Ascochlorin derivatives from the leafhopper pathogenic fungus Microcera sp. BCC 17074

Article abstract of DOI:10.1038/ja.2014.90

Two new ascochlorin derivatives, nectchlorins A (1) and B (2), together with eight known compounds (3-10), were isolated from cultures of the leafhopper pathogen Microcera sp. BCC 17074. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. The absolute configuration of 2 was determined by application of the modified Mosher's method. The absolute configuration of LL-Z 1272α epoxide (9), which is a plausible biosynthetic precursor of ascochlorins, was established by chemical correlations. Cytotoxic activities of these ascochlorin derivatives were evaluated.

Full text of DOI:10.1038/ja.2014.90

The Journal of Antibiotics (2014), 1–5
2014 Japan Antibiotics Research Association All rights reserved 0021-8820/14
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&
ORIGINAL ARTICLE
Ascochlorin derivatives from the leafhopper
pathogenic fungus Microcera sp. BCC 17074
Masahiko Isaka, Arunrat Yangchum, Sumalee Supothina, Pattiyaa Laksanacharoen, J Jennifer Luangsa-ard
and Nigel L Hywel-Jones
Two new ascochlorin derivatives, nectchlorins A (1) and B (2), together with eight known compounds (3–10), were isolated from
cultures of the leafhopper pathogen Microcera sp. BCC 17074. The structures were elucidated on the basis of NMR
spectroscopic and mass spectrometry data. The absolute configuration of 2 was determined by application of the modified
Mosher’s method. The absolute configuration of LL-Z 1272a epoxide (9), which is a plausible biosynthetic precursor of
ascochlorins, was established by chemical correlations. Cytotoxic activities of these ascochlorin derivatives were evaluated.
The Journal of Antibiotics advance online publication, 2 July 2014; doi:10.1038/ja.2014.90
INTRODUCTION
NMR spectrum showed resonances of two phenolic protons at dH
Invertebrate pathogenic fungi have recently been proved to be unique
12.67 (s) and 6.40 (s). The 5-chloroorcylaldehyde unit was revealed by
and prolific sources of structurally diverse bioactive compounds.1
the HMBC correlations from a downfield phenolic proton at dH 12.67
Although a number of compounds with significant biological
(2-OH) to C-1, C-2 and C-3, from another phenolic proton at dH
activities have been isolated from several genera such as Cordyceps,
6.40 (4-OH) to C-3, C-4 and C-5, from H3-7 to C-1, C-5 and C-6 and
Beauveria, Aschersonia, Isaria and Hirsutella,2 there are still many
from H-8 to C-1 and C-2. The planar structure of the sesquiterpene
entomopathogenic genera/species in the order Hypocreales that
unit from C-9 to C-23, similar to ascochlorin, was elucidated by
remain chemically unexplored. As part of our research on bioactive
interpretation of COSY, HMQC and HMBC spectroscopic data. The
secondary metabolites of invertebrate pathogenic fungi collected in
linkage of the side chain to C-3 of 5-chloroorcylaldehyde was
Thailand, a strain of leafhopper pathogen, Microcera sp. BCC 17074 of
confirmed by the HMBC correlations from H2-9 to C-2, C-3 and
the family Nectriaceae, has been chemically investigated. An extract
C-4 and the correlation from H-10 to C-3. The propionyloxy
from this strain exhibited cytotoxicity to KB (oral cavity cancer) cells
substituent at C-12 was demonstrated by the HMBC correlation
with an MIC value of 24mg mlꢀ1 and its 1H NMR spectrum
from H-12 (dH 5.37) to the ester carbonyl carbon C-10 (dC 173.7).
suggested the presence of a known antibiotic ascochlorin3 and
The (E)-geometry of the trisubstituted olefin (C-10/C-11) was evident
several minor derivatives. To our knowledge, there has been no
from the NOESY correlations H2-9/H3-23 and H-10/H-12. The
previous report on the bioactive secondary metabolites from this
relative configuration of the tetrasubstituted cyclohexanone was
genus. Scale-up fermentation and chemical analysis led to the
proved to be identical to that of ascochlorin (7) and other known
isolation and structure elucidation of two new ascochlorin-type
analogs on the basis of the NOESY correlations H-15/H-19, Hb-
metabolites, nectchlorins A (1) and B (2), along with eight known
16(axial)/H3-20, H3-20/H3-21 and H3-20/H3-22.
compounds 3–10 (Figure 1).
The structure of compound 3 was elucidated by similar spectro-
scopic analysis, and it was identified as the known compound,
chloronectrin.4 Therefore, compounds 1 and 3 were assigned as
RESULTS AND DISCUSSION
Nectchlorin A (1) was obtained as a pale yellow solid, and the
propionate and acetate derivatives of the known co-metabolite 4,5
respectively. Nectchlorin A (1) is also closely related to cylindrols A4
molecular formula was established as C26H35ClO6, from the [Mþ
and A5,6,7 which are isovalerate and butanoate variants, respectively.
Among these analogs, the 12R configuration was previously
determined for 4 and cylindrol A4.6,8 Alkaline hydrolysis (1 M
NaOH/dioxane) of chloronectrin (3) gave alcohol 4, which
confirmed the 12R configuration of 3. Hydrolysis of a less reactive
ester 1 did not take place under the same alkaline conditions.
Hydrolysis of 1 under stronger basic conditions or acidic conditions
H]þ ion peak in the HRESIMS. The 1H and 13C NMR spectroscopic
data showed resemblance to those of ascochlorin (7).3 The 13C NMR,
DEPT135 and HMQC spectroscopic data revealed that it contained
an aliphatic ketone (dC 213.5), a conjugated aldehyde (dC 193.5, dH
10.12), an ester carbonyl carbon (dC 173.7), seven sp2 quaternary
carbons, an sp2 methine, an sp3 quaternary carbon, three methines,
1
five methylenes and six methyl groups (Table 1). In addition, the H
National Center for Genetic Engineering and Biotechnology (BIOTEC), Klong Luang, Pathumthani, Thailand
Correspondence: Dr M Isaka, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani
12120, Thailand.
E-mail: isaka@biotec.or.th
Received 14 March 2014; revised 25 April 2014; accepted 1 June 2014

Ascochlorin derivatives from the leafhopper fungus
M Isaka et al
2
23
11
22
O
OH
O
OH
R3
20
O
OH
Cl
9
13
21
2
12
O
3
O
1
6
O
8
19
16
H
H
14
10
H
18
15
R2
O
1'
O
17
4
OH
OH
5
OH
7
Cl
R1
2'
3'
1 R1 = CH2CH3
4 R2 =OH, R3= Cl
5 R2 = H, R3 = Cl
6 R2 = H, R3 = H
7
3 R1 = CH3
22
21
23
O
OH
Cl
O
OH
Cl
O
OH
Cl
OR4
O
9
18
19
H
H
H
20
OH
OH
10
OH
OH
2 R4 = CH3
8 R4 = H
9
Figure 1 Structures of compounds 1–10.
Table 1 NMR spectroscopic data for 1 and 2 in CDCl3
1a
2b
Position
dC, mult.
dH, mult. (J in Hz)
HMBC
dC, mult.
dH, mult. (J in Hz)
HMBC
1
113.5, qC
162.4, qC
113.6, qC
162.1, qC
2
2-OH
3
12.67, s
6.40, s
1, 2, 3
3, 4, 5
12.69, s
6.79, s
1, 2, 3
3, 4, 5
113.8, qC
156.3, qC
114.5, qC
156.7, qC
4
4-OH
5
113.3, qC
138.1, qC
14.7, CH3
193.5, CH
21.8, CH2
124.9, CH
135.8, qC
75.9, CH
39.9, CH2
44.3, qC
113.3, qC
137.6, qC
14.4, CH3
193.2, CH
22.0, CH2
121.1, CH
136.6, qC
39.6, CH2
26.3, CH2
124.5, CH
134.9, qC
36.7, CH2
29.5, CH2
76.3, CH
6
7
2.59, s
10.12, s
1, 5, 6
1, 2
2.60, s
10.13, s
1, 5, 6
1, 2
8
9
3.39-3.37, m
5.56, br t (7.0)
2, 3, 4, 10, 11
3, 9, 12, 23
3.39, d (7.0)
5.20, br t (7.0)
2, 3, 4, 10, 11
3, 9, 12, 23
10
11
12
13
14
15
16
17
18
19
19-OCH3
20
21
22
23
10
5.37, dd (7.4, 4.0)
1.81, m; 1.54, m
10, 11, 13, 14, 23, 10
2.01, m
2.08, m
10, 11, 13, 14, 23
11, 12, 14, 15
12, 13, 16, 22
11, 12, 14, 15, 19, 20
5.12, br t (6.5)
36.8, CH
31.4, CH2
41.7, CH2
213.5, qC
50.6, CH
1.91, m
20, 21
1.78, m; 1.52, m
2.24, m; 2.17, m
14, 15, 21
2.24, m; 2.02, m
1.50, m; 1.39, m
3.42, dd (10.2, 1.4)
14, 15, 17, 18, 22
15, 16, 18, 19
16, 19, 21
15, 16, 18, 19
2.53, q (6.6)
14, 15, 17, 18, 20, 22
77.5, qC
49.1, CH3
20.7, CH3
18.9, CH3
16.0, CH3
16.1, CH3
3.22, s
1.11, s
1.09, s
1.58, s
1.78, s
19
15.6, CH3
15.8, CH3
8.2, CH3
0.53, s
13, 14, 15, 19
14, 15, 16
14, 18, 19
10, 11, 12
18, 19, 21
18, 19, 20
14, 15, 16
10, 11, 12
0.95, d (6.7)
0.79, d (6.6)
1.80, br s
12.0, CH3
173.7, qC
28.2, CH2
9.3, CH3
20
30
2.30-2.29, m
1.13, t (7.6)
10, 30
10, 20
a400 MHz for 1H and 100 MHz for 13C.
b500 MHz for 1H and 125 MHz for 13C.
had been unsuccessful, giving polymeric products. However, close
resemblance of the NMR spectroscopic data of 1 with those of 3, and HRESIMS as C24H35ClO5. The H and 13C NMR spectra suggested
the co-production with 3 and 4 by the fungus BCC 17074 strongly close structural resemblance to the known co-metabolite, chlorocy-
The molecular formula of nectchlorin B (2) was determined by
1
suggested that 1 should have the same absolute configuration.
lindrocarpol (8),9 although 2 additionally bore a methoxy group
The Journal of Antibiotics

Ascochlorin derivatives from the leafhopper fungus
M Isaka et al
3
(dH 3.22; dC 49.1). Detailed interpretation of 2D NMR spectroscopic
Ascochlorin and its analogs have been known to exhibit broad
data revealed that the 5-chloroorcylaldehyde was identical to other range of biological activities including antiviral,3,16 antifungal7 and
ascochlorin derivatives. The sesquiterpene side chain structure was cytotoxic16,17 activities. A variety of specific biological functions
also deduced from 2D NMR (COSY, HMQC and HMBC) data. The related to cancer chemotherapy have also been reported: inhibition
location of the methoxy group was assigned by the HMBC correlation of Ras farnesyl-protein transferase,6 inhibition of mitochondrial
from OCH3 to the quaternary carbon C-19 (dC 77.5). The cytochrome bc1 complex,18 inhibition of matrix metalloproteinase-9
neighboring secondary alcohol functionality was demonstrated by expression19 and activation of p53 in a manner distinct from DNA
HMBC correlations from H2-16, Hb-17 (dH 1.39), H3-20 and H3-21 damaging agents.20 New compounds 1 and 2, their closely related
to the oxymethine at dC 76.3 (C-18), and the correlations from H-18 analogs 3 and 8 and ascochlorin (7; for comparison) were tested for
to C-16, C-19 and C-21. The absolute configuration of this secondary cytotoxic activities against cancer cell-lines, NCI-H187 (small-cell
alcohol carbon (C-18) was determined by application of the modified lung cancer), MCF-7 (breast cancer) and KB (oral cavity cancer), and
Mosher’s method.10 Compound 2 was methylated (MeI, K2CO3, 2- nonmalignant Vero cells (African green monkey kidney fibroblasts)
butanone) to give its 2,4-O-dimethyl derivative, which was then (Table 2). Cytotoxic activities of 1 and 2 were weaker when compared
acylated with (R)- and (S)-MTPA-Cl in pyridine to obtain (S)- and with ascochlorin (7).
(R)-MTPA ester derivatives 11a and 11b, respectively. The Dd-values
indicated the 18S-configuration (Figure 2), which is the same as 8.9
Although the absolute configuration of 8 was previously reported,9 we
METHODS
General experimental procedures
confirmed it with our sample from the fungus Microcera sp. BCC
17074 using the same method as performed for 2.
Melting points were measured with an IA9100 digital melting point apparatus
(Electrothermal, Essex, UK). Optical rotations were measured with a P-1030
digital polarimeter (JASCO, Tokyo, Japan). UV spectra were recorded on a
Cintra 404 spectrophotometer (GBC Scientific Equipment, Braeside, VIC,
Australia). Fourier transform infrared spectra were taken on an ALPHA
spectrometer (Bruker, Bremen, Germany). NMR spectra were recorded on
AV500D and DRX400 spectrometers (Bruker). ESI-time-of-flight mass spectra
were measured with a micrOTOF mass spectrometer (Bruker).
LL-Z 1272a epoxide (9) was previously isolated from a mutant of
Ascochyta viciae,11 but its absolute configuration of the epoxy methine
carbon (C-18) remain unassigned. In earlier reports,12–14 it has been
proposed that farnesyl chain of LL-Z 1272a is epoxidized by a specific
enzyme and then cyclized to a cyclohexanone ring to finally convert
into ascochlorin (7). Since the cyclohexanone ring moiety of all
known ascochlorin derivatives share the same absolute configuration,
it is not unreasonable to assume an enantioselective enzymatic
epoxidation of the achiral biosynthetic precursor, LL-Z 1272a,
which should be the origin of the absolute configuration of
ascochlorin. This hypothesis is consistent with the evidence that 2
and 8, which are probably derived from 9, shared the 18S-
configuration. Nectchlorin B (2) could be an artifact from 9 during
the isolation procedure using MeOH, especially silica gel column
chromatography (CC), although we did not notice such an event. In
this context, we examined conversion of 9 into 2 to determine the
Fungal material
The fungus used in this study was isolated from a leafhopper (Hemiptera)
collected in Khao Sok National Park, Surat Thani Province, Thailand, by one of
the authors (NLH-J). The fungus was deposited in the BIOTEC Culture
Collection as BCC 17074. On the basis of the internal transcribed spacer
sequence data (GenBank accession number, KF564779) and the results of the
BLAST search, this strain was assigned to the genus Microcera, within the
family Nectriaceae.
Fermentation, extraction and isolation
absolute configuration of 9. Expected regioselective epoxide cleavage The fungus BCC 17074 was maintained on potato dextrose agar at 251C. The
agar was cut into small plugs and inoculated into 3 ꢁ 250 ml Erlenmeyer flasks
occurred when 9 was treated with p-TsOH in MeOH at room
temperature for 16h. The major reaction product (2) was purified
containing 25ml of potato dextrose broth (potato starch, 4.0g lꢀ1; dextrose,
20.0g lꢀ1) and incubated at 25 1C for 6 days on a rotary shaker (200r.p.m.).
and it was methylated and then acylated with (R)-MTPA-Cl
Each primary seed culture was transferred into a 1-l Erlenmeyer flask
by following the same procedures as described above. The 1H
containing 250 ml of potato dextrose broth, and incubated at 251C for 5 days
NMR spectrum of the crude acylation product indicated the
presence of a (S)-MTPA ester 11a and the absence of ent-11b,
on a rotary shaker (200 r.p.m.). The seed cultures were combined and a 700-ml
portion was transferred into a 10-l bioreactor containing 6.3 l of potato
which revealed the 18S absolute configuration of the product
from acidic epoxide cleavage. Since the C-18–O bond of 9 was
retained in the reaction, the absolute configuration of 9 was assigned
dextrose broth, and the final fermentation was carried out at 251C for 3 days
under agitation at 120r.p.m. and 0.3 vvm aeration rate, then for 2 days under
agitation at 250 r.p.m. and 1 vvm aeration rate. The cultures were filtered to
to be 18S. Other ascochlorin-type metabolites isolated from BCC separate broth (filtrate) and mycelia (residue). The broth was extracted with
17074 were identified as LL-Z 1272d (5),12 LL-Z 1272e (6)12 and
colletochlorin B (10).15
Table 2 Cytotoxic activities of 1–3, 7 and 8
Cytotoxicity (IC50, mg mlꢀ1
)
0.00
0.00
+0.04
+0.03
-0.04
Compound
NCI-H187
MCF-7
KB
Vero
O
OCH3
+0.04
OCH3
Nectchlorin A (1)
Nectchlorin B (2)
Chloronectrin (3)
Ascochlorin (7)
Chlorocylindrocarpol (8)
Doxorubicina
450
40
450
450
39
17
25
35
26
0.00
H
0.00
-0.04
+0.06
O
46
5.9
30
21
OCH3
0.00
MTPA
0.00
1.6
26
27
3.3
17
Cl
6.2
8.6
—
26
0.10
1.2
0.46
0.55
—
11a (S)-MTPA ester
11b (R)-MTPA estert
Ellipticinea
0.36
Figure 2 Dd-values (dS–dR) of the Mosher esters 11a and 11b.
aStandard compounds for cytotoxicity assays.
The Journal of Antibiotics

Ascochlorin derivatives from the leafhopper fungus
M Isaka et al
4
EtOAc (3ꢁ 6.5l) and concentrated under reduced pressure to obtain a brown
(S)-MTPA ester 11a: colorless gum; 1H NMR (400MHz, CDCl3) partial
gum (extract A, 371 mg). The wet mycelia were macerated in MeOH (1.1l, assignments, d 10.41 (1H, s, H-8), 5.17 (1H, tm, H-18), 5.16 (1H, m, H-10),
room temperature, 2 days) and filtered. Hexane (1.0l) and H2O (300ml) were
added to the MeOH solution and the layers were separated. The hexane
5.02 (1H, m, H-14), 3.86 (3H, s, 2-OCH3 or 4-OCH3), 3.81 (3H, s, 4-OCH3 or
2-OCH3), 3.57 (3H, s, CH3 of MTPA), 3.40 (2H, d, J ¼ 6.2Hz, H-9), 3.19 (3H,
(upper) layer was concentrated under reduced pressure to give a pale brown s, 19-OCH3), 2.63 (3H, s, H-7), 1.79 (3H, s, H-23), 1.50 (3H, s, H-22), 1.13
viscous oil (extract B, 380mg). The H2O/MeOH (bottom) layer was partially (3H, s, H-20), 1.11 (3H, s, H-21).
concentrated by evaporation, and the residue was extracted with EtOAc
(R)-MTPA ester 11b: colorless gum; 1H NMR (400MHz, CDCl3) partial
(3ꢁ 700 ml). The combined EtOAc layer was concentrated under reduced assignments, d 10.41 (1H, s, H-8), 5.16 (1H, tm, H-10), 5.14 (1H, m, H-18),
pressure to obtain a brown gum (extract C, 1.23 g). Extract C was subjected to 5.06 (1H, m, H-14), 3.86 (3H, s, 2-OCH3 or 4-OCH3), 3.81 (3H, s, 4-OCH3 or
CC on silica gel (3.0 ꢁ 16cm, MeOH/CH2Cl2, step gradient elution from 0:100 2-OCH3), 3.55 (3H, s, CH3 of MTPA), 3.40 (2H, d, J ¼ 6.1Hz, H-9), 3.15 (3H,
to 5:97) to afford six pooled fractions, fraction C-1–C-6. Fraction C-1 s, 19-OCH3), 2.63 (3H, s, H-7), 1.79 (3H, s, H-23), 1.54 (3H, s, H-22), 1.09
(626mg) was further fractionated by preparative HPLC using a reversed phase (3H, s, H-20), 1.05 (3H, s, H-21).
column HPLC (SunFire Prep C18 OBD, 19ꢁ 250 mm, 10mm, Waters
Corporation, Milford, MA, USA; mobile phase MeCN/H2O, 70:30, flow rate
Transformation of 9 into 2
15mlminꢀ1) to furnish 7 (282mg), 5 (145mg), 9 (16.6 mg) and 2 (6.4mg).
Fraction C-4 (178mg) was also purified by preparative HPLC (gradient elution
To a solution of 9 (5.0mg) in MeOH (0.5ml) was added p-TsOH ꢂ H2O
(25mg) and the mixture was stirred at room temperature for 16 h. The reaction
was terminated by addition of 1-M NaHCO3, and the mixture was partially
concentrated by evaporation. The residual aqueous solution was extracted with
with MeCN/H2O from 30:70 to 80:20 over 30min) to afford 4 (10.4 mg), 8
(16.1 mg), 6 (12.9mg), 3 (23.1 mg) and 1 (13.1mg). Extract B was also
fractionated by CC on silica gel and preparative HPLC to furnish 5 (62.5 mg),
EtOAc and the organic phase was concentrated under reduced pressure to
10 (7.3mg), 7 (78.5 mg), 9 (31.8 mg), 8 (7.5mg), 3 (7.0mg) and 1 (5.3mg).
obtain a yellow gum, which was purified by CC on silica gel (0–3 % acetone/
CH2Cl2) to afford 8 (1.6mg). The 1H NMR spectroscopic and ESI-MS data of
No unique metabolite was isolated by chromatographic fractionation of
extract A.
this reaction product were consistent with those of the natural product 8.
Nectchlorin A (1): pale yellow solid; mp 134–135 1C; [a]26D þ 25 (c 0.20,
MeOH); UV (MeOH) lmax (log e) 229 (4.33), 292 (4.17), 345 (4.02)nm; IR
(ATR) nmax 1728 sh, 1710, 1629, 1421, 1249, 1188, 755 cmꢀ1 1H NMR
;
Biological assays
(500MHz, CDCl3) and 13C NMR (125MHz, CDCl3) data, see Table 1; HR-MS
(ESI-time-of-flight, positive) m/z 479.2184 [Mþ H]þ (calculated for
C26H36ClO6, 479.2195).
Cytotoxic activities against KB cells (oral cavity cancer), MCF-7 cells (breast
cancer) and NCI-H187 cells (small-cell lung cancer) were evaluated using the
resazurin microplate assay.21 Cytotoxicity to Vero cells (African green monkey
kidney fibroblasts) were performed using the green fluorescent protein
microplate assay.22
Nectchlorin B (2): colorless oil; [a]26D–12 (c 0.32, MeOH); UV (MeOH)
l
max (log e) 228 (4.58), 294 (4.43), 345 (4.14)nm; IR (ATR) lmax 1629, 1420,
1284, 1249, 1079, 751 cmꢀ1 1H NMR (500MHz, CDCl3) and 13C NMR
;
(125MHz, CDCl3) data, see Table 1; HR-MS (ESI-time-of-flight, positive) m/z
461.2064 [Mþ Na]þ (calculated for C24H35ClNaO5, 461.2065).
ACKNOWLEDGEMENTS
We gratefully acknowledge Financial support from the National Center for
Genetic Engineering and Biotechnology (BIOTEC). We also thank Ms
Donnaya Thanakitpipattana for assistance in identification of the fungus.
Alkaline hydrolysis of chloronectrin (3)
To a solution of 3 (1.0mg) in dioxane (0.5ml) was added 1-M aqueous NaOH
(0.1ml) and the mixture was stirred at room temperature for 2 h. The mixture
was neutralized to pH 3 by addition of 0.1-M HCl and partially concentrated
by evaporation. The residual aqueous solution was extracted twice with EtOAc,
and the combined organic layer was dried over anhydrous MgSO4 and
concentrated in vacuo. The residue was purified by CC on silica gel (0–4 %
acetone/CH2Cl2) to furnish a pale yellow gum (0.7mg), whose 1H NMR
(CDCl3) spectroscopic and HR-MS data were identical to those of 4.
´
1
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Synthesis of the 2,4-O-dimethyl derivative of 2 and application
of the modified Mosher’s method
A mixture of compound 2 (1.5mg), MeI (20 ml) and K2CO3 (20 mg) in
2-butanone (0.2ml) was stirred at room temperature for 15 h. The mixture
was diluted with EtOAc and washed with H2O, and the organic layer was
concentrated in vacuo to afford the 2,4-O-dimethyl derivative (1.6mg, pale
7
8
1
yellow gum): H NMR (400 MHz, CDCl3) d 10.41 (1H, s, H-8), 5.16 (1H, t,
J ¼ 6.3 Hz, H-10), 5.13 (1H, t, J ¼ 7.0 Hz, H-14), 3.87 (3H, s, 2-OCH3 or 4-
OCH3), 3.82 (3H, s, 4-OCH3 or 2-OCH3), 3.40 (2H, d, J ¼ 6.3 Hz, H-9), 3.39
(1H, m, H-18), 3.21 (3H, s, 19-OCH3), 2.63 (3H, s, H-7), 2.24 (1H, m, Ha-16),
2.07 (2H, m, H-13), 2.02 (2H, m, H-12), 2.01 (1H, m, Hb-16), 1.79 (3H, s,
H-23), 1.58 (3H, s, H-22), 1.49 (1H, m, Ha-17), 1.35 (1H, m, Hb-17), 1.11
(3H, s, H-20), 1.08 (3H, s, H-21). A small portion (0.5mg) of this reaction
product was treated with (ꢀ)-(R)-MTPA-Cl (10 ml) in pyridine (0.2ml) at
room temperature for 15h. The mixture was diluted with EtOAc and washed
with H2O and 1-M NaHCO3, and the organic layer was concentrated in vacuo.
The residue was purified by preparative HPLC (MeCN/H2O) to afford a
(S)-MTPA ester derivative 11a (0.2mg). Similarly, (R)-MTPA ester derivative
11b was prepared using (þ )-(S)-MTPA-Cl. It should be noted that the
definition of R and S at C-2 of MTPA switches by esterification of MTPA-Cl,
due to the priority order of -COCl4-CF34-COOR.
9
12 Ellestad, G. A., Evans, R. H. Jr
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Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics