An approach to 8 stereoisomers of homonojirimycin from d-glucose via kinetic & thermodynamic azido-γ-lactones

Article abstract of DOI:10.1039/c3ob41334a

Crystal structures were obtained for the two C2 epimeric azido-γ-lactones 2-azido-2-deoxy-3,5:6,7-di-O-isopropylidene-d-glycero-d- ido-heptono-1,4-lactone and 2-azido-2-deoxy-3,5:6,7-di-O-isopropylidene-d- glycero-d-gulo-heptono-1,4-lactone prepared from kinetic and thermodynamic azide displacements of a triflate derived from d-glucoheptonolactone. Azido-γ-lactones are very useful intermediates in the synthesis of iminosugars and polyhydroxylated amino acids. In this study two epimeric azido-heptitols allow biotechnological transformations via Izumoring techniques to 8 of the 16 possible homonojirimycin analogues, 5 of which were isolated pure because of the lack of stereoselectivity of the final reductive amination. A side-by-side glycosidase inhibition profile of 11 of the possible 16 HNJ stereoisomers derived from d-glucose and d-mannose is presented.

Full text of DOI:10.1039/c3ob41334a

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PAPER
An approach to 8 stereoisomers of homonojirimycin from D-glucose via
kinetic & thermodynamic azido-γ-lactones
a,b
b
b
c
Andreas F. G. Glawar, Sarah F. Jenkison, Scott J. Newberry, Amber L. Thompson, Shinpei
d
e
f
e
a
d
Nakagawa, Akihide Yoshihara, Kazuya Akimitsu, Ken Izumori, Terry D. Butters, Atsushi Kato* and
a,b
5
George W. J. Fleet*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Crystal structures were obtained for the two C2 epimeric azido-γ-lactones 2-azido-2-deoxy-3,5:6,7-di-O-
isopropylidene-D-glycero-D-ido-heptono-1,4-lactone and 2-azido-2-deoxy-3,5:6,7-di-O-isopropylidene-
D-glycero-D-gulo-heptono-1,4-lactone prepared from kinetic and thermodynamic azide displacements of
a triflate derived from D-glucoheptonolactone. Azido-γ-lactones are very useful intermediates in the
synthesis of iminosugars and polyhydroxylated amino acids. In this study two epimeric azido-heptitols
allow biotechnological transformations via Izumoring techniques to 8 of the 16 possible homonojirimycin
analogues, 5 of which were isolated pure because of the lack of stereoselectivity of the final reductive
amination. A side-by-side glycosidase inhibition profile of 11 of the possible 16 HNJ stereoisomers
derived from D-glucose and D-mannose is presented.
1
0
1
5
synthesise HNJ 4 itself along with the remaining 7 stereoisomers
Introduction
in an approach to complete the total set of 16 HNJ stereoisomers
Azido-lactones are useful intermediates for the production of
by a combination of chemical and biotechnological techniques.
biologically active molecules including polyhydroxylated amino 35 However an unexpectedly high diastereoselectivity in the
1
2-4
2
2
3
0
5
0
acids and iminosugars. The latter have found application in
formation of the piperidine ring by reductive amination only
5
6
diseases ranging from diabetes and lysosomal storage disorders
allowed isolation of pure samples for 5 of the 8 isomers formed.
7
to cancer. More specifically azido-δ-lactones have been used to
1
80°
8
-10
11
synthesise α-amino acids
and their protected precursors, as
OH
OH
O
1
2
well as spirohydantoins of β-glucopyranose, analogues of
O
HO
OH
HO
OH
N3
O
1
3, 14
15
hydantocidin,
iminosugar-like pipecolic amides and more
O
HOH2C
N
H
CH2OH HOH2C
N
H
CH2OH
recently 8 α-homonojrimycin (HNJ) 4 analogues from D-
O
1
6
O
mannose (Figure 1). Similarly azido-γ-lactones were employed
1
7-20
21
to generate α-amino acids
and analogues , as well as a
Azido-δ-lactone 1
from D-mannose
α−Homomannojirimycin HMJ 2
2
2
spirohydantion of glucopyranose and iminosugars in the form of
2
3
pyrrolidine analogues of galactofuranose.
1
80°
O
In this study azido-γ-lactones from D-glucose were used to
OH
OH
O
a
HO
OH
HO
OH
Oxford Glycobiology Institute, University of Oxford, South Parks Road,
Oxford, OX1 3QU, United Kingdom. Fax: +44 (0)1865 275216; Tel: +44
O
O
O
(
0)1865 275342;
HOH2C
N
H
CH2OH HOH2C
N
H
CH2OH
b
Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA,
O
N3
United Kingdom. Fax: +44 (0)1865 285002; Tel: +44 (0)1865 275645;
E-mail: George.Fleet@chem.ox.ac.uk
Azido-γ-lactone 3
from D-glucose
α−Homonojirimycin HNJ 4
c
Chemistry Research Laboratory, Department of Chemical
1
80°
Crystallography, University of Oxford, Mansfield Road, Oxford, OX1
OH
OH
OH
3
TA, United Kingdom.
Department of Hospital Pharmacy, University of Toyama, 2630
d
HO
HO
OH
HO
OH
OH
HO
OH
Sugitani, Toyama 930-0194, Japan; E-mail: kato@med.u-toyama.ac.jp
e
N
H
CH2OH HOH2C
N
H
HOH2C
N
H
Rare Sugar Research Center, Kagawa University, 2393 Ikenobe, Miki-
cho, Kita-gun, Kagawa 761-0795, Japan.
Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho,
f
Nojirimycin 5
Deoxynojirimycin DNJ 6
Kita-gun, Kagawa 761-0795, Japan.
Figure 1. Examples of iminosugar inhibitors from azido-lactones.
†
Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/b000000x/
40
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[journal], [year], [vol], 00–00 | 1

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2
4, 25
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DOI: 10.1039/C3OB41334A
As a result these two
HNJ 4 synthesised
before it was isolated from Omphalea
direct generation over two steps in 39% yield from the
2
6, 27
31, 36, 37
Diandra L, is a potent inhibitor of α-glucosidase (Figure 1).
35 corresponding alcohol (Scheme 1).
2
8
In this respect it closely resembles deoxynojirimycin 6 the
hydroxymethyl deleted analogue of 4, which in turn is a more
methods were contrasted in this study for the triflate generated
from the diacetonide 19, derived from D-glucose, to synthesise
the two epimeric azides 3 and 21, with the equilibration
producing the higher yield (Scheme 2). Their structures were
40 confirmed using X-ray crystallography (Figure 2 and 3).
2
9
5
stable analogue of the antibiotic nojirimycin 5.
Results and discussion
3
8
Glucoheptonolatone, available via the Kiliani ascension from D-
Synthesis of epimeric azido-γ-lactones
39
40
glucose on scales larger than 200 kg, reacts with acetone
under acid catalysed conditions to give the highly crystalline
The displacement of the C2 triflate 8 in the benzylidene protected
ribono-δ-lactone by sodium azide did not give the expected
4
1-43
diacetonide 19 in 87% yield on scales up to 500 g.
4
4-50
1
0
N
inverted S 2 product 7 but occurred with overall retention of 45 Diacetonide 19 has been used as a chiron in many syntheses.
3
0
configuration to give 9. Such equilibrations of C2 azides in
lactones to the thermodynamically preferred all cis configuration
under the reaction conditions is common in many γ- and δ-
lactones (Scheme 1); the reaction, where necessary, is buffered
For the equilibration route, OH2 in 19 was converted into the
corresponding triflate, which was subjected to the azide
displacement at low temperatures to give the kinetic ido-azide 3
in quantitative yield (Scheme 2). In the presence of sodium azide
3
1
1
5
by pyridinium toluene-p-sulfonate (PPTS). Thus the triflate 11 50 and PPTS the ido-azide 3 was equilibrated over 4 days to the
gives 10 with inversion of configuration at C2 under kinetic
reaction conditions but yields the epimeric azide 12 with overall
retention of configuration under thermodynamic conditions.
thermodynamically more stable all cis gulo-azide 21 in a yield of
94% with 6% of ido-azide being recovered.
For the double inversion route the triflate of alcohol 19 was
treated directly with sodium trifluoroacetate, which yields the
Kinetic
Starting
Material
Thermodynamic
Product
Product
N
55 inverted trifluoroacetate ester via an S 2 reaction. On work-up
O
O
O
with methanol the ester was hydrolysed to give the epimeric
alcohol 20 in 62% yield. Alcohol 20 was treated with
trifluoromethanesulfonic (triflic) anhydride to yield the
corresponding triflate, which on subsequent reaction with sodium
azide gave the thermodynamic gulo-azide 21 in 58% over two
i)
i) 64% ref 30
O
O
O
O
O
7
O
O
Ph
Ph
O
O
N3
Ph
O
OTf
Ph
O
N3
8
9
60
ii) 92% ref 34
O
iii) 86% ref 35
O
O
O
O
O
O
O
steps via a concomitant S 2 displacement. Overall, starting from
N
diacetonide 19, the double displacement yielded the
Ph
O
Ph
O
1
0
N3
11
OTf
12
N3
thermodynamic gulo-azide 21 in 36% over 4 steps, while the
iv) 86% ref 31 (79%)
equilibration gave the same compound in 94% over 3 steps.
O
O
O
O
O
O
O
O
O
O
O
O
O
O
v) 79% ref 31
vi) 66'% ref 31
O
O
O
O
O
O
O
O
O
O
i), ii)
O
Ph
O
Ph
O
Ph
O
1
3
14
OTf
15
N3
O
O
N3
OH
N3
1
9
3
vii) 88% ref 31 (70%)
Kinetic Ido-Azide
O
O
O
O
O
O
iv), v)
iii)
O
O
O
O
O
O
O
O
O
viii) 92% ref 36, 37
N3
17
x) 56% ref 31 (52%)
ix) 39% ref 31
1
6
OTf
18
N3
O
O
O
O
O
2
0
Scheme 1. Thermodynamic (magenta) and kinetic (blue) azido-lactones;
yields given above arrows (over two steps); Conditions: i) NaN , DMF,
3
O
O
3
0
32
33
vi), vii)
O
RT; ii) NaN
3 3
NaN , PPTS, DMF; v) NaN , DMF, RT, 1h; vi) NaN , DMF, RT,
31 34, 35
3
, DMF, RT, 1h; iii) NaN
3
, PPTS, DMF, RT, 20h; iv)
O
O
3
1
31
3
O
O
3
1
2
2h; vii) NaN
3
, PPTS, DMF; viii) NaN
3
, DMF, -30°C, 1.5h;
ix)
OH
N3
2
0
21
3
1
31
2
5
NaN , DMF, 0°C, 2.5h; x) NaN , PPTS, DMF.
3
3
Thermodynamic
Gulo-Azide
65
In the triflate 14, which is closely related to the structure of this
study, the most effective way of generating thermodynamic azide
Scheme 2. i) Tf
2
O, DCM, Pyridine, - 30 °C, 30 min; ii) NaN
(1.4 eq.), PPTS
O, DCM, Pyridine, -40 to -20 °C, 70
COONa (3 eq.), DMF, 60 °C, 72 h then MeOH 16 h, 62%
over two steps; vi) Tf O, DCM, Pyridine, -30 °C, 30 min; vii) NaN (1.1
eq.), DMF, -18 to +5 °C, 17 h, 58% over two steps.
3
(1.5 eq.),
DMF, -15 to +6 °C, 15.5 h, quant over 2 steps; iii) NaN
(1.1 eq), DMF, RT, 4d, 94%; iv) Tf
min; v) CF
3
1
5 was to isolate the kinetic product 13 first, followed by
2
equilibration in the presence of PPTS yielding 15 in 70% over
two steps (in comparison to 66% via direct conversion from the
triflate). However, for the acetonide of D-glucuronolactone 17 it
is more efficient to perform a double inversion to generate the
thermodynamic azide 18 in 84% over 4 steps rather than via
3
70
2
3
3
0
2
| Journal Name, [year], [vol], 00–00
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DOI: 10.1039/C3OB41334A
1
9 as the starting material and was consistent with refinement of
5
1
the Flack parameter.
The gulo-azide 21 (Figure 3) adopts a similar conformation with
the 6-ring acetonide in a chair conformation with the fused 5-ring
lactone in an envelope conformation with C3 out of the plane.
There is significant disorder in the 5-ring acetonide which adopts
an envelope conformation with either O11 or O110 out of the
plane.
20
Synthesis of epimeric azido-heptitols
2
3
3
5
0
5
On a multi gram scale the epimeric ido- and gulo-azides 3 and 21
were converted into their corresponding azido-heptitols in the L-
gulo 24 and D-gulo 25 configuration (Scheme 3).
For the synthesis of the L-gulo-6-azido-heptitol 24 the kinetic
ido-azido-γ-lactone 3 was subjected to a two step reduction using
DIBAL to form the corresponding lactol, which on treatment with
sodium borohydride gave diol 22 in 91% yield on scales up to 9
g. Formation of diol 22 from lactone 3 using lithium borohydride
proved to be very unreliable. Direct reductions of such base
sensitive α-azidolactones, to diols are inefficient, whereas two
3
6
step reductions are generally effective. The diacetonide 22 was
TM
stirred in methanol and water along with acidic Dowex 50WX8
Figure 2. ORTEP plot of 2-azido-2-deoxy-3,5:6,7-di-O-isopropylidine-D-
resin to afford the unprotected L-gulo-6-azido-heptitol 24 in 95%
glycero-D-ido-1,4-heptono-lactone 3. Anisotropic displacement ellipsoids
are represented at the 50% probability level. CCDC number 917203.
yield after recrystallization.
O
O
O
O
O
O
O
OH
N3
O
i), ii)
OH
O
O
N3
Ido: 3
22
23
Gulo: 21
iii)
OH
OH
N3
OH
OH
N3
HO
OH
HO
OH
Or
OH
OH
OH
OH
L
-Gulo-6-azido-heptitol: 24
D-Gulo-2-azido-heptitol: 25
4
0
Scheme 3. Synthesis of epimeric azido-heptitols. From ido-azide 3: i)
DIBAL, CH Cl , -78°C; ii) NaBH , MeOH, -30°C, 3h, 91% over two
steps; iii) Dowex 50WX8 H , 1:4 MeOH: H O, 40°C, 48h, 95%. From
gulo-azide 21: i) DIBAL, CH Cl , -78°C; ii) NaBH , MeOH, -30°C, 8h,
1% over two steps; iii) Dowex 50WX8 H , 3:16 MeOH:H O, 40°C, 48h,
85%.
2
2
4
+
2
2
2
4
+
8
2
45
The epimeric D-gulo-2-azido-heptitol 25 was obtained from the
thermodynamic gulo-azido-γ-lactone 21 by a similar procedure.
A two step reduction protocol of 21 using DIBAL and sodium
5
Figure 3. ORTEP plot of 2-azido-2-deoxy-3,5:6,7-di-O-isopropylidine-D-
glycero-D-gulo-1,4-heptono-lactone 21. Anisotropic displacement
TM
borohydride gave diol 23 in a 81% yield. Dowex 50WX8 resin
ellipsoids are represented at the 50% probability level. CCDC number 50 in methanol/water removed the acetonide protecting groups from
17202.
9
2
3 to give, after recrystallization from isopropanol, the D-gulo-2-
azido-heptitol 25 in 85% yield.
1
0
X-ray analysis
Biosynthetic conversion of azido-heptitols
For the ido-azide 3 (Figure 2) the 6-ring acetonide adopts a chair
conformation with the fused 5-ring lactone adopting an envelope
The generated azido-heptitols 24 and 25, gave access to 8 of the
conformation with C3 out of the plane. The 5-ring acetonide also 55 16 possible stereoisomers of homonojirimycin using the
adopts an envelope conformation with O9 out of the plane. The
absolute configuration was determined from the use of acetonide
biotechnological approach of Izumoring (Scheme 4), 5 of which
were isolated in sufficient quantity and purity for biological
1
5
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Journal Name, [year], [vol], 00–00 | 3

Organic & Biomolecular Chemistry
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5
2, 53
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DOI: 10.1039/C3OB41334A
evaluation.
both carbon branched
recently being used to give access to the other eight stereoisomers
of HNJ from two epimeric azidoheptitols derived from D- 30 be observed that the axial substituent wins out over the torsional
This powerful approach has been used to generate
face, while torsional strain reduction would favour addition from
5
4, 55
56, 57
and deoxy
sugars, as well as
the top face. This system therefore shows the lowest selectivity of
5
8, 59
the four under investigation and, similar to Wong et al.,
it can
1
6
5
0
5
0
5
mannose.
strain reductions (32/4 ratio 3:1). On the other hand in imine 39
both effects complement each other with both an axial substituent
blocking the bottom face and with torsional strain reduction
favouring addition to the top face, hence we observe a high
Gluconobacter thailandicus NBRC 3254 is able to recognize
selectively the R,R configuration of a diol adjacent to a terminal
alcohol in the two azido-heptitols 24/25 and its action on these
substrates led to the formation of azido-ketoses 26/28. In order to 35 degree of selectivity in this system for the formation of 34 (33/34
1
1
2
2
get access to the other pair of azido-ketoses 27/29 the enzyme D-
tagatose-3-epimerase was used which is able to epimerize the C3
carbon, in the ketose starting materials 26/28, adjacent to the
carbonyl group. With the four required azido-ketoses in hand
reductive aminations with Raney nickel in water led to a mixture
of the corresponding HNJ isomers. Each of the reductions
favoured the formation of one of the two possible isomers, and as
a result it was possible to isolate clean samples of 31, 32, 4, 34
and 35, the properties of which are reported in Table 1.
ratio 1:14). With all substituents being equatorial in imine 40
torsional strain again dictates the reaction outcome and hence the
major addition from the top face leading to the formation of 35
(35/36 ratio 11:1).
40
45
50
The selectivity of the reductions can be explained by the presence
of torsional strain and axial substituents in the imine intermediate
(
Figure 4). In the case of imine 37 there are two axial substituents
for either conformer, so the selectivity of the reaction is dictated
by the reduction in torsional strain. Hence the hydrogen is added
from the bottom face leading to the formation of 31 preferentially
(30/31 ratio 1:25). For imine 38 the two effects oppose each other
with the presence of the axial hydroxymethyl blocking the top
OH
OH
HO
OH
HO
OH
3
0
36
iv)
2*
6
iv)
6*
HOH2C
N
H
CH2OH
HOH2C
N
H
2
CH2OH
O
OH
N3
O
OH
N3
1
1
:
2
7
3
:
28
HOH2C
iv)
CH2OH
HOH C
CH OH
2
2
2
5
+
+
11
OH
OH
OH
OH
ii)
OH
OH
iv)
OH
OH
N3
HO
OH
HO
OH
3
1
(R)
35
2
5
HOH2C (R)
CH2OH
6*
2*
2*
2
6
6
6*
2
HOH2C
N
H
CH2OH
HOH2C
N
H
CH2OH
OH
OH
i)
D-glucose
i)
iii)
iii)
OH
OH
OH
OH
N3
HO
OH
HO
OH
2
4
(R)
3
2
HOH C (R)
CH OH
34
2
2
2*
HOH2C
N
H
CH2OH
OH
OH
HOH2C
N
H
6
CH2OH
ii)
iv)
iv)
2
3
14
:
O
OH
N3
O
OH
N3
:
+
+
3
1
26
29
1
HOH2C
CH2OH
HOH C
CH OH
2
OH
OH
OH
OH
OH
OH
HO
OH
HO
2
OH
2
4
iv)
33
iv)
6*
2
HOH2C
N
H
CH2OH
HOH C
N
H
CH OH
Scheme 4. Reagents and conditions: i) chemical transformation; ii) Gluconobacter thailandicus NBRC 3254, H
O; iv) H , Ni, H O; stereocenters from D-glucose shown in red; dashed arrow indicates minor product in reductive amination; asterix indicates carbonyl
carbon of azidoketose precursor in the reduced products; numbering according to IUPAC naming.
2
O, 30°C; iii) D-Tagatose-3-epimerase,
5
5
H
2
2
2
4
| Journal Name, [year], [vol], 00–00
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Table 1 Properties of HNJ Diastereoisomers
DOI: 10.1039/C3OB41334A
OH
OH
OH
OH
OH
HO
OH
HO
OH
HO
OH
HO
OH
HO
OH
Compound
6*
2
2*
2*
2*
6*
CH OH HOH C
HOH C
N
H
CH OH HOH C
N
H
6
CH OH
HOH C
N
H
6
CH OH
HOH C
N
H
6
N
H
2
CH OH
2
2
2
2
2
2
2
2
2
2
3
1
32
4
34
35
‡
-
44.1 (c 1.09, H
2
O)
0.00 (c 1.27, MeOH)
+61.0 (c 0.56, H
2
O)
+2.47 (c 0.55, MeOH)
[Lit. +4.0 (c 1.0,
-0.84 (c 0.36, H O)
[Lit. -1.7 (c 0.35, H O)]
2
2
2
5
60
61
62
60
63
[
α]
D
[Lit. - 41.0 (c 0.69, [Lit. 0.0 (c 0.8, MeOH)] [Lit. +77.2 (c 0.57, H
2
O)]
H
2
O)]
-
MeOH)]
-
62.0
m.p.
169-172 °C
62.4
200-202 °C
(C7) 56.8
-
#
C1
62.1
62.0
60.3
72.0
78.8
72.0
60.3
62.0
C2
C3
C4
C5
C6
C7
H1
H1’
H2
H3
H4
H5
54.4
68.0
71.6
66.7
55.8
55.6
68.9
69.4
68.9
55.6
62.4
(C6) 57.4
(C5) 72.1
(C4) 74.9
(C3) 72.7
(C2) 54.6
(C1) 62.6
58.5
69.6
75.6
69.2
60.7
61.6
δ
C
(D
2
O,
1
00 MHz)
62.1
3.62 (dd, J 6.7, 11.2)
3.66 (dd, J 6.7, 11.2)
3.05 (dt, J 1.3, 6.7)
3.91(dd, J 1.3, 3.8)
3.99 (a-t, J 3.5)
3.75 (dd, J 3.3, 10.6)
3.65 (dd, J 6.9, 11.1)
3.72 (dd, J 6.4, 11.1)
3.07 (dt, J 1.5, 6.6)
3.78 (dd, J 1.5, 3.2)
4.01 (t, J 3.2)
3.78 (dd, J 1.5, 3.2)
3.07 (dt, J 1.5, 6.6)
3.65 (dd, J 6.9, 11.1)
3.72 (dd, J 6.4, 11.1)
(H7) 3.77-3.84 (m)
(H7’) 3.77-3.84 (m)
(H6) 3.27 (dt, J 5.5, 9.4)
(H5) 3.74 (dd, J 5.8, 10.0)
(H4) 3.48 (t, J 9.5)
3.65 (dd, J 6.7, 11.1)
3.66 (dd, J 6.7, 11.2)
2.84 (dt, J 1.4, 6.7)
3.99 (dd, J 1.3, 2.6)
3.54 (dd, J 2.8, 9.8)
3.54-3.59 (m)
3.62 (dd, J 6.7, 11.6)
3.88 (dd, J 2.9, 11.6)
2.64 (ddd, J 2.9, 6.7, 9.7)
3.24 (t, J 9.5)
δ
H
(D
2
O,
†
3.38 (t, J 9.1)
3.24 (t, J 9.5)
4
00 MHz)
(H3) 3.19 (t, J 9.6)
H6 2.86 (ddd, J 3.2, 5.3, 10.5)
H7
H7’
(H2) 2.84 (ddd, J 2.9, 7.2, 10.0) 2.56 (ddd, J 2.8, 5.4, 9.8) 2.64 (ddd, J 2.9, 6.7, 9.7)
3.70 (dd, J 5.6, 11.6)
3.79 (dd, J 3.0, 11.6)
(H1) 3.56 (dd, J 7.1, 11.4)
(H1’) 3.90 (dd, J 2.9, 11.4)
3.72 (dd, J 5.5, 11.6)
3.82 (dd, J 3.0, 11.6)
3.62 (dd, J 6.7, 11.6)
3.88 (dd, J 2.9, 11.6)
IR and MS data is given in the supporting information; * indicates carbonyl carbon of azidoketose precursor in the reduced products; † a-t = apparent
triplet; ‡ optical rotation of HCl salt for comparison to literature; # Previous numbering of α-HNJ in literature given in brackets.
is a good and selective inhibitor of α-galactosidases albeit not as
H2
torsional strain
H2
torsional strain
60, 68
active as 30.
35 the C6 epimer of 4 displays similar
1
2
2
3
3
4
5
0
5
0
5
0
behaviour, in having β-gluco configuration, but not inhibiting β-
6
3
OH
30
36
glucosidases. However it is a good inhibitor of α-glucosidases.
1
1
The isolated α-homonojirimycin 4 and its four isomers were
subjected to a set of enzymatic inhibition assays including α/β-
glucosidase, α/β-galactosidase, trehalase and α-L-rhamnosidase
(Table 2). As a result it was possible to confirm the strong and
selective α-galactosidase inhibition against the coffee bean
enzyme previously reported by us for 31 (1 µM) and 34 (3.4
R
R
HO
HO
N
N
R
HO
OH
2
5
R
11
3
7
40
OH
3
1
35
H2
H2
6
0
µM). The same was true for the same enzyme isolated from
human lysosome, which was also inhibited well by these two
H2
axial substituent
H2
torsional strain
axial substituent
61, 69, 70
compounds. Despite having been synthesized previously,
3
2 had not been subjected to enzymatic inhibition assays, and in
3
2
34
R
this study was found to be a good and selective α-glucosidase
inhibitor (Rice) with a IC50 of 75 µM. The well known α-
glucosidase inhibitor α-homonojirimycin 4 was confirmed to be
active against this enzyme and therefore all compounds retaining
HO
3
HO
14
HO
N
N
R
OH
OH
R
R
1
OH
1
3
8
39
4
33
3
stereocentres from D-glucose were good α-glucosidase
inhibitors. 4 also showed good inhibition against β-glucosidase
A. niger, 60 µM) and threhalase (20 µM) and weak inhibition of
α-galactosidase and α-L-rhamnosidase and is therefore less
H2
H2
Figure 4. Selectivity of imine reductions
(
Biological evaluations
6
3
selective. It had previously been reported that 35 was a good
inhibitor of α-glucosidase derived both from rice and rat
intestinal maltase, this activity was confirmed in this study with
an IC50 of 48 µM and 109 µM respectively. 35, however, turned
out to be inactive against α-glucosidase derived from yeast and
5
HNJ and isomers from D-glucose
5
of the 7 isomers of HNJ 4 under investigation in this study have
previously shown interesting bioactivity. 30 is a potent inhibitor
6
0, 64
of α-galactosidase
and hence has been included in studies on
71
the same result was found for human fibroblasts by Yu et al.
6
5
Fabry disease and along with 31 and 33 has recently been
shown to be a possible immunosuppressant acting via reduction
The previously reported inhibition of almond β-glucosidase, Jack
bean α-mannosidase and snail β-mannosidase was not confirmed
1
0
6
6, 67
of secreted cytokines.
34 the C6 epimer of 30 despite having
72
in this study.
the β-galacto configuration, does not inhibit β-galactosidases but
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5

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Page 6 of 14
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DOI: 10.1039/C3OB41334A
HNJ isomers derived from D-mannose
tolerant to changes in substituents at C1 and C5 of α-L-fucose.
4
5
With this behaviour it parallels the previously mentioned 41 and
it would be feasible to predict, that the C6 epimer of 44, which
would share the same configuration of all the stereocentres with
α-L-fucose would be an even more potent inhibitor of the
respective enzyme. Polyol 42, the second compound from the
In order to allow for a uniform cross-comparison of 11 of the 16
possible isomers of HNJ the enzymatic evaluation against the
same extended panel of glycosidases is given in Table 3 for HNJs
2, 41-45 derived from D-mannose, which were produced in a
similar chemical/biotechnological approach in our previous
5
1
6
50 HNJ analogues derived from D-mannose which had not been
previously subjected to an enzymatic inhibition study, was found
to be a selective and good to moderate inhibitor of α-glucosidases
publication. 41 has previously been shown to be an inhibitor of
α-L-fucosidase and this activity could be confirmed in this study
6
3, 73, 74
(
Bovine kidney, 126 µM).
It was furthermore a reasonable
(
Rice, 54 µM; rat intestinal maltase, 180 µM). These results on
1
1
2
0
5
0
inhibitor of α-galactosidase (Coffee bean, 194 µM) and in this
regard was comparable to its C2 epimer 2 which also inhibited
this enzyme from human lysosome weakly (424 µM). HMJ 2 had
previously been screened against a panel of enzymes,
however no significant inhibition was found. Later studies
the HNJs derived from D-mannose (Table 3), together with the
HNJs from D-glucose from this study (Table 2) allow for a true
cross-comparison of glycosidase inhibition by 11 of the 16
isomers of HNJ.
55
7
4, 75
6
3
showed that it was a good inhibitor of rice and rat intestinal
maltase α-glucosidases, an observation which is also found in
this study (Rice, 161 µM; rat intestinal maltase, 250 µM). It was
this activity that got this compound included in the previously
Conclusions
In summary this study presents synthesis of the corresponding
6
0
epimeric
azido-γ-lactones
2-azido-2-deoxy-3,5:6,7-di-O-
6
5
mentioned study on Fabry disease. Inversion of the C3 centre of
2 does give 43 an HNJ analogue which shares the configuration
at 4 stereocenters with α-D-galacto-pyranose, which could
explain its strong inhibition of α-galactosidase (Human
lysosome, 5.5 µM; coffee bean, 93µM) and moderate inhibition
of β-galactosidase (Bovine liver, 271µM) confirming previous
isopropylidene-D-glycero-D-ido-heptono-1,4-lactone 3 and 2-
azido-2-deoxy-3,5:6,7-di-O-isopropylidene-D-glycero-D-gulo-
heptono-1,4-lactone 21 in quantitative and 94% yield starting
form the readily available 3,5:6,7-di-O-isopropylidene-D-
glycero-D-gulo-heptono-1,4-lactone 19. To confirm the
configuration of the azides at C2 X-ray crystal structures were
obtained. Both azides were converted into their corresponding
azidoheptitols 24 and 25 in good yields of 86% over 5 steps and
65
6
3-65
2
3
3
4
5
0
5
0
observations.
HNJ analogue 45, which to our knowledge
hasn’t been subjected to enzymatic inhibition studies previously,
turned out to being a strong inhibitor of α-galactosidase (Human
lysosome, 8.1 µM; coffee bean, 26 µM). In this regard it behaves
6
5% over 6 steps from the starting lactone 19 respectively. These
just like 43 and we can conclude that the all cis arrangement of 70 azidoheptitols were suitable substrates for conversion into 7 of 15
the stereocentres C6-C4 is crucial for inhibition of this enzyme,
whilst the configuration C2 and C3 is not as important. However
the same isn’t true for β-galactosidase, as 45 shares the
configuration of 4 stereocentres of the ring with β-D-galacto-
pyranose, and still doesn’t inhibit the enzyme, proving it to be
quite sensitive to substitutions at the anomeric position. A similar
sensitivity is displayed by α- and β-mannosidase, as HNJ 2 and
possible HNJ stereoisomers along with HNJ itself using the
biotechnological Izumoring approach. The 5 pure samples
obtained from this method along with 6 HNJ isomers previously
synthesized were subjected to systematic characterization and a
wide panel of glycosidase enzymes confirming the previously
reported inhibition profiles of 31, 4, 34, 35, 41, 2, 43 and 44. HNJ
isomers 32, 42 and 45 were subjected to a biological evaluation
for the first time; 32 and 42 were identified as a good and specific
inhibitor of α-glucosidase, while 45 turned out to be a strong α-
galactosidase inhibitor. Overall this is the first time a systematic
cross comparison of glycosidase inhibition data of 11 of the 16
isomers of HNJ is possible.
75
4
4 display the same configuration of all stereocentres on the ring
compared with α- and β-D-manno-pyranose, but still show no
inhibition of the respective enzymes. However, 44 was identified
6
3
80
in this study, in accordance with previous results, to be a potent
inhibitor of α-L-fucosidase (Bovine kidney, 21 µM), which in
turn again could be rationalized with its matching stereochemical
configuration at 4 stereocentres of the ring, but the enzyme is
8
9
9
5
0
5
6
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

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Table 2. Concentration of homonojirimycin isomers derived from D-glucose giving 50% of inhibition
IC50 (µM)
DOI: 10.1039/C3OB41334A
OH
OH
OH
OH
OH
HO
OH
HO
OH
HO
OH
HO
OH
HO
OH
6*
Lit.
‡
2
2*
2*
2*
6*
CH OH HOH C
HOH C
N
H
CH OH HOH C
N
H
6
CH OH HOH C
N
H
6
CH OH HOH C
N
H
6
N
H
2
CH OH
2
2
2
2
2
2
2
2
2
2
3
1
32
-
4
34
35
71
†
60, 66, 67
27, 64, 65, 71
60, 68
References:
Enzyme
Lit.
Lit.
Lit.
α-Glucosidase
Yeast
#
NI (2%)
NI (5%)
NI (15%)
NI (14%)
75
294
99
0.1
0.2
NI (24%)
196
369
NI (45%)
48
109
Rice
Rat intestinal
maltase
β-Glucosidase
Almond
NI (7%)
NI (1.8%)
NI (4%)
NI (7%)
NI (11%)
NI (3%)
NI (17%)
60
NI (0%)
NI (20%)
NI (8%)
NI (2%)
NI (31%)
17
NI (0%)
A. niger
Bovine liver
α-Galactosidase
Coffee bean
1.0
8.0
NI (35%)
NI (20%)
722
NI (24%)
3.4
10
560
NI (31%)
Human lysosome
β-Galactosidase
Bovine liver
Rat intestinal
lactase
NI (24%)
NI (21%)
NI (12%)
NI (25%)
NI (11%)
NI (28%)
NI (36%)
NI (20%)
NI (32%)
NI (13%)
α-Mannosidase
Jack bean
NI (2%)
NI (0%)
NI (8%)
NI (4%)
NI (38%)
NI (0%)
NI (17%)
NI (4%)
NI (8%)
NI (13%)
NI (0%)
NI (6%)
NI (17%)
20
NI (14%)
NI (28%)
NI (0%)
NI (2%)
NI (49%)
NI (11%)
NI (0%)
NI (0%)
NI (21%)
121
β-Mannosidase
Snail
α-L-Fucosidase
Bovine kidney
Trehalase
Porcine kidney
α-L-Rhamnosidase
P. decumbens
116
Stereocenters from D-glucose shown in red; * indicates carbonyl carbon of azidoketose precursor in the reduced products; numbering according to IUPAC
naming; † References containing biological evaluations; ‡ NI: No inhibition (less than 50% inhibition at 1000 µM); # ( ): inhibition % at 1000 µM.
5
0
5
1
1
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Table 3. Concentration of homonojirimycin isomers derived from D-mannose giving 50% of inhibition
IC50 (µM)
DOI: 10.1039/C3OB41334A
OH
OH
OH
OH
OH
OH
HO
OH
HO
OH
HO
OH
HO
OH
HO
OH
HO
OH
2*
6
2*
6
2
* N
6
6
* N
2
6
* N
2
* N
6 2
HOH2C
N
H
CH2OH HOH2C
N
H
CH2OH HOH2C
CH2OH HOH2C
CH2OH HOH2C
CH2OH HOH2C
CH2OH
H
42
-
H
H
H
45
-
4
1
2
43
44
15, 63
Lit.
†
63, 73, 74
63, 65, 74, 75
63-65
References:
Enzyme
Lit.
Lit.
Lit.
α-Glucosidase
Yeast
Rice
Rat intestinal
maltase
‡
#
NI (16%)
NI (27%)
NI (9%)
NI (39%)
161
250
NI (4%)
54
180
NI (8%)
NI (37%)
NI (34%)
153
NI (9.7%)
140
NI (11.4%)
NI (1.6%)
NI (34.5%)
β-Glucosidase
Almond
A. niger
NI (0%)
NI (19%)
NI (16%)
NI (0%)
NI (15%)
NI (10%)
NI (3%)
NI (15%)
NI (20%)
NI (34%)
NI (13%)
576
NI (2.4%)
NI (10.0%)
NI (9.0%)
NI (11.6%)
NI (15.9%)
NI (4.1%)
Bovine liver
α-Galactosidase
Coffee bean
Human lysosome
194
NI (39%)
NI (17%)
424
NI (17%)
NI (28%)
93
5.5
NI (41.3%)
476
26
8.1
β-Galactosidase
Bovine liver
Rat intestinal
lactase
NI (19%)
NI (20%)
NI (29%)
NI (2%)
NI (17%)
NI (28%)
271
NI (45%)
NI (0%)
NI (16%)
NI (1.7%)
NI (10%)
α-Mannosidase
Jack bean
NI (4%)
NI (8%)
126
NI (33%)
NI (5%)
NI (10%)
NI (10%)
NI (16%)
NI (13%)
NI (18%)
NI (36%)
NI (4%)
NI (13%)
NI (5%)
NI (25%)
NI (6.7%)
NI (2.8%)
21
NI (10.3%)
NI (2.5%)
NI (42.0%)
NI (0%)
β-Mannosidase
Snail
α-L-Fucosidase
Bovine kidney
NI (38%)
NI (11%)
NI (11%)
Trehalase
Porcine kidney
NI (5%)
NI (23%)
NI (38.0%)
NI (14.5%)
α-L-Rhamnosidase
P. decumbens
NI (42.9%)
Stereocenters from D-mannose shown in green; * indicates carbonyl carbon of azidoketose precursor in the reduced products; numbering according to
IUPAC naming; † References containing biological evaluations; ‡ NI: No inhibition (less than 50% inhibition at 1000 µM); # (): inhibition % at 1000 µM.
were recorded on a Bruker Tensor 27 FT IR spectrophotometer
Experimental
20 using thin films on either NaCl or Ge plates. Only the
characteristic peaks are quoted. Nuclear magnetic resonance
All commercial reagents were used as supplied. Pyridine and
DMF were purchased dry from the Aldrich chemical company in
Sure-Seal™ bottles. All other solvents were used as supplied,
without prior purification. Reactions performed under an
atmosphere of argon gas were maintained by an inflated balloon.
All solutions are saturated unless otherwise stated. Thin layer
chromatography (TLC analysis) was performed on aluminum
sheets coated with 60 F254 silica supplied by Merck. Sheets were
visualized using either a dip of 0.2% w/v cerium (IV) sulfate and
1
(
NMR) spectra were record on a Bruker AMX500 ( H: 500 MHz
1
3
1
and C: 125.7 MHz) or a Bruker AV400 ( H: 400.2 MHz and
1
5
0
5
3
C: 100.6 MHz) spectrometer in the deuterated solvent stated.
All chemical shifts (δ) are quoted in ppm and coupling constants
J) in Hz. Residual signals from the solvents were used as an
internal reference. Acetonitrile were used as internal references
where D O was used as the solvent. Low resolution mass spectra
m/z) were recorded on a Micromass LCT (ESI) spectrometer.
2
5
(
2
1
(
30
High resolution mass spectra (HRMS m/z) were carried out on a
5
% ammonium molybdate in 2 M sulfuric acid, or potassium
Bruker MicroTof (Resolution = 10,000 FWHM). Electrospray
permanganate (0.5% in 1 M NaOH). Flash chromatography was
performed either on Sorbsil C60 40/60 silica or Merck grade
9385, 230-400 mesh, 60Å. Melting points were recorded on a
Kofler hot block and are uncorrected. Optical rotations were
recorded on a Perkin-Elmer 241 polarimeter with a path length of
(
ESI) was used throughout.
1
-1
1
dm. Concentrations are quoted in g 100 mL . Infrared spectra
8
| Journal Name, [year], [vol], 00–00
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C3OB41334A
Synthesis of kinetic and thermodynamic azides
was diluted in DCM (850 mL) and washed with 2 M aq HCl (20
mL). The aqueous layer was extracted with DCM (2 x 30 mL)
3
,5:6,7-Di-O-isopropylidene-D-glycero-D-ido-heptono-1,4-
60
and the combined organic fractions were washed with 50% brine
lactone 20
(
4
100 mL), dried (MgSO ) and concentrated in vacuo to yield the
3
,5:6,7-Di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-
crude triflate as a white crystalline solid (726 mg, quant) which
was used without further purification in the following reaction.
The triflate (727 mg, 1.72 mmol) was dissolved in DMF (5 mL)
5
0
5
0
5
0
5
0
5
lactone 19 (1.5 g, 5.21 mmol) was dissolved in DCM (15 mL),
pyridine (13.0 mmol, 1.05 mL) was added and the mixture was
cooled to -40 °C. Trifluoromethanesulfonic anhydride (7.3 mmol, 65 and cooled to -18 °C in an ice/salt bath. Sodium azide (124 mg,
.2 mL) was added dropwise and the reaction was stirred 1.90 mmol) was added to the reaction and it was left to react for
17 at -18 to +5 °C. TLC analysis (1:2 ethyl
acetate/cyclohexane) indicated complete consumption of starting
triflate (R 0.67) and the presence of two major species and a
been formed. The reaction mixture was diluted with DCM (135 70 minor impurity. The major spots are the ido-azide 3 (R 0.58) and
0.41) with minor amounts of
0.5). The reaction mixture was diluted with ethyl
acetate (50 mL) and extracted with 50% aq brine (4x 50 mL). The
organic fraction was dried (MgSO ) and evaporated to dryness on
rotary evaporator at RT. Purification by column
1
vigorously as it warmed to -20 °C. After 70 min TLC analysis
(1:2 ethyl acetate/cyclohexane) showed all the starting material
h
1
1
2
2
3
3
4
4
(
R
f
0.11) had been consumed and a single product (R
f
0.50) had
f
f
mL) and washed with 2 M aq HCl (45 mL). The aqueous layer
was extracted with DCM (60 mL) and the combined organic
the title compound gulo-azide 21 (R
gulo-triflate (R
f
f
4
layers were washed with brine (80 mL), dried (MgSO ), filtered
and concentrated in vacuo at RT. The crude triflate (2.39 g) was
4
dissolved in DMF (30 mL) and sodium trifluoroacetate (2.12 g,
75
a
1
5.6 mmol) was added and the reaction mixture stirred at 60 °C.
chromatography (5:1 cyclohexane/ethyl acetate) yielded the ido-
azide 3 (201 mg, 0.64 mmol, 37%) along with the title compound
gulo-azide 21 (313 mg, 1.0 mmol, 58%).
After 72 h MeOH (1.5 mL) was added to the reaction and the
mixture was stirred for another 16 h. After this time TLC analysis
(
1:3 ethyl acetate/cyclohexane) indicated a major product (R
f
Method B (via equilibration): Pyridinium p-toluenesulfonate
0
.13) and the reaction mixture was diluted with ethyl acetate (200 80 (201 mg, 0.80 mmol) and sodium azide (62 mg, 0.96 mmol) was
mL) and washed with 50% brine (3 x 90 mL). The organic layer
was dried (MgSO ), filtered and evaporated to dryness. NMR
added to a stirred solution of 2-O-azido-2-deoxy-3,5:6,7-di-O-
isopropylidene-D-glycero-D-ido-heptono-1,4-lactone 3 (220 mg,
0.70 mmol) in dry DMF (2 mL, 100 mg/mL) at RT and stirred
vigorously. The reaction was monitored via TLC (1:2 ethyl
4
analysis of the crude product still indicated the presence of DMF
and hence the crude was dissolved in ethyl acetate (20 mL) and
4
extracted with 50% aq brine (3 x 20 mL), dried (MgSO ), filtered 85 acetate/cyclohexane) and after 97.5 h the major species was the
and evaporated to dryness to yield the crude product as a off
white solid (1.10 g, 3.82 mmol, 74%). The crude was purified via
flash column chromatography (1:4 ethyl acetate/cyclohexane) to
yield the product 20 as white crystalline solid (0.931 g, 3.23
mmol, 62%).
title compound 21 (R
epimer remaining 3 (R
f
0.41) with trace amounts of the starting
0.58). The reaction mixture was diluted
f
with ethyl acetate (20 mL) and washed with 50% aq brine (4x 20
mL). The organic layer was dried (MgSO ) and evaporated to
4
90 dryness to yield the crude product (227 mg, quant). Column
chromatography (6:1 ⇒ 3:1 cyclohexane/ethyl acetate) yielded
the title compound 21 (206 mg, 0.66 mmol, 94%) along with
some retained starting azide 3 (16 mg, 0.05 mmol, 6%).
m.p. 160-162 °C (ethylacetate/cyclohexane), [lit. m.p. 180 °C
5
0
25
(
ethylacetate/cyclohexane)]; [α]
D
-123.2 (c 1.1 in CHCl
max (thin film): 3376 (br, m,
(400 MHz, CDCl ) 4.67 (1 H, a-t, H4,
3,4 2.2 J4,5 2.2), 4.42 (1 H, dd, H3, J3,4 2.4, J2,3 0.6), 4.31 (1 H, 95 1.09 in CHCl
ddd, H6, J5,6 8.4, J6,7 6.2, J6,7’ 4.0), 4.16 (1 H, d, H2, J2,OH 2.9), (400 MHz, CDCl
.10 (1 H, dd, H7, J7,7’ 8.9, J6,7 6.2), 3.94 (1 H, dd, H7, J7,7’ 8.9,
3
), [lit.
2
0
50
[α]
D
–99.0 (c 1.07 in CHCl
3
)];
ν
2
D
5
OH), 1783 (s, C=O); δ
H
3
m.p. 153-155°C (Isopropyl alcohol/cyclohexane); [α]
); νmax (thin film): 2115 (s, N ), 1777 (s, C=O); δ
) 4.70 (1 H, dd, H3, J2,3 3.9, J3,4 2.1), 4.35 (1 H,
-91.8 (c
J
3
3
H
3
4
a-t, H4, J4,5=J3,4= 2.1), 4.32 (1 H, ddd, H6, J5,6 8.6, J6,7 6.0, J6,7’
3.8), 4.11 (1 H, dd, H7, J7,7’ 9.0, J6,7 6.0), 3.95 (1 H, d, H2, J2,3
3.9), 3.93 (1 H, dd, H7’, J7,7’ 9.0, J6,7’ 3.8), 3.83 (1 H, dd, H5, J5,6
J
J
6,7 4.0), 3.86 (1 H, dd, H5, J5,6 8.4, J4,5 2.0), 3.36 (1 H, d, OH,
2,OH 3.3), 1.47 (3 H, s, Me), 1.44 (3 H, s, Me), 1.36 (3 H, m,
Me), 1.35 (3 H, s, Me); δ
C
(101 MHz, CDCl
), 98.2 (3,5-CMe ), 73.9 (C6), 73.5 (C2), 72.4 (C4),
2.1 (C3), 69.5 (C5), 67.0 (C7), 28.9 (Me), 27.0 (Me), 25.0 (Me),
3
) 175.3 (C1), 109.6 100 8.6, J4,5 2.1), 1.50 (3 H, s, Me), 1.43 (6 H, s, 2x Me), 1.35 (3 H, s,
Me); δ (101 MHz, CDCl ) 170.6 (C1), 109.9 (6,7-CMe ), 99.1
(3,5-CMe ), 73.3 (C6), 70.1 (C4), 70.0 (C3), 69.7 (C5), 67.1
(C7), 61.7 (C2), 28.8 (Me), 27.1 (Me), 25.0 (Me), 19.6 (Me); m/z
(
6,7-CMe
2
2
C
3
2
7
2
+
+
19.5 (Me); m/z (ESI+ve): 311 ([M+Na] , 82%), 599 ([2M+Na] ,
+
+
+
1
00%); HRMS (ESI+ve): Found 311.1107 ([M+Na] );
requires 311.1101.
(ESI+ve): 336 ([M+Na] , 100%), 649 ([2M+Na] , 99%); HRMS
+
C
13
H
20NaO
7
105 (ESI+ve): Found 336.1162 ([M+Na] ); C13
36.1166.
19 3 6
H N NaO requires
3
2
-Azido-2-deoxy-3,5:6,7-di-O-isopropylidene-D-glycero-D-gulo-
heptono-1,4-lactone 21
2-Azido-2-deoxy-3,5:6,7-di-O-isopropylidene-D-glycero-D-ido-
heptono-1,4-lactone 3
5
0
Method A (via inverted alcohol): 3,5:6,7-Di-O-isopropylidene-D-
glycero-D-ido-heptono-1,4-lactone 20 (500 mg, 1.73 mmol) was
3,5:6,7-Di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-
dissolved in DCM (5 mL) and pyridine (0.35 mL, 4.33 mmol) 110 lactone 19 (1g, 3.47 mmol) was dissolved in DCM (10 mL) and
and cooled to –30 °C. Trifluoromethanesulfonic anhydride (0.41
mL, 2.43 mmol) was added dropwise and the reaction was stirred
vigorously at –30 °C. After 30 min TLC analysis (1:2 ethyl
acetate/cyclohexane) indicated the conversion of the starting
pyridine (0.8 mL, 9.9 mmol) and cooled to -30 °C.
Trifluoromethanesulfonic anhydride (0.8 mL, 4.86 mmol) was
added dropwise and the reaction was stirred vigorously at -30°C.
After 30 min TLC analysis (1:2 ethyl acetate/cyclohexane)
5
5
f f f
material (R 0.18) into a major product (R 0.67). The reaction 115 indicated the conversion of the starting material (R 0.11) into a
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DOI: 10.1039/C3OB41334A
major product (R
f
0.50). The reaction was diluted in DCM (100
virtually no remaining starting material, glacial acetic acid was
mL) and washed with 2 M aq HCL (30 mL). The aqueous layer
slowly added until the reaction mixture was pH < 6. The solvent
was extracted with DCM (2 x 40 mL) and the combined organic 60 was removed under reduced pressure and the resulting residue
fractions were washed with 50% brine (60 mL), dried (MgSO
4
)
was purified by column chromatography (1:3 ⇒ 1:1 ethyl
acetate/cyclohexane) yielding 2-azido-2-deoxy-3,5:6,7-di-O-
isopropylidene-D-glycero-D-ido-heptitol 22 (9.07 g, 91%) as a
white solid.
5
0
5
0
5
0
and concentrated in vacuo at 14°C to yield the crude triflate as a
white crystalline solid (1.48 g, quant) which was used without
further purification in the following reaction. The triflate was
dissolved in DMF (6 mL, 240 mg/mL), cooled to -15°C in a 65 m.p. 100-103°C (from cyclohexane/ethyl acetate); [α]
2
5
D
+37.4 (c
);
3
, 400 MHz): 4.23 (1H, ddd, H2, J2,3 8.2, J2,1’ 6.4, J2,1
ice/salt bath and sodium azide (329 mg 5.1 mmol) was added
portionwise. Over 15.5 h the reaction warmed to +6°C and TLC
analysis (1:2 ethyl acetate/cyclohexane) showed that the starting
1.09 in CHCl
(CDCl
3 3
); νmax (thin film): 3449 (br, m, OH), 2103 (s, N
1
1
2
2
3
δ
H
4.7), 4.08-4.12 (1H, m, H5), 4.09 (1H, dd, H1’, J1’,1 8.7, J1’,2 6.3),
3.86 (1H, dd, H1, J1,1’ 8.7, J1,2 4.7), 3.73-3.80 (3H, m, H7, H7’,
material 19 (R
f
0.50) had been converted into a major species 3
(
R
f
0.58) with trace amounts of the gulo-azide 21 (R 0.41) being 70 H6), 3.66-3.70 (2H, m, H4, H3), 2.43 (1H, br-d, OH-4, JOH,4
f
present. The reaction mixture was diluted with ethyl acetate (120
mL) which had been cooled to +4°C, washed quickly with 50%
11.4), 1.99-2.04 (1H, br-m, OH-7), 1.50 (3H, s, Me), 1.45 (3H, s,
Me), 1.43 (3H, s, Me), 1.36 (3H, s, Me); δ (CDCl , 100.6 MHz):
109.5 (1,2-CMe ), 99.9 (3,5-CMe ), 74.2 (C4 or C3), 73.9 (C5),
73.0 (C2), 67.1 (C1), 63.6 (C6), 62.9 (C4 or C3), 60.8 (C7), 29.4,
C
3
aq brine (4x 120 mL), dried (MgSO
to give the crude product as an off white solid (1.106 g, quant).
Column chromatography (8:1 cyclohexane/ethyl acetate) was 75 26.9, 25.0, 19.1 (4 × Me); m/z (ESI+ve): 340 ([M+Na]
4
) and evaporated to dryness
2
2
+
,
50%),
381 ([M+MeCN+Na] , 98%), 657 ([2M+Na] , 100%); HRMS
+
+
used to purify the crude reaction residue and the title compound 3
was yielded as a white crystalline solid (1.01 g, quant) along with
a trace of the gulo-azide 21 (29 mg, 3%).
+
(ESI+ve): found 340.1471 [M+Na] ; C13
340.1479.
23 3 6
H N NaO requires
2
3
m.p. 102-104 °C (Diethylether/cyclohexane), [lit. m.p. 102°C] ,
6
-Azido-6-deoxy-D-glycero-L-gulo-heptitol 24
2
5
22
[
α]
CHCl
MHz, CDCl
D
-216.0 (c 1.78 in CHCl
3
), [lit. [α]
)] ; νmax (thin film): 2116 (s, N ), 1787 (s, C=O); δ
) 4.51 (1 H, a-t, H4, J3,4=J4,5=2.2), 4.33 – 4.25 (2 H,
D
-224 (c 1.0 in
2
2
3
3
H
(400
OH
2
OH
4
N3
6
3
HO
1
3
5
7
OH
m, H3, H6), 4.09 (1 H, dd, H7, J7,7’ 8.9, J6,7 6.2), 4.06 (1 H, s,
H2), 3.92 (1 H, dd, H7’, J7,7’ 8.9, J6,7’ 4.0), 3.81 (1 H, dd, H5, J5,6
OH
OH
80
8
.5, J4,5 2.0), 1.45 (3 H, s, Me), 1.44 (3 H, s, Me), 1.36 (3 H, s,
Me), 1.35 (3 H, s, Me); δ (101 MHz, CDCl ) 171.1 (C1), 109.9
(6,7-CMe ), 98.8 (3,5-CMe ), 73.8 (C6), 72.4 (C4), 70.8 (C3),
9.5 (C5), 67.1 (C7), 63.4 (C2), 29.0 (Me), 27.1 (Me), 25.0 (Me),
To a solution of 6-azido-6-deoxy-1,2:3,5-di-O-isopropylidene-D-
glycero-L-gulo-heptitol 22 (5.91 g, 18.6 mmol) in H O (60 mL)
and methanol (10 mL) at 40 °C was added Dowex 50WX8 (H
form) (11.8 g). After 48 h, TLC analysis (9:1 ethyl
acetate/methanol) indicated the consumption of starting material
C
3
2
2
2
TM
+
6
1
8
+
+
9.6 (Me); m/z (ESI+ve): 336 ([M+Na] , 100%), 649 ([2M+Na] ,
85
+
0%); HRMS (ESI+ve): Found 336.1167 ([M+Na] );
f f
(R 0.80) and the presence of a major product (R 0.05). The
C
13
H
19
N
3
NaO
6
requires 336.1166.
reaction mixture was filtered and the solvent removed. The crude
residue was co-evaporated with MeOH (3 × 15 mL) to give a
white solid (4.42 g). The solid was heated in IPA (90 mL) at
reflux until complete dissolution occurred and the solution was
allowed to cool slowly over 2 h and then was stirred at 0 °C for 1
h. The resulting white crystals were filtered and washed with
3
5
Synthesis of 6-azidoheptitols
6
-Azido-6-deoxy-1,2:3,5-di-O-isopropylidene-D-glycero-L-gulo-
90
heptitol 22
2
-Azido-2-deoxy-3,5:6,7-di-O-isopropylidene-D-glycero-D-ido-
heptono-1,4-lactone 3 (9.80 g, 31.3 mmol) was dissolved in DCM
Et
2
O (15 mL), yielding 6-azido-6-deoxy-D-glycero-L-gulo-
4
4
5
5
0
5
0
5
(100 mL) and was cooled to -78°C under argon. DIBAL (1.5 M
heptitol 24 (4.20 g, 95%) as a white solid.
2
5
in toluene) (22.0 mL, 33.0 mmol) was added dropwise over 15 95 m.p. 81-83 °C (from methanol); [α]
D
-0.74 (c 1.04 in H
2
O); νmax
min and the mixture stirred at -78°C for 2 h. TLC analysis (98:2
chloroform/methanol) indicated the consumption of starting
(thin film, Ge): 3385 (br, s, OH), 2115 (s, N ); δ (D O, 400
3
H
2
MHz): 3.95 (1H, dd, H2, J1,2 6.3, J2,3 2.2), 3.91 (1H, dd, H7’, J7,7’
11.8, J6,7’ 4.4), 3.78-3.88 (4H, m, H1’, H4, H5, H7), 3.73 (1H, a-
dd, H6, J5,6 7.7, J6,7’ 4.2), 3.70 (1H, dd, H3, J3,4 8.1, J2,3 2.2), 3.67
material (R
f f
0.75) and the formation of a major product (R 0.40).
The reaction mixture was diluted with DCM (100 mL), to which
C 2
was then added potassium sodium tartrate (200 mL) and the 100 (1H, dd, H1, J1,1’ 11.6, J1,2 6.1); δ (D O, 100.6 MHz): 72.3, 71.6
resulting mixture was stirred vigorously for 20 h. The phases
were then separated, and the aqueous phase was extracted with
DCM (4 × 50 mL). The combined organic phases were dried
(C4, C5), 71.5 (C3), 70.6 (C2), 64.6 (C6), 63.4 (C1) , 62.4 (C7);
+
m/z (ESI+ve): 260 ([2M+Na] , 100%); HRMS (ESI+ve): found
+
260.0852 [M+Na] ; C
7
H
15
N
3
NaO
6
requires 260.0853.
(MgSO
pressure. The resulting colourless oil was dissolved in MeOH
100 mL) and cooled to -30°C under nitrogen. Sodium
4
), filtered, and the solvent removed under reduced
2
-Azido-2-deoxy-3,5:6,7-di-O-isopropylidene-D-glycero-D-gulo-
1
05 heptitol 23
(
2
-Azido-2-deoxy-3,5:6,7-di-O-isopropylidene-D-glycero-D-gulo-
borohydride (315 mg, 8.33 mmol) was added at -30°C. After 1.5
h, TLC analysis (97:3 chloroform/methanol) indicated some
heptono-1,4-lactone 21 (5.86 g, 18.7 mmol) was dissolved in
DCM (60 mL) and cooled to -78 °C. DIBAL (1.5 M in toluene)
starting material (R
.20). Additional sodium borohydride (160 mg, 4.23 mmol) was
then added at -30°C. After 1.5 hours, TLC analysis indicated
f f
0.60) and the presence of a major product (R
(
19 mL, 28.5 mmmol) was added dropwise and the mixture
0
1
10 stirred at -78 °C for 4 h. The reaction was monitored by IR,
1
0 | Journal Name, [year], [vol], 00–00
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-1
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DOI: 10.1039/C3OB41334A
looking for the disappearance of a lactone peak at 1794 cm . The 55 MHz): 3.97 (1H, dd, H1’, J1’,1 11.9, J1’,2 3.0), 3.96 (1H, a-t, H4,
reaction mixture was diluted with DCM (150 mL) and saturated
aq potassium sodium tartrate solution (200 mL) was then added
and the mixture was allowed to warm to RT and stirred
vigorously for 18 h. The aqueous phase was extracted with DCM
J4,3 = J4,5 = 3.2), 3.80 (1H, dd, H7’, J7’,7 11.5, J7’,6 3.0), 3.73-3.80
(3H, m, H1, H5, H6), 3.72 (1H, dd, H3, J3,2 7.4, J3,4 3.2), 3.69
(1H, ddd, H2, J2,1 7.6, J2,3 7.4, J2,1’ 3.0), 3.65 (1H, dd, H7, J7,7’
5
0
5
0
5
0
5
C 2
11.5, J7,6 5.9); δ (D O, 125.8 MHz): 73.2 (C3), 72.2 (C5 or C6),
(
(
3 × 60 mL) and the combined organic phases were dried 60 71.7 (C5 or C6), 69.4 (C4), 64.6 (C2), 63.1 (C7), 61.6 (C1); m/z
-
MgSO
4
), filtered, and concentrated under reduced pressure. The
(ESI-ve): 236 ([M-H]
,
100%); HRMS (ESI-ve): found 236.0886
-
resulting colourless oil was dissolved in MeOH (85 mL), cooled
to -30 °C and treated with sodium borohydride (382 mg, 10.1
[M-H] ; C H N O requires 236.0888.
7 14 3 6
Characterization of HNJ and four isomers:
1
1
2
2
3
3
mmol) at -30 °C for
chloroform/methanol) indicated the presence of starting material
0.55) and the presence of a major product (R
f
0.25). Sodium 65 optical rotations are given in Table 1. Full experimental data is
4
h. TLC analysis (97:3
Proton and carbon NMR data along with melting points and
(
R
f
borohydride (192 mg, 5.06 mmol) was then added and the
mixture was stirred at -30 °C for a further 2 h. TLC analysis still
indicated residual starting material, so additional sodium
borohydride (383 mg, 10.1 mmol) was then added and the
mixture stirred for a further 2 h. No starting material was then
observable by TLC so acetic acid was added dropwise until the
pH < 6. The solvent was removed under reduced pressure and the
resulting orange oil was purified by column chromatography (1:4
available in the supporting information.
54,56
Biotechnological protocols
Microbial Oxidation of 24/25
Gluconobacter thailandicus NBRC 3254 was used for
biotransformation of 24 to 26 and 25 to 28. This bacteria was
grown aerobically in Erlenmeyer flasks (500 mL) containing
medium (0.5 % D-glucose, 0.5 % polypepton, 0.5 % yeast extract
7
0
⇒
1:2 ethyl acetate/cyclohexane) yielding 2-azido-2-deoxy-
and 0.1 % MgSO
4 2
.7H O) at 30 °C for 48 h. The collected cells
3
,5:6,7-di-O-isopropylidene-D-glycero-D-gulo-heptitol 23 (4.82
were washed twice with deionized water by centrifugation at
g, 81%) as a white solid.
2
5
75 9000 rpm for 10 min. The washed cells were suspended in 50
mM sodium phosphate buffer (pH 7.0) for carrying out the
biotransformations. The transformation reaction was carried out
in a 500 mL Erlenmeyer flask containing a solution of 3-5%
m.p. 92-94 °C (from cyclohexane/ethyl acetate); [α]
D
-16.5 (c
);
, 400 MHz): 4.25 (1H, ddd, H6, J6,5 8.3, J6,7’ 6.3, J6,7
.6), 4.09 (1H, dd, H7’, J7’,7 8.7, J7’,6 6.3), 3.90-3.94 (1H, br-m,
3 3
1.10 in CHCl ); νmax (thin film): 3449 (br, s, OH), 2104 (s, N
δ
H 3
(CDCl
4
2
4/25 at 30 °C, with continuous shaking at 120 rpm with the cells
H1’), 3.87 (1H, dd, H7, J7,7’ 8.7, J7,6 4.6) , 3.79-3.80 (2H, m, H2,
H4), 3.70-3.77 (2H, m, H1, H3), 3.67 (1H, dd, H5, J5,6 8.3, J5,4
1.2), 2.39 (1H, br-d, OH-4, JOH,H 10.7), 2.15 (1H, br-s, OH-1),
80
(Cell OD A600=30). The formation of product (26/28) was
monitored by HPLC. At the end of the reaction, the cells were
removed by centrifugation at 10,000 rpm for 10 min. The
supernatant was decolorized using activated charcoal, which was
then removed using centrifugation and filtration. The decolorized
supernatant was deionized using ion exchange resin DIAION
1
(
(
.44 (6H, s, 2 × Me), 1.40 (3H, s, Me), 1.37 (3H, s, Me); δ
CDCl , 100.6 MHz):, 109.6 (6,7-CMe ), 98.3 (3,5-CMe ), 74.2
C5), 73.2 (C6), 71.9 (C4), 67.0 (C7), 62.3, 62.4 (C2 and C3),
2.3 (C1) 29.4, 26.9, 25.0, 19.1(4 × Me); m/z (ESI+ve): 657
C
3
2
2
85
6
2
-
+
+
+
3
(SKIB, H+) and Amberlite (IRA-411, CO ).
([2M+Na] , 100%), 381 ([M+MeCN+Na] , 41%), 340 ([M+Na] ,
+
1
8%); HRMS (ESI+ve): found 340.1475 [M+Na] ;
NaO requires 340.1479.
Epimerization of 26/28
C
13
H
23
N
3
6
Epimerization of 26 to 27 and 28 to 29 was carried out using
immobilized D-tagatose-3-epimerase. This reaction was carried
out by shaking at 40 °C in a 500 mL flask containing 10 g of
immobilized enzyme (2,000 U) and 200 mL of 1-2% solution of
26/28. Conversion of 26/28 in the reaction mixture was analysed
by high performance liquid chromatography (HPLC). This
reaction reached its equilibrium after 24 h.
2
-Azido-2-deoxy-D-glycero-D-gulo-heptitol 25
90
OH
6
OH
4
N3
2
HO
7
5
3
1
OH
OH
OH
4
4
5
0
5
0
To a solution of 2-azido-2-deoxy-3,5:6,7-di-O-isopropylidene-D-
glycero-D-gulo-heptitol 23 (3.92 g, 12.4 mmol) in H O (40 mL)
9
5
Separation of 26 and 27 or 28 and 29
2
TM
+
and MeOH (7.5 mL) Dowex 50WX8 (H form) resin (14.7 g)
was added and the mixture stirred at 40 °C for 48 h. TLC analysis
Immobilized enzyme was removed from the reaction mixture by
filtration. The supernatant was deionized using ion exchange
resin and concentrated under rotatory evaporation. The products
(
9:1 ethyl acetate/methanol) indicated the consumption of starting
2
+
material (R 0.85) and the formation of a major product (R 0.10).
f
f
were then separated using Dowex 50W-X2 resin (Ca ). The
The reaction mixture was filtered, and the solvent removed under 100 purity of the product was established by HPLC.
reduced pressure. The resulting pale orange solid was heated in
IPA (50 mL) at reflux until complete dissolution had occurred.
Preparation of catalyst
The solution was allowed to cool slowly to RT over 2 h and then
was stirred at 0°C for 1 h. The resulting white crystals were then
Raney nickel (30 g) was dissolved in 300 mL of 20% aqueous
sodium hydroxide (300 mL). The catalyst was heated at 80 °C for
6 h and then washed several times with distilled water to obtain
105 pH 9.2. The resultant precipitate was used for hydrogenation of
all the azidoketoses.
filtered, washed with Et
2
O (30 mL), yielding 2-azido-2-deoxy-D-
glycero-D-ido-heptitol 25 (2.49 g, 85%) as a white solid.
2
1
m.p. 110-111 °C (from methanol); [α]
D
19.1 (c, 0.985 in H
2
O);
ν
max (thin film, Ge): 3357 (br, s, OH), 2111 (s, N
3
); δ (D O, 500
H
2
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DOI: 10.1039/C3OB41334A
Hydrogenation of 26 and 27
were assayed at pH 5.8 for rat intestinal maltase and lactase using
maltose and lactose. For the activity of rice α-glucosidase and rat
intestinal maltase and lactase, the reaction mixture (0.2 mL)
contained 25 mM maltose and the appropriate amount of enzyme,
and the incubations were performed for 10-30 min at 37 °C. The
reaction was stopped by heating at 100 °C for 3 min. After
centrifugation (600 g; 10 min), 0.05 mL of resulting reaction
mixture was added to 3 mL of Glucose CII-test Wako (Wako
Pure Chemical Ind., Osaka, Japan). The absorbance at 505 nm
was measured to determine the amount of the released D-glucose.
Other glycosidase activities were determined using an appropriate
p-nitrophenyl glycoside as substrate at optimum pH of each
enzyme. The reaction mixture (1 mL) contained 2 mM of the
substrate and the appropriate amount of enzyme. The reaction
The catalyst was added to a solution of 26, 27, 28, or 29 in water
5
00 mL (final concentration 0.02-0.2%). The reactor was charged
with hydrogen at 1.2 MPa and the contents heated to 50 °C for 24
h. The catalyst was then removed by centrifugation and filtration,
and the supernatant fluids concentrated by evaporation under
6
6
7
0
5
0
5
vacuum at 37 °C. The resulting crude mixtures were
+
chromatographed over an Amberlite CG-50 column (NH
with H O as eluent.
4
form)
2
1
0
Separation of HNJ mixtures
The mixtures of HNJ isomers were separated by ion exchange
-
chromatography by Dowex 1x2 (OH form).
X-ray structure determination
2 3
was stopped by adding 2 mL of 400 mM Na CO . The released p-
nitrophenol was measured spectrometrically at 400 nm.
X-Ray diffraction data was collected on either a Nonius Kappa-
7
6
1
2
2
3
3
4
5
0
5
0
5
0
CCD Diffractometer (21) or a Oxford Diffraction/Agilent
7
7
SuperNovae A (3) using molybdenum or copper radiation
Acknowledgment
respectively. The crystals were placed in the cold stream of an
7
8
Oxford Cryosystems open-flow nitrogen cryostat
with
a
This work was supported by Cancer Research UK (AFGG),
nominal stability of 0.1K. Cell refinement and data reduction was 75 EPSRC (SJN) and the Leverhulme Trust (GWJF). We would like
7
9
80
performed using either Denzo/scalepack or CrysAlis RED.
to thank the Oxford Chemical Crystallography Service for the use
of their instruments.
8
1
The SIR92 program was used to solve the structures and the
8
2
structures were refined in CRYSTALS. For the gulo 21
compound in the absence of significant anomalous scattering,
Friedel pairs were merged and the absolute configuration was
assigned from the use of D-glycero-D-gulo-heptonolactone as the
starting material. In the case of the ido 3 compound the absolute
configuration was determined by refinement of the Flack
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