N-Butyl-l-deoxynojirimycin (l-NBDNJ): Synthesis of an Allosteric Enhancer of α-Glucosidase Activity for the Treatment of Pompe Disease

Article abstract of DOI:10.1021/acs.jmedchem.7b00646

The highly stereocontrolled de novo synthesis of l-NBDNJ (the unnatural enantiomer of the iminosugar drug Miglustat) and a preliminary evaluation of its chaperoning potential are herein reported. l-NBDNJ is able to enhance lysosomal α-glucosidase levels in Pompe disease fibroblasts, either when administered singularly or when coincubated with the recombinant human α-glucosidase. In addition, differently from its d-enantiomer, l-NBDNJ does not act as a glycosidase inhibitor.

Full text of DOI:10.1021/acs.jmedchem.7b00646

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Article
N-Butyl-L-Deoxynojirimycin (L-NBDNJ): Synthesis of an Allosteric
Enhancer of #-Glucosidase Activity for the Treatment of Pompe Disease
Daniele D'Alonzo, Maria De Fenza, Caterina Porto, Roberta Iacono, Mylene
Huebecker, Beatrice Cobucci-Ponzano, David Priestman, Frances M. Platt,
Giancarlo Parenti, Marco Moracci, Giovanni Palumbo, and Annalisa Guaragna
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00646 • Publication Date (Web): 07 Nov 2017
Downloaded from http://pubs.acs.org on November 8, 2017
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NꢀButylꢀ
L
ꢀDeoxynojirimycin ( ꢀNBDNJ): Synthesis
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of an Allosteric Enhancer of αꢀGlucosidase Activity
for the Treatment of Pompe Disease
Daniele D’Alonzo,*,‡ Maria De Fenza,‡ Caterina Porto,† Roberta Iacono,§ Mylene Huebecker,#
Beatrice CobucciꢀPonzano,§ David Priestman,# Frances Platt,# Giancarlo Parenti,†,≠ Marco
Moracci,§,¶ Giovanni Palumbo‡ and Annalisa Guaragna‡
‡Department of Chemical Sciences, Università degli Studi di Napoli Federico II, via Cintia,
80126 Napoli (Italy); †Department of Translational Medical Sciences, Section of Pediatrics,
Università degli Studi di Napoli Federico II, Via S. Pansini 5, 80131 Napoli (Italy); §Institute of
Biosciences and Bioresources, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131
Napoli (Italy); #Department of Pharmacology, University of Oxford, Mansfield Road, Oxford
OX1 3QT, UK; ≠Telethon Institute of Genetics and Medicine, Via Campi Flegrei 34, 80078
Pozzuoli (Italy); ¶Department of Biology, Università degli Studi di Napoli Federico II, via Cintia,
80126 Napoli (Italy).
dandalonzo@unina.it
KEYWORDS: iminosugars; de novo synthesis; acid αꢀglucosidase; pharmacological chaperone;
Pompe disease.
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ABSTRACT: The highly stereocontrolled de novo synthesis of
LꢀNBDNJ (the unnatural
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enantiomer of the iminosugar drug Miglustat) and a preliminary evaluation of its chaperoning
potential are herein reported. ꢀNBDNJ is able to enhance lysosomal αꢀglucosidase levels in
Pompe disease fibroblasts, either when administered singularly or when coꢀincubated with the
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recombinant human αꢀglucosidase. In addition, differently from its
not act as a glycosidase inhibitor.
Dꢀenantiomer, LꢀNBDNJ does
INTRODUCTION
Acid αꢀglucosidase (GAA; EC 3.2.1.20) is a lysosomal enzyme enabling the degradation of
glycogen to glucose (glycosyl hydrolase family: GH31).1 A deficiency in GAA activity leads to
intralysosomal glycogen storage, causing extensive and progressive damages to cardiac and
skeletal muscles.2 The genetic disorder due to mutationsꢀinduced functional defects of GAA is
known as Pompe disease (PD; glycogen storage disease type II) and represents one of the most
common lysosomal storage disorders (LSDs).3,4 To date, the only clinically approved strategy for
the treatment of the disease is the enzyme replacement therapy (ERT), based on intravenous
administration of recombinant human GAA (rhGAA).5 Even though ERT has shown to stabilize
the disease course, use of rhGAA has some major limitations, regarding, inter alia, its adequate
delivery to lysosomes and the stability to nonꢀacidic conditions.6 In the search for alternative
approaches, the identification of small molecule chaperones able to restore functions and
properties of the mutated enzyme – the core of the soꢀcalled pharmacological chaperone therapy
(PCT) – is representing an emerging strategy and a formidable challenge of the modern
biomedical research.7,8 According to PCT, the use of suitable GAA ligands enables stabilization
of protein conformation, inhibits premature misfolding and facilitates enzyme translocation into
the lysosome, thereby preventing ERꢀassociated degradation processes.7 Among
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pharmacological chaperones,9ꢀ11 the iminosugar drug12 Nꢀbutylꢀ
Dꢀdeoxynojirimycin (1) (Dꢀ
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NBDNJ; Figure 1) currently represents one of the most promising candidates under development
for the treatment of PD.4 In early studies, 1 has been found to improve residual activity and
lysosomal trafficking of mutated GAA in cultured fibroblasts from Pompe patients.13 Even more
remarkably, the chaperone has subsequently demonstrated to enhance the physical stability and
therapeutic efficacy of rhGAA itself.14 This finding has represented the first documented
example of a synergistic effect deriving from the combined use of PCT and ERT, which has
opened the way to the soꢀcalled “combination therapy” for the treatment of various LSDs.7,8
Higher lysosomal GAA levels in blood as resulting from coꢀadministration of rhGAA and 1 have
been recently found in animal models and in PD patients.15 Further trials aimed at addressing the
clinical efficacy of the combination protocol are currently in progress.16
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Figure 1. Dꢀ and Lꢀiminosugars 1 and entꢀ1.
Despite these promising data, it must be noted that 1, like most iminosugar chaperones, is an
example of activeꢀsite specific chaperone (ASSC). Accordingly, in its protonated form 1
resembles the oxocarbenium ion produced during glucopyranoside hydrolysis and therefore acts
as a competitive inhibitor of GAA as well as of a variety of other glucosidases.17 Even though
chaperoning activity typically occurs at subꢀinhibitory concentrations,13 GAA inhibition may
clearly represent a limitation to clinical use, as it could hamper to reach the optimal drug dosage.
Hence, the identification of newꢀgeneration chaperones, able to protect GAA from degradation
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without interfering with its activity (allosteric chaperones), is likewise being pursued.6 In this
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context, novel therapeutic opportunities are recently being offered by
although typically acting as weaker20 and nonꢀcompetitive18 glycosidase inhibitors, several
ꢀenantiomers)21 have displayed
L
ꢀiminosugars.18,19 Indeed,
Lꢀ
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iminosugars (either alone or in combination with their
D
noteworthy chaperoning activities toward the same enzymes in their mutated forms.21,22 Based
on these combined data, our research program aimed at exploring the stereoselectivity of
biomolecular recognition processes18,23ꢀ26 took us to study the role of iminosugar chirality in PCT
through the analysis of the chaperoning potential of the unnatural enantiomer of 1, i.e. Nꢀbutylꢀ
Lꢀ
deoxynojirimycin (entꢀ1) ( ꢀNBDNJ; Figure 1). In this communication, the stereoselective
L
synthesis of entꢀ1, the analysis of its glycosidase inhibition properties and a preliminary
evaluation of its potential as enhancer of GAA activity both in its mutated and recombinant form
are reported.
RESULTS AND DISCUSSION
Chemistry. To the best of our knowledge, the synthesis of entꢀ1 has never been reported
before.27 More generally, the access to
commercial availability of almost all ꢀhexoses, which hampers their use as starting materials.
Lꢀiminosugars is often problematic, due to the limited
L
Alternatively, in an extension of a long studied28 de novo methodology, already devised to obtain
unnatural carbohydrates28 and biomimetics25,26,29ꢀ31 including iminosugars32 and their
precursors,33 the synthesis of entꢀ1 was explored from the synthetically available32 alcohol 2
(Scheme 1). Particularly, having previously fixed the stereochemistry of C4 and C5
stereocentres,34 we have herein addressed the synthetic problem of the stereoselective
introduction of the two remaining stereocentres at C2 and C3 positions having a transꢀ
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diequatorial orientation of the substituents. In early attempts, inspired by previous results on Lꢀ
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glucose synthesis,35 the preparation and stereoselective functionalization of 1,6ꢀanhydroꢀ
iminosugars was considered (Scheme 1). Secꢀalcohol 2 was first benzylated under common
conditions (NaH, BnBr). The resulting benzyl ether 3 was then treated with a DCE/MeOH
solution of 2,3ꢀdichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone (DDQ) directly providing, after 48h at rt
and then 8h at 60 °C, the bicyclic compound 4. As already observed for similar substrates,28 the
reaction was the result of a multistep process, which was enabled by the excellent electronꢀ
donating properties of the bisꢀthioenol ether moiety.31 Spontaneous cyclization of 4 into 1,6ꢀ
anhydroꢀiminosugar precursor 5 was even observed during chromatographic purification
procedures. In spite of the synthetic utility of the reaction, 5 could not be further functionalized
as the subsequent double bond unmasking step (RaꢀNi) did not provide the expected olefin 6; the
corresponding overꢀreduced compound was instead obtained. Looking for alternative strategies,
we then investigated the reactivity of oxirane 7, obtained from alcohol 2 as previously
described32 (Scheme 1). Epoxide ring opening of 7 with refluxing NaOH directly produced the
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unprotected iminosugar with Lꢀaltro configuration 10, additionally in a highly stereoselective
fashion (altro:gluco > 20:1). Conversely, treatment of bisꢀMOM acetal 9 (from 7: MeONa, then
MOMCl/DIPEA) under the same conditions gave, after the subsequent HCl addition to the crude
mixture, the desired 11 as the only detected stereoisomer (d.r. > 99:1).
Scheme 1. Stereocontrolled de novo synthesis of ent-1.
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The stereodivergent outcome of the reactions was assumed to rely on the conformational
preferences of epoxides 7 and 9 (Scheme 2). We observed that diacetate 7 adopted a sugar
conformation having both C4 and C6 substituents in pseudoꢀdiaxial orientations.32 In line with
previous findings, this behavior was judged to be a consequence of the steric clash occurring
between the NꢀBoc and the pseudoꢀequatorially oriented C6 acetate groups.32 Accordingly, the
formation of 10 from 7 was hypothesized to take place via the baseꢀlabile cyclic carbamate36
intermediate 12 (Scheme 2a). In agreement with literature data,37 an energyꢀminimized structure
of 12 suggested that it was firmly locked in a quasiꢀplanar conformation close to a 5E form with a
pseudoꢀequatorially oriented methyleneoxy moiety (Scheme 2a). The conformational inversion
from epoxide 7 to 12 justified the formation of 10 after transꢀdiaxial epoxide ring opening of 12.
Conversely, when the Ac group was replaced by the baseꢀstable MOM group, the oxirane ring
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cleavage of bisꢀacetal 9 only provided the glucoꢀconfigured diol 13 (Scheme 2b). Even though
the broadness of most NMR signals of epoxide 9 hampered the identification of its preferred
conformation, NMR analysis of diol 13 indicated a conformation with all substituents in near
axial orientations (J1,2 2.5 Hz; J3,4 1.8 Hz; J4,5 2.6 Hz). Close agreement between these J values
and the theoretical values38 obtained from an energyꢀminimized structure (Scheme 2b) suggested
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that a 4C1 chair was adopted in this case. Eventually, the “allꢀequatorial”
by the concurrent removal of NꢀBoc and MOM groups, enabling as expected a conformational
LꢀDNJ 11 was obtained
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inversion from C1 to C4 chair (Scheme 2b). With the latter in hand, the access to the
corresponding entꢀ1 was achieved under standard Nꢀalkylation conditions (Scheme 1).
Scheme 2. Reactivity and conformational preferences of iminosugar intermediates 7-9.
Biological evaluation. Glycosidase inhibition studies of entꢀ1 were first carried out. The
selected glycosidases were representative examples of various GH families (Table 1 and
Supporting Information, Figures S1ꢀS3). No inhibitory effect up to 1 mM was found for entꢀ1 in
most cases, except for the very weak inhibition of intestinal sucrase/isomaltase (38% inhibition,
IC50 2 mM, entry 2), lactase (30% inhibition, IC50 >5 mM, entry 4) mixed GBA1/GBA2 (18%
inhibition, entry 7) and αꢀfucosidase (19%, entry 14). Instead, rhGAA and βꢀglucosidases from
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P. furiosus and S. solfataricus P2 even showed a 20ꢀ25% activation (entries 3, 5 and 7), even
though no significant enhancement in the melting temperature (Tm) of rhGAA by entꢀ1 was
indicated by differential scanning fluorimetry experiments (Supporting Information, Figure S4).
Conversely, 1 displayed inhibition of rice αꢀglucosidase (IC50 275 ꢁM, entry 1),
sucrase/isomaltase (IC50 1.5 ꢁM, entry 2), GBA1/GBA2 (IC50 180 ꢁM, entry 7) and recombinant
human βꢀglucocerebrosidase (rhGBA1 or CerezymeTM: IC50 0.3 mM, entry 8). Inhibition of
rhGAA was also observed (IC50 158 ꢁM, entry 2). In addition, 1 is reported to be a much more
effective inhibitor of endogenous GAA (IC50 5.3 ꢁM).39
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Table 1. Residual activity of various glycosidases in the presence of 1 and ent-1 (1 mM).
Residual activity (%)
Entry
Enzyme
GH Family
entꢀ1
98
1
1
Rice αꢀglucosidase
GH31
GH31
GH31
GH1
17
3
2
Intestinal sucrase/isomaltasea
62
3
rhꢀGAA
19
62
81
94
24
35
98
82
100
98
125
70
4
Intestinal lactasea
5
βꢀGlucosidase from P. furiosus
βꢀGlucosidase from S. solfataricus P2
Human GBA1/GBA2b,c
rhꢀGBA1
GH1
120
124
82
6
GH1
7
GH30/116
GH30
GH20
GH36
GH27
GH42
8
97
9
Human βꢀHexosaminidaseb
αꢀGalactosidase from T. maritime
rhꢀGal A
98
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12
100
104
102
βꢀGalactosidase from A. acidocaldarius
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Human βꢀGalactosidaseb
αꢀFucosidase from S. solfataricus
GH35
GH29
87
83
98
81
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a
Inhibition studies were conducted on purified enzymes unless otherwise specified. Enzyme
source: small intestine of WT BL6 mice. bEnzyme source: HL60 cells. cMeasure of the overall βꢀ
glucosidase activity in HL60 cells.
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The effect of entꢀ1 in cell lines deriving from Pompe patients was then explored (Figures 2ꢀ3).
In a first set of experiments, PD fibroblasts from a patient carrying the mutation
p.L552P/p.L552P (omozygous mutation) were incubated in the absence or in the presence of 20
ꢁM entꢀ1 or its D
ꢀenantiomer40 (Figure 2). The administration of entꢀ1 effectively increased the
residual activity of mutated GAA (Figure 2). The activating effect (1.5ꢀfold increase) was
slightly higher than that observed for 1 under the same experimental conditions (1.3ꢀfold
increase).
Figure 2. Effect of 1 and entꢀ1 (20 ꢁM) on residual activity of mutated GAA in fibroblasts from
a PD patient carrying the p.L552P/p.L552P mutation.
The chaperoning activity of an
Lꢀpiperidine iminosugar has been reported only seldom
before;21 in addition, to the best of our knowledge the higher enhancing effect of an
Lꢀpiperidine
iminosugar compared to that of the corresponding Dꢀenantiomer is even unprecedented. The lack
of inhibition of the deficient enzyme and of the other glycosidases further augments the
activating potential of entꢀ1, especially if compared with its enantiomer. It must be noted that,
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however, under these conditions the enhancement of GAA residual activity was observed only in
cells with entꢀ1 and it was not sufficiently high to be considered of therapeutic relevance.
Potential synergistic effects deriving from coꢀadministration of entꢀ1 and rhGAA in PD
fibroblasts were also considered. PD fibroblasts from three patients carrying various mutations
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(Figure 3) were incubated with rhGAA and entꢀ1 or its Dꢀenantiomer (20 ꢁM). In all cases, coꢀ
incubation of entꢀ1 and rhGAA provided a higher increase in GAA levels than those provided by
rhGAA when incubated singularly. Especially when PD fibroblasts carrying the mutation
p.L552P/p.L552P were evaluated, a 2ꢀfold increase of GAA activity was found; this value was
the same observed by treatment of the cells with 1/rhGAA. In the remaining cases, the enhancing
effect was less marked (and lower than that found for 1/rhGAA) (Figure 3).
Figure 3. Synergy between rhGAA and 1 or entꢀ1 in PD fibroblasts.
Western blot analysis was eventually performed in entꢀ1ꢀtreated and untreated fibroblasts
carrying the mutation p.L552P/p.L552P (Figure 4), to provide first clues on the mechanisms
enabling the observed enhancement of GAA activity. In this case, density scans of rhGAA bands
indicated that the mature 76 KDa isoform was more represented in the presence of both
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enantiomers, even though the increase with rhGAA/entꢀ1 was less marked with respect to that
observed in cells treated with rhGAA/1.
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Figure 4. Western blot analysis of rhGAA processing in Pompe fibroblasts carrying the
p.L552P/p.L552P mutation incubated with rhGAA alone, rhGAA + 1, or rhGAA + entꢀ1.
CONCLUSION
The synthesis and a preliminary evaluation of the potential of entꢀ1 as enhancer of GAA
activity have been herein reported. The access to entꢀ1 has been performed by a highly
stereocontrolled de novo strategy, exploiting the different reactivity and conformational
preferences of epoxide intermediates 7-9. Differently from its Dꢀenantiomer, entꢀ1 did not act as
a glycosidase inhibitor; however, it was found to enhance the activity of lysosomal αꢀglucosidase
in PD fibroblasts. Especially when coꢀincubated with rhGAA, we found a synergistic enhancing
effect, which was comparable to that observed for rhGAA/1. From these early studies, entꢀ1
seems not to affect the stability of the enzyme, while enhancing the activity of the latter in cells.
Thus, although the molecule cannot be considered a pharmacological chaperone of GAA, it
represents a promising new candidate for the combination therapy of Pompe disease. Further
studies aimed at elucidating the nature of the enhancing effect of entꢀ1 are currently ongoing and
will be published in due course.
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EXPERIMENTAL SECTION
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1) Chemical synthesis
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General information. All chemicals and solvents were purchased with the highest degree of
purity (SigmaꢀAldrich, Alfa Aesar, VWR) and used without further purification. All moistureꢀ
sensitive reactions were performed under nitrogen atmosphere using ovenꢀdried glassware. The
reactions were monitored by TLC (precoated silica gel plate F254, Merck) and the products were
detected by exposure to ultraviolet radiation, iodine vapor and chromic mixture. Column
chromatography: Merck Kieselgel 60 (70ꢀ230 mesh); flash chromatography: Merck Kieselgel 60
(230ꢀ400 mesh). The purity of the synthetic intermediates and the final compounds was
determined either by HPLC, NMR, CHNS analysis or by optical rotation and was ≥ 95% in all
cases. HPLC analysis was performed using an Agilent 6420 Triple Quadrupole LCꢀMS/MS
system with a HPLC 1100 series binary pump. HRMS analysis was performed on a Thermo LTQ
Orbitrap XL mass spectrometer coupled to a Thermo U3000 HPLC system. Monoꢀ and
bidimensional NMR spectra were recorded on NMR spectrometers operating at 200 MHz
(Varian), 400 MHz (Bruker DRX, Bruker AVANCE) or 500 MHz (Varian Inova), using CDCl3
solutions unless otherwise specified. Combustion analyses were performed using a CHNS
analyzer. Optical rotations were measured at 25 ± 2 °C in the stated solvent.
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Benzyl ether 3. NaH (31.5 mg, 1.31 mmol) was added to a solution of 2 (0.50 g, 1.01 mmol)
in anhydrous DMF (10 mL) at 0 °C under nitrogen atmosphere. After 10 min, BnBr (0.14 mL,
1.21 mmol) was added in one portion. The reaction mixture was warmed to room temperature,
stirred for 2h, then carefully quenched with 10% aq NH4Cl. The mixture was extracted with
EtOAc, the combined organic phases washed with brine, dried (Na2SO4) and evaporated under
reduced pressure. Chromatography of the crude residue over silica gel (hexane/Et2O = 7:3)
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afforded the pure 3 (0.48 g, 80% yield). Oily; H NMR (400 MHz): δ 1.42 (s, 9H), 1,44 (s, 3H),
5
6
7
8
1,51 (s, 3H), 3.09ꢀ3.28 (m, 4H), 3.79 (s, 3H), 3.80ꢀ3.92 (m, 2H), 4.18ꢀ4.40 (m, 4H), 4.42ꢀ4.47
(m, 4H), 6.85 (d, 2H, J = 8.2 Hz), 7.28ꢀ7.40 (m, 7H). 13C NMR (100 MHz): ppm 27.1, 28.3,
29.1, 30.1, 55.2, 59.6, 63.1, 64.1, 69.8, 70.7, 71.6, 80.0, 94.5, 113.7, 126.5, 127.9, 128.4, 128.9,
129.4, 129.6, 133.2, 137.5, 159.3, 162.4. Anal. calcd for C31H41NO6S2: C, 63.34; H, 7.03; N,
2.38 S, 10.91. Found: C, 63,22; H, 7.05; N, 2,39; S, 10,94.
9
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Tricyclic compound 5. To a stirring solution of alcohol 3 (0.58 g, 0.99 mmol) in a
DCE/MeOH solution (3/1 v/v, 20 mL), DDQ (0.56 g, 2.50 mmol) was added in one portion at rt.
The resulting mixture was stirred at the same temperature in the dark. After 48h, the reaction was
heated to 60 °C and stirred at the same temperature for 8 h. The mixture was then cooled to rt,
transferred into a separatory funnel, washed with brine and extracted repeatedly with DCM. The
collected organic layers were dried (Na2SO4) and the solvent evaporated under reduced pressure.
The crude residue was dissolved in DCM (10 mL) and treated with silica gel (2 g) at rt for 30
min. The mixture was then filtered, and the filtrate was concentrated under reduced pressure.
Chromatography of the crude residue over silica gel (hexane:acetone = 8:2) gave the pure 5 (0.37
1
g, 91% overall yield). Oily; H NMR (200 MHz): δ 1.43 (s, 9H), 3.08ꢀ3.32 (m, 4H), 3.45 (dd,
1H, J = 8.0, 1.2 Hz), 3.69 (d, 1H, J = 2.1 Hz), 3.85 (dd, 1H, J = 8.0, 6.6 Hz), 4.62 (d, 1H, J =
11.6 Hz), 4.76 (d, 1H, J = 11.6 Hz), 4.89 (bd, 1H, J = 6.2 Hz), 5.46 (s, 1H), 7.22ꢀ7.45 (m, 5H).
13C NMR (50 MHz): ppm 27.6, 28.0, 28.2, 64.2, 70.5, 79.0, 80.1, 81.6, 85.6, 120.9, 126.2, 127.8,
128.1, 128.4, 137.8, 153.4. Anal. calcd for C20H25NO4S2: C, 58.94; H, 6.18; N, 3.44; S, 15.74.
Found: C, 59.08; H, 6,16; N, 3.43; S, 15.69.
Diol 8. Zemplén deacetylation was accomplished by treatment of a solution of 7 (0.24 g, 0.76
mmol) in MeOH (5 mL) with MeONa (41 mg, 0.76 mmol) for 4h at room temperature. The
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mixture was neutralized with few drops of acetic acid, then the volatiles were removed under
reduced pressure. The crude residue was dissolved in CHCl3 and filtered through a short pad of
silica gel. The resulting filtrate was eventually concentrated to dryness, giving 8, which was used
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in the following step without further purification. Oily; H NMR (400 MHz, DMSOꢀd6): δ 1.39
(s, 9H), 3.14 (bs, 1H), 3.35 (bs, 1H), 3.41ꢀ3.57 (m, 1H), 3.87 (bt, 1H, J = 7.2 Hz), 3.91ꢀ3.98 (m,
1H), 4.02ꢀ4.13 (m, 1H), 4.14ꢀ4.23 (m, 1H), 4.62 (bt, 1H, J = 4.9 Hz). 1H NMR (400 MHz, 55 °C,
DMSOꢀd6): δ 1.40 (s, 9H), 3.11ꢀ3.20 (m, 2H), 3.32ꢀ3.41 (m, 2H), 3.46ꢀ3.55 (m, 1H), 3.84ꢀ3.96
(m, 2H), 4.13 (bs, 1H), 4.47 (t, 1H, J = 5.7 Hz), 5.19 (d, 1H, J = 5.2 Hz). 13C NMR (100 MHz):
ppm 28.4, 43.1, 50.2, 53.6, 60.0, 61.1, 63.1, 81.2, 156.9. Anal. calcd for C11H19NO5: C, 53.87; H,
7.81; N, 5.71. Found: C, 53.94; H, 7.79; N, 5.73. HRMS: m/z [M + Na]+, calcd: 268.1155; found:
268.1144.
Bis-acetal 9. To a stirring solution of the crude diol 8 (26 mg, 0.1 mmol) in DCM (2 mL),
DIPEA (35 ꢁL, 0.2 mmol) and MOMCl (15 ꢁL, 0.2 mmol) were sequentially added at room
temperature. The resulting mixture was stirred at the same temperature for 48 h; then the reaction
was quenched by the addition of H2O (1 mL). The mixture was diluted with DCM and washed
with brine (pH 7). The organic phases were dried (Na2SO4), filtered, and concentrated under
reduced pressure. Chromatography of the crude residue gave the bisꢀacetal 9 (25 mg, 76% yield)
as a colourless oil. 1H NMR (400 MHz): δ 1.45 (s, 9H), 3.31 (bs, 1H), 3.45 (s, 3H), 3.37ꢀ3.46 (m,
4H), 3.56ꢀ3.70 (m, 4H), 4.18 (bs, 1H), 4,30 (bd, 1H, J = 13.3 Hz), 4.54ꢀ4.68 (m, 2H), 4,71 (d,
1H, J = 6.7 Hz), 4.79 (d, 1H, J = 6.7 Hz). 1H NMR (400 MHz, 45°C): δ 1.53 (s, 9H), 3.31ꢀ3.35
(m, 2H), 3.38 (t, 3H), 3.45 (t, 3H), 3.47ꢀ3.53 (m, 1H), 3.60ꢀ3.64 (m, 1H), 3.68 (t, 2H, J = 7.9
Hz), 4.20ꢀ4.45 (m, 2H), 4.61 (d, 1H, J = 6.6 Hz), 4.63 (d, 1H, J = 6.7 Hz), 4.73 (d, 1H, J = 6.6
Hz), 4.82 (d, 1H, J = 6.8 Hz). 13C NMR (100 MHz): ppm 28.3, 50.4, 51.1, 53.8, 55.2, 55.7, 65.1,
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69.7, 72.8, 80.2, 96.0, 155.5. Anal. calcd for C15H27NO7: C, 54.04; H, 8.16; N, 4.20. Found: C,
C, 54.20; H, 8.14; N, 4.19.
7
8
9
L
-Altro-deoxynojirimycin (10). Acetate 7 (33 mg, 0.1 mmol) was suspended in a 1M NaOH
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solution (3 mL). The resulting suspension was warmed to reflux and stirred at the same
temperature for 1 h. Temperature was then cooled to rt; the mixture was eluted through a short
pad containing a Dowex 50WX8 (H+ form) resin. The resulting filtrate was concentrated;
chromatography of the crude residue provided 10 (9.0 mg, 55% yield) as an oil. NMR spectra of
10 were fully in line with those reported elsewhere.32 HRMS: m/z [M + H]+, calcd: 164.085;
found: 164.0902.
L
-Deoxynojirimycin hydrochloride (11). Oxirane 9 (25 mg, 75 ꢁmol) was suspended in a
NaOH 1M solution (2 mL). The reaction mixture was warmed to reflux for 1 h; then temperature
was cooled to rt. The mixture was eluted through a short pad containing a Dowex 50WX8 (H+
form) resin. The resulting filtrate was concentrated, acidified with HCl 2M (3 mL) and refluxed
for 1 h. Removal of the volatiles under reduced pressure yielded 11 (15 mg, 91% over two steps)
as a white solid. 1H NMR (400 MHz, D2O): δ 3.14 (dd, 1H, J = 12.6, 11.6 Hz), 3.37 (ddd, 1H, J
= 3.2, 5.2, 10.5 Hz), 3.67 (dd, 1H, J = 12.6, 5.4 Hz), 3.76 (dd, 1H, J = 10.5, 9.3 Hz), 3.87ꢀ3.98
13
(m, 2H), 4.03 (dd, 1H, J = 5.2, 12.8 Hz), 4.11 (dd, 1H, J = 3.2, 12.8 Hz). C NMR (100 MHz,
D2O): ppm 41.5, 53.6, 55.8, 62.8, 63.7, 72.2. Anal. calcd for C6H14ClNO4: C, 36.10; H, 7.07; N,
7.02. Found: C, 36.22; H, 7.05; N, 7.00. HRMS: m/z [M + H]+, calcd: 164.0917; found:
164.0910.
Diol 13. A small amount of the crude residue deriving from oxirane ring cleavage of 9 was
subjected to column chromatography (hexane:ethyl acetate, from 100:0 to 70:30), to provide the
pure 13 as a pale yellow oil. 1H NMR (400 MHz): δ 1.47 (s, 9H), 3.36 (s, 3H), 3.39 (bd, 1H, J =
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14.3 Hz), 3.43 (s, 3H), 3.71 (bd, 1H, J = 2.7 Hz), 3.77 (dd, 1H, J = 4.8, 10.3 Hz), 3.85 (dd, 1H, J
= 5.5, 10.3 Hz), 3.88 (bt, 1H, J = 2.0 Hz), 3.91 (bt, 1H, J = 3.3 Hz), 4.07 (dd, 1H, J = 3.0, 14.3
Hz), 4.47 (bt, 1H, J = 5.1 Hz), 4.62 (d, 1H, J = 6.6 Hz), 4.65 (d, 1H, J = 6.6 Hz), 4.71 (d, 1H, J =
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13
7.0 Hz), 4.74 (d, 1H, J = 7.0 Hz). C NMR (100 MHz, acetoneꢀd6): ppm 27.8, 51.6, 51.8, 54.3,
55.0, 64.8, 69.3, 69.6, 74.3, 78.7, 95.4. 95.9, 155.3. Anal. calcd for C15H29NO8: C, 51.27; H,
8.32; N, 3.99. Found: C, 51,40; H, 8.30; N, 3.98. HRMS: m/z [M + Na]+, calcd: 374.1785; found:
374.1769.
N-Butyl-
L
-deoxynojirimycin (entꢀ1). Butanal (16.4 ꢁL, 0.18 mmol) was added to a stirring
solution of
L
ꢀDNJ (11, 28 mg, 0.14 mmol) in an acetic acid/methanol (2 mL, 1:200 v/v) solution.
After a few minutes, NaBH3CN (8.8 mg, 0.14 mmol) was added. The resulting mixture was
stirred at the same temperature for 16 h; then the volatiles were removed under reduced pressure.
Flash chromatography of the crude residue (DCM:MeOH:NH4OH = 70:28:2) yielded the pure
entꢀ1 (30 mg, 96% yield) as a colorless oil. [α]D +17.0, c 0.27 (1: ꢀ15.9, c 0.77).39 1H NMR (400
MHz, D2O): δ 0.92 (t, 3H, J = 7.3 Hz), 1.31 (sext, 2H, J = 7.3 Hz), 1.47ꢀ1.59 (m, 2H), 2.40ꢀ2.55
(m, 2H), 2.68ꢀ2.80 (m, 1H), 2.82ꢀ2.95 (m, 1H), 3.10ꢀ3.21 (m, 1H), 3.32 (t, 1H, J = 9.3 Hz), 3.45
(t, 1H, J = 9.5 Hz), 3.61 (ddd, 1H, J = 10.7, 9.3, 4.9 Hz), 3.89 (dd, 1H, J = 12.9, 2.5 Hz), 3.94
(dd, 1H, J = 12.9, 2.5 Hz). 13C NMR (100 MHz, CD3OD): ppm 14.2, 21.4, 27.0, 53.7, 56.5, 57.7,
67.4, 69.6, 70.8, 79.6. Anal. calcd for C10H21NO4: C, 54.77; H, 9.65; N, 6.39. Found: C, 54.88;
H, 9.62; N, 6.37. HRMS: m/z [M + H]+, calcd: 220.1471; found: 220.1533.
2) Computational studies
The optimal conformation of compounds 12-13 was simulated using the semiempirical method
PM3 (software package Hyperchem 8.0).
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3) Biochemical studies
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Glycosidase inhibition experiments. Glycosyl hydrolase activities were assayed by
spectroscopic techniques using artificial sugarꢀsubstrates containing 2ꢀnitrophenyl (2Np), 4ꢀ
nitrophenyl (4Np) or 4ꢀmethylumbelliferyl (4ꢀMU) moieties.
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The inhibition assays of rice αꢀglucosidase (8 ꢁg) and recombinant human αꢀglucosidase
(rhGAA; Myozyme, 5 ꢁg) were performed on 4Npꢀαꢀ ꢀglucopyranoside (4NpꢀαꢀGlc, 25 and 20
D
mM, respectively), in 100 mM sodium acetate buffer, pH 4.0, at 37 °C. The inhibition assays of
the βꢀglucosidases from Pyrococcus furiosus (0.15 ꢁg) and Sulfolobus solfataricus P2 (0.1 ꢁg),
and the βꢀgalactosidase from Alicyclobacillus acidocaldarius (0.05 ꢁg) were performed on 2.5
mM 4Npꢀβꢀ
D
ꢀglucopyranoside (4NpꢀβꢀGlc), 6 and 20 mM, respectively, 2NpꢀβꢀDꢀ
galactopyranoside (2NpꢀβꢀGal), in 50 mM citrate/phosphate buffer, pH 5.5, at 65 °C. The
inhibition assays of the recombinant human αꢀgalactosidase A (Fabrazyme, 0.4 ꢁg) were
performed on 30 mM 4Npꢀαꢀ ꢀgalactopyranoside (4NpꢀαꢀGal) in 50 mM citrate/phosphate pH
D
4.5, at 37°C. The inhibition assays of the αꢀgalactosidase from Thermotoga maritima (0.1 ꢁg)
were performed on 2.5 mM 4NpꢀαꢀGal, in 50 mM sodium acetate pH 5.0, at 65 °C. The
inhibition assays of the αꢀfucosidase from Sulfolobus solfataricus (2 ꢁg) were performed on 1
mM 4NpꢀαꢀLꢀfucopyranoside (4NpꢀαꢀFuc), in 50 mM sodium phosphate buffer, pH 6.5, at 65
°C. For all the above enzymes, the % of inhibition was measured in the presence of 1 and entꢀ1
(1 mM). The reactions were stopped in ice by adding 0.8 mL of 1 M sodium carbonate pH 10.2.
The absorbance values were measured by a Varian Cary 50 Bio UVꢀvisible spectrophotometer at
420 nm at room temperature and the molar extinction coefficients were 17.2 mM− 1 × cm− 1
(4Np) and 4.83 Mꢀ1 × cmꢀ1 (2Np). The activities of the enzymes without inhibitors were reported
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as 100% (Figure S2). The IC50 values of 1 were measured for rice αꢀglucosidase and rhGAA at
their standard conditions, in the range 25ꢀ500 ꢁM and 1ꢀ1000 ꢁM inhibitor, respectively (Figure
S3). All kinetic data were calculated as the average of the two experiments and were plotted and
refined with the programme GraphPad Prism.
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The remaining assays were performed as follows. βꢀGlucosidase, βꢀgalactosidase and βꢀ
hexosaminidase activity: the enzyme sources were HL60 cell pellets (human promyelocytic
leukemia cells) homogenised in PBS with 0.1% TritonXꢀ100. HLꢀ60 cells were cultured in
RPMIꢀ1640 supplemented with 20% fetal bovine serum, 1% penicillin/streptomycin and 1% Lꢀ
glutamine. Cultures were maintained at 37 °C with 5% CO2. For the enzyme assays, 30 x 106
cells were collected, centrifuged, washed with PBS, centrifuged again and stored as dry pellets at
ꢀ20°C. For the determination of βꢀgalactosidase activity, 1 mM 4ꢀMU βꢀ
Gal), in assay buffer (200 mM acetate, pH 4.3, 100 mM NaCl, 0.1% TritonXꢀ100) was used. The
substrate for βꢀglucosidase and Cerezyme™ activity was 4.5 mM 4ꢀMU βꢀ ꢀglucoside (4ꢀMUꢀ
Glc) in assay buffer (200 mM sodium citrate, pH 5.5, 0.1% TritonXꢀ100). For the determination
of βꢀhexosaminidase activity, 3 mM 4ꢀMU Nꢀacetylꢀβꢀ ꢀglucosaminide (4ꢀMUꢀGlcNAc) in
Dꢀgalactoside (4ꢀMUꢀ
D
D
assay buffer (200 mM sodium citrate, pH 4.5, 0.1% TritonXꢀ100) was used. Assays (in triplicate)
containing homogenate, different concentrations of iminosugar inhibitors and 4ꢀMU substrates
were incubated at 37 °C for 30 min. The reaction was stopped by adding cold 0.5 M Na2CO3 pH
10.7, and released 4ꢀMU was measured with a FLUOstar OPTIMA plate reader (BMG Labtech,
Ortenberg, Germany) with excitation at 360 nm and emission at 460 nm. Sucrase/isomaltase and
lactase activity: supernatant from homogenised small intestines of C57BL/6 control mice41 (5
weeks old) was used as the enzyme source. Small intestines of C57BL/6 control mice were
dissected (2ꢀ3cm), carefully washed in PBS and centrifuged in PBS for 10 min at 3.000 rpm. The
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supernatant was discarded and clean intestines were set up at 100 mg wet weight/ml 100 mM
sodium citrate / 100 mM citric acid buffer, pH 6. The intestines were homogenised by using an
Ultraturrax T25 probe homogeniser (Janke&Kunkel, IKAꢀLabortechnik, Germany). The
homogenate underwent three repeated cycles of freezing and thawing before second
centrifugation for 10 min at 3.000 rpm. Finally, the pellet was discarded and the supernatant was
kept at ꢀ20 °C for enzyme assays. Sucrase/isomaltase and lactase activities were assayed by
measuring glucose released from sucrose or lactose substrate using a Glucose Assay Kit (Sigmaꢀ
Aldrich). Assays (in triplicate) contained a range of concentrations of iminosugar inhibitors and
were incubated at 37 °C for 30 min. Absorbance at 340 nm was measured with a FLUOstar
OPTIMA plate reader (BMG Labtech, Ortenberg, Germany).
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Differential scanning fluorimetry (DSF) of rhGAA. Thermal stability scans of rhGAA were
performed as already described.6 Briefly, 2.5 ꢁg of enzyme were incubated in the absence and in
the presence of 1 and entꢀ1 (0.1 mM and 1 mM in both cases), SYPRO Orange dye, 25 mM
sodium phosphate buffer and 150 mM NaCl, pH 7.4. Thermal stability scans were performed at 1
°C/min in the range 25–95 °C in a RealꢀTime Light Cycler (Biorad, Milan, Italy). SYPRO
Orange fluorescence was normalized to maximum fluorescence value within each scan to obtain
relative fluorescence.
4) Biological studies
Cell lines. Fibroblasts from the PD patients carrying different mutations (pt. 1: phenotype,
intermediate; genotype, p.L552P/p.L552P; pt. 2: phenotype, severe; genotype, aberrant
splicing/unknown; pt. 3: phenotype, juvenile; genotype, p.R375L/p.V755SfsX41) were available
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in the laboratory of Department of Translational Medical Sciences, section of Pediatrics,
Federico II, University of Naples, Italy. All cell lines were grown at 37 °C with 5% CO2 in
Dulbecco’s modified Eagle’s medium (Invitrogen, Grand Island, NY) and 20% fetal bovine
serum (SigmaꢀAldrich, St Louis, MO), supplemented with 100U/ml penicillin and 100 mg/ml
streptomycin.
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Reagents. rhGAA (Myozyme) was purchased from Genzyme Co. (Naarden, Netherlands).
Compound 1 was purchased from (SigmaꢀAldrich, St Louis, MO). The primary antibodies used
for western blot analysis were antiꢀhuman GAA, purchased from (PRIMM s.r.l., Milan, Italy);
antiꢀβꢀactin, mouse monoclonal antibody, purchased from (SigmaꢀAldrich, St Louis, MO). The
secondary antibodies were goat antiꢀrabbit IgG (H + L)ꢀHRP conjugated and goat antiꢀmouse
IgG (H + L)ꢀHRP conjugated purchased from Biorad (Hercules, CA).
Treatment of fibroblasts with iminosugars. Fibroblasts were incubated for 4 days with 1 or
entꢀ1 (20 ꢁmol/L). The same cell lines were cultured in parallel in the absence of iminosugars for
comparison. After each experiment, the cells were harvested and the cell extracts were used for
the assay of GAA activity.
Uptake of recombinant enzyme. To study rhGAA uptake and correction of GAA activity, PD
and control fibroblasts were incubated with 50 ꢁmol/L rhGAA for 24 hours in the absence and in
the presence of 1 or entꢀ1 (20 ꢁmol/L). The cells were then harvested and cell pellets were
washed twice with phosphateꢀbuffered saline, resuspended in water and disrupted by five cycles
of freezeꢀthawing. GAA activity was assayed as described above. Protein concentrations were
measured according to Lowry et al.42
GAA enzyme assay. Fibroblasts were harvested by trypsinization and disrupted by freezing
and thawing (3x). Cell homogenates (10ꢀ20 ꢁg of proteins) were incubated at 37°C for 60 min
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with 2 mM 4 methylumbelliferyl αꢀDꢀglucopyranoside as substrate in a 0.2 acetate buffer pH 4 in
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an incubation mixture of 20 ꢁl. Reactions were stopped with 1 ml glycine carbonate buffer pH
10.7 and fluorescence was read on a Turner biosystem fluorometer Modulus 9200 (360 nm
excitation, 450 nm emission). Protein concentration in cell homogenates was measuring
according to Lowry et al.42
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Western blot analysis. To study GAA processing fibroblast extracts were subjected to
western blot analysis. The cells were harvested, washed in phosphateꢀbuffered saline,
resuspended in water, and disrupted by five cycles of freezeꢀthawing. Equal amounts (20 ꢁg
protein) of fibroblast extracts were subjected to sodium dodecyl sulfate polyacrylamide gel
electrophoresis (7.5% acrylamide) and proteins were transferred to PVD membrane (GE
Healthcare). An antiꢀhuman antiserum GAA was used as primary antibody to detect GAA
polypeptides; to detect βꢀactin, a monoclonal mouse antibody was used. Immunoreactive
proteins were detected by chemiluminescence (ECL; GE Healthcare). Quantitative analysis of
band intensity was performed using ImageJ.
ASSOCIATED CONTENT
Copies of NMR spectra, LCꢀMS data, glycosidase inhibition data and DSF experiments.
Molecular formula strings of compounds 1 and entꢀ1 (CSV). This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: dandalonzo@unina.it.
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ACKNOWLEDGMENT
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Financial support from MIUR, Italy (PRIN 2010–2011, prot. 20109Z2XRJ) to G.P. and from the
Mizutani Foundation for Glycoscience to D.P. is gratefully acknowledged. F.P. is a Royal
Society Wolfson Research Merit Award holder and Wellcome Trust Investigator in Science.
Support to M.H. was provided by the Erasmus Placement Program.
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ABBREVIATIONS
ASSC, activeꢀsite specific chaperone; Bn, benzyl; DCE, dichloroethane; DCM, dichloromethane;
DDQ, 2,3ꢀdichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone; DIPEA, N,Nꢀdiisopropylethylamine; DMF,
N,Nꢀdimethylformamide; ECL, enhanced chemiluminescence; ERT, enzyme replacement
therapy; Fuc, fucopyranoside; GAA, acid αꢀglucosidase; Gal, galactopyranoside; GBA,
glucocerebrosidase; GH, glycosyl hydrolase; Glc, glucopyranoside; HRP, horseradish
peroxidase; IgG, immunoglobulin G; LSD, lysosomal storage disorder; MOM, methoxymethyl;
MU, methylumbelliferyl; NBDNJ, Nꢀbutyldeoxynojirimycin; NMR, nuclear magnetic resonance;
NP, nitrophenyl; PBS, phosphateꢀbuffered saline; PCT, pharmacological chaperone therapy; PD,
Pompe disease; PVD, polyvinylidene.
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(11) Mention must also be given to DNJ (Duvoglustat), which currently represents the most
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Barth, J.; Barlow, C.; Lockhart, D. J.; Valenzano, K. J.; Boudes, P.; Johnson, F. K.; Byrne, B.
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(24) D’Alonzo, D.; Guaragna, A. Stereoselective methods in the synthesis of bioactive
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(27) It must be observed that a few synthetic routes to LꢀDNJ have been reported, see for
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nojirimycin and their 1ꢀdeoxyderivatives from myoꢀinositol. Carbohydr. Res. 1992, 237, 185ꢀ
194. (b) Best, D.; Wang, C.; WeymouthꢀWilson, A. C.; Clarkson, R. A.; Wilson, F. X.; Nash, R.
J.; Miyauchi, S.; Kato, A.; Fleet, G. W. J. Looking glass inhibitors: scalable syntheses of DNJ,
DMDP, and (3R)ꢀ3ꢀhydroxyꢀ
and (3S)ꢀ3ꢀhydroxyꢀ ꢀbulgecinine from
βꢀgalactosidases whereas ꢀDMDP is a potent and specific inhibitor of αꢀglucosidases.
Lꢀbulgecinine from
Dꢀglucuronolactone and of LꢀDNJ, LꢀDMDP,
D
Lꢀglucuronolactone. DMDP inhibits βꢀglucosidases and
L
Tetrahedron: Asymmetry 2010, 21, 311ꢀ319.
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containing chiral building blocks: new avenues in glycochemistry. In Domino and
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Intramolecular Rearrangement Reactions as Advanced Synthetic Methods in Glycoscience, 1st
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(29) Guaragna, A.; D’Alonzo, D.; Paolella, C.; Napolitano, C.; Palumbo, G. Highly
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stereoselective de novo synthesis of Lꢀhexoses. J. Org. Chem. 2010, 75, 3558ꢀ3568.
(30) D’Alonzo, D.; Van Aerschot, A.; Guaragna, A.; Palumbo, G.; Schepers, G.; Capone, S.;
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L
L
(31) Paolella, C.; D’Alonzo, D.; Palumbo, G.; Guaragna, A. Sulfurꢀassisted domino access to
bicyclic dihydrofurans: case study and early synthetic applications. Org. Biomol. Chem. 2013,
11, 7825ꢀ7829.
(32) Guaragna, A.; D’Errico, S.; D’Alonzo, D.; Pedatella, S.; Palumbo, G. A general approach
to the synthesis of 1ꢀdeoxyꢀLꢀiminosugars. Org. Lett. 2007, 9, 3473ꢀ3476.
(33) Guaragna, A.; Dell’Isola, A.; D’Errico, S.; Palumbo, G.; D’Alonzo, D. Beyond
Achmatowicz reaction: DDQꢀmediated chemoꢀ and stereoconvergent dominoꢀone pot
cyclization/rearrangement of bisꢀthioenol etherꢀcontaining chiral building blocks. Tetrahedron
Lett. 2014, 55, 7007ꢀ7010.
(34) As already reported (see ref. 32), a carbohydrateꢀlike numbering was applied for entꢀ1 and
its precursors.
(35) D’Alonzo, D.; Guaragna, A.; Napolitano, C.; Palumbo, G. Rapid access to 1,6ꢀanhydroꢀβꢀ
Lꢀhexopyranose derivatives via domino reaction: synthesis of
Lꢀallose and
Lꢀglucose. J. Org.
Chem. 2008, 73, 5636ꢀ5639.
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(36) The formation of 12 allows explaining the NaOHꢀmediated cleavage of the NꢀBoc group,
despite the recognized stability of the latter to alkaline conditions, see: Agami, C.; Couty, F. The
reactivity of the NꢀBoc protecting group: an underrated feature. Tetrahedron 2002, 58, 2701ꢀ
2724.
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(37) Singh, A.; Kim, B.; Lee, W. K.; Ha, H.ꢀJ. Asymmetric synthesis of 1ꢀdeoxyazasugars
from chiral aziridines. Org. Biomol. Chem. 2011, 9, 1372ꢀ1380.
(38) Theoretical J values were obtained by applying the Haasnoot–de Leeuw–Altona equation:
NavarroꢀVázquez, A.; Cobas, J. C.; Sardina, F. J.; Casanueva, J.; Díez, E. A graphical tool for
the prediction of vicinal protonꢀproton 3JHH coupling constants. J. Chem. Inf. Comput. Sci. 2004,
44, 1680ꢀ1685.
(39) Asano, N.; Kizu, H.; Oseki, K.; Tomioka, E.; Matsui, K.; Okamoto, M.; Baba, M. Nꢀ
Alkylated nitrogenꢀinꢀtheꢀring sugars: conformational basis of inhibition of glycosidases and
HIVꢀ1 replication. J. Med. Chem. 1995, 38, 2349ꢀ2356.
(40) For a comparative analysis of the biological activity of the two enantiomers, experiments
using 20 ꢁM solutions of entꢀ1 – the optimal concentration value found for 1 (ref. 13) – were
conducted in this study.
(41) All animal studies were approved by the UK Home Office for the conduct of regulated
procedures under licence (Animal Scientific Procedures Act, 1986).
(42) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the
Folin phenol reagent. J. Biol. Chem. 1951, 1, 265ꢀ275.
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