The selective enzymatic synthesis of lipophilic esters of swainsonine

Article abstract of DOI:10.1016/S0968-0896(98)00271-5

The potent and specific inhibitor of Golgi α-mannosidase II, swainsonine (SW) has been isolated in high yield from Swainsona procumbens and derivatised by regiospecific enzymatic reactions. In this study the regioselectivity of three commercially available enzymes, subtilisin Carlsberg, porcine pancreatic lipase (PPL) and Candida cylindracea lipase was determined for the acylation of swainsonine in predominantly anhydrous organic medium. The use of subtilisin in pyridine facilitated the single step synthesis of 2-O-butyryl-SW in a 23% yield, whilst catalysis by PPL in tetrahydrofuran gave 2-O-butyryl-SW (6%) and 1,2-di-O-butyryl-SW (31%). Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00271-5

Bioorganic & Medicinal Chemistry 7 (1999) 831±835
The Selective Enzymatic Synthesis of Lipophilic Esters of
Swainsonine
Gabriel G. Perrone,* Kevin D. Barrow and Ian J. McFarlane
School of Biochemistry & Molecular Genetics, University of New South Wales, Sydney, NSW, 2052, Australia
Received 27 August 1998; accepted 23 October 1998
AbstractÐThe potent and speci®c inhibitor of Golgi a-mannosidase II, swainsonine (SW) has been isolated in high yield from
Swainsona procumbens and derivatised by regiospeci®c enzymatic reactions. In this study the regioselectivity of three commercially
available enzymes, subtilisin Carlsberg, porcine pancreatic lipase (PPL) and Candida cylindracea lipase was determined for the
acylation of swainsonine in predominantly anhydrous organic medium. The use of subtilisin in pyridine facilitated the single step
synthesis of 2-O-butyryl-SW in a 23% yield, whilst catalysis by PPL in tetrahydrofuran gave 2-O-butyryl-SW (6%) and 1,2-di-O-
butyryl-SW (31%). # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
day.9 Reported side e€ects include edema, elevated
serum aspartate amino transferase, anorexia, abdominal
pains and fatigue. Previous research on swainsonine
found that substitution of the C-2 hydroxyl with
hydrophobic functional groups such as 2-O-butyryl and
2-O-p-nitrobenzoyloxy produced compounds with 300-
fold lower inhibition of Jack Bean a-mannosidase in
vitro.10 In contrast, the in vivo biological activity of the
compounds were comparable to that of swainsonine
when measured as inhibitors of oligosaccharide proces-
sing. Indirect evidence suggests that the hydrolysis of
the swainsonine derivatives by esterases was essential
for their in vivo activity.10
Swainsonine (I), an indolizidine alkaloid ®rst isolated
from Swainsona canescens,1 has received interest due to
its potentially therapeutic biological activities. Swainso-
nine is a potent and speci®c inhibitor of lysosomal acid
and cytosolic a-mannosidases2 as well as Golgi a-manno-
sidase II.3 Its inhibition of the latter enzyme leads to the
accumulation of hybrid-type oligosaccharides and a
decrease in glycoproteins containing complex side chains.
During the malignant transformation of murine and
human cells the degree of GlcNAc-b1-6Man-a1-6Man-b-
branching in asparagine linked oligosaccharides increa-
ses. Swainsonine a€ects the expression of b1±6 branched
chain oligosaccharides leading to the inhibition of
tumour cell invasion and metastasis.4 Its activity has
been shown to reduce the growth of human melanoma
cells (MeWo) by 50%.5 Swainsonine modulates the
e€ect of immuno-suppressive factors produced in the
serum of tumour bearing mice and reverses the e€ect of
Concanavalin A leading to stimulated lymphocyte pro-
liferation.6 The alkaloid confers a protective e€ect
against high-dose chemotherapy in mice by inducing
recovery from myelosuppression,7 and enhances natural
killer (NK) cell activity in vivo leading to the inhibition
of metastasis.8
Although biological availability data for swainsonine or
its derivatives has not been reported, uptake studies of
the related alkaloid castanospermine have been pub-
lished. Using 14C-labelled castanospermine and 6-O-
butyrylcastanospermine it was found that the lipophilic
properties of the later compound facilitated its absorp-
tion into mouse melanoma and human T cells (30-fold
higher than castanospermine).11 Studies in mice given a
single oral dose of either compound showed that 6-O-
butyrylcastanospermine was preferentially absorbed
from the gastrointestinal tract and the compound was
rapidly converted to castanospermine in the blood and
intracellularly.12 Multiple oral doses were also e€ective
in obtaining higher concentrations of the prodrug in the
target organs. Similarly, the increased lipid solubility of
some esters of swainsonine would be expected to
increase their intestinal and cellular absorption. There-
fore, lower oral doses of prodrug, compared to swain-
sonine, would achieve equivalent plasma and tissue
levels of swainsonine. In addition, the lower inhibition
by swainsonine esters prior to hydrolysis, may help
A recent, phase IB clinical trial of swainsonine estab-
lished the maximum tolerated oral dose as 300 mg/kg/
Key words: Swainsonine; enzymatic synthesis; regioselective synthesis;
mannosidase inhibitor; Swainsona procumbens.
*Corresponding author. Tel.: +61-(2)-9385-2037; fax: +61-(2)-9385-
1483; e-mail: g.perrone@unsw.edu.au
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(98)00271-5

832
G. G. Perrone et al. / Bioorg. Med. Chem. 7 (1999) 831±835
alleviate some of the dose dependent side e€ects that
occur with oral swainsonine treatment, thereby making
them attractive candidates for prodrug development.
The aim of this study was to develop enzymatic meth-
ods for the regioselective synthesis of ester derivatives of
swainsonine. Three commercially available enzymes,
Subtilisin Carlsberg, porcine pancreatic lipase and Candida
cylindracea lipase were used in a predominantly anhy-
drous medium. The use of enzymes in synthetic organic
chemistry has been recently reviewed.13 In addition, we
provide conclusive evidence of considerable accumulation
of swainsonine in the seeds of Swainsona procumbens.
the addition of 1 M hydrochloric acid (55 mL) and
extracted with diethyl ether (3Â50 mL). The combined
organic phase was washed with 5% (w/v) sodium
hydrogen carbonate (50 mL) followed by water
(3Â50 mL) and dried over anhydrous sodium sulphate.
The diethyl ether was evaporated under reduced pres-
sure (40 ꢀC) leaving a light yellow oil. Vacuum distilla-
tion gave 4.58 g (48%) of 2,2,2-trichloroethylethanoate,
a colourless liquid; bp 72 ꢀC at 20 mmHg, lit. 71 ꢀC at
18 mmHg.16 2,2,2-Trichloroethylbutanoate (TCEB) was
synthesised as for 2,2,2-trichloroethylethanoate but with
butyric anhydride. Yield of 2,2,2-trichloroethyl-
butanoate was 7.56 g (69%); bp 110±112 ꢀC at
20 mmHg, lit. 119.8±120 ꢀC at 48 mmHg.17
Experimental
Enzymes were conditioned prior to use in organic sol-
vent.18 Subtilisin Carlsberg (100 mg) was lyophilised
from 1 ml of 0.1 M potassium phosphate bu€er, pH 7.8.
C. cylindracea lipase (1 g) was dissolved in 2 mL of
0.05 M potassium phosphate bu€er, pH 7.0 and cooled
to 4 ꢀC. The mixture was dried in vacuo at room
temperature for 72 h. Porcine pancreatic lipase (5 g)
was dissolved in 15 mL of 0.05 M glycine bu€er, pH
8.4 and cooled to 4 ꢀC. An equal volume of chilled
( 20 ꢀC) acetone was added, and the mixture stirred at
4 ꢀC for 30 min. The precipitated enzyme was recovered
by centrifugation (14 000 g; 20 min) and washed with
cold ( 20 ꢀC) acetone, dried under a stream of nitrogen,
with further drying in vacuo at room temperature for
72 h.
Swainsona procumbens seeds were obtained from Nin-
dethana Seed Service, Woogenilup, Western Australia.
Subtilisin Carlsberg, porcine pancreatic lipase, C.
cylindracea lipase, Jack Bean a-mannosidase, 2,2,2-tri-
chloroethanol, acetic anhydride and butyric anhydride
were purchased from Sigma; bovine serum albumin was
obtained from Miles Laboratories. All reagents were of
analytical grade unless otherwise stated. Organic sol-
vents were redistilled, then dried by standing for at least
24 h with 3A molecular sieves prior to use.
Analytical TLC was performed using 0.2 mm silica gel
60 F254 (Machery-Nagel, Germany) and compounds
were visualised using permanganate.14 Centrifugal silica
gel chromatography was performed on a Chromatotron
(Model 7924T, Harrison Research, USA) using 2 mm
silica gel PF254 (Merck) on a glass rotor. RP-HPLC was
performed on a Waters system consisting of two pumps
(Model 6000A and M45), solvent programmer (Model
660), injector (Model U6K) and detector (Model 450).
Analytical separations were achieved on a Waters C-18
Nova-Pak column (8Â100 mm). Preparative separations
used a Waters C-18 Prep Nova-Pak HR column
(25Â100 mm). 1H NMR spectra were recorded at
300 MHz on a Bruker AC 300 spectrometer or at
500.1 MHz on a Bruker DMX 500 spectrometer. Che-
mical shifts (d) are given with respect to the chemical
shift of TMS or residual CHCl3 for samples dissolved in
CDCl3. Infrared analyses were performed on a dual
beam Perkin±Elmer 298 infrared spectrophotometer in
chloroform solution. Optical rotations were recorded
on a Jasco dip-1000 digital polarimeter with readings
measured at 589 nm (Na) and 25.0 ꢀC using a 1mL solu-
tion cell in either dichloromethane or water depending
on solubility. Mass measurements were made using a
VG Autospec Q mass spectrometer operating at a
resolving power of 6000 (10% valency de®nition). The
instrument was operated in the positive electron ionisa-
tion mode and samples were introduced using a heated
direct insertion probe. In vitro activity of swainsonine,
and related compounds were determined using the
method previously described.15
Isolation of swainsonine
Powdered S. procumbens seed (200 g) was extracted
with methanol for 48 h. Following evaporation of
methanol, the residue was suspended in water (200 mL)
and extracted three times with an equal volume of pet-
roleum ether (bp 80±100 ꢀC). The aqueous fraction was
passed through a SP Sephadex-C25 ion exchange col-
umn in the ammonium form (bed volume 400 mL). The
column was washed with three volumes of water and
eluted with three volumes of 1 M ammonium hydroxide.
The eluate was evaporated and the residue dried in
vacuo over silica gel. The residue was redissolved in
water (50 mL) and extracted with chloroform in a
liquid:liquid extractor for 72 h. The organic phase was
evaporated and the residue vacuum sublimed (90 ꢀC,
0.1 mmHg). The sublimate was crystallised from
chloroform to give colourless needles of swainsonine
(342 mg; 0.17% w/w). Mp 144±145 ꢀC, [a]d25 89.3ꢀ (c 1,
H2O), [lit. mp 144±146 ꢀC, [a]2d0 87.2ꢀ19]. 1H NMR
(D2O, 500 MHz) d 4.39 (ddd, J=2.2, 6.3, 7.8 Hz, H-2),
4.30 (dd, J=4.2, 6.2 Hz, H-1), 3.87 (apptd, J=4.7,
10.3 Hz, H-8), 2.95 (m, H-5eq), 2.93 (m, H-3), 2.60 (1H,
dd, J=7.8, 11.1 Hz, H-30), 2.11 (m, H-7eq), 2.01 (td,
J=2.9, 11.9 Hz, H-5ax), 1.96 (dd, J=4.0, 13.9 Hz, H-
8a), 1.78 (m, H-6eq), 1.59 (qt, J=4.2, 13.5 Hz, H-6ax),
1.29 (dq, J=4.5, 12.2 Hz, H-7ax). Proton assignments
are based on those given by Kardono et al.20; EIMS
m/z (relative intensity) 173 (27.5), 155 (36.0), 138 (18.8),
129 (6), 113 (100), 100 (17.9), 96 (56), 84 (8), 72 (26);
high-resolution mass spectra m/z 173.105044, calcd for
C8H15NO3, 173.105194.
2,2,2-Trichloroethylethanoate was prepared by mixing
2,2,2-trichloroethanol (50 mmol), acetic anhydride
(105 mmol) and pyridine (80 mmol) in a screw capped
vessel for 24 h (25 ꢀC). The reaction was terminated by

G. G. Perrone et al. / Bioorg. Med. Chem. 7 (1999) 831±835
833
Synthesis of 1,2,8-tri-O-acetylswainsonine
rate of 1 mL/90 s. Selected fractions were further pur-
i®ed by analytical HPLC using an aqueous acetonitrile
gradient (acetonitrile, linear 0±100% in 30 min; 1 mL/
min) to give 6 mg (25%) of 2-O-butyrylswainsonine (II)
(Rf 0.50) as a waxy semi-solid. [a]2d5 49.0ꢀ (c 0.46,
H2O), 1H NMR (CDCl3, 300 MHz): d 5.09 (ddd, J=2.0,
6.0, 7.7 Hz, H-2), 4.46 (dd, J=4.1, 6.2 Hz, H-1), 3.87
(ddd, J=4.6, 10.0, 12.3 Hz, H-8), 3.10 (dd, J=2.0,
11.1 Hz, H-3), 2.97 (m, H-5eq), 2.51 (dd, J=7.6,
11.0 Hz, H-30), 2.07 (m, H-7eq), 1.90 (td, J=3.6,
11.1 Hz, H-5ax), 1.81 (dd, J=4.1, 8.9 Hz, H-8a), 1.69
(m, H-6eq), 1.60 (m, H-6ax) 1.20 (m, H-7ax), 2.37 (t,
To swainsonine (0.1 mmol) dissolved in pyridine (3 mL)
was added acetic anhydride (2.5 mmol). The reaction
was stirred in a screw capped vessel (25 ꢀC) under
nitrogen for 48 h at which time the reaction was termi-
nated by the addition of water (5 mL) and extracted
with ethyl acetate (4Â5 mL). The combined organic
phase was washed with an equal volume of water, dried
over anhydrous sodium sulfate, and evaporated under
reduced pressure (40 ꢀC). The residue was puri®ed by
analytical HPLC using acetonitrile:water (20:80; v/v) at
1 mL/min, giving 25 mg (84%) of swainsonine triacetate
J=7.4 Hz,
CH3CH2CH2CO)
1.68
(sextet,
1
as a colourless oil. H NMR (CDCl3, 300 MHz) d 5.53
CH3CH2CH2CO), 0.96 (t, J=7.4 Hz, CH3CH2CH2CO).
IR, n, cm 1 (CHCl3), 3650, 3600±3300 (OH), 2780±2820
(Bohlmann bands), 1730 (CˆO), 1375 (CH3), 1250 (C±
O), 1135. EIMS: m/z (relative intensity) 243 (1.8), 172
(5), 155 (95.9), 138 (100.0), 120 (13.4), 113 (13.4); high-
resolution mass spectra m/z 243.146016; calcd for
C12H21NO4, 243.147058.
(dd, J=4.3, 6.5 Hz, H-1), 5.22 (ddd, J=2.0, 6.2, 7.8 Hz,
H-2), 4.96 (ddd, J=4.8, 10.3, 12.7 Hz, H-8), 3.17 (dd,
J=2.1, 11.2 Hz, H-3) 3.06 (m, H-5eq), 2.59 (dd J=7.6,
11.2 Hz, H-30), 2.14 (dd, J=4.3, 9.6 Hz, H-8a), 1.97±
1.85 (m, H-7eq, H-5ax), 1.80±1.75 (m, H-6ax, H-6eq),
1.24 (m, H-7ax), 2.09 (s, CH3CO-2), 2.06 (s, CH3CO-1),
2.00 (s, CH3CO-8); EIMS m/z (relative intensity) 256
(2), 239 (40), 180 (37), 137 (45), 120 (100); high resolu-
tion mass spectra m/z 256.118327; calcd for C12H18NO5
[M Ac]+, 256.118498. Proton assignments are based
on those given by Kardono et al.20
Synthesis of 2-O-butyrylswainsonine and 1,2-di-O-
butyrylswainsonine using porcine pancreatic lipase
To swainsonine (0.1 mmol) dissolved in tetrahydrofuran
(3 mL) was added 2,2,2-trichloroethylbutanoate
(1 mmol) and porcine pancreatic lipase (690 units). The
reaction was shaken at 175 rpm and 37 ꢀC with product
formation monitored by silica gel TLC (dichloro-
methane:ethanol, 85:15 (v/v)). The reaction was termi-
nated (16 h) by centrifugation (3500 g; 10 min) and
removal of the enzyme, followed by evaporation of the
supernatant under a stream of nitrogen. The residue was
puri®ed by centrifugal silica gel chromatography using
250 mL of dichloromethane:ethanol (80:20; v/v), col-
lecting 5 mL fractions at a ¯ow rate of 1 mL/min. Two
products were isolated:
Synthesis of 1,2,8-tri-O-butyrylswainsonine
This synthesis was as for swainsonine triacetate but,
with butyric anhydride. The residue was chromato-
graphed by preparative HPLC using aqueous 75%
methanol at 4 mL/min. The solvent was removed under
reduced pressure (40 ꢀC) to yield 35 mg (92%) of 1,2,8-
tri-O-butyrylswainsonine as a pale-yellow oil. [a]d25
5.5ꢀ (c 1, CH2Cl2); H NMR (CDCl3, 300 MHz) 5.40
1
(dd, J=4.1, 7.1 Hz, H-1), 5.16 (ddd, J=2.3, 6.2, 7.2 Hz,
H-2), 4.85 (ddd, J=4.7, 10.3, 10.3 Hz, H-8), 3.04 (dd,
J=2.3, 11.1 Hz, H-3) 2.96 (m, H-5eq), 2.52 (dd, J=7.8,
11.1 Hz, H-30), 2.15 (m, H-8a) 2.07 (m, H-7eq), 1.84 (m,
H-5ax), 1.64 (m, H-6ax, H-6eq), 1.17 (m, H-7ax), 2.12
(t, J=7.4 Hz, CH3CH2CH2CO), 1.52 (sextet,
Product 1. 2-O-Butyrylswainsonine (II) (Rf 0.50) was
isolated as a waxy semi-solid in a 6% (w/w) yield (25%
with subtilisin). [a]2d5 49.0ꢀ (c 0.46, H2O), H NMR
1
CH3CH2CH2CO), 0.84 (t, 7.4 Hz, CH3CH2CH2CO);
1
(CDCl3, 500 MHz) d 5.09 (ddd, J=1.8, 6.1, 7.7 Hz, H-
2), 4.47 (dd, J=4.3, 6.2 Hz, H-1), 3.86 (ddd, J=4.6,
10.1, 12.3 Hz, H-8), 3.09 (dd, J=1.6, 11.1 Hz, H-3), 2.96
(m, H-5eq), 2.50 (dd, J=7.5, 11.0 Hz, H-30), 2.07 (m, H-
7eq), 1.89 (td, J=3.1, 11.5 Hz, H-5ax), 1.81 (dd, J=4.2,
9.0 Hz, H-8a), 1.69±1.60 (m, H-6eq, H-6ax), 1.25 (m, H-
7ax), 2.37 (t, J=7.4 Hz, CH3CH2CH2CO), 1.67 (dd,
J=7.4, 14.4 Hz, CH3CH2CH2CO), 0.95 (t, J=7.4 Hz,
IR, n, cm
(CHCl3), 3600±3400, 2800 (Bohlmann
bands), 1740 (CˆO), 1350 (CH3), 1275 (C±O), 950;
EIMS m/z (relative intensity) 384 (0.7), 383 (0.5), 312
(1.2), 296 (14.5), 295 (50.1), 208 (37.3), 137 (65), 136
(14.9), 120 (100); high resolution mass spectra m/z
383.223779; calcd for C20H33NO6, 383.230788.
1
CH3CH2CH2CO); IR, n, cm (CHCl3), 3650, 3600±
Synthesis of 2-O-butyrylswainsonine using subtilisin
Carlsberg
3300 (OH), 2780±2820 (Bohlmann bands), 1730 (CˆO),
1375 (CH3), 1250 (C±O), 1135. EIMS m/z (relative
intensity) 243 (2.1), 225 (2.7), 172 (6.2), 156 (33.1), 155
(85.3), 138 (100), 120 (13.4), 113 (18.5); high-resolution
mass spectra m/z 243.146926; calcd for C12H21NO4,
243.147058.
To swainsonine (0.1 mmol) dissolved in pyridine (3 mL)
was added 2,2,2-trichloroethylbutanoate (1 mmol) and
subtilisin (232 units). The reaction was shaken at
200 rpm and 45 ꢀC with product formation monitored
by silica gel TLC (dichloromethane:ethanol, (85:15; v/
v). The reaction was terminated (72 h) by centrifugation
(3500 g; 10 min) and removal of the enzyme, followed by
evaporation of the supernatant under a stream of nitro-
gen. The residue was puri®ed by centrifugal silica gel
chromatography using 150 mL of dichloromethane:
ethanol (8.5:1.0; v/v), collecting 2 mL fractions at a ¯ow
Product 2. 1,2-di-O-Butyrylswainsonine (III) (Rf 0.74):
Following silica gel chromatography selected fractions
were further puri®ed by preparative HPLC using aqu-
eous 85% methanol (v/v) at a ¯ow rate of 5 mL/min.
One peak was collected and evaporation of the solvent
gave 9.6 mg (31%) of 1,2-di-O-butyrylswainsonine as a

834
G. G. Perrone et al. / Bioorg. Med. Chem. 7 (1999) 831±835
pale-brown residue. Mp 60±62 ꢀC, [a]d25 50.0ꢀ (c 1,
CH2Cl2); 1H NMR (CDCl3, 300 MHz) d 5.47 (dd,
J=3.4, 6.3 Hz, H-1), 5.15 (ddd, J=2.6, 6.1, 7.4 Hz, H-
2), 3.40 (ddd, J=4.7, 9.8, 12.2 Hz, H-8), 3.19 (dd,
J=2.7, 11.0 Hz, H-3), 3.01 (ddd, J=3.4, 7.1, 10.8 Hz,
H-5eq), 2.62 (dd, J=8.4, 11.0 Hz, H-30), 2.06 (m, H-
7eq), 1.95 (td, J=3.5, 11.1 Hz, H-5ax), 1.88 (dd, J=3.5,
8.9 Hz, H-8a), 1.74±1.57 (m, H-6eq, H-6ax) 1.23 (m, H-
7ax), 2.31 (t, J=7.4 Hz, CH3CH2CH2CO), 1.65 (sextet,
demonstrates a selectivity for the C-2 hydroxyl group of
swainsonine and reaction with TCEB a€orded 2-O-
butyrylswainsonine (II) in an overall yield of 23%. No
other substitutions were detected with subtilisin.
The use of tetrahydrofuran as a reaction medium facili-
tated catalysis by porcine pancreatic lipase (PPL) and
C. cylindracea lipase (CCL). In the presence of
TCEB, both lipases catalysed the mono and diester-
i®cation of swainsonine. However, in comparison to
PPL the activity of the CCL was far lower in tetra-
hydrofuran, as indicated by the TLC monitoring of the
reaction. As the activity of PPL was higher it was
deemed to be the most appropriate biocatalyst for the
preparative scale acylation of swainsonine. In anhy-
drous tetrahydrofuran PPL acylates the C-2 position of
swainsonine (6%). In contrast to catalysis by subtilisin,
the use of PPL also resulted in the esteri®cation of the
C-1 hydroxyl group to give 1,2-di-O-butyrylswainsonine
(III) (31%). No other products were detected by silica
gel TLC (Fig.1).
CH3CH2CH2CO), 0.94 (t, J=7.4 Hz, CH3CH2CH2CO);
1
IR, n, cm
(CHCl3), 3650, 3600±3400 (O±H), 1725
(CˆO), 1300 (CH3), 1150 (C±O),1100; EIMS m/z (rela-
tive intensity) 313 (1.8), 225 (14), 138 (100), 120 (15);
high resolution mass spectra m/z 313.175789; calcd for
C16H27NO5, 313.188844.
Results and Discussion
To date there has only been limited evidence for the
presence of swainsonine in S. procumbens.15 The
extraction of S. procumbens seeds led to the isolation of
swainsonine (I) in a 0.17% (w/w) yield. As anticipated,
the isolated sample was a potent inhibitor of Jack Bean
a-mannosidase (IC50=5.8 mM15), which is consistent
with the documented biological activity of swainso-
nine.2 The identi®cation of the alkaloid was con®rmed
by the preparation and characterisation of peracylated
derivatives of the alkaloid. The isolation of a signi®cant
quantity of swainsonine provided an opportunity for
the novel synthesis of 2-O-butyrylswainsonine. In this
study the regioselectivity of three commercially avail-
able enzymes, subtilisin Carlsberg, porcine pancreatic
lipase and C. cylindracea lipase, was investigated with
respect to swainsonine.
Initial reactions with PPL and swainsonine were
attempted in pyridine at 45 ꢀC and 200 rpm. Under
these conditions products were not detected by TLC
after four days, but swainsonine was still detected. This
suggests the catalytic eciency of PPL is e€ected more
by the nature of the organic solvent than by subtle
changes in temperature and mixing conditions.
In vitro analysis of 2-O-butyrylswainsonine and 1,2-di-
O-butyrylswainsonine determined that both compounds
were less active than swainsonine at inhibiting Jack
Bean a-mannosidase (data not shown). However, in
vivo studies of 6-O-butyryl-castanospermine have
shown that it is both better tolerated than castanos-
permine and is taken up more rapidly by cells.11 Pre-
viously, Dennis and colleagues10 reported the potential
Subtilisin has a broad substrate speci®city in many
organic solvents.13 In anhydrous pyridine subtilisin
Figure 1. Enzymatic conversion of swainsonine to 2-O-butyrylswainsonine and 1,2-di-O-butyrylswainsonine.

G. G. Perrone et al. / Bioorg. Med. Chem. 7 (1999) 831±835
835
therapeutic value of derivatives of swainsonine, parti-
cularly 2-O-butyrylswainsonine. Perhaps some of the
reported in vivo side e€ects of swainsonine treatment9
may be reduced by the administration of lypophilic
esters of swainsonine. The regioselectivity of enzymatic
catalysis in organic medium facilitates the single step
synthesis of 2-O-acyl and 1,2-di-O-acyl analogues of
swainsonine. The procedures described here, including
the isolation of signi®cant quantities of swainsonine
from S. procumbens, are capable of being conveniently
scaled up to produce amounts of compounds for phar-
macological studies.
7. Oredipe, O. A.; White, S. L.; Grzegorzewski, K.; Gause, B.
L.; Cha, J. K.; Miles, V. A.; Olden, K. J. Natl. Cancer Inst.
1991, 83, 1149.
8. Humphries, M. J.; Matsumoto, K.; White, S. L.; Moly-
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