′, 2× H-5′); C NMR (75.5 MHz, CDCl
3
): δ = 157.7, 157.5
], 74.7,
4
2
.30–3.78 (m, 3H, H-2eq, H-3, H-5a), 3.77–3.53 (m, 2H, 2× H-6), 2.91–
(C O, Boc), 109.5 [C(CH ], 97.6, 97.5 (MOM), 80.8 [OC(CH )
=
)
3 2
3 3
.53 (m, 3H, H-2ax, 2× H-1′), 1.76 (bs, 1H, H-5); 13C NMR (75.5 MHz,
74.5 (C-4), 73.4, 73.2 (C-3), 68.4 (C-6), 55.6 (MOM), 52.1, 50.8 (C-
5a), 43.8, 43.1, 40.8, 40.7, 34.8, 34.6 (C-2, C-5, C-6′), 30.5, 30.4 (C-
CDCl
3
): δ = 155.4, 155.1 (C
=
O, Boc), 152.2, 151.78 (C-2′), 117.2, 116.0
], 102.0, 100.9 (C-3′), 96.6 (MOM), 80.1,
(C-4′), 108.6, 108.5 [C(CH
)
3 2
1′), 28.8 (OC(CH
C-5′); MS: Calcd for [C22
[M + H]+ 431.3120; MS: Calcd for [C22
3
)
3
), 28.6, 26.8 [(CH
3
)
2
C], 28.7-26.04 (C-2′, C-3′, C-4′,
+
7
5
3
2
9.8 [OC(CH
0.6, 50.3, 49.6, 49.5 (C-5a), 43.2, 42.7, 42.1, 41.9 (C-2), 39.9, 39.8,
9.5, 38.8 (C-5), 37.9, 37.0, 36.6, 36.1 (C-1′), 28.4, 28.3 [OC(CH ],
6.2, 24.2 [(CH C].
3
)
3
], 73.1-71.5 (C-3, C-4), 67.4, 67.3 (C-6), 55.4 (MOM),
H
42
N
2
O
6
H]: m/z 431.3121 [M + H] ; Found
42 2 6
H N O Na]: m/z 453.2941
+
+
3
)
3
[M + Na] ; Found [M + Na] 453.2926.
3 2
)
4
3
.22. (5aS)-N-tert-Butyloxycarbonyl-5a-C-(4-dansylamino)butyl-
,4-O-isopropylidene-6-O-methoxymethylene-4-epi-isofagomine
4.19. 6-[(5aS)-(N-tert-Butyloxycarbonyl-3,4-O-isopropylidene-6-O-
(39)
methoxymethylene-4-epi-isofagomin-5a-yl)]-hex-4-enoic nitrile
36)
(
Following general procedure D, compound 37 (28 mg, 0.07 mmol)
was treated with Na CO (19 mg, 0.18 mmol) and dansyl chloride
2
3
A solution of freshly prepared LDA [by dropwise addition of 2.5 M
n-BuLi (0.38 mL) in hexane to diisopropyl amine (0.14 mL, 0.99 mmol,
0% solution in dry THF)] in dry THF was added dropwise at −78 °C
(23 mg, 0.08 mmol) to give crude compound 39 which was imme-
diately taken to the next step.
2
under an atmosphere of nitrogen to a suspension of triphenyl-(3-
cyano)propyl phosphonium bromide (421 mg, 1.03 mmol). After
stirring at −40 °C for 60 min, a solution of compound 34 (110 mg,
4.23. (5aS)-N-tert-Butyloxycarbonyl-5a-C-(6-dansylamino)hexyl-
3,4-O-isopropylidene-6-O-methoxymethylene-4-epi-isofagomine
(40)
0
.29 mmol, 3% in dry THF) was added dropwise. After having been
stirred for 12 h, the reaction mixture was diluted with CH Cl and
consecutively washed with HCl (6%) and saturated NaHCO . The com-
bined organic layers were dried (Na SO ) and solvents were removed
2
2
Following general procedure D, compound 38 (64 mg, 0.15 mmol)
was treated with Na CO (44 mg, 0.42 mmol) and dansyl chloride
2 3
(52 mg, 0.19 mmol) to give crude compound 40 which was imme-
3
2
4
under reduced pressure. Chromatographic purification (silica gel,
diately taken to the next step.
C/EE 4:1 v/v) of the crude product gave 36 (75 mg, 0.18 mmol, 60.1%)
1
as a mixture of the E/Z-isomers. H NMR (300 MHz, CHCl
5
3
): δ = 5.71–
4.24. (5aS)-5a-C-(4-Dansylamino)butyl-4-epi-isofagomine (41)
Following general procedure A, crude compound 39 was con-
.39 (m, 2H, H-2′, H-3′), 4.51 (m, 1H, H-4), 4.26–3.94 (m, 2H, H-2eq,
H-3), 3.93–3.69 (m, 1H, H-5a), 3.63 (bs, 2H, 2× H-6), 2.86–2.66 (m,
1
1
1
9
H, H-2ax), 2.65–2.08 (m, 6H, 2× H-1′, 2× H-4′, 2× H-5′), 1.86 (bs,
verted into title compound 41. Purification on silica gel (CHCl
MeOH/concd NH OH 8:4:0.01 v/v/v) and subsequent recrystallization
in MeOH in the presence of HCl afforded 41 HCl (21 mg, 0.41 mmol,
3
/
13
H, H-5); C NMR (75.5 MHz, CDCl
28.4, 128.3, 128.2 (C-2′, C-3′), 119.4 (C-6′), 108.5, 108.4 [C(CH
6.5, 96.4 (MOM), 79.8, 79.5 [OC(CH ], 73.3, 73.1, 71.7, 71.5 (C-3,
3
): δ = 155.4, 155.2 (C
=
O, Boc),
4
.
3 2
) ],
20
3
)
3
59.1% over 2 steps) as faintly green fluorescent solid.
[
α
]
=−15.2
D
1
C-4), 67.1, 66.7 (C-6), 55.3, 55.2 (MOM), 50.4, 49.6 (C-5a), 43.7, 42.8
4
(c 1.1, MeOH); H NMR (300 MHz, MeOH-d ): δ = 4.10 (dd, J3,4 = J4,5
(
[
C-2), 38.5, 37.9 (C-5), 30.5, 29.4 (C-1′), 28.4 [OC(CH
(CH C], 23.3 (C-4′), 17.4 (C-5′).
3
)
3
], 26.2, 24.2
2.5 Hz, Hz), 3.75 (ddd, 1H, J2ax,3 10.5 Hz, J2eq,3 6.1 Hz, H-3), 3.65 (bd,
2H, 2× H-6), 3.18 (ddd, 1H, J5,5a 10.9 Hz, J5a,1′ 6.9 Hz, J5a,1′ 3.1 Hz, H-5a),
3 2
)
3
.14–3.00 (m, 2H, H-2eq, H-2ax) 2.85 (m, 2H, 2× H-4′), 1.69–1.155
13
(
m, 8H, H-5, 2× H-1′, 2× H-2′, 2× H-3′); C NMR (75.5 MHz, MeOD):
δ = 153.3–116.5 (dansyl), 69.1 (C-4), 67.6 (C-3), 61.6 (C-6), 53.6 (C-
5a), 45.8 (2xNMe ), 45.1 (C-5), 44.4 (C-2), 43.4 (C-4′), 30.8, 30.5 (C-
′, C-3′), 22.5 (C-2′); MS: Calcd for [C22 SH]: m/z 452.2219
[M + H] ; Found [M + H] 452.2336; MS: Calcd for [C22 SNa]:
4.20. (5aS)-N-tert-Butyloxycarbonyl-5a-C-(4-amino)butyl-3,4-O-
isopropylidene-6-O-methoxymethylene-4-epi-isofagomine (37)
2
1
33 3 5
H N O
+
+
Nitrile 35 (64 mg, 0.16 mmol) was converted into amine 37
33 3 5
H N O
+
+
(
49 mg, 0.12 mmol, 75.4%) employing general procedure C.
m/z 474.2039 [M + Na] ; Found [M + Na] 474.2052.
20
= +33.8 (c 2.36, MeOH); 1H NMR (300 MHz, MeOH-d
[
α
]
4
):
δ = 4.56 (dd, 1H, J3,4 8 Hz, J4,5 2 Hz, H-4), 4.24 (m, 1H, H-3), 3.99 (m,
D
4.25. (5aS)-5a-C-(6-Dansylamino)hexyl-4-epi-isofagomine (42)
Following general procedure A, crude compound 40 was con-
1
3
H, H-2eq), 3.79 (ddd, 1H, J5,5a 10.6 Hz, J5a, 1′ 7.3 Hz, J5a,1′ 3.0 Hz, H-5a),
.72–3.55 (m, 2H, 2× H-6), 2.98–2.78 (m, 3H, H-2ax, 2× H-4′), 1.89
1
3
(
(
9
4
m, 1H, H-5), 1.79–1.30 (m, 6H, 2× H-1′, 2× H-2′, 2× H-3′); C NMR
75.5 MHz, MeOH-d ): δ = 157.7, 157.4 (C O, Boc), 109.4 [C(CH ],
7.5 (MOM), 81.2, 80.9 [OC(CH ], 81.2, 80.9 (C-3), 74.8, 74.6 (C-
), 68.4 (C-6), 55.6 (MOM), 52.0, 50.7 (C-5a), 43.8, 43.2 (C-2), 41.1
verted into title compound 42. Purification on silica gel (CHCl
3
/
4
=
)
3 2
MeOH/concd NH OH 8:4:0.01 v/v/v) provided 42 (35 mg, 0.07 mmol,
4
2
D
0
3
)
3
49.1% over 2 steps) as fluorescent wax.
H NMR (300 MHz, MeOH-d ): δ = 4.11 (dd, 1H, J3,4 = J4,5 2.5 Hz, H-4),
4
[
α
]
=−10.3 (c 2.17, MeOH);
1
(
[
C-4′), 40.4, 40.2 (C-5), 34.3, 34.1 (C-1′), 30.2, 29.4 (C-3′), 28.8
OC(CH ], 26.8, 24.4, 24.2 [(CH C], 23.5, 23.3 (C-2′); MS: Calcd
Na]: m/z 425.2628 [M + Na] ; Found [M + Na]
3.66 (bd, 2H, 2× H-6), 3.62 (ddd, J2ax,3 10.58 Hz, J2eq,3 6.12 Hz, H-3),
2.95–2.78 (m, 5H, H-2ax, H-2eq, H-5a, 2× H-6′), 1.64–1.03 (m, 11H,
H-5, 2× H1′, 2× H-2′, 2× H-3′, 2× H-4′, 2× H-5′); C NMR (75.5 MHz,
3
)
H
3
3 2
)
+
+
13
for [C20
38
N
2
O
6
4
25.2699.
4
MeOH-d ): δ = 153.2–116.4 (dansyl), 70.0 (C-4), 69.6 (C-3), 61.9
Please cite this article in press as: Martin Thonhofer, et al., Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable β-galactosidase inhibitors and activity promotors
of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C, Carbohydrate Research (2016), doi: 10.1016/j.carres.2016.03.020

ARTICLE IN PRESS
10
M. Thonhofer et al./Carbohydrate Research ■■ (2016) ■■–■■
(
6
C-6), 52.6 (C-5a), 46.8 (C-5), 45.9 (C-2), 45.8 (2xNMe
2
), 43.7 (C-
12. Sawkar AR, Adamski-Werner SL, Cheng W-C, Wong C-H, Beutler E, Zimmer K-P,
et al. Chem Biol 2005;12:1235–44.
3. Wennekes T, van den Berg RJBHN, Donker W, van der Marel GA, Strijland A, Aerts
′)32.5 (C-1′), 30.4, 30.1, 27.2, 25.7 (C-2′, C-3′, C-4′, C-5′); MS: Calcd
1
+
+
for [C24
MS: Calcd for [C24
H
37
N
3
O
5
SH]: m/z 480.2532 [M + H] ; Found [M + H] 480.2480;
MFG, et al. J Org Chem 2007;72:1088–97.
14. Jespersen TM, Dong W, Sierks MR, Skrydstrup T, Lundt I, Bols M. Angew Chem
Int Ed Engl 1994;33:1778–9.
+
H
37
N
3
O
5
SNa]: m/z 502.2352 [M + Na] ; Found
+
[
M + Na] 502.2273.
1
1
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Financial support by the Austrian Fonds zur Förderung der
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Appendix: Supplementary material
Supplementary data to this article can be found online at
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Please cite this article in press as: Martin Thonhofer, et al., Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable β-galactosidase inhibitors and activity promotors
of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C, Carbohydrate Research (2016), doi: 10.1016/j.carres.2016.03.020