Stereochemical correction and total structure of roridin J

Article abstract of DOI:10.1016/j.tet.2017.07.048

The (2′Z)-configuration of roridin J (1) was revised to the (2′E) form by conducting NOE experiments. Although the configurations with respect to the stereogenic carbons on the macrocyclic ring of 1 had remained unknown, a combination of NMR analysis and molecular modeling calculations revealed the (2′E,5′R,6′S,7′E,9′Z,13′S) form. In addition, the modeling calculations successfully reproduced the ¹H and ¹³C NMR chemical shifts as well as the ECD spectrum. An X-ray crystallographic analysis verified all the relative configurations.

Full text of DOI:10.1016/j.tet.2017.07.048

Accepted Manuscript
Stereochemical correction and total structure of roridin J
Manami Matsumoto, Atsushi Ito, Akio Tonouchi, Masaaki Okazaki, Masaru Hashimoto
PII:
S0040-4020(17)30790-1
10.1016/j.tet.2017.07.048
TET 28880
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 22 June 2017
Revised Date: 25 July 2017
Accepted Date: 26 July 2017
Please cite this article as: Matsumoto M, Ito A, Tonouchi A, Okazaki M, Hashimoto M, Stereochemical
correction and total structure of roridin J, Tetrahedron (2017), doi: 10.1016/j.tet.2017.07.048.
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Stereochemical Correction and Total
Structure of Roridin J
Leave this area blank for abstract info.
Manami Matsumoto,a Atsushi Ito,a Akio Tonouchi,a
Masaaki Okazaki,b and Masaru Hashimotoa,*
a Faculty of Agriculture and Life Science, Hirosaki University, 3-Bunkyo-cho, Hirosaki, 036-8561, Japan
b Graduate School of Science and Technology, Hirosaki University, 3-Bunkyo-cho, Hirosaki, 036-8561, Japan
1

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Tetrahedron
journal homepage: www.elsevier.com
Stereochemical Correction and Total Structure of Roridin J
Manami Matsumoto,a Atsushi Ito,a Akio Tonouchi,a Masaaki Okazaki,b and Masaru Hashimotoa,*
a Faculty of Agriculture and Life Science, Hirosaki University, 3-Bunkyo-cho, Hirosaki, 036-8561, Japan
b Graduated School of Science and Technology, Hirosaki University, 3-Bunkyo-cho, Hirosaki, 036-8561, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The (2′Z)-configuration of roridin J (1) was revised to the (2′E) form by conducting NOE
experiments. Although the configurations with respect to the stereogenic carbons on the
macrocyclic ring of 1 had remained unknown, a combination of NMR analysis and molecular
modeling calculations revealed the (2′E,5′R,6′S,7′E,9′Z,13′S) form. In addition, the modeling
calculations successfully reproduced the 1H and 13C NMR chemical shifts and the ECD
spectrum. An X-ray crystallographic analysis verified all the relative configurations.
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved
.
roridin J
verrucarol
structural revision
modeling calculation
theoretical spectra
1. Introduction
2. This result allowed us to revise that into the (2′E)
configuration shown as 1. The chemical shift for C12′ (13.26
ppm) supported our revision by taking account of the steric
compression effect due to the C1’ carbonyl group.15 Methyl
carbons on the trisubstituted Z-double bonds usually resonate at
22–27 ppm, while those of corresponding E-isomers appear at
around 5-10 ppm lower frequency. This was further verified
through chemical shift calculations, as described later.
Trichothecenes carrying macrocyclic rings, such as roridins,1,2
verrucarins, baccharins,3,4 and muconomycins5, have been
isolated from fungal sources, such as the Myrothecium and
Fusarium species. In addition to unique structures, these exhibit
potent antifungal,6,7 lethal,8 and/or cytotoxic9-11 properties.
Accordingly, this family has attracted attention of the organic
chemists and biological scientists.1,12,13 Although
a
Although several trichothecenes carrying macrocyclic rings
have been reported, there is a poor consistency in the
configurations of the stereogenic centers on the macrocycles; e.g.,
four diastereomers at C6′ and C13′ are known as roridin E and its
analogs [only epiisororidin E (2) is shown in Figure 1].16 As the
configurations C4′, C5′, C6′, and C13′ of 1 are unknown,
comprehensive biological investigation requires well-established
three-dimensional structures, the configurations of the
macrocyclic moiety in some roridin congeners, e.g., roridin J (1)
were not determined (Figure 1). While exploring the fungal
secondary metabolites, we had an opportunity to isolate 1 from
Calcarisporium arbuscular, which led us to fully determine its
stereostructure using a combination of spectral analysis and
molecular modeling calculations. We succeeded in not only
revising the configuration of the C2′C3′ double bond but also
establishing all configurations of asymmetric centers on the
macrocycles. The proposed configuration was confirmed by an
X-ray crystallographic diffraction analysis.
2. Results and discussion
Roridin J (1) was isolated from the culture broth of C.
arbuscula. The 13C NMR spectral data of our sample were
coincident with those reported by Jarvis in 1980 (max |∆δ13C|:
0.1 ppm, both in CDCl3).14 Although Jarvis reported a potent
NOE between H3-12′ and H-2′ to conclude the (2′Z) form for the
original structure of roridin J (1′), irradiation of H-2′ resulted a
remarkable signal enhancement at H-4′ (13%) in our experiment,
while that at H3-12′ was not obvious (1.3%), as shown in Figure
Figure 1. Structures of roridin J (1), its original structure (1′), and
2

isolation of 1 allows us to experimentally deduce them.
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Prior to these investigations, the relative and absolute
Figure 3, Relative configuration of the macrocyclic moiety and the
characteristic NOEs as well as the 1H coupling constants.
Figure 2. Medium-frequency region of the difference NOE spectrum of
1 obtained by irradiating H-2′.
moiety as shown in Figure 3. In addition, NOESY correlations
are found at H-4′/H-13′ and H-5′/H-6′, establishing a rel-
(5′R,6′S,13′S) configuration of the 1,3-dioxolane ring moiety on
the macrocycle.
configurations of the C1–C16 trichothecene moiety were
experimentally confirmed. The NOESY spectrum afforded the
correlations at Hα-3/H-4, Hα-3/H-11, H-4/H-11, H-4/H-14, H-
4/H-15, and Hβ-7/H-13, indicating that the relative
stereochemistry of trichothecene part is identical to those of other
roridins.17,18,11 The absolute configuration of this moiety was
established by a chemical correlation with verrucarol (3)19,20
being obtained by a basic methanolysis of 1. Crude sample 3 thus
The remaining structural unknowns were the absolute
configurations of C4′ and C5′, which were investigated with the
1
stable conformation, theoretical H and 13C chemical shifts, and
ECD spectrum based on DFT ωB97X-D/6-31G* (structural
optimization and chemical shift calculations) and BHLYP/def2-
TZVP//ωB97X-D/6-31G* (ECD spectral calculations) by using a
protocol similar to that used in previous studies.21,22 Since the
macrocyclic part of 1 has high conformational flexibility, the
conformational distribution was estimated based on the free
energy (G) by adding the entropic factor (S) and the temperature
term (298 K) to the steric energy (H). The δC and δH root-mean-
square (RMS) values of each isomer were obtained from the
residuals between the experimental and theoretical chemical
shifts. As we have established the rel-(5′R,6′S,13′S) configuration
and the (2'E,7'E,9'Z) geometry via NMR analysis, the (4′R) and
(4′S) epimers of both (2′E,5′R,6′S,7′E,9′Z,13′S) and
(2′E,5′S,6′R,7′E,9′Z,13′R) forms (in total, four isomeric models)
were subjected to the calculations. The models are expressed
using three letters, e.g., ERR, and these three letters refer to the
configurations at the 2′,3′ double bond, the 4′, and 5′ asymmetric
carbons.
1
prepared (1.5 mg) provided an H NMR spectrum that showed
accordance with the data in the literature;19 however, it was both
qualitatively and quantitatively insufficient for measuring the
specific rotation. It was found that the following derivatization
into bis-O-2-naphthoate 4 resulted in giving distinct Cotton
effects at 242 (∆ε +6.8) and 229 nm (∆ε −20.2) in the ECD
spectrum after purification with preparative silica gel TLC.
Fortunately, the producer fungus C. arbuscula afforded a
considerable amount of 2, a congener carrying the same
trichothecene unit.16 Basic methanolysis of 2 under the same
abovementioned conditions afforded authentic 3 (4.5 mg), which
22
allowed us to obtain a reliable specific rotation ([α]D −31.5 (c
The calculations naturally revealed the stable conformers as
shown in Figure 4. First, we analyzed the transannular NOEs
based on their stable conformers suggested by these calculations.
Roridin J (1) shows transannular NOEs at H3-14/H-2′, H3-14/H-
8′, H-2′/H-8′, and H-4'/H-13'. The most stable conformer of
model ERR explains all these NOEs. Since C4′-epimeric model
ESR adopts the similar conformation on the basis of these
calculations, this isomer would also satisfy most of the above
NOEs; however, the distance between H-4′ and H-13′ (3.6 Å) is
slightly large to expect a distinct NOE between those (note that
the distance between the vicinal trans-diaxial protons on a chair
formed cyclohexane is around 3.1 Å). The calculations also
revealed that the C1′–C4′-conjugated planes in the stable
conformers of models ERS and ESS are flipped around from that
in model ERR, which increased the distance between H3-14 and
H-2′ (ERS: 4.2 Å and ESS: 4.0 Å). Thus, the NOE between these
protons cannot be expected in those models. The characteristic
difference between the stable conformers of models ERR and
ESR is the dihedral angle H-4′/H-5′. These hydrogen atoms
adopt a nearly antiperiplanar relationship in the stable conformers
of model ERR, while these in model ESR is synclinal. The
Scheme 1. Chiral assignment of the trichothecene part.
0.41, CHCl3)). The following 2-naphthoylation afforded 4¸ which
gave identical ECD spectrum to that of the sample prepared from
1. Ishihara and Tadano synthesized verrucarol in an optically
21
active form to report its specific rotation ([α]D −40.6 (c 0.13,
CHCl3)).19,20 A combination of these results established the
absolute configuration for the trichothecene moiety in 1, despite
the indirect manner.
3
3
3
Boltzmann distribution weighted JH-4′/H-5′ value of model ERR is
The vicinal spin couplings JH-7′/H-8′ (15.0 Hz) and JH-9′/H-10′
(11.1 Hz) confirm the same (7′E,9′Z) form as Jarvis’s assignment.
The NOESY spectrum affords a correlation between H-7′ and H-
9′, suggesting a transoid conformation for the C7′–C10′ diene
5.9 Hz (Table A in Figure 5) which is in agreement with the
3
experimental coupling constant of 7.0 Hz, when the JH-4′/H-5′
value of each stable conformer was calculated with the Karplus
3

1 is a C4’-hydroxy derivative roridin H.26 Notably, the C12′ in
ACCEPTED MANUSCRIPT
roridin H was reported to resonate at 18.2 ppm, which is 5.1 ppm
Figure 4. The most stable conformers of models ERR, ERS, ESR,
and ESS based on ωB97X-D/6-31G*.
equation modified by Haasnoot.23,24 In contrast, the 3JH-4′/H-5′ value
for model ESR was estimated to be 1.7 Hz. Although model ESS
shows a desirable JH-4′/H-5′ value (6.2 Hz), this model disagrees
with the transannular NOEs, as described. These results
suggested model ERR to be the most plausible isomer as 1.
Figure 5. Summary of molecular modeling calculations. Table A:
configurations of the isomers investigated and 3JH-4′/H-5′ as well as δC-12′
of the models. Bar chart B: δ1H and δ13C root-mean-square (RMS)
values of the models against the experimental data in CDCl3. Spectra C:
Theoretical ECD and UV spectra of the models. Calculations for Table
A and bar charts B were generated with /ωB97X-D/6-31G*, and spectra
C was obtained using BHLYP/def2-TZVP//ωB97X-D/6-31G*.
3
Next, we investigated with the theoretical chemical shifts
employing ωB97X-D/6-31G*. The accuracy levels of the δH and
δC RMS values in these calculations were set to be around 0.20
and 2.0 ppm, respectively, on the basis of our experience.21,22,25
The best candidate model ERR affords agreeable both theoretical
1H and 13C chemical shifts (0.22 and 1.49 ppm, respectively; bar
chart B). Since these values for C4′-epimeric model ESR are also
acceptable (0.22 and 1.78 ppm, respectively), this model can not
be discarded with this argument. However, models ERS and
ESS, the diastereomer around the C6′OC7′C12′O dioxolane ring
larger than that of 1. We assume that the C4’-hydroxy group
contributes to a magnetic shielding of the C12′ in addition to the
steric compression by C1’ carbonyl. This shielding can be
explained by the γ-gauche effect.27 In fact, the C12’-C3’-C4’-O
adopts gauche conformation in the most stable conformer of
model ERR. Similarly, the C12’ resonance in model ESS can be
explained. However, quantitative argument such as additivity of
these shielding effects would be improper, because the C4’OH
also affects to the distance between the C12’ and the C1’-
carbonyl.
1
moiety, yields large unacceptable H RMS values (0.43 and 0.31
ppm, respectively).
Because we revised the (2′Z) double bond of 1′ to the (2′E)
with the NOE experiments, a series of the (2′Z) isomeric models
was also calculated. As expected, the C12′ resonances of the
(2′Z) isomers would appear at a remarkably higher frequency
(22.3–26.2 ppm) than the experimental data (13.26 ppm), while
those in all (2′E) isomeric models are agreeable (14.7–16.4 ppm;
Table A). Furthermore, none of the (2′Z) isomeric models satisfy
Roridin J (1) shows negatively split Cotton effects (∆ε −6.7 at
251 nm and +11.8 at 218 nm) in CH3CN. The theoretical ECD
spectra for models ERR and ESR are similar each other and
show accordance with the experimental spectrum (spectra C).
Models ERS and ESS also afford resembled theoretical ECD
spectra to each other, however, these do not match to the
1
the H or 13C RMS values (>0.29 and >2.84 ppm, respectively).
These results verified our previous conclusion, and revealed that
4

experimental spectra at all. Molecular modeling calculations have
deposited in the NITE Biological Resource Center (NBRC) under
accession number NBRC 112400.28
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suggested that the configurations at the C4’ hardly contribute to
the conformation of the macrocyclic ring. Since models giving
resembled theoretical ECD spectra were found to adopt the
resembling conformations around the macrocyclic moiety, it is
likely that the experimental Cotton effects are attributed to an
exciton coupling due to chiral torsion between the C1′–C3′
enonate and the C7′–C11′ dienoate chromophores.
Isolation
The fungus C. arbuscula was cultured with a potato–dextrose
medium [prepared from potato (2.0 kg), glucose (200 g), and
H2O (10.0 L)] at 25 °C by shaking (110 rpm) for 14 days. After
mycerium was removed by filtration in suction, the filtrate was
concentrated until the whole volume became 2.0 L. The resulting
broth was shaken with EtOAc (1.0 L ×3), and the EtOAc layer
was concentrated in vacuum to give the crude residue (3.0 g).
The crude residue was suspended with 20% aqueous MeOH (10
mL) and loaded on Sep-Pack ODS (10 g). After washing with
20% aqueous MeOH (60 mL), elution with 40% aqueous MeOH
As described, only model ERR satisfies spectroscopic
3
1
properties of 1, such as the NOEs, the JH-4'/H-5' value, the H and
13C NMR chemical shifts, and the ECD spectrum, and shows no
inconveniences so far we investigated.
Fortunately, standing a solution of 1 in 1:1 mixture of hexane
and ethyl acetate afforded yellowish plate crystals (m.p. 267–
270 °C), which enabled an X-ray crystallographic analysis
(Figure 6). Although the absolute structure could not be
discussed because of high standard uncertainty in the Flack
parameter (χ = 0.0(7)), that confirmed not only the identical
relative configuration but also nearly the same conformation as
that of the most stable conformer obtained by the calculations.
This result also demonstrated the high quality of molecular
afforded
a
fraction containing roridins (280 mg after
concentration). Preparative HPLC [Sunfire Prep C18 OBD 19
mm × 150 mm, CH3CN-H2O (containing 0.1% TFA), 10 mL/min
flow, detected at 254 nm] gave roridin J (1, 15 mg) and
epiisororidin E (2, 62 mg).
Roridin J (1): m.p. 267-270 °C (from EtOAc/hexane), UV (6.3
× 10−5 mol/L, CH3CN) λmax 205 nm (ε 15000), 260 nm (ε 12000),
ECD (6.3 × 10-5mol/L, CH3CN) ∆ε235 0, ∆ε217 +11.5 ∆ε251 –6.9,
1H NMR (500 MHz, CDCl3) δ 0.85 (3H, s, H3-14), 1.37 (3H, d, J
= 6.0 Hz, H3-14′), 1.71 (3H, s, H3-16), 1.81–2.05 (4H, H2-7, H2-
8), 2.18 (1H, dt, J = 4.5, 15.3 Hz, Hβ-3), 2.27 (3H, d, J = 1.0 Hz,
H3-12′), 2.47 (1H, dd, J = 8.5, 15.3 Hz, Hα-3), 2.82, 3.13 (each
1H, d, J = 4.0 Hz, H2-13), 3.65 (1H, brd, J = 4.3 Hz, H-11), 3.65
(1H, dq, J = 8.9, 6.0 Hz, H-13′), 3.84 (2H, H-2, H-4′), 4.02 (1H,
d, J = 12.5 Hz, H2-15), 4.09 (1H, dt, J = 2.0, 8.9 Hz, H-6′), 5.25
(1H, d, J = 7.0 Hz, H-5′), 5.44 (1H, brd, J = 4.3 Hz, H-10), 5.76
(1H, brq, J = 1.2 Hz, H-2′), 5.80 (1H, d, J = 11.5 Hz, H-10′), 5.89
(1H, dd, J = 4.5, 8.5 Hz, H-4), 5.92 (1H, dd, J = 2.0, 15.5 Hz, H-
7′), 6.55 (1H, t, J = 11.5 Hz, H-9′), 7.65 (1H, dddd, J = 0.9, 2.0,
11.5, 15.5 Hz, H-8′) 13C NMR (125 MHz, CDCl3) δ 7.36 (C14),
13.05 (C12′), 15.94 (C14′), 20.34 (C7), 2193.34 (C16), 27.60
(C8), 34.69 (C3), 43.20 (C6), 47.96 (C13), 49.15 (C5), 63.40
(C15), 65.55 (C12), 67.82 (C11), 73.80 (C4), 76.42 (C13′), 79.16
(C2), 79.74 (C4′), 82.23 (C6′), 103.29 (C5′), 118.61 (C10),
118.75 (C10′), 119.91 (C2′), 126.07 (C8′), 134.48 (C7′), 140.50
(C9), 143.11 (C9′), 155.26 (C3′), 165.90 (C1′), 166.21 (C11′).
1
The H and 13C data were coincident with those in the literature.
Figure 6. The X-ray crystallographic structural image of 1 (CPK
colored ellipsoid model) overlaid on the most stable conformer of model
RRR based on wB97X-D/6-31G* (green colored shadowed model).
The X-ray image was inversed from the original data.
ESIMS (rel. int. %) m/z 551.2282 (25, calcd. for C29H36O9Na
[M+Na]+: 551.2257, 546.2731 (100, calcd. for C29H40NO9
[M+NH4]+: 546.2703), and 529.2467 (67, calcd. for C29H37O9
1
[M+H]+: 529.2438). The H and 13C NMR data of this sample
were coincident with those reported by Jarvis.14
modeling in the present study.
Epiisororidin E (2): UV (5.1 × 10−5 mol/L, CH3CN) λmax 220
nm (ε 22000), 258 nm (ε 14000), ECD (5.1 × 10−5 mol/L,
3. Conclusion
1
CH3CN) ∆ε233 0, ∆ε218 +13.8, ∆ε249 −11.3, H NMR (500 MHz,
As described, we revised a geometry at the 2’,3′-double bond
in the original structure of roridin J (1’) reported in 1980.
Although the configurations of the stereogenic centers on the
macrocyclic ring had remained unknown, this study established
them to disclose the whole structure as 1 by a combination of a
comprehensive NMR analysis and modeling calculations. All of
the configurations were confirmed by an X-ray diffraction
analysis.
CDCl3) δ 0.80 (3H, s, H3-14), 1.15 (3H, d, J = 6.5 Hz, H3-14′),
1.55 (1H, dd, J = 4.9, 13.0 Hz, H-7), 1.71 (3H,s, H3-16), 2.02
(2H, m, Hβ-3, H-8), 2.10 (1H,m, H-8), 2.12 (1H, dd, J = 6.0, 13.0
Hz, H-7), 2.23 (3H, d, J = 1.2 Hz, H3-12′), 2.34 (1H, dt, J = 7.6,
13.0, H-4′), 2.56 (1H, dd, J = 8.2, 14.9, Hα-3), 2.56 (1H, m, H-
4′), 2.84, 3.15 (each 1H, d, J = 4.0 Hz, H2-13), 3.6 (1H, ddd, J =
5.0, 7.6, 10.2 Hz, H-5′), 3.75 (1H, dt, J = 7.6, 10.2 Hz, H-5′),
3.85 (1H, d, J = 5.0 Hz, H-2), 3.92 (1H, ddd, J = 1.0, 3.2, 6.1, H-
6'), 4.01 (1H, dq, J = 3.2, 6.5 Hz, H-13′), 4.05, 4.20 (each 1H, d,
J = 12.5 Hz, H2-15), 4.06 (1H, brd, J = 5.8 Hz, H-11), 5.50 (1H,
brdd, J = 1.2, 5.8 Hz, H-10), 5.82 (1H, d, J = 11.1 Hz, H-10'),
5.86 (1H, d, J = 1.2 Hz, H-2′), 5.9 (1H, dd, J = 6.1, 18.0 Hz, H-
7′), 6.32 (1H, dd, J = 4.1, 8.2 Hz, H-4), 6.62 (1H, t, J = 11.1 Hz,
H-9′), 7.56 (1H, dd, J = 11.1, 16.0 Hz, H-8′) 13C NMR (125
MHz, CDCl3) δ 6.48 (C14), 17.87 (C14′), 19.23 (C12′), 22.35
(C7), 23.23 (C16), 27.68 (C8), 36.40 (C3), 40.32 (C4′), 42.59
4. Experimental
Fungus.
Calcarisporium arbuscula strain 1310-04 was isolated from a
fruit body of Gymnopilus junonius which was collected at
Aomori prefecture Japan in September 2013. It has been
5

(C6), 47.81 (C5), 48.48 (C13), 64.29 (C15), 65.65 (C12), 66.28
prepared from epiisororidin E (2). The obtained sample was 2-
naphthoylated under the conditions similar to those mentioned
above by employing 2-naphthoyl chloride (2.0 mg), DMAP (2.0
mg), and CH2Cl2 (0.5 mL). Then, a workup similar to that
discussed above and the following preparative silica gel TLC
under the same conditions afforded the bis(2-naphthoate) 4 (0.6
mg, the quantity was estimated by assuming its ε value at 236 nm
to be 135000). ECD (7.0 × 10−6 mol/L, CH3CN) ∆ε242 +6.8, ∆ε229
−20.2. The chromatographic property and the ECD profile of this
sample were identical to those derived from 2.
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(C5′), 67.06 (C11), 68.48 (C13′), 75.06 (C4), 79.24 (C2), 81.83
(C6'), 117.24 (C2′), 118.82 (C10), 119.06 (C10′), 131.05 (C8′),
134.68 (C7′), 140.26 (C9), 142.33 (C9′), 157.99 (C3′), 166.35
(C1′), 166.42 (C11′), ESIMS (rel. int. %) m/z 537.2460 (45,
calcd. for C29H38ONa [M+Na]+: 537.2464), 532.2905 (100, calcd.
for C29H42NO8 [M+NH4]+: 529.2910), 515.2640 (27, calcd. for
1
C29H39O8 [M+H]+: 515.2645). The H and 13C NMR data of this
sample were coincident with those reported by Jarvis.16
Verrucarol (4,15)-O-bis(2-naphthoate) 4 from epiisororidin E (2)
Modeling calculations
Epiisororidin E (2, 10 mg) was stirred with K2CO3 (20 mg) in
MeOH (1.0 mL) at room temperature for 2 h. The suspension
was diluted with diethyl ether (5.0 mL) and then filtered. The
filtrate was concentrated in vacuum and then subjected to
preparative silica gel TLC. Development with EtOAc:hexane
(80:20) afforded 3 (4.5 mg; Rf = 0.2 under the above conditions).
Eight isomeric models, namely ERR, ESR, ERS, ESS, ZRR,
ZSR, ZRS, and ZSS, were constructed using Spartan’14. The
three letters for the models refer to the configurations at the 2′, 4′,
and 5′ positions, respectively. A conformational search for each
model was performed using MMFF by setting the rotation at
C15-O bonding, and the flips at C1′, C2′, C3′, C4′, C5′, C7′, C8′,
C9′, C10′, and C11′, which theoretically generate 354294 initial
conformers and the search gave 100 candidates of stable
conformers in each model. In some models, the program
automatically judged the completion of the search to terminate
after checking approximately 1 × 104 conformers. The candidates
were refined with HF/STO-3G. After removing the overlaps,
conformers (4–9 conformers) within 10 kJ/mol from the global
minimum conformer were further optimized stepwise by
EDF2/6-31G* and ωB97X-D/6-31G* on Spartan’14 and
Spartan’16, respectively, considering the entropy term using a
vibrational analysis (the software was updated during the
research period). The optimized conformers were subjected to
chemical shift calculations with the ωB97X-D/6-31G*.
Distributions of the conformers were estimated based on the
relative free energies (∆G) and the Boltzmann distribution law.
Theoretical chemical shifts were corrected based on the
distributions of the conformers. Major conformers existing in
cumulatively 90 % populations of models ERR, ESR, ERS, and
ESS were subjected to ECD calculations on Turbomole X
(version 7.1) with def2-TZVP/BH-LYP//ωB97X-D/6-31G*. Fifty
excitations were examined to express the UV and ECD spectra in
the range 200–600 nm. The UV and ECD spectra of each
conformer were constructed based on the frequencies as well as
the oscillator strength and the rotary strength, respectively, by
employing the NORMDIST function in Microsoft Excel® 2016
(standard deviation = 14 nm for UV spectra; 15 nm for ECD
spectra). The wavelengths for these spectra were corrected (+14
nm) based on the experimental UV absorption of 1. Theoretical
UV and ECD spectra were obtained after correction based on the
conformational distributions. The theoretical UV intensity of
model ERR was normalized with experimental data at 260 nm,
and the same parameter was used for the other models. The
intensities of the theoretical ECD spectra were appropriately
adjusted.
22
21
[α]D = −31.5° (c 0.41 CHCl3), (lit: [α]D −40.6 (c 0.13,
CHCl3).19 1H NMR (500 MHz, CDCl3) δ 0.95 (3H, s, H3-14),
1.36 (1H, br, alcoholic proton at C15O), 1.66 (1H, brd. J = 9.1
Hz, alcoholic proton at C4O), 1.72 (3H, s, H3-16), 1.74 (1H, dt, J
= 1.8, 5.5 Hz, H-7), 1.91 (1H, ddd, J = 3.0, 5.5, 15.5 Hz, Hβ-3),
1.96 (1H, dd, J = 6.0, 12.3 Hz, H-7), 2.05 (2H, m, H-8), 2.58
(1H, dd, J = 7.5, 15.5 Hz, Hα-3), 2.81, 3.11 (each 1H, d, J = 3.9
Hz, H2-13), 3.57, 3.76 (each 1H, d, J = 11.7 Hz, H2-15), 3.62
(1H, d, J = 5.4 Hz, H-11), 3.82 (1H, d, J = 5.5 Hz, H-2), 4.63
(1H, br, H-4), 5.43 (1H, d, J = 5.4 Hz, H-10), 13C NMR (125
MHz, CDCl3) δ 7.29 (C14), 20.99 (C7), 23.28 (C16), 28.25 (C8),
39.96 (C3), 43.89 (C6), 47.64 (C13), 49.00 (C5), 62.55 (C15),
65.71 (C12), 66.54 (C11), 74.56 (C4), 78.67 (C2), 118.77 (C10),
141.05 (C9), ESIMS (rel. int. %) m/z 289.1408 (66, calcd. for
C15H22O4Na [M+Na]+: 289.1416), 267.1585 (60, calcd. for
C15H23O4 [M+H]+: 267.1596), and 249.1485 (100, calcd. for
C15H21O3 [MH-H2O]+: 249.1491). The 1H and 13C data were
coincident with those in the literature.19
Verrucarrol (3) (1.0 mg) thus prepared was stirred with 2-
naphthoyl chloride (5.0 mg) and N,N-dimethyl-4-aminopyridine
(DMAP, 4.0 mg) in CH2Cl2 (0.2 mL) at room temperature for 30
min. Diethyl ether (5.0 mL) was added to the mixture, and the
resulting suspension was filtered through a cotton filter. After
concentration in vacuo, the residue was purified using silica gel
column chromatography (EtOAc:hexane = 33:67) to give the
bis(2-naphthoate) 4 (1.6 mg, Rf = 0.45 under the above
conditions, and the quantity of the sample was estimated by
assuming its ε value at 236 nm to be 135000). ECD (7.0 × 10−6
1
mol/L, CH3CN) ∆ε242 +6.8, ∆ε229 −20.2, H NMR (500 MHz,
CDCl3) δ 1.13 (3H, s, H3-14), 1.75 (3H, s, H3-16), 1.98–2.12 (3H,
m, H-7, H-8), 2.18 (1H, dd, J = 6.0, 11.6 Hz, H-7), 2.26 (1H,
ddd, J = 3.5, 5.3, 15.5 Hz, Hβ-3), 2.77 (1H, dd, J = 7.5, 15.5 Hz,
Hα-3), 3.00, 3.26 (each 1H, d, J = 4.1 Hz, H2-13), 3.91 (1H, d, J
= 5.4 Hz, H-11), 3.97 (1H, d, J = 5.3 Hz, H-2), 4.42, 4.64 (each
1H, d, J = 12.3 Hz, H2-15), 5.53 (1H, dd, J = 1.5, 5.4 Hz, H-10),
6.05 (1H, dd, 3.5, 7.5 Hz, H-4), 7.50–7.60 (4H, aromatic
protons), 7.83–7.88 (4H, aromatic protons), 7.94, 7.97 (each 1H ,
brd, J = 8.0 Hz, aromatic protons), 8.01, 8.06 (each 1H, dd, J =
1.4, 8.4 Hz, aromatic protons), 8.58, 8.60 (each 1H, brs,
aromatic protons). ESIMS (rel. int. %) m/z 597.2245 (33, calcd.
for C37H34O6Na [M+Na]+: 597.2253), 592.2683 (54, calcd. for
C37H38NO6 [M+NH4]+: 592.2699), and 575.2421 (100, calcd. for
C37H35O6 [M+H]+: 575.2434).
X-ray diffraction analysis25
A solution of 1 (approximately 10 mg) in a mixture of 1:1
EtOAc and hexane (0.3 mL) was stood at room temperature for a
week to precipitate the crystalline. After the mother liquid was
pipetted off, diethyl ether (0.2 mL) was added and the mixture
was gently agitated for 10 second at room temperature. The
ethereal liquid was quickly removed by pipetting to give the
yellowish single crystals. Data were collected at 123 K on a
Rigaku RAXIS RAPID II imaging plate diffractometer using
graphite-monochromated Mo Ka radiation (λ = 0.71075 Å) and
corrected by the Lorentz, polarization, and absorption effects.
The structure was solved by direct methods (SHELXS-97) and
refined on F2 using the full-matrix least-squares method
Preparation of 4 from roridin J (1)
In the same manner as above, roridin J (1, 1.5 mg) was treated
with K2CO3 (10 mg) in MeOH (0.5 mL). The similar workup and
purification gave 3 (approximately 1.5 mg). The 1H NMR
spectrum of the crude sample agreed with that of the sample
6

(SHELXS-97).29 Anisotropic refinement was applied to all non-
hydrogen atoms. All hydrogen atoms were located at the
calculated positions. Crystallographic data were deposited at the
Cambridge Crystallographic Data Centre (CCDC- 1556402). The
data can be obtained free of charge from the Cambridge
Crystallographic Data Centre.
21. Tanaka, S.; Honmura, Y.; Uesugi, S.; Fukushi, E.; Tanaka, K.;
ACCEPTED MANUSCRIPT
Maeda, H.; Kimura, K.-i.; Nehira, T.; Hashimoto, M. J. Org.
Chem. 2017, 82, 5574-5582.
22. Kusakabe, K.; Honmura, Y.; Uesugi, S.; Tonouchi, A.; Maeda,
H.; Kimura, K.-i.; Koshino, H.; Hashimoto, M. J. Nat. Prod.
2017, 80, 1484-1492.
23. Haasnoot, C. A. G.; de Leeuw, F. A. A. M.; Altona, C.
Tetrahedron 1980, 36, 2783-2792.
24. Stenutz, R., Conformational analysis of natural products,
http://www.stenutz.eu/conf/index.html
25. Honmura, Y.; Uesugi, S.; Maeda, H.; Tanaka, K.; Nehira, T.;
Kimura, K.-i.; Okazaki, M.; Hashimoto, M. Tetrahedron 2016,
72, 1400-1405.
26. Traxler, P.; Tamm, C. Helvetica Chimica Acta 1970, 53, 1846-
1869.
Acknowledgment
Part of this research was supported by a Grant-in-Aid for
Scientific Research (B) (15H04491) and a Grant-in-Aid for
Challenging Exploratory Research (16K14910) from the Japan
Society for the Promotion of Science (JSPS). We would like to
thank Enago (www.enago.jp) for the English language review.
27. Jacobsen, N. E. In NMR Data Interpretation Explained:
Understanding 1D and 2D NMR Spectra of Organic
Compounds and Natural Products; John Wiley & Sons Inc.:
Hoboken, New Jersey, 2016; pp. 143-146.
Supplementary Material
Supplementary Material associated with this article can be
found in the online version at http:
28. http://www.nbrc.nite.go.jp/e/index.html.
29. (a) Sheldrick, G. M. SHELXS-97, Programs for Solving X-ray
Crystal Structures; University of Göttingen, 1997. (b)
Sheldrick, G. M. SHELXL-97, Programs for Refining X-ray
Crystal Structures; University of Göttingen, 1997.
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7