Isolation synthesis and glycosidase inhibition profile of 3-epi-casuarine

Article abstract of DOI:10.1016/j.tetasy.2006.10.005

3-epi-Casuarine, the first naturally occurring stereoisomer of casuarine, was isolated from Myrtus communis L. The glycosidase inhibition profile and NMR spectra of 3-epi-casuarine are compared with those of casuarine; the change in configuration at one of the six stereogenic centres causes a dramatic change in the conformation of the bicyclic system. The key step in the 6% overall yield synthesis of 3-epi-casuarine from d-gluconolactone is the efficient cyclization of a completely unprotected pentahydroxyaminomesylate to the pyrrolizidine nucleus. A low yield synthesis of casuarine is also reported.

Full text of DOI:10.1016/j.tetasy.2006.10.005

Tetrahedron: Asymmetry 17 (2006) 2702–2712
Isolation synthesis and glycosidase inhibition profile of 3-epi-casuarine
Jeroen Van Ameijde,a Graeme Horne,a,b Mark R. Wormald,c Raymond A. Dwek,c
Robert J. Nash,b Paul Wyn Jones,b Emma L. Evinsonb and George W. J. Fleeta,*
aDepartment of Organic Chemistry, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK
bInstitute of Grassland & Environmental Research, Plas Gogerddan, Aberystwyth SY23 3EB, Dyfed, Wales, UK
cGlycobiology Institute, Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, UK
Received 20 September 2006; accepted 5 October 2006
Abstract—3-epi-Casuarine, the first naturally occurring stereoisomer of casuarine, was isolated from Myrtus communis L. The glycosi-
dase inhibition profile and NMR spectra of 3-epi-casuarine are compared with those of casuarine; the change in configuration at one of
the six stereogenic centres causes a dramatic change in the conformation of the bicyclic system. The key step in the 6% overall yield syn-
thesis of 3-epi-casuarine from D-gluconolactone is the efficient cyclization of a completely unprotected pentahydroxyaminomesylate to
the pyrrolizidine nucleus. A low yield synthesis of casuarine is also reported.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
from Casuarina equisetifolia, is the most heavily oxygen-
ated of the naturally occurring pyrrolizidine alkaloids with
six adjacent stereogenic centres and is a potent and specific
a-glucosidase inhibitor;1 its 6-a-glucopyranosyl derivative
has been isolated from C. equisetifolia and Eugenia jam-
bolana.2 None of its 63 stereoisomers have been hitherto
identified as a natural product.
Herein, we report the isolation of 3-epi-casuarine 1 from
the shrub Myrtus communis L. (Myrtle), and comparison
of its glycosidase inhibition profiles and its NMR spectra
with those of casuarine 2. The synthesis of 1 from D-gluc-
onolactone 11 depends on the clean cyclization of the com-
pletely unprotected aminomesylate 6; in spite of possible
competitive formation of epoxides, the ring closure to the
pyrrolizidine ring is very efficient. Less than 5% of casuar-
ine 2 is formed, presumably via epoxide 7. Casuarine 2,
Polyhydroxylated monocyclic and bicyclic alkaloids can be
viewed as sugar mimics in which the ring oxygen is replaced
by nitrogen;3 the biological properties and uses as chemo-
7a
HO
OH
HO
OH
HO
OH
HO
OH
HO
OH
H
N
H
H
N
H
N
H
N
7
HO
OH
CH2OH
HO
OH
CH2OH
OH
OH
OH
3
N
CH2OH
CH2OH
CO2H
1
2
4
5
3
HO
OH
HO
OH
OH
OH
OH
H
H
H
N
HO
HO
OH
OH
O
OH
OH
HO
OH
CH2OH
HN
HN
HO
HO
NH
OMs
OH
NH
CH2OH
CH2OH
6
9
7
8
10
*
Corresponding author. E-mail: george.fleet@chem.ox.ac.uk
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.10.005

J. V. Ameijde et al. / Tetrahedron: Asymmetry 17 (2006) 2702–2712
2703
therapeutic agents have ensured an escalating interest in
synthesizing both naturally occurring and synthetic com-
pounds.4 Alexine 3, isolated from Alexa leiopetala, was
the first pyrrolizidine alkaloid found with a carbon substi-
tuent at C-3.5 The 7a epimer of alexine, australine 4, was
found in Castanospermum australe;6 the corresponding
amino acid, 7a-epialexaflorine 5 was isolated from the
leaves of Alexa grandiflora.7 Alexine 3 and australine 4
may be viewed as bicyclic analogues of the monocyclic pyr-
rolidine natural products DGDP 8 and DMDP 9. Alexines
have five chiral centres; seven of the 32 stereoisomers have
been isolated as natural products from plants.8 The natural
products and the synthetic analogues have significant gly-
cosidase inhibition9 and antiviral properties.10 Alexine 3
and some of its stereoisomers were first made from glu-
cose;11 the biological potential and the complexity of five
adjacent stereogenic centres have led to a large number of
subsequent papers on their synthesis.12 The isomeric tetra-
hydroxypyrrolizidine 7-deoxycasuarine 10 has also been
identified as a specific inhibitor of amyloglucosidase.13
castanospermine (the benchmark for b-D-glucosidase inhi-
bition) in the same assay has an IC50 of 2 · 10ꢁ5 M. Gluco-
sidase inhibition by 3-epi-casuarine 1 is very different than
that by casuarine 2.19
Table 1. Results of the glycosidase inhibition study
Enzyme
% Inhibition
% Inhibition
(3-epi-Casuarine (Casuarine 2,
1, 700 lM)
700 lM)
a-D-Glucosidase (yeast)
a-D-Glucosidase (rice)
0
13
12
56
4
6
0
0
0
64
76
86
0
4
0
5
0
14
a-D-Glucosidase (Bacillus)
b-D-Glucosidase (almond)
a-D-Galactosidase (coffee bean)
b-D-Galactosidase (bovine liver)
a-D-Mannosidase (Jack bean)
a-L-Fucosidase (bovine kidney)
b-N-Acetyl-D-glucosaminidase
(bovine kidney)
Naringinase (Pencillium)
21
0
In contrast to the synthetic endeavours on the alexines,
there are only two syntheses of casuarine 214 and very lim-
ited studies on its stereoisomers.15 The isolation of 3-epi-
casuarine 1 as a natural product indicates that, similar to
the alexines, many stereoisomers may exist in Nature.
The X-ray crystal structure of 1 firmly established the rela-
tive stereochemistry of 3-epi-casuarine;16 the absolute con-
figuration is shown by the synthesis reported herein from
D-gluconolactone 11. Casuarines possess six contiguous
stereogenic centres; the challenge of devising strategies
for the efficient synthesis of reasonable amounts for biolog-
ical evaluation of such compounds remains.
1.2. NMR Studies on 3-epi-casuarine 1
The 1H NMR spectra of 1 and 2 are shown in Figure 1 and
the resonance assignments are given in Table 2. Despite the
structural similarities of the two compounds, their NMR
spectra are dramatically different. 1H NMR Studies on five
alexines and casuarine have shown that changes at one
sterocentre can lead to substantial conformational changes
of the bicyclic pyrrolizidine ring system and hence signifi-
cant changes in chemical shifts.20 Differences in 13C chem-
ical shifts are helpful in fingerprint analysis for identifying
known alexines.21
1.1. Isolation and glycosidase inhibition of 3-epi-casuarine 1
3-epi-Casuarine 1 was isolated as a natural product from
the European shrub M. communis L. (Myrtle) growing in
the grounds of the Institute of Grassland and Environmen-
tal Research in Aberystwyth. The isolation was conducted
using ion exchange chromatography and fractionation was
followed by GC–MS of the pertrimethylsilyl-derivatives.17
Casuarine 2 was the major alkaloid present in M. communis
(eluted first with water from the anion exchange resin Dow-
ex 1 in the hydroxyl form) and 3-epi-casuarine 1 was the
only epimer detected in the plant (eluted after casuarine
from Dowex 1). Casuarine 2 and 3-epi-casuarine 1 were
crystallized from warm 95% aqueous ethanol by layering
1
with acetone. The H and 13C NMR of the natural 3-epi-
casuarine were identical to those of the synthetic material;
23
the natural sample had a specific rotation, ½aꢀD ¼ þ5:7 (c
22
Figure 1. 1H NMR spectra in 2H2O at 30 ꢁC of (a) casuarine 2, pH 8.3,
and (b) 3-epi-casuarine 1, pH 9.3. The ring numbering scheme is shown
top right.
0.5, H2O) in comparison to ½aꢀD ¼ þ5:8 (c 0.69, H2O) for
the synthetic material.
The glycosidase inhibition properties of 1 and 2 were
determined by the methods previously described and using
commercially available enzymes and 5 mM para-
nitrophenylsubstrates.18
For bicyclic pyrrolizidines, the proton coupling constants
are a characteristic of the relative orientations of the ring
protons.20 For 3-epi-casuarine 1, the very small C7aH–
C1H and C1H–C2H couplings are only consistent with
these two proton pairs being trans but not di-axial. The rel-
atively large C6H–C7H and C7H–C7aH couplings suggest
that C6H, C7H and C7aH are all trans and all axial. The
Casuarine 2 is a good inhibitor of a-D-glucosidases (Table
1). In contrast, the epimer 1 only significantly inhibited b-
D-glucosidases with an IC50 just below 700 lM; by contrast

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J. V. Ameijde et al. / Tetrahedron: Asymmetry 17 (2006) 2702–2712
Table 2. NMR resonance assignments in 2H2O at 30 ꢁC for 3-epi-casuarine 1, pH 9.3, and casuarine 2, pH 8.3
3-epi-Casuarine 1
Casuarine 2
3JHH (Hz)
1H
13C
1H
13C
d (ppm)a
3JHH (Hz)
d (ppm)b
d (ppm)a
d (ppm)c
C5
3.09
Overlapping
51.6
3.270
2.911
4.21
12.2/4.7
12.2/4.0
4.7/4.0/3.1
3.1/3.5
3.5/8.0
8.0/8.0
59.1
C6
C7
C7a
C1
C2
C3
C8
4.098
4.029
3.087
4.261
4.169
3.262
3.984
3.926
8.5/7.7/7.7
7.7
7.7/1.6
1.6/1.4
1.4/3.3
3.3/6.4/7.3
ꢁ11.8/6.4
ꢁ11.8/7.3
75.9
79.2
75.5
80.4
79.7
65.0
57.4
78.5
79.9
73.2
78.9
77.7
71.1
63.3
4.19
3.071
4.162
3.796
3.036
3.771
3.611
8.0/8.0
8.0/3.8/6.6
ꢁ11.9/3.8
ꢁ11.9/6.6
a Referenced to trimethylsilylpropanesulfonic acid at 0.000 ppm.
b Reference to dioxane at 67.2 ppm.
c Referenced to acetone at 30.9 ppm.
Figure 2. Conformations of (a) casuarine 2 and (b) 3-epi-casuarine 1 in water determined by analysis of the proton coupling constants and NOE results.
large C6H–C5H/H0 couplings are also consistent with a cis
axial–equatorial and a trans di-axial arrangement for C6H
with the two C5 protons, confirming C6H as axial. As
C7aH is axial, C1H must be equatorial. The all trans
C7aH–C1H–C2H configuration is consistent with the rela-
tively weak C7aH–C1H and C1H–C2H NOEs. A strong
C1H–C7H NOE indicates that these two protons are on
the same side of the ring, consistent with a trans C7H–
C7aH and a trans C7aH–C1H configurations. A strong
C2H–C3H NOE indicates that these two protons are on
the same side of the ring, the medium C2H–C3H coupling
also being consistent with a cis arrangement for these pro-
tons. These relative configurations confirm 1 as 3-epi-casu-
arine or its enantiomer.
Wittig extension at C-1 to give, after osmium tetroxide
hydroxylation, an 8 carbon sugar derivative with all the
correct stereochemistry for the formation of the target;
the overall yield was 6.0% over 15 steps (83% per step)
(Scheme 1).
The reaction of lactone 11 with dimethoxypropane gave an
open chain diacetonide22 in which the C-2 hydroxyl func-
tion was subsequently esterified with trifluoromethanesulf-
onic (triflic) anhydride, as previously described,23 to give
the stable crystalline triflate 12 in 72% overall yield on a
multigram scale. Displacement of the triflate in 12 with so-
dium azide in dimethyl formamide gave azide 13 (97%
yield)24 in which a nitrogen function has been introduced
at C-2 of the gluconolactone.
Although 1 and 2 only differ at one stereocentre, the proton
coupling constants show the ring conformations to be very
different. Casuarine 2 has an exo-/endo-buckled conforma-
tion (C6 exo relative to the nitrogen lone pair, C2 endo)1
resulting in C6H and C7H being equatorial and C1H and
C2H being axial; 3-epi-casuarine has an endo-/exo-confor-
mation, with C6H and C7H being axial and C1H and C2H
being equatorial (Fig. 2).
Reduction of the azidoester 13 with diisobutylaluminium
hydride (DIBAL) gave the corresponding azidoaldehyde
with no significant over-reduction to the corresponding
alcohol. The isolation of the free aldehyde was extremely
cumbersome and led to a significant loss of material; how-
ever treatment of the crude aldehyde in situ with the stabi-
lized Wittig reagent Ph3P@CHCOOMe 21 afforded the
unsaturated eight carbon carbohydrate 14 in 75% yield
over the two steps. An E:Z ratio of 10:1 was typically
observed for this reaction, provided that the reaction
mixture containing the crude aldehyde was allowed to
warm to room temperature before the addition of the
ylid; when the Wittig step was carried out at lower temper-
atures, significant amounts of the Z-isomer of 14 were
isolated.
1.3. Syntheses of 3-epi-casuarine 1 and casuarine 2 from
D-gluconolactone
The conversion of inexpensive and easily available D-gluc-
onolactone 11—a rare example of a thermodynamically
stable 1,5-lactone—to 3-epi-casuarine 1 (Scheme 1) in-
volved the introduction of nitrogen at C-2, a subsequent

J. V. Ameijde et al. / Tetrahedron: Asymmetry 17 (2006) 2702–2712
2705
O
OH
O
O
F3CO2SO
MeO2C
O
O
O
O
(iii)
HO
O
OH
(i)
(ii)
O
O
MeO2C
O
CH2OH
MeO2C
N3
N3
O
O
O
12
13
11
14
(iv)
O
O
O
O
O
O
[Si]O
[Si]O
[Si]O
HO
MeO2C
O
O
HO
MeO2C
(v)
(vi)
O
O
+
[Si]O
N3
NH
NH
N3
OH
OH
O
O
O
OH
OH
O
O
O
17
16
18
15
(vii)
O
OH
HO
OH
O
[Si]O
HO
H
N
O
OH
(viii)
[Si]O
[Si]O
O
(ix)
(x)
HO
OH
[Si]O
HO
NH
NH
OMs
O[Si]
OMs
OH
NH
OMs
O[Si]
CH2OH
O
1
20
6
19
(x)
OH
HO
OH
H
N
HO
OH
[Si] = tBuMe2Si
HO
OH
HO
NH
O
CH2OH
7
2
Scheme 1. Reagents and conditions: (i) Me2C(OMe)2, p-TsOH, MeOH; then (CF3SO2)2O, pyridine, CH2Cl2, 72%; (ii) NaN3, DMF, 97%; (iii) 3 equiv
iBu2AlH, ꢁ78 ꢁC; then Ph3P@CHCO2Me 21, toluene (75% over two steps); (iv) cat. OsO4, NMO, tBuOH/H2O, 72%; (v) H2, Pd/C, THF; then toluene, D;
then tBuMe2SiCl, imidazole, THF (70% over three steps); (vi) 60% HOAc, H2O/MeOH, 69%; (vii) tBuMe2SiCl, pyridine; then MeSO2Cl, Et3N, CH2Cl2,
66%; (viii) BH3ÆTHF, THF, 57%; (ix) 90% CF3CO2H, H2O; (x) NaOAc, H2O (89% over two steps).
The unsaturated ester 14 was subjected to a dihydroxyla-
tion using catalytic osmium tetroxide and a stoichiometric
amount of N-methylmorpholine N-oxide (NMO) as reoxi-
dant to afford an inseparable 4:1 mixture of 16:15 isomers
in a total yield of 72%; the isomer with the required stereo-
chemistry 16 thus greatly predominated. Hydrogenation of
the mixture of azides 16 and 15 gave the corresponding
amines, which on heating in toluene, resulted in the forma-
tion of the first ring of the pyrrolizidine framework. The
two diastereomeric polar lactams were difficult to separate,
so the mixture was treated with tert-butyldimethylsilyl
(TBDMS) chloride in the presence of imidazole to allow
the isolation of the protected lactam 17 as a single diaste-
reomer in 70% over the three steps (93% calculated from
the original isomer content).
atures gave the pyrrolidine 20 in 57% yield. No
spontaneous cyclization of 20 occurred because the result-
ing pyrrolizidine would contain a trans-diol protected as a
strained ketal. Accordingly, all the protecting groups in 20
were removed by heating with 90% aqueous trifluoroacetic
acid to give the pentahydroxyaminomesylate 6 as its triflu-
oroacetate salt. Treatment of the salt 6 with sodium acetate
as a base resulted in a clean cyclization step to 3-epi-casu-
arine 1 in 89% yield over the last two steps. A small amount
of casuarine 2 (less than 5% yield) was formed, presumably
via an initial epoxide. GC–MS analysis of trimethylsilyl-
derivatives of polyhydoxylated alkaloids is an exquisitely
sensitive method16 of detecting impurities; no other stereo-
isomers were formed.
D-Gluconolactone 11 could also be used for a synthesis of
casuarine but would require an additional inversion of con-
figuration at C-7 of the diol 18 (Scheme 2).
The terminal acetonide in 17 was then removed by treat-
ment with aqueous acetic acid to afford 18 in 69% yield.
Selective protection of the primary hydroxyl group in 18
with TBDMS chloride in pyridine allowed the esterification
of the remaining secondary alcohol with mesyl chloride
and triethylamine to afford 19 in 66% overall yield. Reduc-
tion of the lactam 19 with THF:borane at elevated temper-
TBDMS chloride in pyridine reacted with the diol 18 selec-
tively at C-8 to afford the primary silyl ether 22 in 81%
yield in which the hydroxyl at C-7 is unprotected. A variety
of strategies for inversion of the configuration at C-7 were

2706
J. V. Ameijde et al. / Tetrahedron: Asymmetry 17 (2006) 2702–2712
O
O
O
O
[Si]O
[Si]O
[Si]O
[Si]O
[Si]O
[Si]O
[Si]O
[Si]O
O
O
O
O
((iii)
(i)
(ii)
NH
NH
NH
NH
OH
OH
OH
O[Si]
OSO2CF3
O[Si]
OH
O[Si]
O
O
O
O
22
18
23
24
(iv)
O
O
OH
[Si]O
[Si]O
[Si]O
HO
O
O
OH
HO
OH
(vi)
(v)
(vii)
H
N
[Si]O
HO
HO
OH
NH
NH
OMs
O[Si]
OMs
O[Si]
NH
OMs
OH
O
CH2OH
2
26
27
25
(vii)
OH
HO
OH
H
N
HO
OH
HO
OH
HO
NH
28
O
CH2OH
1
Scheme 2. Reagents and conditions: (i) tBuMe2SiCl, pyridine, 81%; (ii) (CF3SO2)2O, pyridine, CH2Cl2; (iii) CF3CO2Cs, 2-butanone; then K2CO3, MeOH
(20% from 22); (iv) CH3SO2Cl, Et3N, CH2Cl2, 90%; (v) BH3ÆTHF, THF, 57%; (vi) 90% CF3CO2H, H2O; (vi) NaOAc, H2O (91% over two steps).
attempted, though none proceeded in high yield. A method
involving displacement of a triflate by trifluoroacetate was
successful though the yield was poor.25 The free hydroxyl
group in 22 was esterified by treatment with triflic anhy-
dride and pyridine to afford the unstable triflate 23, which
was treated with caesium trifluoroacetate in butanone;
reaction of the resulting trifluoroacetate ester with potas-
sium carbonate in methanol afforded the inverted alcohol
24 in 20% yield over the three steps. The low yield is due
to decomposition of the triflate 23 to uncharacterized base-
line material. The free hydroxyl group in 24 was esterified
with methanesulfonyl chloride in pyridine (90% yield) to
give the lactam mesylate 25, which was reduced with
THF:borane to the protected amine 26 (57% yield). Com-
plete deprotection of 26 by aqueous trifluoroacetic acid
gave the unprotected aminomesylate 27, which with so-
dium acetate gave essentially pure casuarine 2 (91% yield
over two steps) except for a very small amount of 3-epi-
casuarine 1. The closure to epoxide 28 followed by a 5-
exo-tet closure to the pyrrolizidine would account for the
tiny amount of 3-epi-casuarine 1 that is formed. Again
GC–MS analysis of pertrimethylsilyl ethers from the crude
reaction mixture showed that only a trace amount of 1 was
produced and that no other polyhydroxylated alkaloid was
formed. The overall yield of this synthesis of casuarine 2
from D-gluconolactone 11 was 1.4% over 18 steps (78.8%
per step); unless a better method can be found for the
inversion of the alcohol 22 to 24, this route would not
allow the preparation of significant amounts of casuarine
2. The efficient high yield closure of another unprotected
pentahydroxyamino mesylate 27 to a complex pyrrolizi-
dine, with no significant competition from alternative ring
closures, suggest that this may be a general strategy for the
synthesis of casuarines.
2. Conclusion
3-epi-Casuarine 1 is the first stereoisomer of casuarine 2 to
be isolated as a natural product. A practical synthesis of
3-epi-casuarine 1 from D-gluconolactone 11 in an overall
yield of 6% relies on the efficient cyclization of an unpro-
tected polyhydroxyaminomesylate 6. The synthesis of casu-
arine 2 gives an overall yield of 1.4%, in which the only low
yielding step is an inversion of configuration of a secondary
alcohol; however, the cyclization of another unprotected
polyhydroxyaminomesylate 27 to casuarine is also very effi-
cient. It may be that green strategies for the synthesis of
pyrrolizidine alkaloids in which none of the hydroxyl
groups is protected may allow easy access to alexines and
casuarines.
3. Experimental
3.1. General procedures
All solvents were used as supplied (Analytical or HPLC
grade), without further purification, except for DMF and
pyridine, which were dried on Molecular Sieves prior to
use. Reactions performed under a hydrogen atmosphere
were maintained by an inflated balloon. Flash chromato-
graphy was performed using Sorbsil C60 40/60 silica. Thin
layer chromatography (TLC) was carried out on alumin-

J. V. Ameijde et al. / Tetrahedron: Asymmetry 17 (2006) 2702–2712
2707
ium backed sheets coated with 60 F254 silica from Merck.
Plates were developed using a spray of 0.2% w/v cerium
sulfate and 5% ammonium molybdate in 2 M sulfuric acid
with subsequent heating. Melting points were recorded on
a Ko¨fler hot block and are corrected. NMR spectra for 1
and 2 were recorded on a Varian UnityINOVA 500
(1H—500 MHz: 13C—125 MHz) spectrometer, in 2H2O
with a probe temperature of 30 ꢁC. Chemical shifts were
measured relative to internal standards (1H—trimethylsil-
ylspronesulfonic acid at 0.000 ppm: 13C—either dioxane
at 67.2 ppm or acetone at 30.9 ppm). COSY and HSQC
spectra were used to aid assignment of 1H and 13C spectra,
respectively. NOESY spectra were recorded with a 400 ms
mixing time. NMR spectra for all other samples were
recorded on a Bruker DQX 400 (1H—400 MHz: 13C—
100.6 MHz) instrument, with residual signals from the sol-
vents used as the internal references. ES mass spectra were
measured on a Micromass BioQ II-ZS mass spectrometer.
High resolution mass spectra (HRMS) were recorded on a
Micromass LCT (ESI) spectrometer. HRMS spectra were
correlated relative to PEG with tetraoctylammonium bro-
mide as internal lock mass. Infrared spectra (IR) were
recorded on a Bruker Tensor Fourier Transform IR
spectrometer using thin film on NaCl plates. Optical rota-
tions were measured on a Perkin–Elmer 241 polarimeter
with a path length of 1 dm. Concentrations are quoted in
g/100 ml. Microanalyses were carried out by the analytical
service of the ICL in Oxford. The protected triflate 12 was
prepared from D-gluconolactone 11 in 71% yield, as previ-
ously described.23
solvent was removed in vacuo. The residue in ethyl acetate
(100 ml) was washed with water (100 ml), dried (sodium
sulfate) and purified by column chromatography (EtOAc/
cyclohexane 1/4 v/v), to give the azide 13, as a clear, col-
ourless oil (10.96 g, 97%); Rf 0.36 (EtOAc/cyclohexane 1/
22
3 v/v); ½aꢀD ¼ þ21:5 (c 0.69, CHCl3) {lit.15 +21.3 (c 1.30,
CHCl3)}; IR m 2114 (N3), 1754 (C@O); dH (400 MHz,
CDCl3) 1.33, 1.37, 1.41, 1.46 (12H, 4 · s, C(CH3)2), 3.81
(3H, s, OCH3), 3.97 (1H, dd, H-60, J 4.8, 8.0 Hz), 4.04
(2H, m, H-4, H-5), 4.15 (1H, dd, H-6, J 5.2, 8.0 Hz), 4.31
(1H, d, H-2, J 3.5 Hz), 4.38 (1H, dd, H-3, J 3.5, 6.6 Hz);
dC (100 MHz, CDCl3) 25.3, 26.4, 26.9, 27.3 (C(CH3)2),
52.6 (OCH3), 63.0 (C-2), 67.6 (C-6), 76.8 (C-5), 77.6 (C-
4), 80.7 (C-3), 110.0, 110.4 (C(CH3)2), 167.7 (C@O); MS
m/z (ESI +ve) 316.21 (M+H+), 288.01 (MꢁN2+H+); for
C13H21N3O6 calcd C 49.52, H 6.71, N 13.33, found C
49.44, H 6.69, N 13.12.
3.3. Methyl 4-azido-5,6:7,8-di-O-isopropylidene-2,3,4-tri-
deoxy-D-manno-oct-2-enoate 14
Diisobutylaluminium hydride in toluene (35 ml, 1.5 M) was
added dropwise to a solution of azide 13 (10.96 g,
34.8 mmol) in toluene (200 ml) at ꢁ78 ꢁC; the reaction mix-
ture was stirred for 3 h at ꢁ78 ꢁC under a nitrogen atmo-
sphere when excess hydride was quenched by dropwise
addition of methanol (10 ml) and then allowed to warm to
room temperature. Methyl (triphenylphosphoranylidene)-
acetate (12.8 g, 38.3 mmol) was added in one portion and
the resulting mixture was stirred at room temperature
overnight under a nitrogen atmosphere. The solvents were
removed in vacuo. The residue was dissolved in diethyl
ether (100 ml), and the resulting solution extracted with so-
dium hydroxide (100 ml, 2 M) and the organic phase dried
(sodium sulfate). The solvent was removed and the residue
was purified by chromatography (EtOAc/cyclohexane 1/6
v/v) to afford the unsaturated E-ester 14 (8.95 g, 75%), Rf
GC–MS was carried out using a Perkin–Elmer Autosystem
XL gas chromatograph with a high polarity fused-silica
column (Varian ‘Factor Four’ VF-5 ms column,
25 m · 0.25 mm i.d., 0.25 lm phase thickness). The carrier
gas (helium) flow rate was 1 ml minꢁ1. Trimethylsilyl-
(TMS) derivatives were separated using a temperature
programme that started at 160 ꢁC and was held for
5 min, followed by a linear increase to 300 ꢁC at a rate of
10 ꢁC minꢁ1. The temperature was held at 300 ꢁC for an
additional 10 min; the total analysis time was 29 min. Elec-
tron impact mass spectrometry of the column eluant was
carried out using a Perkin–Elmer TurboMass Gold mass
spectrometer, with a quadrupole ion filter system, which
was run at 250 ꢁC constantly during analysis. The detector
mass range was set to 100–650 amu. The temperature of the
transfer line (GC to MS) was held at 250 ꢁC. Samples were
injected onto the column via a split vent (split ratio 50:1)
through a fused silica narrow bore injection liner packed
with deactivated quartz wool; the injection port tempera-
ture was maintained at 200 ꢁC. The injection volume was
1 ll. System control, data collection and mass spectral
analysis were carried out using Perkin–Elmer TurboMass
software (TurboMass v. 4.4).
0.89 (EtOAc/cyclohexane 1/1 v/v) as a white crystalline
22
solid; mp 45–46 ꢁC; ½aꢀD ¼ þ35:5 (c 0.71, CHCl3); IR m
2095 (N3), 1725 (C@O); dH (400 MHz, CDCl3) 1.36, 1.38,
1.51 (12H, 3 · s, C(CH3)2), 3.73 (1H, m, H-7), 3.78 (3H,
s, OCH3), 3.93 (1H, dd, H-80, J 5.2, 8.4 Hz), 4.05 (1H, m,
H-7), 4.14 (1H, dd, H-8, J 4.0, 8.4 Hz), 4.17 (1H, dd, H-
5, J 3.0, 3.6 Hz), 4.33 (1H, ddd, H-4, J 1.2, 3.6, 7.2 Hz),
6.11 (1H, dd, H-2, J 1.2, 15.6 Hz), 6.96 (1H, dd, H-3, J
7.2, 15.2 Hz); dC (100 MHz, CDCl3) 25.1, 26.4, 26.9, 27.2
(C(CH3)2), 51.8 (OCH3), 63.1 (C-4), 67.7 (C-8), 76.8 (C-
7), 78.3 (C-6), 81.4 (C-5), 109.9, 110.6 (C(CH3)2), 125.2
(C-2), 140.3 (C-3), 165.7 (C@O); HRMS m/z (ESI +ve):
Found 683.3251 (2M+H+); C30H47N6O12 requires
683.325; for C15H23N3O6 calcd C 52.78, H 6.79, N 12.31,
found C 52.78, H 6.78, N 12.10.
3.4. Methyl 4-azido-4-deoxy-5,6:7,8-di-O-isopropylidene-D-
erythro-L-altro-octonate 16 and methyl 4-azido-4-deoxy-
5,6:7,8-di-O-isopropylidene-D-erythro-L-gluco-octonate 15
3.2. Methyl 2-azido-2-deoxy-3,4:5,6-di-O-isopropylidene-
D-mannonate 13
A solution of azido ester 14 (6.85 g, 20.0 mmol) in a mix-
ture of acetone (50 ml) and water (10 ml) was treated with
N-methylmorpholine oxide (4.69 g, 40.0 mmol) and os-
mium tetroxide (50 mg). The reaction mixture was stirred
overnight at room temperature under a nitrogen atmo-
Sodium azide (2.55 g, 39.2 mmol) was added to a solution
of the protected triflate 12 (15.1 g, 35.7 mmol) in DMF
(30 ml). The reaction mixture was stirred at room temper-
ature under a nitrogen atmosphere for 12 h, after which the

2708
J. V. Ameijde et al. / Tetrahedron: Asymmetry 17 (2006) 2702–2712
sphere and then treated with ethyl acetate (100 ml) and so-
dium bicarbonate (100 ml, 5% w/v in H2O). The organic
layer was dried (sodium sulfate), the solvent removed and
the residue purified by column chromatography (EtOAc/
cyclohexane 1/1 v/v) to afford an inseparable mixture of
diols 16 and 15 in a ratio of approximately 4:1, respectively
(5.37 g, 72%), Rf 0.36 (EtOAc/cyclohexane 1/3 v/v); IR m
3465 (OH), 2111 (N3), 1748 (C@O); dH (400 MHz, CDCl3)
1.35, 1.38, 1.40, 1.43, 1.47, 1.52 (15.0H, 6 · s, maj/min
C(CH3)2), 2.91 (0.25H, min (C-3)OH), 3.03 (1.00H, maj
(C-2)OH), 3.19 (0.25H, min (C-2)OH), 3.73 (1.00H, dd,
maj H-3), 3.81 (3.25H, m, maj OCH3, min (C-3)OH),
3.85 (0.75H, s, min OCH3), 3.95 (0.25H, m, min H-4),
4.05 (4.00H, m, maj H-6, maj H-7, maj H-80, min H-3,
min H-6, min H-7, min H-80), 4.24 (2.50H, maj H-4, maj-
H-8, min H-5, min H-8), 4.48 (1.25H, m, maj H-2, min
H-2), 4.55 (1.00H, m, maj H-5); dC (100 MHz, CDCl3)
25.0, 25.7, 26.9, 27.2 (maj C(CH3)2), 25.4, 26.5, 27.3 (min
C(CH3)2), 52.5 (maj OCH3), 52.8 (min OCH3), 61.5 (maj
C-4), 65.0 (min C-4), 68.0 (min C-8), 68.6 (maj C-8),
70.9, 71.1 (maj C-2, C-3), 71.1, 71.3 (min C-2, C-3), 76.9
(maj C-6), 77.0 (min C-6), 77.5 (maj C-7), 78.6 (min C-7),
80.2 (min C-5), 80.7 (maj C-5), 110.0, 110.1, 110.3 (maj/
min C(CH3)2), 173.1 (min C@O), 173.3 (maj C@O);
HRMS m/z (ESI +ve): Found 398.1528 (M+Na+);
C15H25N3O8Na requires 398.1539; for C15H25N3O8
calcd C 47.99, H 6.71, N 11.19, found C 48.39, H 6.79,
N 10.87.
(C-8), 75.8 (C-2), 76.5 (C-3), 79.5 (C-7), 81.7 (C-6), 83.5
(C-5), 109.8, 110.4 (C(CH3)2), 172.5 (C@O); HRMS m/z
(ESI +ve): Found 546.3265 (M+H+); C26H52NO7Si2 re-
quires 546.3282; for C26H51NO7Si3 calcd C 57.21, H 9.42,
N 2.57, found C 57.20, H 9.68, N 2.26.
3.6. 4-Deoxy-2,3-di-O-tert-butyldimethylsilyl-5,6-O-isoprop-
ylidene-D-erythro-L-altro-octono-1,4-lactam 18
A solution of the fully protected lactam 17 (3.76 g,
6.89 mmol) in methanol (6 ml), acetic acid (12 ml) and
water (2 ml) was stirred at reflux temperature for 5 h under
a nitrogen atmosphere. Sodium bicarbonate was then
added as a solid until all acetic acid had been neutralized.
The reaction was then treated with diethyl ether (100 ml)
and water (50 ml); the organic layer was dried over sodium
sulfate and the solvent removed. Purification of the residue
by chromatography (EtOAc/cyclohexane 1/1 v/v) gave diol
18 (2.39 g, 69%), colourless oil Rf 0.17 (EtOAc/cyclohexane
23
1/1 v/v); ½aꢀD ¼ þ2:9 (c 0.41, CHCl3); IR m 3336 (OH),
1701 (C@O); dH (400 MHz, CDCl3) 0.14, 0.17, 0.19 (12H,
3 · s, SiCH3), 0.91, 0.92 (18H, 2 · s, SiC(CH3)3), 1.36,
1.40 (6H, 2 · s, C(CH3)2), 2.98 (1H, br s, OH), 3.37 (1H,
dd, H-4, J 2.8, 9.2 Hz), 3.66 (1H, dd, H-6, J 7.2,
15.2 Hz), 3.73 (2H, m, H-7, H-80), 3.82 (1H, dd, H-5, J
7.2, 9.2 Hz), 3.87 (1H, dd, H-8, J 5.6, 13.2 Hz), 4.04 (1H,
d, H-2, J 2.8 Hz), 4.26 (1H, app. t, H-3, J 2.8 Hz), 4.99
(1H, s, OH), 7.48 (1H, s, NH); dC (100 MHz, CDCl3)
ꢁ4.8, ꢁ4.7, ꢁ4.4, ꢁ4.2 (SiCH3), 17.9, 18.1 (SiC(CH3)3),
25.8 (SiC(CH3)3), 26.6, 26.8 (C(CH3)2), 63.4 (C-4), 64.7
(C-8), 72.9 (C-7), 77.9 (C-7), 80.9 (C-6), 82.0 (C-5), 109.1
(C(CH3)2), 175.3 (C@O); HRMS m/z (ESI +ve): Found
506.2970 (M+H+); C23H48NO7Si2 requires 506.2969; for
C23H47NO7Si2 calcd C 54.62, H 9.37, N 2.77, found C
54.85, H 9.64, N 2.54.
3.5. 4-Deoxy-2,3-di-O-tert-butyldimethylsilyl-5,6:7,8-di-O-
isopropylidene-D-erythro-L-altro-octono-1,4-lactam 17
A solution of a 4:1 mixture of diols 16 and 15 (5.28 g,
14.07 mmol) in tetrahydrofuran (50 ml) was stirred for
48 h at room temperature under a hydrogen atmosphere
in the presence of a palladium catalyst (20% Pd(OH)2 on
carbon, 150 mg). The catalyst was removed by filtration
and the solvent removed in vacuo. A solution of the residue
in toluene (200 ml) was heated under reflux for 3 h under a
nitrogen atmosphere. The solvent was then removed in va-
cuo and a solution of the crude product in tetrahydrofuran
(50 ml) was treated with imidazole (4.22 g, 62.04 mmol),
and tert-butyldimethylsilyl chloride (4.68 g, 31.02 mmol)
was added. The reaction mixture was stirred overnight at
reflux temperature under a nitrogen atmosphere, the
solvent removed in vacuo and the residue redissolved in
dichloromethane (50 ml) and washed with aqueous hydro-
chloric acid (50 ml, 1 M), over sodium bicarbonate (50 ml,
5% w/v in H2O), and brine (50 ml). The organic layer was
dried over sodium sulfate and the solvent removed to give a
residue, which was purified by column chromatography
(EtOAc/cyclohexane 1/6 v/v) to afford the protected lac-
3.7. 4-Deoxy-5,6-O-isopropylidene-7-O-methanesulfonyl-
2,3,8-tri-O-tert-butyldimethylsilyl-D-erythro-L-altro-
octono-1,4-lactam 19
tert-Butyldimethylsilyl chloride (784 mg, 5.20 mmol) was
added to a solution of diol 18 (2.39 g, 4.73 mmol) in dry
pyridine (10 ml). The reaction mixture was stirred at room
temperature for 5 h under a nitrogen atmosphere after
which dichloromethane (100 ml) was added; the organic
layer was washed with aqueous hydrochloric acid
(100 ml, 1 M), sodium bicarbonate (50 ml, 5% w/v in
H2O) and brine (50 ml). The organic layer was dried
over sodium sulfate and the solvent reduced in vacuo
until a volume of 10 ml remained. Then, triethylamine
(1.0 ml, 7.1 mmol) and methanesulfonyl chloride (439 ll,
5.68 mmol) were added to the solution of the crude trisilyl
ether 22 (see below for the preparation of pure 22). The
reaction mixture was stirred for 30 min at room tempera-
ture under a nitrogen atmosphere, washed with aqueous
hydrochloric acid (10 ml, 1 M), sodium bicarbonate
(10 ml, 5% w/v in H2O) and brine (10 ml). The organic
layer was dried (sodium sulfate) and the solvent removed
to give a residue, which was purified by chromatography
(EtOAc/cyclohexane 1/4 v/v) to give the trisilyl mesylate
tam 17 as a single diastereomer (5.41 g, 70%), colourless
22
oil; Rf 0.48 (EtOAc/cyclohexane 1/4 v/v); ½aꢀD ¼ þ1:8
(c 1.59, CHCl3); IR m 1725 (C@O); dH (400 MHz, CDCl3)
0.18, 0.20 (12H, 2 · s, SiCH3), 0.95 (18H, s, SiC(CH3)3),
1.36, 1.38, 1.40, 1.46 (12H, 4 · s, C(CH3)2), 3.27 (1H, dd,
H-4, J 8.3, 6.8 Hz), 3.62 (1H, app. t, H-6, J 8.1 Hz), 3.76
(1H, app. t, H-5, J 8.2 Hz), 3.99 (2H, m, H-7, H-80), 4.20
(2H, m, H-3, H-8), 4.28 (1H, d, H-2, J 8.1 Hz); dC
(100 MHz, CDCl3) ꢁ5.1, ꢁ4.6 (SiCH3), 18.4 (SiC(CH3)3),
24.9 (SiC(CH3)3), 26.6, 26.7 (C(CH3)2), 27.5 (C-4), 26.8
19 (2.18 g, 66%), colourless oil, Rf 0.23 (EtOAc/cyclohex-
23
ane 1/4 v/v); ½aꢀD ¼ þ28:9 (c 0.37, CHCl3); IR m 1720

J. V. Ameijde et al. / Tetrahedron: Asymmetry 17 (2006) 2702–2712
2709
(C@O); dH (400 MHz, CDCl3) 0.11, 0.12, 0.17, 0.18 (18H,
4 · s, SiCH3), 0.87, 0.90, 0.91, 0.92, 0.92 (27H, 5 · s
(SiC(CH3)3), 1.40, 1.46 (6H, 2 · s, C(CH3)2), 3.09 (3H, s,
SO2CH3), 3.41 (1H, d, H-4, J 8.0 Hz), 3.81 (1H, dd, H-
80, J 4.0, 12.0 Hz), 3.85 (1H, s, H-2), 3.93 (1H, dd, H-8, J
7.1, 12.0 Hz), 4.07 (1H, app. t, H-5, J 8.0 Hz), 4.16 (1H,
dd, H-6, J 3.7, 8.0 Hz), 4.23 (1H, s, H-3), 4.64 (1H, ddd,
H-7, J 3.7, 4.0, 7.1 Hz), 6.09 (1H, s, NH); dC (100 MHz,
CDCl3) ꢁ5.5, ꢁ5.5, ꢁ5.2, ꢁ4.8, ꢁ4.7, ꢁ4.4 (SiCH3),
17.8, 18.0, 18.2, 18.3 (SiC(CH3)3), 25.7, 25.7, 25.8
(SiC(CH3)3), 26.9, 27.1 (C(CH3)2), 38.4 (SO2CH3), 61.5
(C-8), 65.5 (C-4), 76.0 (C-6), 77.3 (C-3), 78.1 (C-2), 79.2
(C-5), 82.0 (C-7), 110.4, 110.5 (C(CH3)2), 175.0 (C@O);
HRMS m/z (ESI +ve): Found 698.3615 (M+H+);
C30H64NO9SSi3 requires 698.3210; for C30H63NO9SSi3
calcd C 51.61 H 9.10 N 2.01, found C 52.00 H 9.05 N 2.33.
(2H, m, H-6, H-8), 4.10 (1H, dd, H-5, J 1.2, 6.5 Hz), 4.28
(1H, app. p, H-2, J 2.1, 4.0, 3.6 Hz), 4.34 (1H, app. t, H-
3, J 3.6 Hz), 4.80 (1H, m, H-7); dC (100 MHz, D2O) 27.5
(SO2CH3), 39.9 (C-1), 49.4 (C-8), 56.0 (C-5), 56.7 (C-4),
57.8 (C-6), 64.0 (C-2), 65.1 (C-3), 71.0 (C-7); MS m/z
(ESI +ve): 301.82 (M+H+).
3.10. 3-epi-Casuarine 1
3.10.1. Synthesis. A solution of the amino mesylate 6 in
water (20 ml) in the presence of sodium acetate (423 mg,
5.16 mM) was stirred overnight at room temperature under
a nitrogen atmosphere. The solvents were removed in
vacuo; the residue was subjected to ion-exchange chroma-
tography on Amberlite CG120 (H+), using 1 M NH4OH
as eluent, to afford 3-epi-casuarine 1 (314 mg, 89%), white
22
solid; ½aꢀD ¼ þ5:8 (c 0.69, H2O); NMR data see Table 2;
3.8. 1,4-Dideoxy-1,4-imino-5,6-O-isopropylidene-7-O-meth-
anesulfonyl-2,3,8-tri-O-tert-butyldimethylsilyl-D-erythro-L-
altro-octitol 20
HRMS m/z (ESI +ve): Found 206.1030 (M+H+);
C8H16NO5 requires 206.1028. GC–MS analysis of the
TMS product showed only a trace (<2%) of casuarine 2.
Pure 3-epi-casuarine 1 was obtained as its monohydrate
by crystallization of the crude product from warm 95%
aqueous ethanol by adding a layer of acetone. The minor
synthetic epimer, casuarine 2, was purified by chromatog-
raphy on Dowex 1 strongly basic anion exchange resin in
the OHꢁ form. Casuarine is eluted with water before the
major 3-epi product 1.
A solution of borane in tetrahydrofuran (21 ml, 1 M) was
added to a solution of the protected lactam 19 (2.87 g,
4.11 mmol) in tetrahydrofuran (40 ml) and the resulting
mixture stirred at reflux temperature for 1 h under a nitro-
gen atmosphere. The reaction mixture was cooled to room
temperature and treated with methanol (5 ml). Removal of
solvents gave a residue, which was purified by chromatog-
raphy (EtOAc/cyclohexane 1/8 v/v) to afford the protected
3.10.2. Isolation. 3-epi-Casuarine was isolated as a natu-
ral product from the shrub M. communis L. (Myrtle) grow-
ing in the grounds of the Institute of Grassland and
Environmental Research in Aberystwyth. The aerial parts
of the plant were shredded and the alkaloids were extracted
into 50% aqueous ethanol. Alkaloids and amino acids were
bound on to strongly acidic cation exchange resin (IR-120
H+ form) and after washing with copious water they were
displaced with 2 M ammonium hydroxide. Basic amino
acids were removed using weakly acidic cation exchange
resin (Amberlite CG50 in the ammonium form) and the un-
bound material was fractionated in water on strongly basic
anion exchange resin (Dowex 1 in the hydroxide form).
Fractions were analyzed by GC–MS of trimethylsilyl-deriv-
atives prepared using 200 ll of Pierce TriSil per mg of
material. Casuarine was the major alkaloid present in M.
communis (displaced first from Dowex 1), and 3-epi-casuar-
ine was the only epimer detected in the plant (displaced
after casuarine from Dowex 1). Casuarine 2 and 3-epi-casu-
arine 1 were crystallized from warm 95% aqueous ethanol
by layering with acetone. The 1H and 13C NMR of the nat-
ural 3-epi-casuarine were identical to those of the synthetic
amino mesylate 20 (1.60 g, 57%), as a colourless oil,
½aꢀD ¼ ꢁ67:1 (c 0.38, CHCl3); IR m 3394 (NH); dH
24
(400 MHz, CDCl3) 0.10, 0.11, 0.11, 0.12, 0.17, 0.18 (18H,
6 · s, SiCH3), 0.90, 0.92, 0.94 (27H, 3 · s, SiC(CH3)3),
1.39, 1.44 (6H, 2 · s, C(CH3)2), 3.06 (1H, dd, H-4, J 6.4,
8.5), 3.25 (3H, s, SO2CH3), 3.63 (1H, app. d, H-10, J
10.4), 3.90 (2H, m, H-1, H-80), 4.04 (1H, dd, H-8, J 2.0,
12.0), 4.16 (1H, br m, H-3), 4.28 (1H, dd, H-6, J 6.3,
9.2), 4.43 (1H, dd, H-5, J 6.3, 8.5), 4.66 (1H, ddd, H-7, J
2.0, 7.6, 9.2), 4.85 (1H, ddd, H-2, J 2.8, 8.0, 10.4), 5.06
(1H, br s, NH); dC (100 MHz, CDCl3) ꢁ5.5, ꢁ5.4, ꢁ4.9,
ꢁ4.8, ꢁ4.8, ꢁ4.7 (SiCH3), 17.7, 18.3, 18.4 (SiC(CH3)3),
25.6, 25.8, 25.9, 26.0 (SiC(CH3)3), 26.5, 27.1 (C(CH3)2),
39.2 (SO2CH3), 63.8 (C-8), 75.1 (C-4), 76.0 (C-6), 76.2
(C-1), 77.2 (C-5), 77.8 (C-3), 85.0 (C-7), 92.9 (C-2), 110.4
(C(CH3)2); HRMS m/z (ESI +ve): Found 684.3804
(M+H+); C30H66NO8SSi3 requires 684.3817; for
C30H65NO8SSi3 calcd C 52.67 H 9.58 N 2.05, found C
52.54 H 9.41 N 1.99.
3.9. 1,4-Dideoxy-1,4-imino-7-O-methanesulfonyl-D-erythro-
L-altro-octitol 6
material; the natural sample had a specific rotation,
23
½aꢀD ¼ þ5:7 (c 0.5, H2O).
A solution of the protected amine 20 (1.18 g, 1.72 mmol) in
trifluoroacetic acid (4.5 ml) and water (0.5 ml) was stirred
for 3 h at reflux temperature under a nitrogen atmosphere.
The solvents were evaporated in vacuo and the residue co-
evaporated with toluene, to afford the fully deprotected
amino mesylate 6 (978 mg), a light brown oil, which was
used without further purification. dH (400 MHz, D2O)
3.19 (3H, s, SO2CH3), 3.35 (1H, dd, H-10, J 2.1, 12.4 Hz),
3.49 (1H, dd, H-1, J 4.0, 12.4 Hz), 3.64 (1H, dd, H-4, J
3.6, 6.5 Hz), 3.85 (1H, dd, H-80, J 5.2, 13.2 Hz), 3.98
3.11. 4-Deoxy-5,6-O-isopropylidene-2,3,8-tri-O-tert-butyl-
dimethylsilyl-D-erythro-L-altro-octono-1,4-lactam 22
A solution of the diacetonide 17 (2.70 g, 4.76 mmol) in
methanol (1.5 ml), acetic acid (6 ml) and water (0.5 ml)
was stirred at reflux temperature for 5 h under a nitrogen
atmosphere. Ethyl acetate (60 ml) and aqueous sodium
hydroxide (60 ml, 2 M) were then added and the layers
were separated. The organic layer was dried (sodium sul-

2710
J. V. Ameijde et al. / Tetrahedron: Asymmetry 17 (2006) 2702–2712
fate) and the solvents were evaporated. The resulting crude
diol 18 was dissolved in pyridine (10 ml) and treated with
tert-butyldimethylsilyl chloride (860 mg, 5.71 mmol) over-
night at room temperature under a nitrogen atmosphere.
Then, dichloromethane (60 ml) was added and the resulting
mixture washed with aqueous hydrochloric acid (60 ml,
2 M), sodium bicarbonate (60 ml, 5% w/v in H2O) and
brine (60 ml). The organic layer was dried over sodium sul-
fate and the solvent removed to give a residue, which was
purified by chromatography (EtOAc/cyclohexane 1/6 v/v)
17.9, 18.1, 18.3 (SiC(CH3)3), 25.7, 25.7, 25.9 (SiC(CH3)3),
26.9, 27.0 (C(CH3)2), 63.7 (C-4), 63.8 (C-8), 71.3 (C-7),
76.8 (C-3), 77.1 (C-5), 77.5 (C-2), 79.3 (C-6), 109.0
(C(CH3)2), 174.5 (C@O); HRMS m/z (ESI +ve): Found
642.3685 (M+Na+); C29H61NO7Si3Na requires 642.3654;
for C29H61NO7Si3 calcd C 56.17, H 9.92, N 2.26, found
C 56.22, H 9.59, N 2.31.
3.13. 4-Deoxy-5,6-O-isopropylidene-7-O-methanesulfonyl-
2,3,8-tri-O-tert-butyldimethylsilyl-L-threo-L-altro-octono-
1,4-lactam 25
to afford the trisilyl ether 22 (2.38 g, 81%), a colourless
23
oil, Rf 0.35 (EtOAc/cyclohexane 1/4 v/v); ½aꢀD ¼ þ13:5 (c
0.65, CHCl3); IR
m
3306 (OH), 1716 (C@O); dH
Triethylamine (308 ll, 2.19 mmol) and methanesulfonyl
chloride (69 ll, 0.88 mmol) were added to a solution of
the inverted alcohol 24 (452 mg, 0.73 mmol) in dichloro-
methane (10 ml) and the reaction mixture was stirred for
2 h at room temperature under a nitrogen atmosphere.
More dichloromethane (40 ml) was added and the solution
was successively washed with aqueous hydrochloric acid
(50 ml, 2 M), sodium bicarbonate (50 ml, 5% w/v in H2O)
and brine (60 ml). The organic layer was dried over sodium
sulfate and the solvent was removed to give a residue,
which was purified by chromatography (EtOAc/cyclohex-
(400 MHz, CDCl3) 0.10, 0.10, 0.12, 0.17, 0.19 (18H, 5 · s,
SiCH3), 0.90, 0.91, 0.92 (27H, 3 · s, SiC(CH3)3), 1.34,
1.37 (6H, 2 · s, C(CH3)2), 3.07 (1H, d, OH, J 2.8 Hz),
3.35 (1H, dd, H-4, J 9.0, 2.0 Hz), 3.50 (1H, app. t, H-6, J
3.8 Hz), 3.60 (2H, br m, H-7, H-8), 3.80 (1H, dd, H-5,
J 9.0, 7.6 Hz), 3.85 (1H, dd, H-80, J 2.8, 9.6 Hz), 3.97
(1H, d, H-2, J 2.8 Hz), 4.27 (1H, app. t, J 2.8 Hz); dC
(100 MHz, CDCl3) ꢁ5.5, ꢁ4.9, ꢁ4.7, ꢁ4.5, ꢁ4.2 (SiCH3),
17.9, 18.2, 18.2 (SiC(CH3)3), 25.6, 25.8, 25.8 (SiC(CH3)3),
26.7 (C(CH3)2), 63.1 (C-4), 64.6 (C-8), 71.9 (C-7), 77.5
(C-3), 77.7 (C-2), 80.7 (C-6), 82.4 (C-5), 108.8 (C(CH3)2),
174.4 (C@O); HRMS m/z (ESI +ve): Found 620.3857
(M+H+); C29H62NO7Si3 requires 620.3834; for C29H61-
NO7Si3 calcd C 56.17, H 9.92, N 2.26, found C 56.09,
H 10.05, N 2.25.
ane 1/4 v/v) to give the mesylate 25 (461 mg, 90%), as an
22
oil, Rf 0.37 (EtOAc/cyclohexane 1/4 v/v); ½aꢀD ¼ þ6:0
(c 0.57, CHCl3); IR m 1715 (C@O), 1177 (SO3); dH
(400 MHz, CDCl3) 0.11, 0.11, 0.12, 0.14, 0.18, 0.18 (18H,
6 · s, SiCH3), 0.87, 0.89, 0.91 (27H, 3 · s, SiC(CH3)3),
1.38, 1.40 (6H, 2 · s, C(CH3)2), 3.50 (1H, app. d, H-4, J
6.4 Hz), 3.88 (3H, m, H-5, H-8, H-80), 4.03 (2H, m, H-3,
H-6), 4.24 (1H, app. s, H-2), 4.66 (1H, m, H-7), 6.61
(1H, br s, NH); dC (100 MHz, CDCl3) ꢁ5.4, ꢁ5.4, ꢁ5.1,
ꢁ4.8, ꢁ4.7, ꢁ4.4 (SiCH3), 17.8, 18.2, 18.3 (SiC(CH3)3),
25.7, 25.8, 25.9 (SiC(CH3)3), 27.1 (C(CH3)2), 38.8
(SO2CH3), 62.9 (C-8), 64.5 (C-4), 75.7 (C-2), 77.4, 77.6
(C-3, C-5, C-6), 82.9 (C-7), 109.5 (C(CH3)2), 174.9
(C@O); HRMS m/z (ESI +ve): Found 720.3432
(M+Na+); C30H63NO9SSi3Na requires 720.3429; for
C30H63NO9SSi3 calcd C 51.61 H 9.10 N 2.01, found C
51.60 H 9.04 N 2.28.
3.12. 4-Deoxy-5,6-O-isopropylidene-2,3,8-tri-O-tert-butyl-
dimethylsilyl-L-threo-L-altro-octono-1,4-lactam 24
Trifluoromethanesulfonic anhydride (1.29 ml, 7.66 mmol)
was added dropwise to a solution of the trisilyl ether 22
(2.38 g, 3.83 mmol) and pyridine (1.87 ml, 23.0 mmol) in
dry dichloromethane (10 ml) at ꢁ50 ꢁC, after which time
the reaction mixture was stirred for 5 h under a nitrogen
atmosphere while slowly warming to 0 ꢁC. The reaction
mixture was treated with dichloromethane (40 ml) and
aqueous hydrochloric acid (60 ml, 1 M); the organic layer
was then washed with water (50 ml) and dried over sodium
sulfate. The solvent was removed; the crude triflate was
dissolved in dry butanone (10 ml), treated with caesium tri-
fluoroacetate (922 mg, 3.75 mmol) and stirred overnight
at 50 ꢁC under a nitrogen atmosphere. Then, potassium
carbonate (500 mg) was added and the mixture stirred for
15 min. The solvent was removed and the residue dissolved
in ethyl acetate (50 ml); the organic layer was washed with
water (50 ml), dried over sodium sulfate and the solvent re-
moved. The residue was purified by chromatography
(EtOAc/cyclohexane 1/4 v/v), to give the inverted alcohol
3.14. 1,4-Dideoxy-1,4-imino-5,6-O-isopropylidene-7-O-
methanesulfonyl-2,3,8-tri-O-tert-butyldimethylsilyl-L-
threo-L-altro-octitol 26
A solution of borane in tetrahydrofuran (5.9 ml, 1 M) was
added to lactam 25 (411 mg, 0.59 mmol) dissolved in tetra-
hydrofuran (5 ml) and the reaction mixture was stirred at
reflux temperature for 1 h under a nitrogen atmosphere.
Methanol (5 ml) was then added and the combined sol-
vents evaporated. The residue was purified by chromato-
graphy (EtOAc/cyclohexane 1/8 v/v) to afford the amine
24 (469 mg, 20%), a light yellow oil, Rf 0.25 (EtOAc/cyclo-
23
hexane 1/4 v/v); ½aꢀD ¼ þ11:1 (c 0.55, CHCl3); IR m 3269
26 (231 mg, 57%), as a colourless oil, Rf 0.60 (EtOAc/cyclo-
23
(OH), 1715 (C@O); dH (400 MHz, CDCl3) 0.09, 0.10,
0.12, 0.12, 0.17, 0.17 (18H, 6 · s, SiCH3), 0.90, 0.90, 0.91
(27H, 3 · s, SiC(CH3)3), 1.38, 1.40 (6H, 2 · s, C(CH3)2),
2.66 (1H, d, OH, J 3.6 Hz), 3.42 (1H, dd, H-4, J 1.6,
8.0 Hz), 3.64 (1H, dd, H-8, J 6.2, 9.8 Hz), 3.76 (2H, m,
H-7, H-80), 3.92 (1H, d, H-2, J 2.4 Hz), 3.94 (1H, app. t,
H-6, J 3.8 Hz), 4.04 (1H, app. t, H-5, J 8.0 Hz), 4.17
(1H, app. s, H-3), 6.36 (1H, br s, NH); dC (100 MHz,
CDCl3) ꢁ5.4, ꢁ5.3, ꢁ4.9, ꢁ4.7, ꢁ4.5, ꢁ4.3 (SiCH3),
hexane 1/4 v/v); ½aꢀD ¼ ꢁ8:5 (c 1.32, CHCl3); IR m 3487
(NH), 1771 (SO3); dH (400 MHz, CDCl3) 0.07, 0.08, 0.10,
0.11, 0.17, 0.18 (18H, 6 · s, SiCH3), 0.88, 0.90, 0.93 (27H,
3 · s, SiC(CH3)3), 1.39, 1.43 (6H, 2 · s, C(CH3)2), 3.08
(1H, app. dd, H-4, J 6.8, 9.6 Hz), 3.15 (3H, s, SO2CH3),
3.44 (1H, app. d, H-1, J 8.8 Hz), 3.86 (2H, m, H-3, H-8),
4.03 (1H, dd, H-80, J 8.8, 11.6 Hz), 4.07 (1H, dd, H-6, J
7.4, 8.2 Hz), 4.24 (1H, dd, H-5, J 8.2, 9.6 Hz), 4.27 (1H,
app. s, H-2), 4.71 (1H, br s, NH), 4.85 (1H, app. dt, H-

J. V. Ameijde et al. / Tetrahedron: Asymmetry 17 (2006) 2702–2712
2711
10, J 2.4, 8.4 Hz), 5.09 (1H, app. d, H-7, J 7.6 Hz); dC
(100 MHz, CDCl3) ꢁ5.4, ꢁ5.0, ꢁ4.9, ꢁ4.8, ꢁ4.8, ꢁ4.7
(SiCH3), 17.9, 18.1, 18.5 (SiC(CH3)3), 25.6, 25.7, 25.8,
25.9, 26.1, 26.9 (SiC(CH3)3), 26.9 (C(CH3)2), 38.8
(SO2CH3), 64.1 (C-8), 74.8 (C-5), 75.5 (C-4), 76.4 (C-2),
77.2 (C-3), 80.1 (C-6), 82.2 (C-7), 93.0 (C-1), 109.0
(C(CH3)2); HRMS m/z (ESI +ve): Found 684.3810
(M+H+); C30H66NO8SSi3 requires 684.3817; for
C30H65NO8SSi3 calcd C 52.67 H 9.58 N 2.05, found C
52.49 H 9.65 N 1.84.
reader (Benchmark). Water was substituted for the inhibi-
tors in controls.
References
1. Nash, R. J.; Thomas, P. L.; Waigh, R. D.; Fleet, G. W. J.;
Wormald, M. R.; Lilley, P. M. de Q.; Watkin, D. J.
Tetrahedron Lett. 1994, 35, 7849–7852.
2. Wormald, M. R.; Nash, R. J.; Watson, A. A.; Bhadoria, B.
K.; Langford, R.; Sims, M.; Fleet, G. W. J. Carbohydr. Lett.
1996, 2, 169–174.
3. (a) Asano, N.; Nash, R. J.; Molyneux, R. J.; Fleet, G. W. J.
Tetrahedron: Asymmetry 2000, 11, 1645–1680; (b) Winches-
ter, B.; Fleet, G. W. J. Glycobiology 1992, 2, 199–210.
4. Watson, A. A.; Fleet, G. W. J.; Asano, N.; Molyneux, R. J.;
Nash, R. J. Phytochemistry 2001, 56, 265–295.
5. Fellows, L. E.; Nash, R. J.; Dring, J. V.; Derome, A. E.;
Hamor, T. A.; Scofield, A. M.; Watkin, D. J.; Fleet, G. W. J.
Tetrahedron Lett. 1988, 29, 2487–2490.
3.15. 1,4-Dideoxy-1,4-imino-7-O-methanesulfonyl-L-threo-
L-altro-octitol 27
The protected mesylate 26 (138 mg, 0.20 mmol) was stirred
in a mixture of trifluoroacetic acid (1.8 ml) and water
(0.2 ml) for 5 h at reflux temperature under a nitrogen
atmosphere. The solvents were removed; the residue was
dissolved in water (10 ml) and the resulting solution
washed with ethyl acetate (10 ml). The water layer was sub-
sequently evaporated to dryness to give crude amine 27
(105 mg), oil, which was used without further purification,
dH (400 MHz, D2O) 3.18 (3H, s, SO2CH3), 3.29 (1H, dd,
H-1, J 2.8, 12.4 Hz), 3.47 (1H, dd, H-10, J 3.2, 12.4 Hz),
3.66 (1H, app. t, H-4, J 5.2 Hz), 3.78 (1H, dd, H-8, J 5.8,
13.3 Hz), 3.85 (1H, dd, H-80, J 3.2, 13.3 Hz), 3.92 (1H,
dd, H-6, J 4.2, 5.8 Hz), 4.02 (1H, dd, H-5, J 4.2, 5.4 Hz),
4.25 (2H, m, H-2, H-3), 4.76 (1H, m, H-7); dC (100 MHz,
D2O) 38.2 (SO2CH3), 50.4 (C-1), 60.8 (C-8), 66.4 (C-4),
67.5 (C-5), 69.2 (C-6), 74.7, 75.5 (C-2, C-3), 84.3 (C-7);
MS m/z (ESI +ve): 301.76 (M+H+).
6. Molyneux, R. J.; Benson, M.; Wong, R. Y.; Tropea, J. E.;
Elbein, A. D. J. Nat. Prod. 1988, 51, 1198–1206.
7. Pereira, A. C. de S.; Kaplan, M. A. C.; Maia, J. G. S.;
Gottlieb, O. R.; Nash, R. J.; Fleet, G.; Pearce, L.; Watkin, D.
J.; Scofield, A. M. Tetrahedron 1991, 47, 5637–5640.
8. (a) Nash, R. J.; Fellows, L. E.; Girdhar, A.; Fleet, G. W. J.;
Peach, J. M.; Ramsden, N. G.; Hegarty, M. P.; Scofield, A.
M. Phytochemistry 1990, 29, 111–117; (b) Yamashita, T.;
Yasuda, K.; Kizu, H.; Kameda, Y.; Watson, A. A.; Nash, R.
J.; Fleet, G. W. J.; Asano, N. J. Nat. Prod. 2002, 65, 1875–
1881; (c) Yasuda, K.; Kizu, H.; Yamashita, T.; Kameda, Y.;
Kato, A.; Nash, R. J.; Fleet, G. W. J.; Asano, N. J. Nat.
Prod. 2002, 65, 198–202; (d) Asano, N.; Kuroi, H.; Ikeda, K.;
Kizu, H.; Kameda, Y.; Kato, A.; Adachi, I.; Watson, A. A.;
Nash, R. J.; Fleet, G. W. J. Tetrahedron: Asymmetry 2000,
11, 1–8; (e) Kato, A.; Adachi, I.; Miyauchi, M.; Ikeda, K.;
Komae, T.; Kizu, H.; Kameda, Y.; Watson, A. A.; Nash, R.
J.; Wormald, M. R.; Fleet, G. W. J.; Asano, N. Carbohydr.
Res. 1999, 316, 95–103.
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Mitchell, M.; Elbein, A. D. Biochemistry 1989, 28, 2027–2034;
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C.; Choi, S. S.; Fairbanks, A. J.; Fleet, G. W. J. Biochem. J.
1993, 290, 743–749.
10. Taylor, D. L.; Nash, R. J.; Fellows, L. E.; Kang, M. S.; Tyms,
A. S. Antivir. Chem. Chemother. 1992, 3, 273–277.
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Tetrahedron Lett. 1988, 29, 5441–5444.
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5517–5520; (b) Donohoe, T. J.; Sintim, H. O. Org. Lett. 2004,
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3.16. Casuarine 2
A solution of the crude amino mesylate 27 (105 mg) and so-
dium acetate (49 mg, 0.6 mmol) in water (5 ml) was stirred
at room temperature for 3 h under a nitrogen atmosphere.
The solvents were removed in vacuo; the residue was
subjected to ion-exchange chromatography on Amberlite
CG120 (H+) using 1 M aqueous ammonium hydroxide as
eluent to yield casuarine (37 mg, quantitative). The syn-
thetic product run as the trimethylsilyl-derivative, have
the same GC–MS retention time (10.27 min) and mass
23
spectrum as authentic natural casuarine. ½aꢀD ¼ þ16:8 (c
24
0.33, H2O) {lit.1 ½aꢀD ¼ þ16:9 (c 0.80, H2O)}; NMR data
see Table 2; MS m/z (ESI +ve) 205.95 (M+H+).
3.17. Glycosidase inhibition assay
All enzymes and para-nitrophenyl substrates were pur-
chased from Sigma. Enzymes were assayed at 20 ꢁC in
0.1 M citric acid/0.2 M disodium hydrogen phosphate buf-
fers at the optimum pH for the enzyme. The incubation
mixture consisted of 10 ll enzyme solution, 10 ll inhibitor
solution in water and 50 ll of the appropriate 5 mM para-
nitrophenyl substrate made up in buffer at the optimum pH
for the enzyme. The reactions were stopped by addition of
0.4 M glycine (pH 10.4) during the exponential phase of the
reaction, which was determined at the beginning of the
assay using blanks with a 5 mM substrate solution. Absor-
bances were read at 405 nm using a Biorad microtitre plate

2712
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