Biotransformation of a 4(20),11(12)-taxadiene derivative

Article abstract of DOI:10.1016/S0968-0896(00)00299-6

A 4 20), 11 12)-taxadiene derivative was converted to hydroxylated derivatives by Cunninghamella elegans AS3.2033 and Cunninghamella elegans var chibaensis ATCC 20230. Both microorganisms led to C-1 hydroxylations and conversion to a C-15-hydroxylated abeo-taxane. Additional products from the two fungi differed: a C-14 oxidation and a trans-cis isomerization of the cinnamoyl for one and an unprecedented hydroxylation at C-17 for the other. Copyright

Full text of DOI:10.1016/S0968-0896(00)00299-6

Bioorganic & Medicinal Chemistry 9 (2001) 793±800
Biotransformation of a 4(20),11(12)-Taxadiene Derivative
Di-An Sun,a Francoise Sauriol,b Orval Mamerc and Lolita O. Zamira,d,
*
aHuman Health Research Center, INRS-Institut Armand-Frappier, Universite du Quebec, 531 Boulevard des Prairies,
Laval, Quebec, Canada, H7V 1B7
bDepartment of Chemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6
cBiomedical Mass Spectrometry Unit, McGill University, 1130 Pine Avenue west, Montreal, Quebec, Canada H3A 1A3
dMcGill Centre for Translational Research in Cancer, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote Ste.
Catherine Road, Suite D-127, Montreal, Quebec, Canada, H3T 1E2
Received 21 September 2000; accepted 3 November 2000
AbstractÐA 4(20),11(12)-taxadiene derivative was converted to hydroxylated derivatives by Cunninghamella elegans AS3.2033 and
Cunninghamella elegans var chibaensis ATCC 20230. Both microorganisms led to C-1 hydroxylations and conversion to a C-15-
hydroxylated abeo-taxane. Additional products from the two fungi di€ered: a C-14 oxidation and a trans±cis isomerization of the
cinnamoyl for one and an unprecedented hydroxylation at C-17 for the other. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
®lamentous fungi, C. elegans AS 2033, C. elegans var
chibaensis, (ATCC 20230) Kuwabara and Hoshino (IMI
199846), had been successful in other hydroxylations.
Filamentous fungi are known to carry out regio- and
stereoselective hydroxylations of a wide range of organic
compounds.1À16 Unusual structures can be obtained
which would not be accessible by chemical reactions. The
main disadvantages of this route is that the products are
not predictable and except for a few examples the yields
are low. Despite our greater knowledge of chemical reac-
tions, it seems that taxoids also do not follow expected
routes.17,18 One of the few high yield biotransformations
reported involved Absidia coerula and 5a, 7b, 9a, 10b,
13a-pentaacetoxy-4(20),11-taxadiene, which was hy-
droxylated at C-1 and C-14 with yields of 39% and 26%,
respectively.4 These reactions were also obtained by Cun-
ninghamella echinulata, Cunninghamella elegans, Cun-
ninghamella blakesleana and Rhizopus arrhizus but less
eciently. In addition, these authors found that the
taxoids were not metabolized by more than 25 collected
fungal cultures if they contained a C-5 cinnamoyl group
such as 2-deacetoxytaxinine J, taxinine J, taxinine,
O-cinnamoyl-taxicin I triacetate.5À7 Microbial hydro-
xylation at C-6 was shown to require a C-5-acetyl
group.5 We wanted to verify if this speci®city of the
microorganism would not change when other functional
groups were altered. One possibility was to introduce
multiple OH groups as plausible binding sites. The
In this publication, we have found that with di€erent
varieties of the fungus C. elegans, we could obtain C-1
hydroxylation in high yield (44%) as well as a rear-
ranged abeo-taxane (16%) on a taxoid with C-5-cinna-
moyl present but no acetyls on C-7, C-9 and C-10. A C-14
hydroxylation as well as unprecedented isomerization of
the cinnamoyl groupand C-17 oxygenation were also
observed.
Results and Discussion
Recently, C-1 and C-14 hydroxylations of taxoids were
obtained from microbial transformations, particularly
with A. coerula.4 The structures required for bio-
transformation necessitated a C-5-acetate, and C-5-cin-
namoylated taxanes were not metabolized. We wanted
to investigate the role of C-5 cinnamoyl on these reac-
tions and were surprised to ®nd that the removal of the
acetyls on C-7, C-9 and C-10 were essential for the cat-
alyzed hydroxylations by the fungal species used in this
study when the C-5 cinnamoyl was present.
Initially, the 4(20),11(12)-taxadiene derivatives possess-
ing the 5-cinnamoyl group(taxinine J, 2-deacetoxytaxi-
nine J; Fig. 1) were not metabolized by C. elegans
*Corresponding author. Tel.: +1-450-687-5010; fax: +1-450-686-5501;
e-mail: lolita.zamir@iaf.uquebec.ca
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00299-6

794
D.-A. Sun et al. / Bioorg. Med. Chem. 9 (2001) 793±800
Figure 1. Some derivatives of taxinine J.
AS3.2033 and C. elegans ATCC 20230. However, when
2-deacetyldecinnamoyltaxinine J (Fig. 1) was also
unchanged after incubation with the fungi, we decided to
deacetylate C-7, C-9 and C-10 of 2-deacetoxytaxinine J
which is readily available from Taxus species.19 The
derived 4(20),11(12)-taxadiene derivative (1; Scheme 1)
gave hydroxylated products as well as a rearranged abeo-
taxane upon incubation with C. elegans AS3. 2033 and
C. elegans ATCC 20230. The characterization of these
new taxoids is described in the following sections.
compound 1, suggesting that perhaps 1 had been
1
hydroxylated. Comparison of the H NMR of 1 (see
Experimental) and 2 (Table 1) shows some major dif-
ferences in H-1 and H-14, as well as some detectable
variations in H-2, H-3, H-13 and H-16, that is, all the
protons around C-1. In taxane 2, the H-1 signal (at d 1.80
ppm) has disappeared, H-14a d 2.55ppm has a di€erent
pattern as it is now coupled to only two protons (dd,
J=9.8; 15.0 Hz) instead of three protons (ddd, d=
2.71ppm, J=4.9; 9.8, 14.6Hz) for taxane 1. In addition,
the chemical shift of H-14b is 1.53ppm instead of
0.99ppm for 1. The most convincing evidence for the
oxygenation at C-1 is the additional quaternary carbon at
75.9 ppm obtained from HMBC data, where H-2ab, H-
14ab, H-16 and H-17 correlate to this carbon. This
value is in accordance with C-1: 76.2 ppm for the C-1 b-
OH for the biotransformation product of taxa-4(20),11-
diene-5a, 7b,9a,10b,13a-pentaacetate (Fig. 1).4 The struc-
ture of taxane 2 is therefore 1b-hydroxy-2-deacetoxytax-
C-1ꢀ-Hydroxylation of the 4(20),11(12)-taxadiene
derivative 1 (taxanes 2 and 3; Scheme 1, Table 1)
The same major taxoid was obtained from bio-
transformation with C. elegans AS3. 2033 and ATCC
20230, albeit in di€erent yields (44% for the AS3. 2033
and 20% for ATCC 20230). HPLC and TLC data showed
that this metabolite was more polar than the starting
Scheme 1. Microbial transformation products and yields of taxane 1 by fungi C. elegans AS3.2033 (A) and ATCC 20230 (B): (a) yields of taxane 2 by A:
44%, by B: 20%; (b) yield of taxane 3 by A: 2%, none by B; (c) yields of taxane 4 by A: 16%, by B: 6%; (d) yield of taxane 5 by A: 1%, none by B; (e) yield
of taxane 6 by A: 1%, none by B; taxanes 5 and 6 were dicult to obtain in a pure state; (f) yield of taxane 7 by B: 6%, none by A.

D.-A. Sun et al. / Bioorg. Med. Chem. 9 (2001) 793±800
795
inine J. High resolution mass spectrometry con®rmed
the elemental composition of the potassium quasimole-
cular ion of taxane 2.
C-methyls (1.03, 1.15, 1.32, and 1.93) and one acetyl
(1.49). The 11(15!1)-abeo-taxane structure was con-
®rmed by the newly introduced carbon at 61.9 ppm and
the carbon at 77.1 ppm which are attributed to C-1 and
C-15, respectively. Additional HMBC correlations of
the protons of Me-16/17 (1.15 and 1.32) with C-1 (61.9),
C-15 (77.1), and Me-16/17 (27.5 and 25.0) support this
structure. The positionning of the other groups are very
similar to the starting material 1, that is, an acetyl group
on C-13, a trans-cinnamoyl groupon C-5 and hydroxyls
on C-7, C-9 and C-10. The relative stereochemistry of
taxane 4 was established using the information con-
tained in the NOESY experiment. High-resolution mass
spectrometry con®rmed the elemental composition of
the potassium quasimolecular ion of taxane 4.
A minor metabolite with very similar spectral properties
as taxane 2 was isolated only from the incubation of 1
with C. elegans AS3.2033. The only major di€erences in
the H NMR are in the positions H-20 and H-30 with
1
their coupling constant (J=12.6 Hz) typical of the cis
isomer. In addition, H-30 of taxane 3 is quite shielded
compared to that of taxane 2. The phenyl-ortho protons
as well as H-7, H-5, H-20a, H-13, H-3 and Me-18 are
also a€ected. The only explanation for these results is
that in this metabolite the cinnamoyl is in the cis con-
®guration. The in¯uence on H-13 is not unusual since it
is known that the core skeleton of taxanes is in U
shape20 and the C-5-cinnamoyl can mimic some of the
C-13 side chain of paclitaxel.21 High-resolution mass
spectrometry con®rmed the elemental composition of
the potassium quasimolecular ion of taxane 3. This is
the ®rst microbial trans±cis isomerization of a cinna-
moyl groupreported.
C-14ꢀ-Hydroxylation of the 4(20),11(12)-taxadiene
derivative 1 (taxanes 5 and 6; Scheme 1, Table 3)
Two minor metabolites were obtained on incubation of
1
taxane 1 with C. elegans AS3.2033. The H NMR of
both of these compounds showed that the resonances
corresponding to H-14a and H-14b had disappeared,
while an oxygen-bearing methine signal appeared at
3.52 ppm. Con®rmation was obtained by the resonance
at 78.3 ppm in their 13C NMR (Table 3). The stereo-
chemistry of the 14-hydroxyl groupwas determined to be
b- by the NOESY experiment. Indeed, H-14 correlates
Rerrangement to an abeo-taxane (4; Scheme 1, Table 2)
The second highest yield compound obtained by C. ele-
gans AS3 2033 (16%) and ATCC 20230 (6%) showed
the presence of an abeo-taxane skeleton with four
Table 1. 1H and 13C NMR for taxane 2 in CDCl3
Position
d 1H mult.a (J in Hz)
d
13Cb
HMBC
1, 3, 15
NOESYc
1
75.9
37.0
2a
2b
3
4
5
6a/18
6b
7
8
9
10
11
12
13
14a
14b
15
16
17
18/6a
19
20a
20b
OAc
OCO
20
1.86 m
1.80 m
2.85 d (4.9)
Ð
5.57 br s
2.07 om
1.76 om
4.35 dd (5.6, 11.4)
Ð
4.30 d (10.2)
5.02 od
Ð
9w
39.9
7s, 14bm
146.5
75.2
36.7
165.9, 146.5
6as, 6bs, 20as
See 18
71.2
45.6
79.8
71.3
Me-19
3s, 6as, 6bw, 10m
Me-19, 8, 10/7
2am, 17s, 19m
7m, 18m
138.8
135.8
71.1
Ð
5.95 brt
2.55 dd (9.8, 15.0)
1.53 odd (6.4, 15.0)
Ð
1.24 s
1.54 s
2.08 s
1.04 s
5.37 s
5.03 s
1.74 s
14as, 16s
13m, 14bs
14aw
41.7
1, 2, 12, 13
43.7
27.4
21.6
15.6
12.7
115.4
1, 11, 15, Me-17
1, 11, 15, Me-16
11, 12, 13
13w, 14aw, 17w
2bw, 9w, 16w
3w, 5w, 6bs, 7w, 10s
2aw, 6bw, 9w
3, 7, 8, 9
3, 5
5s, 20bs
2bs, 7w, 9w, 20as
20.7
165.9
118.1
145.6
134.1
127.9
130.4
128.8
170.8
Ð
6.50 d (16.0)
7.75 d (16.0)
Ð
7.47 m
7.40 m
7.40 m
165.9, 134.1
165.9, Ph-o, 134.1
30
Ph
o
m
p
aMult, multiplicity: s, singlet; d, doublet; t, triplet; dd, doublet of doublet; br, broad; m, multiplet; o, overlapping. The precision of the coupling
constants is Æ0.5 Hz.
bThe 13C chemical shifts were extracted from the HMQC and HMBC experiments (for quaternary carbons) (Æ0.2 ppm).
cNOESY intensities are marked as strong (s), medium (m) or weak (w).

796
D.-A. Sun et al. / Bioorg. Med. Chem. 9 (2001) 793±800
Table 2. 1H and 13C NMR for taxane 4 in CDCl3
Position
d 1H mult.a (J in Hz)
d
13Cb
HMBC
NOESYc
1
Ð
1.93 om
1.36 d (14.2)
2.57 d (8.2)
61.9
28.5
2a/18
2b
3
1, 3, 8
1, 3, 8
8
See 18
2as, 3w, 17w, 20bs
7s, 14bs, 10w, 2aw, 2bw
38.4
4
5
6a
6b
7
Ð
5.52 dd (2.1, 3.0)
2.13 ddd (1.9, 4.6, 14.6)
1.82 ddd (3.9, 11.4, 14.6)
4.29 dd (4.9, 11.1)
Ð
4.19 d (9.8)
4.61 d (9.8)
Ð
145.6
74.6
36.2
6as, 6bs, 20as
5s, 7s, 6bs
5s, 6as, 7w, 19s
3w, 6as, 6bs, 10w
71.0
44.0
81.7
69.2
140.4
142.2
79.7
19
8
9
7, 8, 10, 19
1, 9, 11, 12
2as, 19w
3w, 7w, 18w
10
11
12
13
14a
14b
15
16
17
18/2a
19
20a
20b
OAc
CO
2
Ð
5.43 t (7.0)
2.37 dd (6.9, 13.8)
1.09 dd (7.5, 13.8)
Ð
1.15 s
1.32 s
1.93 s
1.03 s
5.29 s
4.84 s
1.49 s
Ð
6.36 d (16.0)
7.63 d (16.0)
Ð
7.49 m
7.38 om
7.38 om
16w, 14as, 18w
13s, 14bs, 17w
2bw, 3w, 13s, 14as
44.2
77.1
27.5
25.0
11.2
12.4
113.4
11, 12, 15
1, 15, Me-17
1, 15, Me-16
11, 12, 13
3, 7, 8, 9
3, 5, 4
13s, 14as, 17w
14aw, 16w
2bs, 3w, 9s, 10s, 13s, 19s
2am, 6bm, 9m
5m, 20bs
3, 5
171.0
2bs, 20as
20.4
165.5
118.4
144.6
134.4
127.8
128.9
130.3
C1(Ph), 165.5
C1 (Ph), CO
3
Ph
o
m
p
aMult, multiplicity: s, singlet; d, doublet; t, triplet; dd, doublet of doublet; br, broad; m, multiplet; o, overlapping. The precision of the coupling
constants is Æ0.5 Hz.
bThe 13C chemical shifts were extracted from the HMQC and HMBC experiments (for quaternary carbons) (Æ0.2 ppm).
cNOESY intensities are marked as strong (s), medium (m) or weak (w).
with H-3, H-2a and H-1. It is interesting to note that the
major di€erence in the NMR data of taxanes 5 (Table 3)
and 6 (Experimental) shows isomerization of the double
bond of the cinnamoyl group. In taxane 5 it is trans,
whereas in taxane 6 it is cis. Indeed, H-30 in 6 is quite
shielded compared to taxane 5 (6.94 ppm instead of
7.76ppm). The core skeleton in both taxanes have the
same NOE correlations therefore the only di€erence is in
the con®guration of the cinnamoyl group. We had
observed the same isomerization between taxanes 2 and 3.
High-resolution mass spectrometry con®rmed the ele-
mental composition of the potassium quasimolecular
ion of taxanes 5 and 6.
3.26ppm and carbons 16 and 11. This rules out methyls-
18 and -19 since it would involve more than 3 bond scalar
couplings. The hydroxylation could therefore be on Me-
16 or Me-17. The NOESY experiment unambiguously
determined that the hydroxylation occurred at Me-17.
Indeed, H-13 has a correlation with Me-16 and not Me-
17. Proton H-17a has a strong NOE with H-2 and H-9
and proton H-17b with H-1. High resolution mass
spectrometry con®rmed the elemental composition of
the potassium quasimolecular ion of taxane 7.
Conclusion
The good yields obtained in some of these reactions
demonstrate the potential use of C. elegans in the bio-
transformation of taxane derivatives. Further work will
be necessary to establish the rules underlying the fungus
substrate selectivity. The metabolites which can be
obtained are not accessible by conventional synthetic
methods. It may be predicted from these ®ndings that
further exploration of this relatively new ®eld may lead
to useful insights into the fungal biotransformation
pathways and, possibly, to a means of controlled bio-
synthetic formation of desired metabolites. The next
stepon this way, will involve isolating and purifying the
enzymes responsible for these hydroxylations.
C-17-Hydroxylation of the 4(5),11(12)-taxadiene
derivative 1 (taxane 7, Scheme 1, Table 4)
An unprecedented hydroxylation was observed in a rea-
sonable yield (6%) on incubation of taxane 1 with C. ele-
gans ATCC 20230. In taxane 7, the original four methyl
singlets in taxane 1 have been replaced by three methyl
singlets and two doublet resonances at 4.60 ppm (11.8 Hz,
1 proton) and 3.26 ppm (11.8 Hz, 1 proton), suggesting
the insertion of an oxygen on one of the methyls. This
was con®rmed by the appearance of a resonance at
70.5 ppm in the 13C NMR of taxane 7. The HMBC
experiment showed a correlation between the proton at

D.-A. Sun et al. / Bioorg. Med. Chem. 9 (2001) 793±800
Table 3. 1H and 13C NMR for taxane 5 in CDCl3
797
Position
d 1H mult.a (J in Hz)
d
13Cb
HMBC
NOESYc
1
1.76 om
1.70±1.81 m
1.96 om
2.68 d (5.6)
Ð
50.1
26.2
2a
2b
3
37.4
7s, 14s, 18m
20as
4
5
5.54 t (2.7)
2.07 om
1.80 om
4.34 dd (4.5, 11.5)
Ð
4.31 d (9.9)
5.03 om
Ð
75.3
36.9
6a
6b
7
8
9
10
11
12
13
14
71.4
45.7
80.2
71.3
141.9
131.9
81.4
3s, 6as, 10/20as, 18s
17s, 19m
7/9m, 18s
Ð
5.65 brm
3.52 d (4.8)
78.3
1, 13, 15
3s, 2am (1.96), 1
15
16
17
18
Ð
39.6
31.6
27.0
15.5
12.5
115.5
1.26 s
1.58 s
2.11 s
1.03 s
5.35 s
5.00 s
1.81 s
Ð
6.48 d (16.0)
7.76 d (16.0)
Ð
7.47 m
7.40 m
7.40 m
1, 11, 15, Me-17
1, 11, 15, Me-16
11, 12, 13
13s, 17s
2b/1 (1.8)s, 9s, 16s
3s, 5w, 6bs, 7s, 10s, 13w
2a/1s, 9s, 16w
5s, 20bs
19
3, 7, 8, 9
20a
20b
OAc
CˆO
20
30
Ph
o
2s, 20as
20.9
165.9
118.3
145.6
173.3
C1-Ph
o-Ph
Ph-os, 18w
127.8
130.5
129.1
m
p
aMult, multiplicity: s, singlet; d, doublet; t, triplet; dd, doublet of doublet; br, broad; m, multiplet; o, overlapping. The precision of the coupling
constants is Æ0.5 Hz.
bThe 13C chemical shifts were extracted from the HMQC and HMBC experiments (for quaternary carbons) (Æ0.2 ppm).
cNOESY intensities are marked as strong (s), medium (m) or weak (w).
Experimental
NMR and mass spectrometry measurement
Instrumentation
All the NMR data were obtained at room temperature on
a
Bruker Avance-500 spectrometer operating at
Flash chromatography was performed on Silica gel 60
(230±400 mesh EM Science). Thin layer chromato-
graphy was conducted on Silica Gel 60 F254 pre-coated
TLC plates (0.25 mm, EM Science). The compounds
were visualized on TLC plates with 10% sulfuric acid in
ethanol and heating on a hot plate. Na2SO4 was the
drying agent used in all work upprocedures. Analytical
HPLC was performed on a Waters 600 FHU delivery
system coupled to a PDA 996 detector. Preparative and
semi-preparative HPLC were carried out on a Waters
Delta Prep3000 instrument coupled to a UV 486 Tunable
Absorbance detector set at 227 nm (Waters, Montreal,
Quebec, Canada). Analytical HPLC was performed with
two Whatman partisil 10 ODS-2 analytical columns
(4.6Â250 mm) in series. Semi-preparative HPLC was
performed with two Whatman partisil 10 ODS-2 Mag-9
semi-preparative columns (9.4Â250 mm) in series. Pre-
parative HPLC was performed with one partisil 10
ODS-2 MAG-20 preparative column (22Â500 mm). The
products were eluted with a 50 min linear gradient of
acetonitrile (25±100%) in water at a ¯ow rate of 18 mL/
min (preparative HPLC) and 3 mL/min (semi-pre-
parative HPLC). All the reagents and solvents were of
the best available commercial quality and were used
without further puri®cation.
500.13MHz for proton and at 125.77MHz for carbon-13.
The solvent was used as an internal reference (7.25 ppm
for proton and 77.0 ppm for carbon-13). The various 2-
D spectra were acquired and processed using standard
procedures. For phase sensitive 2-D experiments
(NOESY and HMQC), the data were acquired using the
TPPI phase mode. The NOESY experiment was
obtained using a mixing time of 0.3 s and a relaxation
delay of 1 s. The intensity of the cross-peaks in the
NOESY experiment is designated as strong (s), medium
(m) and weak (w). Positive ion fast atom bombardment
Mass Spectra (FABMS) were obtained with a Vacuum
Generators ZAB-HS double-focussing instrument using a
xenon beam having 8 kV energy at 1 mA equivalent neu-
tral current. Low resolution mass spectra were obtained in
glycerol. Samples were dissolved in 0.2mL DMSO before
addition of 0.5 mL glycerol. FABHRMS was similarly
obtained in glycerol-DMSO at a resolving power of
12,000.
Substrate: 2-deacetoxy-7,9,10-trideacetyltaxinine J, 1.
2-Deacetoxytaxinine J is abundant in various Taxus spe-
cies.19 It was deacetylated at positions 7, 9, and 10 to give
the substrate 2-deacetoxy-7,9,10-trideacetyl-taxinine J, 1,
following the procedure of Sako et al.22 To a solution of

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D.-A. Sun et al. / Bioorg. Med. Chem. 9 (2001) 793±800
Table 4. 1H and 13C NMR for taxane 7 in CDCl3
Position
d 1H mult.a (J in Hz)
1.86 br
1.70 dd (4.0, 15.2)
1.63 dd (4.9, 15.2)
2.91 br d (4.2)
Ð
5.55 t (2.4)
2.10 ddd (2.2, 4.1, 14.1)
1.78 ddd (3.5, 11.7, 14.7)
4.40 dd (11.4, 4.8)
Ð
4.20 br d (9.7)
4.97 d (10.3)
Ð
d
13Cb
HMBC
3, 8
NOESYc
1
37.0
26.7
2a
2b
3
4
5
6a
6b
7
8
9
10
11
12
13
14a
14b
15
16
17a
17b
18
19
20a
20b
OAc
CˆO
2
3m, Me-17m, Me-19m, 20bm
3m, Me-17m, Me-19m, 20bm
7s, 14bm, 18m
36.2
75.3
36.8
6as, 6bs, 20as
71.1
45.6
81.3
71.3
Me-19
3s, 6am, 6bw, 18m
Me-17s, Me-19w
7w, 6bw, Me-18s, 20as
137.0
136.5
70.5
Ð
5.82 t (8.4)
2.74 dt (14.8, 9.7)
0.95 dd (14.8, 7.2)
Ð
14as, 18m, 16s
3s, 14as
31.9
2, 12, 13
1, 13, 15
45.4
26.6
70.5
1.34 s
1, 11, 15, Me-17
1m, 13s, 14aw, 17b(3.2)m
2s, 9s, 17bs
4.60 d (11.8)
3.26 d (11.8)
2.14 s
1.04 s
5.35 s
4.93 s
1.72 s
Ð
6.51 d (16.0)
7.75 d (16.0)
Ð
16, 11
11, 12, 13
3, 7, 8, 9
1s, 16m, 17as
15.8
12.8
1w, 2w
5s, 20bs
2w, 20as
115.3
3, 5
170.6
20.8
166.1
118.2
145.5
134.5
128.0
129.0
130.5
C1-Ph, CˆO
3
Ph
o
m
p
C1- Ph, o-P, CˆO
7.48 m
7.41 om
7.41 om
aMult, multiplicity: s, singlet; d, doublet; t, triplet; dd, doublet of doublet; br, broad; m, multiplet; o, overlapping. The precision of the coupling
constants is Æ0.5 Hz.
bThe 13C chemical shifts were extracted from the HMQC and HMBC experiments (for quaternary carbons) (Æ0.2 ppm).
cNOESY intensities are marked as strong (s), medium (m) or weak (w).
2-deacetoxytaxinine J (1000 mg, 1.54 mmol) in CH2Cl2/
.
1), 39.6 (C-15), 36.9 (C-6), 36.5 (C-3), 32.1 (C-14), 31.7
(C-16), 27.4 (C-2), 27.0 (C-17), 21.0 (OCOMe-13), 15.6
(C-18), 12.9 (C-19).
MeOH 1/1 (180 mL) was added a solution of Ba(OH)2
8H2O (486 mg, 1.54 mmol) in MeOH (20 mL). The
reaction mixture was stirred at 4 ꢀC for 14 h and diluted
with 200 mL H2O and extracted with CH2Cl2 (200
mLÂ3). The CH2Cl2 fraction was washed with brine,
dried, ®ltered and evaporated in vacuo. The residue was
puri®ed by ¯ash chromatography on a silica gel column
(30 g, CH2Cl2/MeOH, 100/0 to 100/2) a€ording 190 mg
2-deacetoxy-7,9,10-trideacetyltaxinine J, 1, and 660 mg
recovered 2-deacetoxytaxinine J. Yield: 69% (based on
recovered starting material). FABHRMS of 1: C31H40
O7K [M+K]+ required 563.2411, found 563.2412; 1H
NMR (500 MHz, CDCl3) d 7.74 (d, J=16 Hz, 1H, H-3),
7.47 (m, 2H, H-Ph-o), 7.39 (m, 3H, H-Ph-m, p), 6.51 (d,
J=16 Hz, 1H, H-20), 5.79 (br. t, J=8.4 Hz, 1H, H-13),
5.55 (br.s, 1H, H-5), 5.33 (s, 1H, H-20a), 5.02 (d,
J=10.1 Hz, 1H, H-10), 4.96 (s, 1H, H-20b), 4.38 (dd,
J=11.1, 4.1 Hz, 1H, H-7), 4.30 (dd, J=10.1, 4.5 Hz, 1H,
H-9), 2.91 (br s, 1H, H-3), 2.70 (m, 1H, H-14b), 2.09 (s,
1H, Me-18), 1.72 (s, 13-Ac), 1.53 (s, Me-16), 1.16 (s,
Me-17), 1.03 (s, Me-19), 0.99 (dd, J=14.7, 6.9 Hz, 1H,
H-14a); 13CNMR (125 MHz, CDCl3) d 170.8 (OCOMe-
13), 166.1(-OCO-), 146.9 (C-4), 145.4 (ˆC-30), 139.2 (C-
11), 134.2 (C-12), 134.2 (Ph-C1), 130.6 (Ph-p), 129.0 (Ph-
m), 128.0 (Ph-o), 118.5 (ˆC-20), 115.2 (C-20), 75.5 (C-5),
71.6 (C-10), 71.5 (C-7), 70.8 (C-13), 45.4 (C-8), 40.2 (C-
Microorganisms and biotransformation procedure
C. elegans AS3.2033 and C. elegans var chibaensis
ATCC 20230 were purchased from the Institute of
Microbiology, Chinese Academy of Science, P. R.
China and American Type Culture Collection (ATCC),
respectively. Cultures were grown in Potato Dextrose
(24 g/L, DIFCO laboratories) broth, and was incubated
at 25 ꢀC and 125 rpm, unless otherwise indicated. C.
elegans AS3.2033 was ®rst preserved on silica gel and
kept at 4 ꢀC to prevent mutation.23 The seed culture was
prepared by the addition of several grains of silica gel
that absorbed the fungus to 30 mL of medium in a
125 mL Erlermeyer ¯ask. The culture was incubated for
3 days. To 1 L of production medium in a 2 L ¯ask was
added 5 mL of the seed culture previously homogenized
with a Polytron homogenizer. The culture was incu-
bated for 3 days after which time it was inoculated with
the substrate 2-deacetoxy-7,9,10-trideacetyltaxinine J 1
(50 mg in 1 mL DMSO). The culture was further incubated
for 12 days, then homogenized and extracted with CH2Cl2
(500 mLÂ3) a€ording 276 mg extract. The exact same
procedure and amounts were used in the incubation of this

D.-A. Sun et al. / Bioorg. Med. Chem. 9 (2001) 793±800
799
substrate 1 with C. elegans var chibaensis ATCC 20230.
The ®nal CH2Cl2 extract obtained weighed 160 mg.
Cultures with the same medium and substrate but with-
out fungi were used as controls in the same experimental
conditions.
14ꢀ-Hydroxy-2-deacetoxy-7,9,10-trideacetyl-taxinine J, 5.
(Colorless gum) FABHRMS: C31H40O8K [M+K]+
required 579.2360, found 579.2361. For 1H and 13C
NMR, HMBC and NOESY spectral data, see Table 3.
14ꢀ-Hydroxy-2-deacetoxy-7,9,10-trideacetyl-5-cis-cinna-
moyl-taxinine J, 6. (Colorless gum) FABHRMS: C31H40
O8K [M+K]+ required 579.2360, found 579.2361. The
NMR data is very similar to taxane 5. However, we
were not able to separate taxanes 5 and 6 the best ratio
we could obtain was 70:30 (5:6). In addition, the
HMQC for 6 was too dilute to get all the shifts. We will
therefore report only the di€erences (H-30; H-20; Ph-o;
H-3; H-5; H-7; H-13; Me-18). 1H NMR (500MHz,
CDCl3) d 6.94 (d, J=12.7 Hz, 1H, H-30), 6.08 (d,
J=12.7 Hz, 1H, H-2), 7.65 (ꢁd, Ph-o), 2.78 (d, J=4.8 Hz,
1H, H-3), 5.46 (br s, 1H, H-5), 4.23 (dd, J=4.4, 11.3Hz,
1H, H-7), 6.05 (o.t. 1H, H-13), 1.97 (s, 3H, Me-18).
Isolation and puri®cation of the taxanes obtained from
incubation of 2-deacetoxy-7,9,10-trideacetyl-taxinine J,
1, with C. elegans AS3.2033
The extract residue (276 mg) was applied on a silica gel
¯ash chromatography column (19 g), eluted with CH2Cl2
(150mL), CH2Cl2/MeOH (100/1, 100/2, 100/4, 100/10,
80/20 each 100 mL) and eleven 60mL fractions were col-
lected. Fractions 7 and 8 (40 mg) containing taxanes were
combined, evaporated and further puri®ed by preparative
HPLC. Two pure compounds were isolated: taxane 2
(tR=26.5 min, 22mg, 44%) and 4 (tR=33.2 min, 8 mg,
16%). A mixture at tR=28.5 min was collected and sepa-
rated by preparative TLC (EtOAc/hexane, 7/1) to give
pure taxane 3 (Rf=0.5, 1 mg, 2%), and a mixture of tax-
anes 5 and 6 (Rf=0.4, 1.0 mg, 2.0%) which was dicult to
obtain in a pure state.
2-Deacetoxy-7,9,10-trideacetyl-17-hydroxy-taxinine J 7.
(colorless gum) FABHRMS: C31H40O8K [M+K]+
required 579.2360, found 579.2361. For 1H and 13C
NMR, HMBC and NOESY spectral data, see Table 4.
Isolation and puri®cation of the taxanes obtained from
incubation of 2-deacetoxy-7,9,10-trideacetyl-taxinine J,
1, with C. elegans var chibaensis ATCC 20230
Acknowledgements
We thank the Natural Science and Engineering
Research Council of Canada and the Canadian Breast
Cancer Research Initiative-Idea grant for support via an
operating grants to L. O. Z. We thank the INRS-Insti-
tut Armand-Frappier for a predoctoral fellowship to
D. A. Sun. Professor Lihe Zhang is gratefully acknow-
ledged for a gift of Cunninghamella elegans AS3.2033 and
Dr. Li Zhichao ((Zhongling (Huizhou) High Science
and Technology Co. Ltd. P. R. China)) for a generous
supply gift of a sample of 2-deacetoxy-taxinine J.
The extract residue (160 mg) was applied on a silica gel
¯ash chromatography column (10 g), eluted with
CH2Cl2 (100 mL), CH2Cl2/MeOH (100/1, 100/2, 100/4,
80/20 each 100 mL) and seven 60 mL fractions were
collected. Fractions 4±6 (35 mg) containing taxanes were
combined, evaporated and further puri®ed by pre-
parative HPLC. Pure taxane 2 was isolated as a white
powder (tR=27.1 min, 10mg, 20%). A mixture at
tR=33.6 min was collected and further separated by pre-
parative TLC (EtOAc/hexane, 7:1) to a€ord taxanes 4
(Rf=0.35, 3 mg, 6%), and 7 (Rf=0.45, 3 mg, 6%).
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NMR, HMBC, and NOESY spectral data see Table 1.
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