Achaetolide-II isolated from Helminthosporium velutinum TS28

Article abstract of DOI:10.1016/j.tet.2015.08.013

A C3-O-acylated analogue of achaetolide (2) was isolated from phytopathogenic fungus Helminthosporium velutinum TS28. The relative configuration involving the C3-O-acyl side chain was determined by NMR spectroscopic analysis combined with conformational analysis computer-assisted chemical shift calculations. The absolute configuration was established through ECD analysis after conversion into the dibenzoate (5).

Full text of DOI:10.1016/j.tet.2015.08.013

Accepted Manuscript
Achaetolide-II isolated from Helminthosporium velutinum TS28
Miki Arayama, Hayato Maeda, Kazuaki Tanaka, Noboru Takada, Tatsuo Nehira,
Masaru Hashimoto
PII:
S0040-4020(15)01190-4
10.1016/j.tet.2015.08.013
TET 27035
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 15 June 2015
Revised Date: 31 July 2015
Accepted Date: 3 August 2015
Please cite this article as: Arayama M, Maeda H, Tanaka K, Takada N, Nehira T, Hashimoto M,
Achaetolide-II isolated from Helminthosporium velutinum TS28, Tetrahedron (2015), doi: 10.1016/
j.tet.2015.08.013.
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Achaetolide-II isolated from
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Helminthosporium velutinum TS28
Miki Arayama,a Hayato Maeda,a Kazuaki Tanaka,a Noboru Takada, a Tatsuo Nehira,b and Masaru Hashimoto*a

1
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Tetrahedron Letters
journal homepage: www.elsevier.com
Achaetolide-II isolated from Helminthosporium velutinum TS28
Miki Arayama,a Hayato Maeda,a Kazuaki Tanaka,a Noboru Takada, a Tatsuo Nehira,b
and Masaru Hashimoto*a
a Faculty of Agriculture and Bioscience, Hirosaki University, 3-Bunkyo-cho, Hirosaki, 036-8561, Japan
b Graduate School of Integrated Arts and Sciences, Hiroshima University, 1-7-1, Kagamiyama, Higashi-Hiroshima, 739-8521, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A C3-O-acylated analogue of achaetolide (2) was isolated from phytopathogenic fungus
Helminthosporium velutinum TS28. The relative configuration involving the C3-O-acyl side
chain was determined by NMR spectroscopic analysis combined with conformational analysis
computer-assisted chemical shift calculations. The absolute configuration was established
through ECD analysis after conversion into the dibenzoate (5).
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Nonalide
Conformational analysis
Chemical shift calculations
Electron circular dichroism analysis
1. Introduction
configurational determination of asymmetric centers in the side
chain based on NOE experiments, theoretical chemical shift
computations, and chemical derivatization. The absolute
configuration was investigated by means of an electronic circular
dichroism (ECD) exciton chirality method after conversion into
the 6,7-O-dibenzoate (5). Degradation of 2 and the following
chemical transformations were performed to verify the
stereochemical conclusion.
Several (E)-olefin-containing ten-membered polyketide
macrolides have been obtained from fungal sources, and some of
these show interesting biological activities, such as antimalarial
activity,1 antifungal activities,2,3 activation of peroxisome
proliferator-activated receptor,4 and cytotoxicities.5 Thus, they
have become attractive synthetic targets for organic chemists.6-8
We have recently isolated achaetolide (1)9 from Ophiobolus sp.
and succeeded in determining its absolute configuration,10 which
had remained unknown. During further exploration of secondary
metabolites from ecologically unique microbes,11-13 we isolated
achaetolide-II (2), a 3-O-acylated analogue of 1, from the
mycelium of Helminthosporium velutinum TS28. We have
revealed the C14-epimeric isomers of cochlioquinones from the
culture broth of this fungus.14 The structural studies involve
2. Results and discussion
Achaetolide-II (2) was found as a major component of the
extract (75 mg/g) from the mycelium of H. velutinum TS28. ESI-
MS analysis showed a protonated molecular ion at m/z =
401.2523, suggesting its molecular formula as C21H36O7 ([M+H]:
401.2539). The 1H NMR spectrum indicated that 2 was
comprised of two inseparable components in an approximately
95:5 ratio. These were assigned as tautomeric isomers, because
the ratio varied in methanol-d4 (ca. 80:20 ratio).15 Structural
determination was performed on the main tautomer in CDCl3.
The NMR data obtained in CDCl3 solution are summarized in
Table 1. The 13C NMR spectrum featured 21 resonances, and
measurement of the DEPT spectrum classified these as two
carbonyl, eight methine (including five oxymethines), eight
methylene, and three methyl carbons, and also revealed 33
protons directly linked to the carbon atoms. The signals of the
last three protons were observed at δ 2.19, 2.26, and 3.96 ppm in
CDCl3. These were assigned as hydroxy protons because no
HMQC correlations were observed and the signals disappeared
upon addition of D2O. The vicinal spin coupling between H-4
and H-5 (J = 15.8 Hz) indicated an (E)-configuration for the
C4,C5-double bond. Detailed analysis revealed very small vicinal
spin couplings (J = 0–1.1 Hz) for H-6/H-7, H-7/Hβ-8, and Hβ-
Figure 1. Structures of achaetolide (1) and achaetolide-II (2)
* Corresponding authors. Tel./fax: +81-172 39-3782 (M.H.)
E-mail address: hmasaru@cc.hirosaki-u.ac.jp (M.H)

2
Tetrahedron
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*
Table 1. NMR spectral data of achaetolide-II (2) in CDCl3
.
position
1
δ13C, type
170.59, C
δ1H (J in Hz)
HMBC
NOESY
α: 2.54, dd (3.5, 12.5)
β: 2.76, dd (3.3, 12.5)
5.68, m
6.03, ddd (2.2, 3.7, 15.8)
5.73, ddd (1.1, 2.2, 15.8)
4.55, m
3.80 br d (10.2)
α: 2.36, ddd (8.3, 10.2, 15.8)
β: 1.53, m
4.80, dt (6.6, 8.3)
1.50, m
1, 3,
1, 3, 4
4
3, 6
3, 6
H-3, H-4
H-3
Hα-2, Hβ-2
Hα-2, Hα-8
H-6, H-7, H-9, H-3', H3-5'
H-5, H-7,
2
41.40, CH2
3
4
5
6
7
68.15, CH
126.00, CH
127.45, CH
73.11, CH
75.23, CH
H-5, H-6, Hβ-8, H-9
H-4,
7, 9
8
36.92, CH2
7, 9, 10
1, 8, 10, 11
7, 8, 9, 11
9
10
73.70
36.79
H-5, H-7, Hβ-8,
24.98, CH2, 29.14, CH2,
29.43, CH2, 31.74, CH2,
22.62, CH2
14.07
11-14
1.25, m
15
16
1'
1.25, m
0.87, t (6.7)
-
16
15
173.71
2'
3'
4'
5'
6-OH
7-OH
3'-OH
49.37, CH
69.26, CH
20.69, CH3
14.67, CH3
2.51, m
1', 4', 5'
4', 5'
2', 3'
1', 2', 3'
5, 6, 7
6, 7, C8
3', 4'
H-3'
3.92,dquint (8.4, 6.2)
1.24, d (6.2)
1.20, d (7.2)
2.26, d (3.3)
2.19, d (6.8)
3.96, d (6.2)
H-5, H-2', H3-4', H3-5'
H-3'
H-5, H-3'
*The spectrum indicated that 2 is composed of two major conformers in a ca. 95:5 ratio. Only the spectral data for the major
conformer .
8/H-9, suggesting almost perpendicular relationship for the
dihedral angles between these protons. The ten-membered
lactone framework was established by observing a HMBC signal
between H-9 and C1. The results suggested that 2 possesses the
same framework as achaetolide (1). The major set of 1H
resonances were in good accordance with those of 1 reported by
Bodo et al.,9 except for the H-3 signal. A NOESY spectrum
indicated characteristic correlations at Hα-2/H-4, H-5/H-7, H-
5/H-9, and H-7/H-9, as shown in Fig. 2, which confirmed the
relative configuration as well as the conformation. It was also
revealed that the minor component evident in the NMR spectrum
was a conformational isomer; although only a few of its signals
were analyzable.
remarkably high frequency for H-3 (5.68 ppm) indicated that C3-
alkoxy group is responsible to an ester linkage with the side
chain. 3-Hydroxy-2-methylbutanoic acid would be readily
eliminated in the mass spectrometer to give a fragment ion at m/z
= 283.1897 ([M–C5H9O3]+) with almost the same intensity as that
of [M+H]+ because of the β-acyloxy carbonyl system.
Interestingly, H-3' gave a NOESY correlation with H-5, as
shown in Fig. 3. This suggested that the C1'-C5' side chain
surrounds the nonalide framework. We assumed a hydrogen
bonding between the C3' alcoholic proton and the C1 carbonyl
oxygen on the basis of the following observations: (a) the C3'-
OH signal appeared at higher frequency (3.96 ppm) than those of
the other two alcoholic protons (2.19 and 2.26 ppm) in CDCl3,16
(b) when 3'-OH was masked in a form of acetate (→ 3), the NOE
between H-5 and H-3’ disappeared, and (c) the above
transformation significantly changed the conformational ratio (to
75:25) in CDCl3. Acetate 3 was prepared from 2 by successive
treatments with (i) p-TsOH, acetone ( → 4), (ii) Ac2O, pyridine,
The COSY spectrum further revealed
a 3-hydroxy-2-
methylbutanoate substructure (C1'-C5' side chain) in 2. A
Figure 2. 3JHH spin couplings (blue arrows) and crucial NOEs
(red arrows) of 2
Figure 3. Middle frequency range of NOESY spectrum of 2

3
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Figure 6. ECD spectrum of dibenzoate 4.
Figure 4. The most stable conformation of the (3S*,2'S*,3'S*)-
isomer of 2 based on EDF2/6-31G*. The C9 side chain was
manually elongated after the calculations.
deviations showed a similar tendency, albeit less markedly than
for the 13C shifts. These observations were in accordance with the
above results. Thus, we determined the total relative
configuration of 2 as the (3S*,6R*,7S*,9R*,2'S*,3'S*)-form.
and (iii) p-TsOH, MeOH.17
Molecular modeling calculations also suggested hydrogen
bonding, irrespective of the configurations at C2' and C3'. The
(3S*,2'S*,3'S*)- and (3S*,2'R*,3'S*)-isomers can explain the
NOE between H-5 and H-3’. Calculations were performed with
EDF2/6-31G*18 after a conformational search with MMFF.19 We
replaced the C10-C16 heptyl side chain with a methyl group in
the calculations in order to reduce the number of conformers to
be considered. Entropic factors (∆S) were taken into
consideration with the vibrational analyses in view of the
conformational flexibility of the C1'-C5' group.20 The most stable
conformation of (3S*,2'S*,3'S*)-isomer is shown in Fig. 4.
Next, we turned our attention to the absolute configuration.
Conversion into 1 under basic conditions was not satisfactory,
because these conditions also hydrolyzed the nonalide ring. We
applied Nakanishi’s dibenzoate rule23 after conversion into
dibenzoate 5. A UV-insensitive acetyl group was furnished at the
C3'-alcohol to avoid undesired ECD interaction.24 Compound 5
was readily prepared by benzoylation of 3. The 1H NMR
spectrum of 5 showed it to be a 5:1 mixture of conformational
3
3
3
isomers. The vicinal spin couplings JH-5/H-6, JH-6/H-7, JH-7/Hβ-8
,
3
and JHβ-8/H-9 for the major conformer of 4 were 0–2 Hz, in good
accordance with those observed in 2. These results suggested that
the conformation of 2 is retained in the major conformer of 5.
Molecular modeling calculations supported this interpretation
(see Supplementary data). Thus, we can simply apply the
benzoate for 5. As shown in Fig. 6, 5 gave a typical split ECD
with a negative Cotton effect at 236 nm (∆ε –19) and a positive
one at 220 nm (∆ε +7.6), revealing a negative chirality for the
C6-C7 diol moiety in 2, that is, a (6R,7S)-configuration. This was
consistent with the absolute configuration of 1.10 Accordingly, we
The δ value of C-5’ (14.67 ppm) empirically suggested threo-
configuration for the 3-hydroxy-2-methylbutanoate moiety,21 i.e.
(2'S*,3'S*)-form in 2. It was further investigated with chemical
shift calculations.11-13 The sets of stable conformers were
subjected to chemical shift calculations with EDF2/6-31G* to
obtain the theoretical chemical shifts after a correction based on
the Boltzmann distribution. Minor conformers of the nonalide
1
moiety were excluded, because their H NMR signals appeared
separately. Deviations between theoretical and experimental
chemical shifts (∆δ = δexperimental – δcalculated, ppm) for the relevant
carbons were plotted to give Fig. 5. The chemical shifts for C1 in
the models were estimated to appear at considerably higher
frequency than that observed experimentally. Replacement of the
C9-heptyl side chain by a non-bulky methyl group presumably
leads to magnetic deshielding of C1 which is a so-called steric
compression effect.22 The (3S*,2'S*,3'S*)-isomer clearly gave the
highest scores among the isomers. The 1H chemical shift
conclude that the absolute configuration of
2
is the
(3S,6R,7S,9R,2'S,3'S)-form.
Methanolysis of 4 under basic conditions afforded methyl
threo-3-hydroxy-2-methylbutanoate (6a)25,26 and the previously
prepared diol 7a.10 These were further converted into (S)- and
(R)-MTPA esters (S)-6b, (R)-6b, (S)-7b and (R)-7b. Application
of the extended-Mosher method27 disclosed (3S,9R,2’S,3’S)-
configuration. These experiments verified the conclusion
described above.
3. Biological properties
In studies carried out to date, achaetolide-II (2) showed no
Figure 5. Chemical shift differences (∆δ, ppm) from the
experimental data for the respective isomers based on EDF2/6-
31G*.
Scheme 1.

4
Tetrahedron
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4.5. 6,7-O-isopropylidene achaetolide-II (4).
antifungal activity against Cochliobolus miyabeanus (IC50
>100 µg/mL). A cytotoxicity assay revealed moderate growth
inhibition of human colon adenocarcinoma (COLO 201) cells
(IC50 370 µg/mL). This molecule also inhibited root growth of
Lactuca sativa at 500 µg/mL.
A solution of 2 (49.0 mg, 122 µmol) in acetone (1.5 mL) was
left to stand in the presence of p-TsOH (1.5 mg) at room
temperature for 3 h. Et3N (one drop) was then added, and the
mixture was concentrated in vacuo. Column chromatography of
the residue on silica gel (hexane/EtOAc, 70:30) gave 4 (45.4 mg,
84%). The 1H NMR spectrum in CDCl3 indicated that the sample
4. Experimental
consisted of three conformers in
a 3:23:74 ratio. Only
representative signals for the major isomer are described: δ 0.87
(3H, t, J = 6.7 Hz), 1.18 (3H, d, J = 7.1 Hz), 1.22 (3H, d, J =
6.4 Hz), 1.25 (10H, m), 1.33, 1.44 (each 3H, s), 1.51 (1H, m),
1.60 (1H, d, J = 16.0 Hz), 1.61 (1H, m), 2.21 (1H, dt, J =16.0,
10.4 Hz), 2.42 (1H, dd, J = 7.8, 13.7 Hz), 2.46 (1H, quint J =
7.1 Hz), 3.12 (1H, dd, J = 7.8, 13.7 Hz), 3.90 (1H, m), 4.13 (1H,
m), 4.61 (1H, dd, J = 5.9, 9.1 Hz), 4.90 (1H, m), 5.57 (1H, q, J =
7.8 Hz), 5.69 (1H, dd, J = 7.8, 16.0 Hz), 5.89 (1H, dd, J = 9.1,
16.0 Hz).
4.1. General
Ultraviolet (UV) spectra were obtained using a Hitachi U-
2010 spectrophotometer. Electronic circular dichroism (ECD)
spectra were measured on a JASCO J-725 spectrometer. NMR
spectra were recorded on a JEOL JNM-ECX500 spectrometer.
Tetramethylsilane was used as internal standard (0 ppm) for both
1H and 13C NMR spectra in CDCl3. When deuteriomethanol was
used as the solvent, the residual proton signal (CD2HOD) was
used as an internal standard (3.31 ppm) in the 1H NMR spectrum.
In 13C NMR spectra recorded in deuteriomethanol, the solvent
signal was employed as an internal standard (13CD3OD:
49.15 ppm). Mass spectra were measured in electrospray
ionization (ESI) mode on a Hitachi NanoFrontier LD mass
spectrometer. In the assay, samples were observed at ×20
magnification using an Olympus CKX31 inverted microscope.
4.6. 3'-O-Acetyl-achaetolide-II (3).
A solution of the acetonide 4 thus obtained (45.4 mg,
103 µmol) in a mixture of pyridine (1.0 mL) and Ac2O (0.1 mL)
was left to stand at room temperature for 12 h. After
concentration in vacuo, column chromatography of the residue
on silica gel (hexane/EtOAc, 90:10) gave the acetate (48.6 mg,
97 %). The 1H NMR spectrum in CDCl3 indicated that the sample
was a mixture of two conformational isomers in a 9:91 ratio.
Only the signals for the major isomer are described: δ 0.88 (3H, t,
J = 6.7 Hz), 1.15 (3H, d, J = 7.2 Hz), 1.22 (3H, d, J = 6.4 Hz),
1.25 (10H, m), 1.33, 1.43 (each 3H, s), 1.51 (1H, m), 1.60 (1H, d,
J = 16.0 Hz), 1.62 (1H, m), 2.01 (3H, s), 2.21 (1H, dt, J = 16.0,
10.4 Hz), 2.33 (1H, dd, J = 8.5, 13.6 Hz), 2.69 (1H, quint, J =
7.2 Hz), 3.13 (1H, dd, J = 8.5, 13.6 Hz), 4.12 (1H, dd, J = 5.9,
10.4 Hz), 4.61 (1H, dd, J = 5.9, 9.1 Hz), 4.91 (1H, m), 5.09 (1H,
dq, J = 7.2, 6.4 Hz), 5.58 (1H, q, J = 8.5 Hz), 5.68 (1H, dd, J =
8.5, 15.9 Hz), 5.89 (1H, dd, J = 9.1, 15.9 Hz). A solution of the
acetate thus obtained (28.6 mg, 59 µmol) in MeOH (1.0 mL) was
left to stand in the presence of p-TsOH (1.0 mg) at room
temperature for 10 h. After neutralization by adding Et3N, the
mixture was concentrated in vacuo. Column chromatography of
the residue on silica gel (hexane/EtOAc, 85:15 → 45:55) gave 3
(13.8 mg, 49%) and recovered alcohol (12.7 mg, 49%),
respectively. [α]D25 −28 (c 0.5, CHCl3); IR (film): ν=3450, 2950,
4.2. Fungus.
The fungus was isolated from the surface of dead branches of
Sambucus sieboldiana around Shinjuku Gyoen National Garden,
Tokyo, Japan in 2002 and was identified as Helminthosporium
velutinum based on its morphological features.28 The fungus has
been deposited at the NIAS (National Institute of Agrobiological
Sciences) Genebank, Japan (ID: MAFF 243854).29
4.3. Calculations.
Conformational searches and chemical shift calculations were
performed using Spartan 14 (Wavefunction, Irvine, CA) installed
on a PC (Operating System: Windows 7 Professional, CPU:
Intel(Xeon) E5-1660 v2 processor 3.70 GHz, RAM 64 GB).
Theoretical ECD spectra were calculated with Gaussian 09
(Revision A.02 by Gaussian Inc., Wallingford, CT) with a PC
(Operating System: CentOS a Linux, CPU: 2 Intel Xeon 3 5550
processors 2.67 GHz, RAM 24 GB).
1
2930, 2860, 1740, 1240, 1160 cm-1. The H NMR spectrum in
CDCl3 indicated that the sample was a mixture of two
conformational isomers in a 3:1 ratio. Only representative signals
are described: (a 0.75, b 0.25) δ 0.87 (3H, m) 1.15 (3H × b, d, J =
7.0 Hz), 1.22 (3H × a, d, J = 7.1 Hz), 1.25 (3H × a, d, J = 6.3 Hz),
1.25 (10H, m), 1.49 (1H × a, d, J = 15.9 Hz), 1.52 (2H, m), 2.00
(3H × a, s), 2.01 (3H × b, s), 2.12 (1H × a, d, J = 6.8 Hz), 2.17
(1H × a, d, J = 3.4 Hz), 2.22 (1H × b, d, J = 6.4 Hz), 2.35 (1H × a,
m), 2.36 (1H × b, m), 2.42 (1H × b, d, J = 1.8 Hz), 2.55 (1H × a,
dd, J = 3.9, 12.7 Hz), 2.67 (1H × a, dd, J = 3.0, 12.7 Hz), 2.70
(1H × b, m), 2.76 (1H × a, quint., J = 7.1 Hz), 3.13 (1H × b, dd, J
= 7.6, 14.1 Hz), 3.60 (1H × b, m), 3.78 (1H × a, br t, J = 8.4 Hz),
4.36 (1H × b, br d, J = 7.0 Hz), 4.53 (1H × a, m), 4.75 (1H × a, dt,
J = 8.2, 6.3 Hz), 5.10 (1H × b, m), 5.12 (1H × a, dq, J = 7.2,
6.3 Hz), 5.48 (1H × b, m), 5.49 (1H × b, m), 5.61 (1H × a, m),
5.70 (1H × a, ddd, J = 1.0, 2.3, 15.8 Hz), 5.79 (1H × b, dd, J =
8.0, 15.5 Hz), 6.04 (1H × a, ddd, J = 2.1, 3.7, 15.8 Hz). Only the
13C NMR signals in CDCl3 for the major conformer are described.
δ 12.88, 14.08, 16.95, 21.24, 22.63, 25.00, 29.17, 29.45, 31.76,
36.79, 36.93, 41.34, 44.74, 68.66, 71.42, 73.03, 73.22, 75.30,
126.63, 127.17, 168.97, 170.15, 172.66. ESI-MS: m/z 443.2643
(C23H38O8, [M+H]+: 443.2639), 465.2459 (C23H38O8, [M+Na]+:
465.2459), 907.5018 (C46H76O16, [2M+Na]+: 907.5018).
4.4. Isolation.
Helminthosporium velutinum TS28 was cultured in potato-
sucrose medium (200 mL in 500 mL baffled Erlenmeyer flask
× 40) on a rotary shaker (110 rpm) at 27 °C for 14 days. After
filtration in suction, the recovered mycelium (820 g, wet weight)
was suspended in MeOH (2.2 L) at room temperature for 24 h.
After filtration in suction, the filtrate was concentrated under
reduced pressure until the volume became 300 mL. Water
(200 mL) was then added, and the resulting suspension was
extracted with EtOAc (2 × 1.0 L). The combined EtOAc extracts
were concentrated to give a crude residue (698 mg), which was
subjected to column chromatography on silica gel. The fraction
eluted with EtOAc/hexane (80:20) was recovered and
concentrated to give partially purified 2 (102 mg). A portion of
the sample (50 mg) was further purified by medium-pressure
column chromatography (Yamazen Universal Column ODS-SM
50 µm 120A, 50–80% MeOH/H2O for 100 min, 8.0 mL/min
flow) to give achaetolide II (2, 32 mg). The H and 13C NMR
1
spectral data are shown in Table 1. ESI-MS data are shown in the
text. Other physical data were follows: [α]D25 −41 (c 1.0, CHCl3);
UV data are shown in the text. IR (film): ν=3480, 2960, 2930,
2860, 1720, 1270, 1160 cm-1.
4.7. 3'-O-Acetyl-6,7-O-benzoyl achaetolide-II (5).

5
A solution of 3 (12.7 mg, 29 µmol) and benzoyl chloride
(20 µL, 0.17 mmol) in CH2Cl2 (0.5 mL) was stirred at room
temperature for 12 h. MeOH (50 µL) was then added and the
mixture was further stirred at room temperature for 1 h. It was
poured into water (15 mL) and extracted with diethyl ether
(15 mL × 2). The combined ethereal extracts were washed with
saturated aqueous NaCl solution, dried over MgSO4, and then
concentrated in vacuo. Column chromatography of the residue on
A solution of 6a (ca. 2.0 mg) in pyridine (0.2 mL) was
ACCEPTED MANUSCRIPT
stirred with (R)-MTPACl (ca. 10 mg) at room temperature for 2 h.
After MeOH (10 mL) was added, the mixture was further stirred
at room temperature for 20 min. Diethyl ether (1.0 mL) was
added and the resulting suspension was filtered through a cotton
pad and the filtrated was concentrated in vacuo. Preparative TLC
(hexane/AcOEt, 90:10) gave the MTPA ester (S)-6b (ca. 2.0 mg).
In the similar manner as described for the preparation of (S)-6b,
(R)-MTPA ester (R)-6b (ca. 1.5 mg) was prepared by employing
(S)-MTPACl in place of (R)-MTPACl. The yields of these
samples were not accurate because of small quantity. The δ∆
values, as shown below, suggested (3S)-configuration.
25
silica gel (hexane/AcOEt, 80:20) gave 5 (15.0 mg, 79%). [α]D
−32 (c 1.0, CHCl3); IR (film): ν=2930, 2860, 1730, 1260,
1
710 cm-1. The H NMR spectrum in CDCl3 indicated that the
sample was a mixture of two conformational isomers in a 5:1
ratio. Only representative signals are described: (a 0.83, b 0.17)
× 0.87 (3H, t, J = 6.7 Hz), 1.17 (3H × b, d, J = 7.1 Hz), 1.26 (3H
× a, d, J = 7.1 Hz), 1.28 (3H × a, d, J = 6.3 Hz), 1.28 (11H, m),
1.60 (1H × a, m), 1.77 (1H × a, d, J = 15.6 Hz), 2.02 (3H, s), 2.50
(1H × b, dd, J = 8.1, 13.4 Hz), 2.61 (1H × a, dd, J = 3.9, 12.6 Hz),
2.69 (1H×a, m), 2.70 (1H × b, m), 2.72 (1H × a, dd, J = 3.2,
12.6 Hz), 2.77 (1H × a, dq, J = 8.3, 7.1 Hz), 3.12 (1H × b, dd, J =
8.1, 13.4 Hz), 4.97 (1H × b, m), 5.08 (1H × a, q, J=8.5 Hz), 5.11
(1H × a, dq, J = 8.3, 6.3 Hz), 5.25 (1H × a, br d, J = 10.1 Hz),
5.56 (1H × b, q, J = 8.1 Hz), 5.62 (1H × a, m), 5.82 (1H × b, dd, J
= 8.1, 16.7 Hz), 5.91 (1H × a, ddd, J = 1.0, 2.8, 16.0 Hz), 5.93
(1H × b, dd, J = 7.5, 16.7 Hz), 5.98 (1H × a, ddd, J = 2.0, 3.5,
16.0 Hz), 6.05 (1H × b, dd, J = 3.3, 7.5 Hz), 6.09 (1H × a, m),
7.38 (2H × a, br t, J = 8.1 Hz), 7.48 (2H × b, br t, J = 8.1 Hz),
7.52 (2H × b, m), 7.53 (2H × a, br t, J = 8.1 Hz), 7.60 (1H × b, br
t, J = 8.1 Hz), 7.65 (1H × a, br t, J = 8.1 Hz), 7.91 (2H × b, br d, J
= 8.1 Hz), 7.93 (2H × a, br dd, J = 1.4, 8.3 Hz), 8.07 (2H × b, br
δ∆
(S)-6b (J , Hz)
CH3O- 3.64 (s)
(R)-6b (J , Hz)
3.54 (s)
(ppm)
+0.10
+0.02
-0.01
-0.08
+0.05
H-2
H-3
2.76 (dq, 7.8, 7.2) 2.74 (dq, 7.9, 7.1)
5.36 (dq, 6.3. 7.8) 5.37 (dq, 6.4, 7.9)
H3-4
1.29 (d, 6.3)
1.19 (d, 7.2)
1.37 (d, 6.4)
1.14 (d, 7.1)
H3-5
CH3O-
(MTPA)
3.50
3.54
-
* common signals: 7.4 (3H, m), 7.51 (2H, m).
4.10. (S)- and (R)-MTPA esters (S)-7b and (R)-7b
In the similar manner as described for the preparation of 6b,
Treatment of 7a (ca. 3.5 mg) with (R)-MTPACl afforded (S)-
MTPA ester (S)-7b (1.9 mg). In the similar manner, (R)-7b (ca.
3.8 mg) was also prepared by employing (S)-MTPACl in place of
(R)-MTPACl. The δ∆ values, as shown below, are consistent
with those we have reported previously. The ∆δ could not
obtained for H2-11 and H2-12 (achaetolide numbering) in the
present study because of small quantity of the samples. Signal
assignment for these protons requires a series of 2D NMR spectra
in high resolution.
d, J = 8.1 Hz), 8.16 (2H × a, br dd, J = 1.4, 8.1 Hz). Only the 13
C
NMR signals in CDCl3 for the major conformer are described: δ
13.27, 14.07, 17.23, 21.24, 22.62, 25.12, 29.14, 29.41, 31.77,
35.61, 36.65, 41.35, 44.90, 68.30, 71.51, 72.65, 73.21, 76.09,
123.23, 128.31, 128.41, 128.69, 129.74, 129.77, 133.23, 133.49,
165.02, 165.47, 168.60, 170.24, 172.65. ESI-MS: m/z 651.3154
(C37H46O10, [M+H]+: 651.3164), 673.2970 (C37H46O10, [M+Na]+:
673.2983), 1323.6039 (C74H92O20, [2M+Na]+: 1323.6074).
δ∆
(S)-7b (J , Hz)
3.59 (s)
(R)-7b (J , Hz)
3.66 (s)
(ppm)
CH3O-
(ester)
-0.07
4.8. Basic methanolysis of 4 giving methyl threo-3-hydroxy-2-
methylbutanoate (6a) and diol 7a.
H-2 2.61 (dd, 4.7, 16.0)
2.77 (dd, 8.9, 16.0)
2.64 (dd, 4.5, 16.5)
2.80 (dd, 9.2, 16.5)
-0.03
-0.03
H-3 5.88 (ddd, 4.7, 6.4, 8.9) 5.86 (ddd, 4.5, 6.5, 9.2) +0.02
A suspension of acetonide 4 (83.0 mg, 189 µmol) and K2CO3
(40 mg, 289 mmol) in methanol (4.0 mL) was stirred at room
temperature for 3 h. The mixture was poured into aqueous 1.0 M
HCl solution (50 mL) and extracted with AcOEt (50 mL ×3). The
organic layer was combined, washed with brine (100 mL),
washed with saturated aqueous NaCl solution, dried over MgSO4,
and then concentrated in vacuo. Silica gel column
chromatography of the residue eluted successively with 80:20
and 60:40 hexane/AcOEt to give 6a (ca. 10 mg) and 7a (32.6 mg).
Taking the volatile property into account, 6a was not fully
concentrated. 1H NMR spectral data 6a: (CDCl3) δ 1.19 (3H, d, J
= 7.2 Hz), 1.23 (3H, d, J = 6.3 Hz), 2.46 (1H, quint, J = 7.2 Hz),
H-4 5.74 (dd, 6.4, 16.0)
H-5 5.80 (dd, 4.9, 16.0)
H-6 4.30 (dd, 4.9, 6.4)
H-7 3.90 (m)
H2-8 1.52 (m)
H-9 5.21 (m)
5.68 (dd, 6.5, 15.5)
5.74 (dd, 5.7, 15.5)
4.47 (t, 5.7)
4.15 (m)
1.61 (m)
5.23 (m)
+0.06
+0.06
-0.17
-0.25
-0.09
-0.02
-
CH3- 1.28 and 1.45
1.32 and 1.43
CH3O-
3.49, 3.57
(MTPA)
3.51, 3.54
-
*common signals: 0.86 (3H, t, J = 7.1 Hz), 1.2 (10H, m), 1.6 (2H,
m), 7.4 (3H, m), 7.51 (2H, m).
1
2.65 (1H, br), 3.72 (3H, s), 3.89 (1H, m). The H NMR spectral
4.11. Inhibition assay against C. miyabeanus.
data of this sample were accorded well with threo-methyl 3-
hydroxy-2-methylbutanoate but not with the corresponding
erythro-isomer.25 1H NMR spectral data 7a: (CDCl3) δ 0.88 (3H,
t, J = 7.1 Hz), 1.28 (10H, m), 1.37 (3H, s), 1.41 (1H, ddd, J = 3.2,
8.8, 14.0 Hz), 1.46 (2H, m), 1.48 (3H, s), 1.63 (1H, ddd, J = 2.8,
10.2, 14.0 Hz), 2.57 (2H, m), 3.02 (1H, brd, J = 4.2 Hz), 3.72
(3H, s), 3.79 (1H, m), 4.45 (1H, ddd, J = 3.4, 6.4, 10.2 Hz), 4.58
(2H, m), 5.72 (1H, dd, J = 6.9, 15.5 Hz), 5.80 (1H, dd, J = 5.2,
15.5 Hz). The spectral data of this sample were accorded well
with that we previously reported.10
A series of suspensions of spores of Cochlibolus miyabeanus
and 0.1% sucrose containing 0.01, 0.1, 1, 10, and 100 µg/mL of
samples in Petri dishes was prepared and incubated at 25 °C for
24 h. These were observed with a microscope.
4.12. Cytotoxicity assay against human colon adenocarcinoma
(COLO 201) cells.
The effect of compounds on human colon adenocarcinoma
(COLO 201) cell proliferation was measured by WST-1 assay.30
COLO 201 cells were cultured in RPMI medium (5×103
cells/mL) containing a series of compounds in 96-well tissue
culture plates with concentrations of 1.0, 5.0, 10, 50, 100,
4.9. (S)- and (R)-MTPA esters (S)-6b and (R)-6b

6
Tetrahedron
ACCEPTED MANUSCRIPT
500 µg/mL. After 24 h, 10 µL of WST-1 reagent was added to
16. The chemical shift for 3’-OH was relatively reproducible (3.96-
each well and the absorbance was measured at 450 nm using a
titer-plate reader.
4.04 ppm), while those of 6-OH and 7-OH varied considerably
(2.26-2.57, 2.18-2.48 ppm, respectively).
17. Interestingly, acetonide formation reversed the conformational
ratio of the nonalide moiety from 95:5 to 24:76.
18. Lee, S. B.; Lin, C. Y.; Gill, P. M. W.; Webster, R. D. J. Org.
Chem. 2005, 70.
19. Halgren, T. A. J. Comp. Chem. 1996, 17, 490-519.
20. Modeling softwares estimate only steric energies (enthalpy) in
the general calculations. Thermodynamic calculations are
required to obtain ∆S. The costs for the thermodynamic
calculations are much higher than those of structural
optimization. In these particular cases, remarkable differences
were not found from those without considering ∆S.
21. Heathcock, C. H.; Pirrung, M. C.; Sohn, J. E. J. Org. Chem.
1979, 44, 4294-4299.
4.13. Growth inhibition assay against Lactuca sativa.
Samples of 0, 25, 50, 100, 250, 500, and 1000 µg were
dissolved in MeOH (1.0 mL). Each solution was loaded on a
55 mmφ filter paper and the solvent was removed at room
temperature. Each filter paper was placed on a Petri dish
(70 mmφ), and distilled water (1.0 mL) and ten sprouts of L.
sativa were added. After incubation for 3 days at 25 °C, the roots
and stems were measured with a ruler.
5. Acknowledgments
Part of this work was supported by a JSPS Grant-in-Aid for
Exploratory Research (26660093, 15H04491). The authors would
like to thank Enago (www.enago.jp) for the English language
review.
22. Prashad, M.; Seth, M.; Bhaduri, A. P.; Banerji, A. Org. Magn.
Resonance 1980, 14, 74-75.
6. Supplementary data
23. Harada, N.; Nakanishi, K.; Berova, N. In Comprehensive
Chiroptical Spectroscopy: Applications in Stereochemical
Analysis of Synthetic Compounds, Natural Products, and
Biomolecules Berova, N.; Polavarapu, P. L.; Nakanishi, K.;
Woody, R. W. Eds.; Wiley: New Jersey, USA, 2012; pp. 115-
166.
Supplementary data associated with this article can be found,
in the online version, at http:
References and notes
24. Hashimoto, M.; Tsushima, T.; Murakami, T.; Nomiya, M.;
Takada, N.; Tanaka, K. Bioorg. Med. Chem. Lett. 2008, 18,
4228-4231.
1.
Rukachaisirikul, V.; Pramjit, S.; Pakawatchai, C.; Isaka, M.;
Supothina, S. J. Nat. Prod. 2004, 67, 1953-1955.
2. Wu, S.-H.; Chen, Y.-W.; Shao, S.-C.; Wang, L.-D.; Li, Z.-Y.;
Yang, L.-Y.; Li, S.-L.; Huang, R. J. Nat. Prod. 2008, 71, 731-
734.
3. Boonphong, S.; Kittakoop, P.; Isaka, M.; Pittayakhajonwut, D.;
Tanticharoen, M.; Thebtaranonth, Y. J. Nat. Prod. 2001, 64,
965-967.
4. Hiep, N. T.; Choi, Y.-h.; Kim, N.; Hong, S. S.; Hong, S.-B.;
Hwang, B. Y.; Lee, H.-J.; Lee, S.-J.; Jang, D. S.; Lee, D. J.
Nat. Prod. 2012, 75, 784-788.
5. Lu, S.; Kurtán, T.; Yang, G.; Sun, P.; Mándi, A.; Krohn, K.;
Draeger, S.; Schulz, B.; Yi, Y.; Li, L.; Zhang, W. Eur. J. Org.
Chem. 2011, 2011, 5452-5459.
25. Tai, A.; Imaida, M. Bull. Chem. Soc. Jpn. 1978, 51, 1114-1117
26. Relative configuration of 6a could be verified by converting
into acetonide 8 in two steps (LiAlH4/THF then p-
TsOH/acetone). Spin coupling between the two methine
protons of 8 was 9.8 Hz to establish its relative configuration.
1H NMR data of 8 (in CDCl3) δ 0.76 (3H, d, J = 6.7 Hz), 1.18
(3H. d. J = 6.0 Hz), 1.40, 1.45 (each 3H, s), 1.57 (1H, m), 3.51
(1H, t, J = 11.6 Hz), 3.60 (1H, dq, J = 9.8, 6.7Hz), 3.68 (1H, dd,
J = 5.0, 11.6 Hz).
6. Ramesh, P.; Chennakesava Reddy, B.; Meshram, H. M.
Tetrahedron Lett. 2012, 53, 3735-3738.
7. Yadav, J. S.; Anantha Lakshmi, K.; Mallikarjuna Reddy, N.;
Swapnil, N.; Prasad, A. R. Tetrahedron: Asymmetry 2012, 23,
1155-1160.
8. Mohapatra, D. K.; Reddy, D. P.; Dash, U.; Yadav, J. S.
Tetrahedron Lett. 2011, 52, 151-154.
27. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.
Chem. Soc. 1991, 113, 4092.
28. Shirozu, T.; Harada, Y. Mycoscience 2004, 45, 17-23.
29. http://www.gene.affrc.go.jp/distribution-micro_en.php.
30. Ishiyama, M.; Shiga, M.; Sasamoto, K.; Mizoguchi, M.; He, P.-
G. Chem. Pharm. Bull. 1993, 41, 1118-1122.
9. Bodo, B.; Molho, L.; Davoust, D.; Molho, D. Phytochemistry
1983, 22, 447-451.
10. Tayone, W. C.; Shindo, S.; Murakami, T.; Hashimoto, M.;
Tanaka, K.; Takada, N. Tetrahedon 2009, 65, 7464-7467.
11. Hirose, A.; Maeda, H.; Tonouchi, A.; Nehira, T.; Hashimoto,
M. Tetrahedron 2014, 70, 1458-1463.
12. Yasumura, R.; Ashtekar, K. D.; Tonouchi, A.; Nehira, T.;
Borhan, B.; Hashimoto, M. Tetrahedron 2013, 69, 9469-9474.
13. Takekawa, H.; Tanaka, K.; Fukushi, E.; Nehira, T.; Hashimoto,
M. J. Nat. Prod. 2013, 76, 1047–1051.
14. Arayama, M.; Nehira, T.; Maeda, H.; Tanaka, K.; Miyagawa,
H.; Ueno, T.; Hosokawa, S.; Hashimoto, M. Tetrahedron 2015,
71, 4788-4794.
15. Hashimoto, M.; Matsumoto, M.; Terashima, S. Tetrahedron
2003, 59, 3041-3062.

ACCEPTED MANUSCRIPT
Supplemental data
Achaetolide-II isolated from
Helminthosporium velutinum TS28
M. Arayama, H. Maeda, K. Tanaka, N. Takada, T. Nehira, and M. Hashimoto*a
INDEX
1H NMR spectrum (500 MHz) of achaetolide-II (2) in CDCl3
13C NMR spectrum (125 MHz) of achaetolide-II (2) in CDCl3
COSY spectrum of achaetolide-II (2) in CDCl3
S-2
S-3
S-4
S-5
S-6
S-7
S-8
S-9
HMQC spectrum of achaetolide-II (2) in CDCl3
HMBC spectrum of achaetolide-II (2) in CDCl3
NOESY spectrum of achaetolide-II (2) in CDCl3
Difference NOE spectra of achaetolide-II (2) in CDCl3
1H NMR spectrum (500 MHz) of 3’-O-acetyl-6,7-O-isopropylidene achaetolide-II in CDCl3
Main conformer of 3’-O-acetyl-6,7-O-isopropylidene-achaetolide-II
and its 1H spin couplings of in CDCl3
1H NMR spectrum (500 MHz) of 3’-O-acetyl-achaetolide-II (3) in CDCl3
13C NMR spectrum (125 MHz) of 3’-O-acetyl-achaetolide-II (3) in CDCl3
1H NMR spectrum (500 MHz) of 3’-O-acetyl-6,7-O-dibenzoyl-achaetolide-II (5) in CDCl3
S-10
S-11
S-12
S-13
13C NMR spectrum (125 MHz) of 3’-O-acetyl-6,7-O-dibenzoyl-achaetolide-II (5) in CDCl3 S-14
1H NMR spectrum (500 MHz) of 6a in CDCl3
S-15
S-16
S-17
S-18
S-19
S-20
1H NMR spectrum (500 MHz) of (S)-MTPA ester (S)-6b in CDCl3
1H NMR spectrum (500 MHz) of (R)-MTPA ester (R)-6b in CDCl3
1H NMR spectrum (500 MHz) of 7a in CDCl3
1H NMR spectrum (500 MHz) of (S)-MTPA ester (S)-7b in CDCl3
1H NMR spectrum (500 MHz) of (R)-MTPA ester (R)-7b in CDCl3
Stable conformation of 3’-O-acetyl-6,7-O-dibenzoyl-achaetolide-II (5) based on 1H spin
coupling and molecular modeling calculation for the model based on EDF2/6-31G*
Results of chemical shift calculations with EDF2/6-31G* for achaetolide-II (2)
S-21
S-22
S-1

ACCEPTED MANUSCRIPT
1H NMR (500 MHz) spectrum of achaetolide-II (2) in CDCl3
S-2

ACCEPTED MANUSCRIPT
13C NMR spectrum (125 MHz) of achaetolide-II (2) in CDCl3
S-3

ACCEPTED MANUSCRIPT
COSY spectrum of achaetolide-II (2) in CDCl3
S-4

ACCEPTED MANUSCRIPT
HMQC spectrum of achaetolide-II (2) in CDCl3
S-5

ACCEPTED MANUSCRIPT
HMBC spectrum of achaetolide-II (2) in CDCl3
S-6

ACCEPTED MANUSCRIPT
NOESY spectrum of achaetolide-II (2) in CDCl3
S-7

ACCEPTED MANUSCRIPT
Difference NOE spectra of achaetolide-II (2) in CDCl3
H
O
O H15C7
H
3'
Me
Me
2'
O
H
H
NOE
O
O
OH
OH
S-8

ACCEPTED MANUSCRIPT
1H NMR spectrum (500 MHz) of 3’-O-acetyl-6,7-O-isopropylidene-achaetolide-II in CDCl3
S-9

ACCEPTED MANUSCRIPT
Main conformer of 3’-O-acetyl-6,7-O-isopropylidene-achaetolide-II and its 1H spin couplings of in CDCl3
10.4 Hz
H
~ 0 Hz
H
H
8.5 Hz
8.5 Hz
O
H
5.9 Hz
~ 0 Hz
H
H
O
H
O
O
8.5 Hz
9.1 Hz
H
H
15.9 Hz
O10.4 Hz
H
CH3
H3C
Conformation of 3'-O-acetyl-6,7-O-isopropylidene achaetolide II
S-10

ACCEPTED MANUSCRIPT
1H NMR spectrum (500 MHz) of 3’-O-acetyl-achaetolide-II (3) in CDCl3
OAc
O
O
C7H15
O
O
OH
OH
S-11

ACCEPTED MANUSCRIPT
13C NMR spectrum (125 MHz) of 3’-O-acetyl-achaetolide-II (3) in CDCl3
OAc
O
O
C7H15
O
O
OH
OH
S-12

ACCEPTED MANUSCRIPT
1H NMR spectrum (500 MHz) of 3’-O-acetyl-6,7-O-dibenzoyl-achaetolide-II (5) in CDCl3
OAc
O
O
C7H15
O
O
OBz
OBz
S-13

ACCEPTED MANUSCRIPT
13C NMR spectrum (125 MHz) of 3’-O-acetyl-6,7-O-dibenzoyl-achaetolide-II (5) in CDCl3
OAc
O
O
C7H15
O
O
OBz
OBz
S-14

ACCEPTED MANUSCRIPT
1H NMR spectrum (500 MHz) of 6a in CDCl3
OH
O
OMe
S-15

ACCEPTED MANUSCRIPT
1H NMR spectrum (500 MHz) of (S)-MTPA ester (S)- 6b in CDCl3
OMTPA
O
OMe
S-16

ACCEPTED MANUSCRIPT
1H NMR spectrum (500 MHz) of (R)-MTPA ester (R)-6b in CDCl3
OMTPA
O
OMe
S-17

ACCEPTED MANUSCRIPT
1H NMR spectrum (500 MHz) of 7a in CDCl3
O
OH
C7H15
O
O
O
OH
S-18

ACCEPTED MANUSCRIPT
1H NMR spectrum (500 MHz) of (S)-MTPA ester (S)-7b in CDCl3
O
OMTPA
C7H15
O
O
O
OMTPA
S-19

ACCEPTED MANUSCRIPT
1H NMR spectrum (500 MHz) of (R)-MTPA ester (R)-7b in CDCl3
O
OMTPA
C7H15
O
O
O
OMTPA
S-20

ACCEPTED MANUSCRIPT
Stable conformation of 3'-O-acetyl-6,7-O-dibenzoyl-achaetolide-II (5) based on 1H spin coupling (left) and molecular modeling calculation for the model based
on EDF2/6-31G* (right)
The most stable conformation based on EDF2/6-31G*
1H spin couplings
O
3.2 Hz
H 2.72
5.08 H
~ 0 Hz
O
O
8.5 Hz
5.91
R
H
5.25
O
~0 Hz
H
2.61
~0 Hz
5.62
H
H
2.8 Hz
3.9 Hz
1.77
H
10.1 Hz
16.0 Hz
OBz
H
H
H
6.09
5.98
3.5 Hz
2.70
OBz
S-21

ACCEPTED MANUSCRIPT
Results of chemical shift calculations with EDF2/6-31G*
position
δ C-1
δ C-2
δ C-3
δ C-4
δ C-5
δ C-1'
δ C-2'
δ C-3'
δ C-4' δ C-5'
20.69 14.67
Boltz.
distr.
(%)
∆H
(kJ/mol) (kJ/mol)
∆Gº
ID
170.59
41.40
68.15 125.99 127.45 173.71
49.37
69.26
M0002
M0001
M0017*
M0018
M0005
M0004
M0016
M001[1]
M0048
M005[1]
0.00
5.70
0.00
6.67
0.862
0.058
0.032
0.005
0.006
0.021
0.007
0.003
0.005
0.001
173.73
173.32
170.28
174.07
170.34
173.92
170.46
172.98
169.39
174.29
173.53
40.15
39.40
39.44
40.95
40.38
38.46
40.64
38.02
40.39
39.50
40.05
68.42 126.99 128.65 173.72
71.08 125.72 130.41 173.60
70.24 125.74 129.47 179.17
68.55 127.39 127.94 171.77
69.78 125.89 129.36 177.52
69.79 128.55 136.69 174.51
73.72 123.92 138.73 178.80
71.26 128.03 137.07 174.29
73.37 125.28 138.77 179.07
70.45 131.95 132.25 174.44
68.74 126.88 129.10 173.98
50.71
47.85
43.67
46.20
47.26
50.55
42.97
50.45
43.04
47.54
50.17
69.07
70.93
70.03
74.08
69.18
68.81
70.47
68.97
70.42
70.76
69.24
20.62 15.99
21.99 16.19
22.34 16.73
22.03 15.56
19.03 15.36
20.38 15.99
22.17 17.72
20.51 16.00
22.16 17.68
22.17 16.18
20.77 16.04
8.46
8.14
11.17
14.21
9.98
12.68
12.44
9.14
12.73
14.81
14.42
14.59
11.97
14.31
12.50
16.88
Boltz Avgs
M0037
M0009
M0021
M0001
M0003
M0033
M0019
M0026
M0006
M0002
M0015
M0041*
M0035
M0004
M0046
0.00
0.19
0.21
0.59
1.13
1.30
2.03
2.86
3.15
3.17
3.32
5.15
5.55
5.56
5.99
0.00
1.94
1.76
(0.66)
0.29
1.52
3.07
9.40
3.12
2.10
5.01
10.61
4.67
4.75
6.27
0.161
0.073
0.079
0.210
0.143
0.087
0.046
0.004
0.045
0.069
0.021
0.002
0.024
0.024
0.013
173.77
173.85
173.73
173.66
173.83
173.97
174.01
173.12
173.36
173.97
174.20
169.24
169.78
169.78
170.01
173.54
40.45
39.96
39.94
39.23
38.96
40.56
39.88
39.12
39.25
38.94
39.77
39.05
38.92
38.90
38.75
39.61
68.03 128.32 124.98 173.93
70.26 127.06 128.73 173.85
70.28 127.01 128.79 173.84
71.09 125.77 130.56 176.11
71.58 127.12 126.89 176.02
67.99 127.85 126.14 173.83
70.56 128.07 125.61 173.78
71.40 124.86 135.69 176.55
71.35 123.46 133.01 176.38
71.87 126.83 127.94 176.18
70.86 127.70 126.61 173.87
71.11 124.31 135.66 179.77
71.97 125.76 129.52 179.23
71.94 125.74 129.53 179.23
72.17 127.00 126.34 179.22
70.37 126.87 128.03 175.26
47.94
45.64
45.65
47.82
47.82
47.94
45.62
47.92
48.06
47.71
45.63
43.67
43.59
43.59
43.57
47.11
72.72
72.28
72.27
69.46
69.57
72.76
72.26
69.98
70.00
69.81
72.26
70.82
71.05
71.06
70.98
71.05
22.94 15.28
23.77 17.49
23.75 17.48
21.74 14.00
21.69 14.15
22.96 15.27
23.79 17.53
21.71 14.23
21.74 14.32
21.67 14.21
23.82 17.49
22.88 17.54
22.96 17.63
22.96 17.64
23.00 17.60
22.55 15.36
Boltz Avgs
M0002
M0016
M0001
M0014
M0004*
M005[1]
M002[1]
(2.06)
0.00
2.28
2.10
4.62
8.87
9.79
9.11
0.488
0.194
0.209
0.075
0.013
0.009
0.012
173.97
173.28
173.38
173.25
169.56
173.93
173.85
173.60
40.16
39.58
39.59
40.96
39.18
38.38
38.46
39.94
69.05 127.28 128.33 174.59
70.76 125.69 130.73 174.23
70.73 125.68 130.70 174.20
68.02 126.95 128.22 174.10
71.19 125.86 129.13 180.13
70.28 127.66 137.29 175.04
70.27 127.84 137.23 175.03
69.71 126.60 129.48 174.49
45.50
46.37
46.42
45.06
42.87
45.22
45.32
45.79
66.81
71.98
71.97
71.32
66.46
66.53
66.53
69.22
20.36
9.40
(0.02)
0.00
3.60
8.99
9.17
9.17
17.19 14.64
17.20 14.68
15.97 13.55
18.71 10.84
20.19
20.17
9.26
9.25
Boltz Avgs
18.73 11.84
M0001
M0002
M0005
M0026
M0044*
M0010
M0019
M0007
M0013
M0039
M0025
M0050
M0043
M0008
M0024
M0011
M0028
M0036
M0029
M0022
M0017
M0035
M0032
M0018
0
2.64
2.7
0.00
1.50
0.331
0.181
0.148
0.020
0.012
0.109
0.049
0.075
0.021
0.001
0.008
0.001
0.008
0.008
0.007
0.005
0.001
0.003
0.003
0.001
0.002
0.003
0.001
0.001
173.31
173.85
172.89
172.95
169.71
170.36
169.92
170.73
173.59
173.52
173.83
169.44
169.98
169.98
169.74
170.49
174.51
168.92
169.42
173.12
169.55
171.73
169.32
170.06
172.48
39.43
39.21
39.54
39.42
39.31
39.31
39.63
39.32
39.88
39.26
39.71
39.39
39.15
39.17
39.07
39.01
40.07
40.42
39.97
38.96
38.46
40.58
38.63
38.32
39.40
70.61 125.86 130.00 175.45
71.11 126.80 127.82 175.46
70.56 123.63 133.09 175.73
70.55 124.85 135.72 175.78
69.88 124.16 135.90 180.65
70.51 126.91 126.25 180.21
69.63 123.19 133.12 180.35
70.53 126.40 127.63 180.30
70.51 126.88 129.00 174.39
72.40 126.19 132.32 175.90
70.89 127.95 125.82 174.22
70.48 124.46 135.71 177.11
71.06 125.81 129.45 176.94
71.14 125.77 129.47 176.97
70.85 123.41 133.14 177.03
71.49 126.58 127.45 176.89
70.44 125.32 137.61 176.37
68.98 125.60 132.26 179.48
70.22 128.13 127.66 176.02
71.02 129.50 132.10 175.35
71.40 126.80 129.40 173.46
72.22 127.26 128.09 170.92
70.71 124.60 133.19 173.70
71.49 127.76 127.45 173.62
70.63 125.72 129.77 176.69
47.46
47.54
47.45
47.64
43.14
43.20
43.24
43.12
47.53
47.69
47.56
44.10
44.16
44.17
44.20
44.10
47.81
43.32
44.42
47.41
45.12
44.08
45.35
45.27
46.29
67.22
67.26
68.07
67.60
66.60
66.59
66.58
66.53
69.16
68.41
69.28
71.37
71.80
71.82
71.77
71.79
68.58
66.50
70.45
67.17
70.19
70.84
70.26
70.30
67.43
21.04
20.96
21.11
20.83
18.92
19.08
19.02
19.04
9.37
9.38
9.12
9.42
9.88
9.89
9.94
9.88
2.00
3.13
3.3
6.98
8.24
3.73
3.76
4.6
2.76
4.73
3.68
6.4
6.80
17.85 14.98
20.99 8.95
7.02
7.72
8.45
8.74
8.76
9.41
10.35
11.13
11.3
13.73
9.16
17.89 15.04
17.76 13.76
17.96 13.77
18.03 13.73
18.23 13.61
18.11 13.70
13.63
9.08
9.11
9.45
10.32
13.55
11.97
11.76
15.52
12.94
11.74
14.23
13.44
21.09
8.96
18.89 10.89
17.19 14.02
11.67
13.14
14.49
14.78
14.88
15.19
21.02
9.57
18.45 14.65
16.84 13.87
18.36 14.69
18.40 14.73
Boltz Avgs
20.32
9.81
*: the extended conformation with the lowest energy
S-22