Synthesis of Isofagomine Derivatives as New Fluorescence pH Indicators/Glycosidase Inhibitors

Article abstract of DOI:10.1002/ejoc.202000522

Inhibitor protonation of azasugars of the isofagomine type when bound to enzyme can be investigated using photon induced electron transfer (PET) quenching of an attached fluorophore. For this purpose, Isofagomine, iso-d-galacto-fagomine, and iso-l-gulo-fagomine were converted to N-(10-chloroanthracenenyl-9-alkyl) derivatives where the alkyl group contained one, two, or three methylene groups. The new derivatives displayed pH dependent fluorescence; as the ammonium forms they were fluorescent, while 90–99 percent of the fluorescence was quenched in the amine forms. The 3 isofagomine derivatives were competitive inhibitors of T. Maritima Ι-glucosidase with K_i values from 0.37–4.6ΤM. Similarly, the iso-d-galacto-fagomines inhibited A. Niger Ι-galactosidase with K_i values from 63–2000 nm. When bound to the enzymes the inhibitors displayed between 1–15 percent fluorescence.

Full text of DOI:10.1002/ejoc.202000522

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Synthesis of isofagomine derivatives as new fluorescence pH
indicators/glycosidase inhibitors.
Authors: Bo Wang, Sidsel Ammitzbøll Bogh, Jens Christian Navarro
Poulsen, Bo W. Laursen, and Mikael Bols
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202000522
Link to VoR: https://doi.org/10.1002/ejoc.202000522
0
1/2020

European Journal of Organic Chemistry
10.1002/ejoc.202000522
Synthesis of isofagomine derivatives as new fluorescence pH
indicators/glycosidase inhibitors.
Dr. Bo Wang, Dr. Sidsel Ammitzbøll Bogh, Dr. Jens Christian Navarro Poulsen, Prof. Bo W. Laursen and Prof. Mikael
Bols*
Department of chemistry, University of Copenhagen, bols@chem.ku.dk, twitter: @MikaelBols
Key topic: Inhibitor protonation
Table of Contents (Graphical Abstract): To check pH conditions in the active site of an enzyme an inhibitor with
on/off fluorescence upon protonation is required. Here the potent glycosidase inhibitor isofagomine is converted
into ‘glycosidase fluorescence indicators.’
Abstract: Inhibitor protonation of azasugars of the isofagomine type when bound to enzyme can be investigated
using photon induced electron transfer (PET) quenching of an attached fluorophore. For this purpose Isofagomine
and iso-D-galacto-fagomine and iso-L-gulo-fagomine were converted to N-(10-chloroantracenenyl-9-alkyl)
derivatives where the alkyl group contained one, two or three methylene groups. The new derivatives displayed
pH dependent fluorescence, as the ammonium forms were fluorescent while 90-99% of the fluorescence was
quenched in the amine forms. The 3 isofagomine derivatives were competitive inhibitors of T. Maritima


-glucosidase with K values from 0.37-4.6M. Similarly the iso-D-galacto-fagomines inhibited A. Niger
i
-galactosidase with K values from 63-2000 nM. When bound to the enzymes the inhibitors displayed between
i
1
-15% fluorescence.
Introduction
Glycosidases or glycoside hydrolases are an extremely common type of enzyme that participates in many different
1
biological events such as degradation of polysaccharides or processing of oligosaccharides. Due to the very
widespread occurrence of glycosidases, glycosidase inhibitors have a large drug potential. Some of the most
widely applicable inhibitors are imino- and azasugars that has the sugar shaped amine general structure of
2
3
1
-deoxynojirimycin (DNJ, 2) or isofagomine (1, Figure 1). The basic nitrogen atom (pK (1)= 8.4; pK (2)= 6.7)
a a
4
which these compounds have in common with many other inhibitors and drugs, means that these compounds
5
are substantially protonated at neutral pH.
1
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European Journal of Organic Chemistry
10.1002/ejoc.202000522
Figure 1: Aza- and iminosugars studied.
+
+
The resemblance of the protonated amines (1,H and 2,H ) with the transition state of glycoside hydrolysis have
6,7
led to discussion of the possible transition state resemblance of these inhibitors. It is clear from the pH
8
dependent inhibition of 1 and 2 that they bind in one distinct protonation state but the actual protonation state
of these inhibitors cannot be elucidated from those data. This can however potentially be done by converting the
inhibitor into a fluorescence indicator where photoinduced electron transfer (PET) quench the fluorescence when
9
the lonepair of the amine is unoccupied by protonation. We have recently shown with DNJ derivatives (i.e. 2)
with fluorescent anthracene groups attached (e.g. 3) that they were unprotonated when bound to Phanerochaete
10
chrysosporium -glucosidase – a type 3 glycosidase. Another smaller study with the isofagomine analogue 4
11
showed that this compound was unprotonated when bound to almond -glucosidase – a type 1 glycosidase. In
the present work we wished to do a larger study with isofagomine derivatives and therefore required more
compounds. We have here expanded the previous study with 4 with longer chain-length analogues and also
prepared analogues of iso-D-galacto-fagomine (5) and iso-L-gulo-fagomine (6).
Scheme 1: Synthesis of 10-chloroanthracenyl substituted isofagomines 9 and 10
Results and discussion
2
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European Journal of Organic Chemistry
10.1002/ejoc.202000522
12
10
Synthesis. The isofagomine-chloroanthracene derivatives were prepared from 1 and the aldehydes 7 and 8 as
outlined in scheme 1: Reductive amination using sodium cyanoborohydride in methanol-chloroform and acetic
o
acid at 60 C gave the products 9 and 10 in yields of 44 and 57%, respectively (Scheme 1). The reaction is sluggish
requiring long time even at elevated temperature which is the reason for the generally modest yields in the
reductive amination reactions with the chloroanthracene aldehydes. The shorter chain analogue 4 and the
11
lactame reference compound 11 (Figure 1) were prepared as previously described.
13
The iso-galacto-fagomine derivatives were made in a similar fashion (Scheme 2). Alkylation of 5 with
chloride 12 and potassium carbonate in DMF gave 13 in 53% yield (Scheme 2). Reductive amination of 7 and 8
o
with 5 using sodium cyanoborohydride in methanol-chloroform and acetic acid at 60 C gave the products 14 and
1
5 in 33 and 35% yield, respectively (Scheme 2).
Scheme 2: Synthesis of 10-chloroanthracenyl substituted iso-galacto-fagomines 13, 14 and 15
Iso-L-gulo-fagomine 6 was obtained from L-xylose as outlined in Scheme 3. Starting from the 1-O-benzylated
14
L-xylopyranoside 16 dibutyltin oxide assisted tosylation of 16 gave the 4-O-tosylate 17 in high yield (Scheme 3).
Treatment of this compound with potassium tert-butoxide gave the 3,4-epoxide 18 also in high yield. Reaction
with triisopropylsilyl triflate and lutidine gave the 2-O-triisopropyl (TIPS) protected derivative 19. Epoxide opening
with cyanide afforded some difficulties but was achieved using cyanodiethyl aluminum which afforded two
regioisomers 20 and 21 in 10% and 40% yield, respectively. The minor regioisomer 20 was desilylated with
tetrabutylammonium fluoride to give 22 and then subjected to hydrogenolysis and hydrogenation to give 6 in
good yield.
With 6 in hand two chloroanthracene derivatives were prepared (Scheme 3). Reaction of 6 with 12 and
potassium carbonate gave N-alkylation to 23. Reductive amination of 7 with 6 and sodium cyanoborohydride gave
2
4.
3
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European Journal of Organic Chemistry
10.1002/ejoc.202000522
Scheme 3: Synthesis of 10-chloroanthracenyl substituted iso-L-gulo-fagomines 23 and 24
Fluorescence measurements. The fluorescence as a function of pH of compound 4 and the new chloroanthracenyl
substituted isofagomine derivatives 9-10, 13-15, 23 and 24 were determined by measuring their fluorescence
spectra at different pH. Due to the poor solubility of these derivatives in pure water these experiments had to be
carried out with 10% DMSO being added as cosolvent. The influence on pK and K values was expected to be
a
i
minor with this low content of DMSO. The excitation wave-length was 358 nm and the fluorescence spectra were
measured from 380-680 nm. From plots of the fluorescence intensity at maximum versus pH (Figure 2) pK values
a
for the compounds were determined as well as relative intensity of fluorescence remaining in the free amine form
(
Table 1). All 8 compounds worked as fluorescence indicators with 76-95% of the fluorescence being quenched in
the amine. The pK values were from 1.6-2.4 units lower than those of 1 and 5 (pK (1)= 8.4; pK (5)= 8.8) which in
a
a
a
part is due to the greater sterical hindrance associated with protonation in these tertiary amines and in part is due
to an inductive electron withdrawing-effect from the anthracene. The basicity increases with increasing chain
length in each series consistent with the decreasing influence of the anthracene on the amine. There is also a
consistent pK difference between stereoisomers, which is due to the higher electron-withdrawing effect of the
a
5
equatorial OH. In otherwise similar compounds the gulo isomer is more basic than the galacto isomer, which is
more basic than the gluco isomer i.e. pK decreases in the methylene series pK (23)> pK (13)> pK (4) and in the
a
a
a
a
ethylene series pK (24)> pK (14)> pK (9).
a
a
a
The effectiveness of photo induced electron transfer (PET) is decreased with chain-length: In the short chain
analogues 4, 13 and 23 quenching is almost complete as the fluorescence is reduced to 5-6 %, while the residual
fluorescence increases to 7-10% in the C2-chain analogues 9, 14 and 24 and to 20-24% in the C3-chain analogues
1
0 and 15.
4
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European Journal of Organic Chemistry
10.1002/ejoc.202000522
Figure 2: Fluorescence titration curves for inhibitors 4, 9-10, 13-15 and 23-24.
Inhibition. The isofagomine analogues 9-10 and 23-24 were investigated for inhibition of Thermotoga maritima
-glucosidase, while the galacto-analogues 13-15 were tested for inhibition of Aspergillus Niger -galactosidase.

Due to the solubility issue discussed above all these assays were done in aqueous buffers containing 10% DMSO.
The added DMSO resulted in homogeneous solutions of the inhibitors and did not disrupt these two enzymes. In
all cases competitive inhibition was found with the listed K values, which mostly are submicromolar (Table 1). The
i
10
gluconolactame 11 was also tested against the -glucosidase and was a reasonably good inhibitor (K 12.1 M).
i
Inhibition was measured at pH 6.5 unless otherwise noted, which is close to the optimum pH of all these
inhibitors.
Table 1. pK values, fluorescence quenching, optimum pH for inhibition and inhibition constants of 4, 9-11 13-15
a
and 23-24. A means 10-Chloro-anthracene, - means not relevant or not tested. Optimum pH was calculated as
8
previously described.
Compound
Structure
n
1
2
3
1
2
pK
a
Fluorescence in amino-form
K
i
(M, pH 6.5) -glucosidase T. Maritima
4
9
5.78
6.00
6.75
6.36
7.09
5%
10%
24%
5%
0.37
4.3
2.3
1.1
4.6
1
2
2
0
3
4
7%
5
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European Journal of Organic Chemistry
10.1002/ejoc.202000522
1
1
-
-
100%
12
K
i
(M, pH 6.5) -galactosidase A. Niger
1
1
1
3
4
5
1
2
3
6.15
6.47
7.16
6%
8%
0.29
2.1
20%
0.063
Fluorescence during inhibition. Investigation of inhibitor fluorescence upon binding to the enzyme was performed.
This was done by recording the fluorescence spectrum of the inhibitor at pH 6.5 in buffer containing 10% DMSO
and adding increasing amounts of excess enzyme. With -glucosidase T. Maritima the glucosidase inhibitors 4, 9
and 11 had a decrease in intensity of the fluorescence proportional to the added glycosidase (Figure 3 and 4). This
means that the fluorescence is quenched when the inhibitor binds to this enzyme. A change in pH to more acidic
pH makes the quenching more apparent (Figure 3 left) as more inhibitor is protonated and hence fluorescent
outside the enzyme. The decrease in fluorescence of one of these inhibitors increase with enzyme concentration
so that the data fit the equation F = Fo[I] + Fx[EI] where [I] is the concentration of free inhibitor, [EI] is the
concentration of inhibitor-enzyme complex, F is the total fluorescence, Fo is the fluorescence from inhibitor with
no enzyme and Fx is fluorescence of the inhibitor when totally bound (Figure 5).
1
000
0
1
uM -glucosidase
.3 uM -glucosidase
2.6 uM -glucosidase
.2 uM -glucosidase
500
8
00
00
00
00
0
5
4
3
2
1
00
00
00
00
0
1
3 uM -glucosidase
6
4
2
26 uM -glucosidase
pH 6.0
pH 6.5
4
00
500
600
400
500
600

(nm)
 (nm)
Figure 3. Difference fluorescence spectra of inhibitor 4 (0.8 M) with increasing concentration of -glucosidase
the corresponding spectra without inhibitor have been subtracted). It is seen that fluorescence decrease with
(
increasing enzyme concentration.
Unfortunately, the fluorescence of lactam 11, a control compound, is also quenched by the enzyme in an
identical manner. This means that this enzyme is inherently quenching the fluorescence of the anthracene, and
the decrease in fluorescence is probably unrelated to the protonation state of the inhibitors. This quenching is
presumably caused by close proximity of tryptophans in the enzyme and the anthracene groups upon binding of
15
the inhibitor as tryptophans are known to be able to quench the fluorescence.
6
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European Journal of Organic Chemistry
10.1002/ejoc.202000522
11
Figure 4. Difference fluorescence spectra of inhibitors 9 and 11 (0.8 M) with increasing concentration of
-glucosidase (T. maritima) at pH 6.5 (the corresponding spectra without inhibitor have been subtracted). It is
seen that fluorescence decrease with increasing enzyme concentration.

8
6
4
2
00
00
00
00
0
0
10
20
30
40
[
-glucosidase] M
Figure 5. Decrease in fluorescence at 410 nm from inhibitor 9 as of result of increasing concentration of
-glucosidase (T. maritima) at pH 6.0. The data fit to the equation F = Fo[I] + Fx[EI] where Fo is the fluorescence

from inhibitor with no enzyme and Fx is fluorescence when totally bound. This shows that the fluorescence
change is a result of the simple binding event E + I → EI.
The fluorescence of the galactosidase inhibitors 13, 14 and 15, when bound to -galactosidase from A. Niger (pH
6
.5 in buffer containing 10% DMSO) was also investigated (Figure 6). In all cases the fluorescence was quenched
when these inhibitors bound to the galactosidase very similar to what was seen above. As the quenching could be
inherent quenching by tryptophanes near the active site no conclusion with regard to protonation of the inhibitors
can be made.
2
2
50
00
8
7
6
00
00
00
6
5
4
3
2
1
00
00
00
00
00
00
0
0
uM -galactosidase
1.3 uM -galactosidase
2.6 uM -galactosidase
5
1
.2 uM -galactosidase
13
14
150
500
3 uM -galactosidase
4
3
2
00
00
00
26 uM -galactosidase
1
00
15
5
0
100
0
0
-100
4
00
450
500
550
600
400
450
500
550
600
400
450
500
550
600

(nm)
 (nm)
 (nm)
Figure 6. Difference fluorescence spectra of inhibitors 13-15 with increasing concentration of - galactosidase at
7
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European Journal of Organic Chemistry
10.1002/ejoc.202000522
pH 6.5 (the corresponding spectra without inhibitor have been subtracted). It is seen that the fluorescence
decrease with increasing enzyme concentration.
Conclusions In summary in this work we have synthesized eight new isofagomine anthracene conjugates and
shown that they function as pH indicators and are good to excellent inhibitors of two glycosidases. In all cases the
enzymes quenched the fluorescence from the protonated inhibitors which could be due to removal of a proton or
quenching of the fluorescence by nearby tryptophan residues in the enzymes. For -glucosidase from T. maritima
it was seen that the enzyme was capable of quenching fluorescence from the pH independent and permanently
fluorescent and related inhibitor 11.
Experimental part
General information
Dry solvents were trapped from a solvent purification system. Reactants were purchased from commercial sources
and used without further purification. HRMS were recorded on a Bruker Solarix XR mass spectrometer analyzing
TOF. NMR spectra were recorded on a Brüker 500 MHz spectrometer. Chemical shifts (δ) are reported in ppm
1
13
relative to the residue solvent signals (CDCl : δ = 7.26 for H-NMR and 77.16 for C-NMR. D O: δ = 4.79 for
3
2
1
1
13
1
H-NMR. DMSO-d : δ = 2.50 for H-NMR and 39.52 for C-NMR. CD OD: δ = 3.31 for H-NMR and 49.00 for
6
3
13
C-NMR). Assignments were aided by COSY and HSQC experiments.
Determination of pKa value of iminosugars by fluorescence titration.
Iminosugar was dissolved in DMSO to give 8 μmol/L stock solution. 0.1 mL was added to 0.9 mL of
citrate-phosphate buffers with different pH values (2.2, 3.0, 4.0, 4.4, 5.0, 5.2, 5.5, 5.8, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, 7.5,
8
.0, 8.5, 9.0, 9.5). The fluorescence spectra were recorded from 380-680 nm with excitation at 358 nm.
Inhibition constants (Ki) of isofagomine derivatives to different glycosidases were measured in
phosphate buffer (pH 6) containing 10% DMSO
Procedure for fluorescence measurements. The inhibitor was dissolved in DMSO to create a stock solution
of approximately 8.0 M. Normally, fluorescence was measured on four types of solutions:
A. Blank (0.45 mL phosphate buffer and 0.05 mL DMSO)
B. Inhibitor (0.45 mL phosphate buffer, and 0.05 mL inhibitor stock solution, [I] = 0.8 M)
C. Enzyme (0.1-0.2 mL enzyme stock solution in phosphate buffer, and 0.25-0.35 mL phosphate
buffer and 0.05 mL DMSO. [E] = 5.8-72 M)
D. Enzyme and inhibitor (0.1-0.2 mL enzyme stock solution in phosphate buffer, and 0.25-0.35 mL
phosphate buffer and 0.05 mL inhibitor stock solution. [I] = 0.8 M, [E] = 5.8-62


M
Fluorescence spectra was taken of samples A-D exciting at 358 nm on a Perkin Elmer LS50 instrument.
The subtracted spectra (B-A), (D-C) gave the net effect of inhibitor fluorescence when free and bound.
Benzyl 3,4-anhydro--D-arabinopyranoside (18)
8
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European Journal of Organic Chemistry
10.1002/ejoc.202000522
14
To a solution of compound 16 (50 mg, 0.208 mmmol, 1 equiv.) in dry methanol (5 mL), Bu SnO (53 mg, 0.208
2
mmol, 1 equiv.) was added. The reaction mixture was refluxed at 66°C overnight. The reaction was then cooled
down to room temperature and concentrated under reduced pressure. After dried under vacuum, the residue of
stannyl acetal was dissolved in 1,4-dioxane (5 mL). TsCl (45 mg, 0.229 mmol, 1.1 equiv.) and DMAP (5 mg, 0.416
mmol, 0.2 equiv.) were added. After being stirred at r.t. for 2 hrs, the reaction mixture was concentrated under
reduced pressure. The flash chromatographical column (PE/EtOAc: 2/1, then 1/2) gave the tosylated xylose 17 (80
mg, 97%) as a white solid (R = 0.25 in DCM/MeOH: 7/1). To a solution of 17 (0.88 g, 2.23 mmol, 1 equiv.) in THF
f
(
1
20 mL), tBuOK (300 mg, 2.68 mmol, 1.2 equiv.) was added at 0 ºC. The reaction mixture was then stirred at r.t. for
hr and sat. NH Cl (15 ml) was added. After being stirred for another 30 min, the mixture was extracted with
4
EtOAc (3×20 mL). The combined organic layer was dried over MgSO , filtered and concentrated in vacuum to give
4
1
8 (0.48 g, 97%) as a colorless oil, which was used in the next step without further purification.
20
1
tert
R = 0.20 (PE/EtOAc: 2/1). [α] =+31.3(c 0.3, CHCl ). H NMR (CDCl , 500 MHz): δ 7.23-7.20 (m, 5H, H-Ar ), 4.78
f
D
3
3
2
2
2
3
(
d, 1H, J = 11.0 Hz, CHPh), 4.44 (d, 1H, J = 11.2 Hz, CHPh), 4.17 (d, 1H, J = 13.7 Hz, H-5a), 4.08 (d, 1H, J = 6.7
5,5’ 1,2
2
3
3
Hz, H-1), 3.82 (d, 1H, J = 13.7 Hz, H-5a), 3.68 (d, 1H, J = 6.7 Hz, H-2), 3.14 (d, 1H, J = 4.1 Hz, H-3), 3.05 (d,
5,5’
1,2
3,4
3
13
quat
1
1
H, J = 4.1 Hz, H-4), 2.31 (b, 1H, OH-2). C NMR (CDCl , 125 MHz): δ 137.05 (C-Ar ), 128.68(2C), 128.34(2C),
3,4 3
tert
28.21 (5×C-Ar ), 101.51 (C-1), 70.44 (CH Ph), 66.97 (C-2), 62.98 (C-5), 54.05 (C-3), 49.84 (C-4). HRMS (ESP):
2
1+
Calcd. For C H O Na [M+Na] : 245.2297; Found: 245.2298
12 14 4
Benzyl 3,4-anhydro-2-O-triisopropylsilyl--D-arabinopyranoside (19)
To a solution of 18 (480 mg, 2.16 mmol, 1 equiv.) in DMF (10 mL), 2,6-lutidine (0.6 mL, 5.18 mmol, 2.4 equiv.) was
added. The reaction mixture was then cooled down to 0ºC. TIPSOTf (0.79 mL, 2.59 mmol, 1.2 equiv.) was added.
The reaction mixture was then stirred at r.t. overnight. The reaction was diluted with EtOAc (10 mL) and washed
with HCl (1 M), (2×10mL), water (2×10mL) and brine. The combined organic layer was dried over MgSO , filtered
4
and concentrated in vacuum. The residue was purified by flash chromatography (PE/EtOAc: 95/5, then 4/1) to give
2
5
1
1
9 (0.65 g, 79%) as a colorless oil. R = 0.8 (PE/EtOAc: 2/1). [α] =+49.3(c 0.3, CHCl ). H NMR (CDCl , 500 MHz): δ
f D 3 3
tert
2
2
2
7
.35-7.27 (m, 5H, H-Ar ), 4.84 (d, 1H, J = 10.6 Hz, CHPh), 4.52 (d, 1H, J = 10.6 Hz, CHPh), 4.22 (d, 1H, J = 12.8
5,5’
3
3
Hz, H-5a), 4.18 (d, 1H, J = 6.4 Hz, H-1), 3.92-3.89 (m, 2H, H-5a, H-2), 3.19 (d, 1H, J = 4.0 Hz, H-3), 3.16 (d, 1H,
1,2
3,4
quat
3
13
J3,4 = 4.1 Hz, H-4), 1.06-1.02 (m, 21H, TIPS). C NMR (CDCl , 125 MHz): δ 137.33 (C-Ar ), 128.41(2C), 128.34(2C),
3
tert
1
1
27.80 (5×C-Ar ), 101.83 (C-1), 70.25 (CH Ph), 67.84 (C-2), 62.64 (C-5), 55.39 (C-3), 50.11 (C-4), 18.10, 18.04,
2
1+
2.39 (TIPS). HRMS (ESP): Calcd. For C H O SiNa [M+Na] : 401.2119; Found: 401.2105
12 34 4
Benzyl
4-cyano-4-deoxy-2-O-triisopropylsilyl--L-xylopyranoside
(20)
and
benzyl
3
-deoxy-cyano-2-O-triisopropylsilyl--D-lyxopyranoside (21)
To a solution of 19 (0.635 g, 1.68 mmol, 1 equiv.) in dry Et O (15 mL) at 0 ºC, Et AlCN (1.85 mL, 1.85 mmol, 1.1
2
2
equiv., 1 M in Et O) was added. The reaction mixture was then refluxed at 45 ºC for 3h30, and stirred at r.t. for
2
another 1 hr. The reaction mixture was cooled down to -40ºC, and sat. NH Cl was added dropwise. After being
4
stirred for 1 hr., the solid was filter and washed with EtOAc. The filtrate was evaporated and the residue was
purified by flash chromatography (PE/EtOAc: 6/1, then 5/1) to give 20 (67 mg, 10%), 21 (270 mg, 40%), and
starting material 19 (120 mg, 19%) as colorless oil.
1
tert
2
2
0: R = 0.55 (PE/EtOAc: 3/1), H NMR (CDCl , 500 MHz): δ 7.38-7.29 (m, 5H, H-Ar ), 4.81 (d, 1H, J = 11.5 Hz,
f 3
3
2
CHPh), 4.64 (d, 1H, J1 = 3.7 Hz, H-1), 4.54 (d, 1H, J = 11.5 Hz, CHPh), 4.18 (dd, 1H, J = 12.1, 3.0 Hz, H-5a), 3.97
,2
(
dd, 1H, J= 12.7, 5.6 Hz, H-3), 3.76 (dd, 1H, J = 12.1, 5.1 Hz, H-5a), 3.71–3.68 (m, 1H, H-2), 3.33 (d, 1H, J = 7.5 Hz,
13
quat
OH-3), 2.81 (m, 1H, H-4), 1.06–0.99 (m, 21H, TIPS). C NMR (CDCl , 125 MHz): δ 136.26 (C-Ar ), 128.70(2C),
3
tert
1
28.53(2C), 128.46 (5×C-Ar ), 128.26 (CN), 100.36 (C-1), 70.95 (C-2), 70.60 (CH Ph), 70.43 (C-3), 57.81 (C-5),
2
9
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European Journal of Organic Chemistry
10.1002/ejoc.202000522
1
+
3
4
2
4
1
2.03 (C-4), 18.10, 18.08, 12.44 (TIPS) HRMS (ESP): Calcd. For C H NO SiNa [M+Na] : 428.5998; Found:
22 35 4
28.6100
1: R = 0.50 (PE/EtOAc: 3/1), [α] =+61.4(c 1.0, CHCl ), H NMR (CDCl , 500 MHz): δ 7.38-7.31 (m, 5H, H-Ar ),
20
1
tert
f
D
3
3
2
3
2
.74 (d, 1H, J = 11.9 Hz, CHPh), 4.64 (d, 1H, J = 2.3 Hz, H-1), 4.50 (d, 1H, J = 11.9 Hz, CHPh), 4.26 (ddd, 1H, J =
1,2
3
5.2, 10.1, 5.2 Hz, H-4), 4.18 (t, 1H, J1,2 = 2.5 Hz, H-2), 3.81 (dd, 1H, J = 11.2, 5.0 Hz, H-5a), 3.56 (t, 1H, J = 10.8 Hz,
13
H-5b), 3.08 (dd, 1H, J = 2.5, 10.0 Hz, H-3), 2.15 (d, 1H, J = 5.9 Hz, OH-4), 1.06–1.03 (m, 21H, TIPS). C NMR (CDCl3,
quat
tert
1
6
25 MHz): δ 136.77 (C-Ar ), 128.71(2C), 128.38(2C), 128.33 (5×C-Ar ), 118.88 (CN), 96.40 (C-1), 69.37 (CH Ph),
2
9.36 (C-2), 63.75 (C-4), 63.21 (C-5), 39.60 (C-3), 18.13, 18.05, 12.65 (TIPS). HRMS (ESP): Calcd. For C H NO SiNa
22
35
4
1+
[
M+Na] : 428.2155; Found: 428.2223
Benzyl 4-deoxy-cyano--L-xylopyranoside (22)
To a solution of 20 (0.067 g, 0.165 mmol, 1 equiv.) in dry THF (5 mL) at 0 ºC, TBAF (0.314 mL, 0.314 mmol, 1.9
equiv., 1 M in THF) was added. The reaction mixture was stirred at r.t. for 3 hrs. The solvent was evaporated and
the residue was purified by flash chromatography (DCM, then DCM/Acetone: 9/1) to give 22 (40 mg, 97%) as a
1
tert
white solid. R = 0.33 (DCM/acetone: 9/1), H NMR (CD OD, 500 MHz): δ 7.42–7.27 (m, 5H, H-Ar ), 4.87–4.84 (d,
f
3
1
H, J = 11.8 Hz, CHPh), 4.64 (d, 1H, J = 11.8 Hz, CHPh), 4.36 (d, 1H, J = 7.6 Hz, H-1), 4.18–4.13 (dd, 1H, J = 4.6, 11.8
Hz, H-5a), 3.67 (dd, 1H, J = 9.0, 10.6 Hz, H-3), 3.60 (t, 1H, J = 11.4, H-5b), 3.21 (dd, 1H, J = 8.7, 7.6 Hz, H-2), 2.87 (td,
1
3
quat
1
H, J = 10.8, 4.8 Hz, H-4)). C NMR (CD OD, 125 MHz): δ 138.90 (C-Ar ), 129.36(2C), 129.21(2C), 128.81
3
tert
(
5×C-Ar ), 118.80 (CN), 104.10 (C-1), 75.58 (C-2), 73.51 (C-3), 71.95 (CH Ph), 63.23 (C-5), 36.88 (C-4), HRMS (ESP):
2
1+
Calcd. For C H NO [M+H] : 250.1079; Found: 250.1080
13
16
4
Iso-L-gulo-fagomine HCl salt (6)
Compound 22 (40 mg, 0.190 mmol) was dissolved in a 5 mL mixture of dioxane/water (4/1). To this mixture, 40 mg
Pd(OH) /C (20% w/w) was added, together with acetic acid (0.016 mL, 1.5 equiv.). The reaction mixture was
2
stirred under hydrogen atmosphere for 48 hrs. The mixture was then filtrated through a Celite pad and the solvent
was evaporated. After co-evaporation with HCl (1 M), iso-L-gulo-fagomine hydrochloride salt 6 (22 mg, 74%) was
16
afforded as a white solid. NMR data was identical to previously published data.
General procedures for N-alkylation of iminosugars
To a solution of iminosugar (0.060 mmol, 1 equiv.) in DMF (5 mL), anhydrous K CO (0.018 mmol, 3 equiv) was
2
3
added, followed by addition of 10-chloro-9-anthracenemethyl chloride (0.066 mmol, 1.1 equiv.). The reaction
mixture was stirred at 80ºC for 4 hrs. The reaction mixture was filtered and the solvent was evaporated. The
residue was purified by flash chromatography (CHCl , then CHCl /MeOH 8/1) to give the corresponding
3
3
1
0-chloroanthracenyl iminosugars as yellow solid.
General procedures for reductive amination of iminosugars
A mixture of iminosugar (0.060 mmol, 1 equiv.) and aldehyde (0.075 mmol, 1.25 equiv.) in methanol (10 mL) and
chloroform (1 mL) was heated to reflux until a homogenous solution was formed. Then NaBH CN (0.15 mmol, 2.5
3
equiv.) and acetic acid (0.075 mmol, 1.25 equiv.) were added. The reaction mixture was refluxed overnight. After
that, the solvent was evaporated and the residue was purified by flash chromatography (CHCl , then CHCl /MeOH
3
3
8
/1) to give the corresponding 10-chloroanthracenyl iminosugars as yellow solid.
N-(2-(10-Chloroanthracen-9-yl)ethyl) isofagomine (9)
1
3
Yield (44%). R = 0.16 (DCM/MeOH: 7/1). H NMR (CD OD, 500 MHz): δ 8.50 (d, 2H, J
= 8.6 Hz, 2×H-a), 8.47 (d,
f
3
Ha,Hb
10
This article is protected by copyright. All rights reserved.

European Journal of Organic Chemistry
10.1002/ejoc.202000522
3
3
2
2
3
2
1
H, J
= 8.7 Hz, 2×H-a’), 7.65-7.61 (m, 4H, 2×H-b, 2×H-c), 3.90 (dd, 1H, J = 4.5 Hz, J = 9.8 Hz, H-5’),
Ha’,Hc
5’,5
3
.89-3.85 (m, 2H, 2×H-d), 3.69-3.62 (m, 2H, H-3, H-5’), 3.37-3.32 (m, 2H, H-2, H-6), 3.22 (t, 1H, J = 8.4 Hz, H-4),
13
.83 (t, 2H, J = 8.5 Hz, 2×H-e), 2.24-2.18 (m, 2H, H-2, H-6), 1.91 (m, 1H, H-5). C NMR (CD OD, 125 MHz): δ 131.12,
3
quat
30.14(2C), 128.47(2C), 127.43 (6×C ), 126.31(2C) (2×C-b), 126.00(2C) (2×C-c), 124.95(2C) (2×C-a), 124.12(2C)
(
2×C-a’), 74.27 (C-4), 71.59 (C-3), 61.10 (C-5’), 58.07 (C-6), 57.80 (C-e), 54.81 (C-2), 43.68 (C-5), 24.72 (C-d). HRMS
1+
(
ESP): Calcd. For C H ClNO [M+H] : 386.1447; Found: 386.1515
22 25 3
N-(3-(10-Chloroanthracen-9-yl)propyl) isofagomine (10)
1
3
Yield (57%). R = 0.23 (DCM/MeOH: 7/1). H NMR (CD OD, 500 MHz): δ 8.51 (d, 2H, J
= 8.7 Hz, 2×H-a), 8.38 (d,
f
3
Ha,Hc
2
3
3
2
2
2
1
H, J
= 8.3 Hz, 2×H-a’), 7.63-7.58 (m, 4H, 2×H-b, 2×H-c), 3.80 (dd, 1H, J = 3.5 Hz, J = 11.0 Hz, H-5’), 3.67 (t,
Ha’,Hc 5’,5
H, J = 7.9 Hz, 2×H-d), 3.60-3.53 (m, 2H, H-3, H-5’), 3.19-3.13 (m, 3H, H-2, H-4, H-6), 2.78 (t, 2H, J = 7.1 Hz, 2×H-f),
1
3
.14-2.00 (m, 4H, H-2, H-6, 2×H-e), 1.81 (m, 1H, H-5) 7.65 (m, 2H,). C NMR (CD OD, 125 MHz): δ 135.38,
3
quat
31.39(2C), 129.87(2C), 128.35 (6×C ), 127.65(2C) (2×C-b), 127.12(2C) (2×C-c), 126.28(2C) (2×C-a), 125.87(2C)
(
(
2×C-a’), 75.24 (C-4), 72.50 (C-3), 62.18 (C-5’), 59.02 (C-6), 58.41 (C-f), 55.99 (C-2), 44.64 (C-5), 29.00 (C-e), 26.08
1+
C-d). HRMS (ESP): Calcd. For C H ClNO [M+H] : 400.1680; Found: 400.1689
23
27
3
N-(10-Chloroanthracen-9-yl)methyl) iso-D-galacto-fagomine (13)
1
3
Yield (53%). R = 0.33 (DCM/MeOH: 8/1). H NMR (CD OD, 500 MHz): δ 8.59 (d, 2H, J
= 8.4 Hz, 2×H-a), 8.54 (d,
f
3
Ha,Hb
3
2
=
H, J
= 8.5 Hz, 2×H-a’), 7.65-7.58 (m, 4H, 2×H-b, 2×H-c), 4.53 (m, 2H, 2×H-d), 3.90 (br, 1H, H-4), 3.63 (dd, 1H, J
Ha’,Hc
10.0 Hz, J =6.6 Hz,H-5’a), 3.55-3.50 (m, 2H, H-5’b, H-3), 2.72-2.70 (m, 2H, H-6a, H-2), 2.58 (t, 1H, J = 10.4 Hz,
13
quat
H-2a), 2.47–2.38 (m, 1H, H-6a), 1.79 (m, 1H, H-5). C NMR (CD OD, 125 MHz): δ 133.12(3C), 129.76(3C) (6×C ),
3
1
27.68(2C) (2×C-b), 127.04(2C) (2×C-c), 126.78(2C) (2×C-a), 125.99(2C) (2×C-a’), 70.88 (C-3), 69.52 (C-4), 63.07
1+
(
C-5’), 54.90 (C-2), 54.65 (C-d), 51.10 (C-6), 43.88 (C-5). HRMS (ESP): Calcd. For C H ClNaNO [M+Na] : 394.1186;
21 22 3
Found: 394.1184
N-(2-(10-Chloroanthracen-9-yl)ethyl) iso-D-galacto-fagomine (14)
1
Yield (33%). R = 0.19 (DCM/MeOH: 7/1). H NMR (CD OD, 500 MHz): δ 8.54 (m, 2H, 2×H-a), 8.38 (m, 2H, 2×H-a’),
f
3
3
3
7
.64-7.62 (m, 4H, 2×H-b, 2×H-c), 4.01 (t, 1H, J = 2.2 Hz, H-4), 3.89 (m, 2H, 2×H-d), 3.78 (ddd, 1H, J= 10.9, 4.7, 2.9
Hz, H-3), 3.73 (dd, 1H, J = 10.8 Hz, J = 6.4 Hz,H-5’), 3.63 (dd, 1H, J = 10.9 Hz, J = 7.2 Hz,H-5’), 3.00-2.94 (m, 2H, H-2,
2
2
13
H-6), 2.81-2.78 (m, 2H, 2×H-e), 2.52 (t, 1H, J = 10.6 Hz, H-2), 2.34 (t, 1H, J = 11.6 Hz, H-6), 1.98 (m, 1H, H-5).
C
quat
NMR (DMSO, 125 MHz): δ 133.05, 131.59(2C), 129.93(2C), 128.70 (6×C ), 127.74(2C) (2×C-b), 127.37(2C)
(
2×C-c), 126.37(2C) (2×C-a), 125.61(2C) (2×C-a’), 70.52 (C-3), 69.16 (C-4), 63.02 (C-5’), 59.62 (C-e), 54.69 (C-2),
1+
5
0.64 (C-6), 43.66 (C-5), 26.12 (C-d). HRMS (ESP): Calcd. For C H ClNO [M+H] : 386.1523; Found: 386.1521
22 25 3
N-(3-(10-Chloroanthracen-9-yl)propyl) iso-D-galacto-fagomine (15)
1
3
Yield (35%). R = 0.13 (DCM/MeOH: 7/1). H NMR (CD OD, 500 MHz): δ 8.52 (d, 2H, J
= 8.1 Hz, 2×H-a), 8.39 (d,
f
3
Ha,Hc
3
3
2
H, J
= 7.8 Hz, 2×H-a’), 7.63-7.61 (m, 4H, 2×H-b, 2×H-c), 3.94 (t, 1H, J = 2.6 Hz, H-4), 3.71-3.63 (m, 4H, H-3,
Ha’,Hc
3
2
2
H-5’, 2×H-d), 3.53 (dd, 1H, J5 = 7.9 Hz, J = 10.9 Hz, H-5’), 2.83-2.74 (m, 4H, H-2, H-6, 2×H-f), 2.40 (t, 1H, 1H, J =
’,5
2
13
1
1.4 Hz, H-2), 2.22 (t, 1H, 1H, J = 11.5 Hz, H-6), 2.07-2.01 (m, 2H, 2×H-e), 1.91 (m, 1H, H-5). C NMR (CD OD, 125
3
quat
MHz): δ 135.59, 131.39(2C), 129.87(2C), 128.29 (6×C ), 127.66(2C) (2×C-b), 127.10(2C) (2×C-c), 126.27(2C)
(
2×C-a), 125.91(2C) (2×C-a’), 70.02 (C-3), 68.87 (C-4), 62.75 (C-5’), 58.81 (C-f), 54.42 (C-2), 50.62 (C-6), 43.19 (C-5),
1+
2
9.11 (C-e), 26.23 (C-d). HRMS (ESP): Calcd. For C H ClNO [M+H] : 400.1680; Found: 400.1679
23 27 3
N-(10-Chloroanthracen-9-yl)methyl) iso-D-galacto-fagomine (23)
11
This article is protected by copyright. All rights reserved.

European Journal of Organic Chemistry
10.1002/ejoc.202000522
1
Yield (25%). R = 0.32 (DCM/MeOH: 8/1). H NMR (CD OD, 500 MHz): δ 8.55-8.52 (m, 4H, 2×H-a, 2×H-a’),
f
3
7
3
2
.64-7.58 (m, 4H, 2×H-b, 2×H-c), 4.52 (m, 2H, 2×H-d), 3.69-3.66 (m, 1H, H-5’), 3.63-3.58 (m, 2H, H-4, H-5’a),
.52-3.49 (m, 1H, H-3), 2.89-2.86 (m, 2H, H-6, H-2), 2.62 (dd, 1H, J = 11.6, 3.5 Hz, H-2), 2.42 (br, 1H, H-6),
1+
.09-2.04 (m, 1H, H-5). HRMS (ESP): Calcd. For C H ClNaNO [M+H] : 372.1366; Found: 372.1358
21
22
3
N-(2-(10-Chloroanthracen-9-yl)ethyl) iso-L-gulo-isofagomine (24)
1
Yield (26%). R = 0.20 (DCM/MeOH: 7/1). H NMR (CD OD, 500 MHz): δ 8.54 (m, 2H, 2×H-a), 8.38 (m, 2H,
f
3
2
1
1
×H-a’), 7.64-7.62 (m, 4H, 2×H-b, 2×H-c), 4.07-3.92 (m, 3H, 2×H-d, H-3), 3.87 (t, 1H, H-4), 3.77 (dd, 1H, J =
0.9, 5.8 Hz, H-5’), 3.66 (dd, 1H, J = 10.9, 7.5 Hz, H-5’), 3.29-3.14 (m, 5H, 2×H-e, 2×H-2, H-6), 3.00 (t, 1H, J =
1+
1.5 Hz, H-6), 2.49 (br, 1H, H-5). HRMS (ESP): Calcd. For C H ClNO [M+H] : 386.1523; Found: 386.1583
22
25
3
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