Structure Determination, Biosynthetic Origin, and Total Synthesis of Akazaoxime, an Enteromycin-Class Metabolite from a Marine-Derived Actinomycete of the Genus Micromonospora

Derived Actinomycete of the Genus Micromonospora

Article

Structure Determination, Biosynthetic Origin, and Total Synthesis of

Akazaoxime, an Enteromycin-Class Metabolite from a Marine-

Yasuhiro Igarashi,* Yoe Matsuyuki, Masayuki Yamada, Nodoka Fujihara, Enjuro Harunari, Naoya Oku,

Md. Rokon Ul Karim, Taehui Yang, Katsuhisa Yamada, Chiaki Imada, Keisuke Fukaya,

and Daisuke Urabe *

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sı Supporting Information

ABSTRACT: A new enteromycin-class antibiotic, akazaoxime (1), possess-

ing an aldoxime functionality in place of O-methyl nitronic acid, was isolated

from the cultured extract of a marine-derived actinomycete of the genus

Micromonospora, along with known A-76356 (2). The structure of 1,

including the absolute stereochemistry of three chiral centers, was

established by comprehensive analysis of nuclear magnetic resonance

(

NMR) and mass spectrometry data coupled with magnetic anisotropy

analysis of its phenylglycine methyl ester derivatives. The stereochemistry of

, not determined previously, was proven to be the same as that of 1 on the

basis of the similarity of their NMR and speciﬁc rotation data. Precursor

feeding experiments using C-labeled compounds elucidated that the

carbon skeletons of 1 and 2 are constructed from propionate (methylmalonate), leucine, and glycine. Establishment of the concise

and ﬂexible synthetic route to 1 enabled us to implement biological evaluation of 1 and its unnatural analogues, demonstrating weak

to moderate antimicrobial activities of 1 against Gram-positive Kocuria rhizophila [minimum inhibitory concentration (MIC) of 50

μg/mL] and those of synthetic analogues against a plant pathogen Glomerella cingulata (MIC of 50 μg/mL) and a human pathogen

Trichophyton rubrum (MIC of 25−50 μg/mL).

INTRODUCTION

validate their biosynthetic potential as alternatives to terrestrial

■

13−16

counterparts.

In the course of our continuous screening

The O-methyl nitronic acid functionality is quite rare in natural

products. Enteromycin is the ﬁrst natural compound that

possesses this functional group. To date, fewer than 10 related

eﬀort, one of the strains isolated from the seawater collected at

a depth of 800 m in Sagami Bay, Japan, was found to produce

two enteromycin-class metabolites. NMR- and MS-based

structural analyses revealed that one was a known antibiotic

A-76356 (2) and the other its new congener designated

akazaoxime (1), bearing the aldoxime functionality instead of

O-methyl nitronic acid (Figure 1). We herein describe the

isolation, structure determination, and biosynthetic origin of 1

and 2 by ¹³C labeling experiments. We also describe a total

synthesis of 1 and its unnatural analogues, and the bioactivity

of these compounds.

compounds have been isolated, exclusively from actino-

−7

mycetes,

and most of them were described to show

antibacterial activity against Gram-positive and/or Gram-

negative bacteria. Despite the rarity of O-methyl nitronic

acid, nothing is known about its biosynthesis and no total

synthesis of this class of compounds has been described.

Additionally, stereochemical assignment has not been made for

any of the previously reported compounds with chiral centers.

Besides the terrestrial microorganisms that have been

intensively screened for bioactive compounds, marine

ecosystems have attracted attention as a reservoir for

unexplored species of microorganisms and secondary metab-

olites thereof. In the past several decades, the number of new

Received: February 12, 2021

Published: April 22, 2021

natural products from marine microbes has been steadily

,10

increasing.

In particular, the marine environment is the

potential niche for new species and/or genera of rare

actinomycetes, from which several hundreds of new secondary

11,12

metabolites were identiﬁed.

Our laboratory has been

investigating secondary metabolites of marine actinomycetes to

2021 American Chemical Society

The Journal of Organic Chemistry

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J. Org. Chem. 2021, 86, 6528−6537

Article

Figure 1. Structures of akazaoxime (1) and A-76356 (2).

Figure 2. NMR analysis of 1. (a) COSY and key HMBC correlations.

(b) NOESY correlations and J_H_,_Hvalues supporting the relative

RESULTS AND DISCUSSION

conﬁguration.

■

The producing strain Micromonospora sp. AKA109 was

cultured in A16 medium, and the whole culture broth was

extracted with 1-butanol. After evaporation, the extract was

successively fractionated by silica gel and ODS column

chromatographies. The ﬁnal puriﬁcation was performed by

reverse-phase HPLC to yield akazaoxime (1) and A-76356 (2).

HRMS (ESI/TOF) analysis of 1 gave a molecular formula of

The geometry of the aldoxime group was determined to be E

^b^y^c^o^m^p^a^rⁱ^s^oⁿ^o^f^t^h^e^c^h^e^mⁱ^c^a^l^s^hⁱ^f^t^o^f^H²^′⁽^δH 7.47) with

^lⁱ^t^e^r^a^t^u^r^e^v^a^l^u^e^s^r^e^p^o^r^t^e^d^f^o^r^p^-^c^h^l^o^r^o^b^eⁿ^z^a^l^d^o^xⁱ^m^e⁽^E^,^δH

.30; Z, δ 8.20) and p-nitrobenzaldoxime (E, δ 7.60; Z, δ

H H H

.36). The relative conﬁgurations of the contiguous

asymmetric centers at C2−C4 were determined on the basis

of J coupling constants and NOESY correlations. A large J

C H N O , which was in good agreement with the H and

value of 9.5 Hz for H2/H3 and a small value of 1.6 Hz for H3/

H4 indicated an anti- and syn-relationship for these proton

pairs, respectively (Figure 2b). Furthermore, NOEs were

observed between H9 and H4, H9 and H3, H3 and H4, and

H3 and H5, thereby establishing a 2R*,3S*,4S* conﬁguration.

C{ H} NMR spectroscopic data. The IR spectrum displayed

absorption bands for hydroxy and carbonyl groups (3268 and

−

710 cm ). Analysis of C{ H} NMR and HSQC spectral

data measured in CD OD allowed the assignment of 11 carbon

resonances to two oxygenated sp carbons (δ 179.5 and

64.8), one sp methine (δ 144.0), four sp methines (δ

5.9, 49.2, 45.4, and 26.1), one sp methylene (δ 43.0), and

Next, the PGME method was applied to determine the

absolute conﬁguration of C2. Compound 1 was reacted with

(

R)- or (S)-phenylglycine methyl ester (PGME) to prepare the

three methyl groups (δ 23.6, 22.8 and 14.7) (Table 1). COSY

corresponding PGME amides (3a and 3b). Negative Δδ

analysis deﬁned the connectivity among eight carbons, starting

from the doublet methyl H9 to the isopropyl terminus

comprising H6 methine and H7/H8 doublet methyls (Figure

S−R

values for protons H3−H5 and a positive value for H9

unambiguously determined a 2R conﬁguration (Figure 3),

thereby establishing a 2 R ,3 S ,4 S conﬁguration.

). An HMBC correlation from H9 to C1 (δ 179.5) indicated

the presence of a carboxy group connecting to C2. H and C

chemical shifts of H3/C3 and H4/C4 were suggestive of the

connection of a hydroxy group at C3 and an amide

functionality at C4, respectively. HMBC correlations from

H4 and H2′ to C1′ connected the two-carbon unit (C1′−C2′)

through the amide bond to C4. The remaining part was

deduced to be an aldoxime group considering the chemical

shift of C2′ and the remaining fragment HNO suggested by

the high-resolution MS analysis, to complete the planar

structure of 1 (Figure 2a).

Figure 3. Δδ_S₋_Rvalues for PGME derivatives (3a and 3b) of 1.

Table 1. H and ¹³C{ H} NMR Data for Akazaoxime (1) and A-76356 (2)

CDCl₃

CD OD

acetone-d₆

b,c

position

δ_C

δ_Hmult [J (Hz)]

HMBC

δ_C

δ_Hmult [J (Hz)]

δ_C

δ_Hmult [J (Hz)]

HMBC

179.5, C

45.4, CH

75.9, CH

49.2, CH

177.1

179.9, C

42.9, CH

74.5, CH

2.41 dq (9.5, 7.0)

3.69 dd (9.5, 1.6)

4.24 ddd (8.9, 5.3, 1.6) 5, 6, 1′

1, 3, 4, 9

1, 2, 5

44.3 2.45 dq (9.5, 7.1)

75.2 3.74 dd (9.5, 1.2)

48.2 4.25 br. dd (7.7, 5.5) 48.4, CH

42.7 1.39 m

1.63 m

25.5 1.63 m

23.4 0.92 d (6.4)

22.6 0.94 d (6.3)

14.4 1.16 d (7.1)

162.6

2.57 dq (8.6, 7.2)

3.75 dd (8.8, 1.8)

4.28 dddd (9.5, 9.5, 5.5, 1.8) 5, 6, 1′

1, 3, 4, 9

1, 2, 5, 9

43.0, CH₂1.37 m

3, 4, 6, 7, 8

4, 5

5, 6, 8

5, 6, 7

41.6, CH₂1.43 m

3, 4, 6, 7, 8

5, 7, 8

5, 6, 8

5, 6, 7

.59 m

1.67 m

1.63 m

26.1, CH

1.61 m

24.8, CH

23.6, CH₃0.946 d (6.3)

22.8, CH₃0.954 d (6.3)

14.7, CH₃1.17 d (7.0)

164.8, C

22.9, CH₃0.94 d (6.4)

22.2, CH₃0.95 d (6.4)

14.1, CH₃1.27 d (7.2)

160.1, C

110.0, CH 6.83 s

54.3, CH₃3.84 s

8.42 d (9.5)

1, 2, 3

1′

2′

3′

144.0, CH 7.47 s

1′

144.5 7.45 s

1′

6.95 d (8.9)

4, 1′

Recorded at 125 MHz (referenced to δ 49.2 for CD OD, δ 29.8 for acetone-d , and δ 77.0 for CDCl ). Recorded at 500 MHz (referenced to

δ 3.31 for CD OD, δ 2.05 for acetone-d , and δ_H7.27 for CDCl ). HMBC correlations are from protons stated to the indicated carbon(s).

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a,b

Table 2. Comparison of the Experimental and Calculated NMR Chemical Shifts of 2 and Truncated Structures 2a and 2b

A-76356 (2)

δ_C

position

δ_H

δ_C

δ_H

δ_C

δ_H

|Δδ_C|

|Δδ_H|

|Δδ_C|

|Δδ_H|

1′

2′

3′

160.1

110.0

54.3

159.0

111.1

52.8

158.4

111.3

52.5

1.1

1.5

1.2

1.7

1.3

1.8

1.6

6.83

3.84

6.53

3.66

6.14

3.71

MAE

0.30

0.18

0.24

0.69

0.13

0.41

NMR chemical shifts were calculated at the mPW1PW91/6-311+G(2d,p)-IEFPCM level of theory and corrected by the scaling facotors. See ref

2. |Δδ | = |δ (2) − δ (2a or 2b)|, and |Δδ | = |δ (2) − δ (2a or 2b)|. MAE is the mean absolute error of the calculated scaled shifts to

experimental shifts.

Figure 4. (a) Incorporation of ¹³C-labeled precursors into akazaoxime (1) and A-76356 (2). (b) Biosynthesis of leucine from isovaleric acid and

acetate.

The planar structure of 2, determined by NMR and mass

spectroscopic analyses (Table 1), was identical to A-76356, an

Enteromycin-class antibiotics are characterized by a rare

nitrogen-containing functional group, O-methyl nitronic acid.

To date, only fewer than 10 related compounds are known and

all but 2 are produced by actinomycetes of the genus

Streptomyces. Among them, U-15774 has an aldoxime

group. Akazaoxime (1) is the second to have this functionality

in this class.

antibacterial metabolite from Micromonospora described in a

patent. Because J information was lacking in the literature

H,H

and H and C chemical shifts were slightly diﬀerent

presumably due to diﬀerent puriﬁcation conditions, their

identity was reasonably aﬃrmed by the similarity in the

Despite the rarity of aldoxime and O-methyl nitronic acid in

microbial metabolites, none of the compounds in this class

have yet to become a subject of biosynthetic studies, which

producing strains and other physicochemical properties. Thus,

was concluded to be A-76356. Analysis of J

coupling

H,H

constants (Table 1) and NOESY correlation data (Figure S21)

supported the same relative conﬁguration of 1. The geometry

of the O-methyl nitronate group was unclear, although a weak

NOESY correlation between H2′ and H3′ was indicative of its

E conﬁguration. To gain further proof for this double bond

geometry, NMR chemical shifts were calculated for the

motivated us to conduct feeding experiments with C-labeled

leucine, propionate, acetate, and glycine, as the plausible

precursors.

For the measurement of C{ H} NMR peak areas, acetone-

d was used as the solvent, because the C4 (δ 49.2) resonance

in 1 (Table 1) was overlapped by a CD OD peak. Feeding of

0,21

truncated model compounds, E-2a and Z-2b.

The

[

1- C]propionate resulted in C enrichment at C1, indicating

experimental NMR data showed better matching to the

the incorporation of methylmalonate into C1, C2, and C9

(

simulated data for 2a, with the MAE values being smaller than

Figure 4a). On the contrary, a DL-[2- C]leucine-fed culture

those of 2b for both H and C{ H} NMR chemical shifts,

which supported the E geometry of the O-methyl nitronate

group of 2 (Table 2).

produced a C4-labeled 1, which suggested leucine to be the

origin of the C unit from C3 to C8. This was supported by

labeling of C3 by [1- C]acetate feeding, because leucine is

Derivatization of 2 with PGME was not successful because

of the instability under the reaction conditions, but it is highly

likely that 1 and 2 possess the same absolute conﬁguration

considering their similar negative speciﬁc rotation values as

well as the production from the same organism.

biosynthesized from α-ketoisovaleric acid and acetic acid in

four steps (Figure 4b). The aldoxime functionality was not

labeled by any of the precursors described above except

[1- C]glycine, which caused 2.5-fold signal enhancement at

C1′ (Table 3). Similarly, the same labeling patterns were

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Table 3. Incorporation of ¹³C-Labeled Precursors into

Akazaoxime (1)

Scheme 1. Synthetic Plan of Akazaoxime (1)

relative enrichment

label

DL-[2- C]

leucine

[1- C]

position

δ_C

propionate

acetate

glycine

177.1

44.3

75.2

48.2

42.7

25.5

23.4

22.6

14.4

162.6

144.5

0.48

0.93

0.95

1.15

1.31

1.24

1.41

1.60

1.15

0.50

1.00

6.75

0.71

1.01

1.17

1.09

0.98

1.05

1.00

0.85

0.84

1.00

1.15

1.11

1.19

0.97

1.10

1.02

1.04

1.23

1.39

2.53

1.00

0.75

0.98

2.61

0.87

1.10

1.08

1.12

0.86

1.00

1.38

3.56

1.12

1.26

1.68

1.09

1.32

1.46

0.58

1.00

Accordingly, we chose known N-Boc-L-leucinal (6) as a

starting compound for the total synthesis.

1′

′

N-Boc-L-leucinal (6) was synthesized by the IBX oxidation

of N-Boc-L-leucinol (7) in reﬂuxing EtOAc (Scheme 2). The

C signal intensity of each peak in labeled 1 divided by that of the

corresponding signal in the unlabeled, normalized to the peak area of

C2′ to give an enrichment ratio for the enriched peak. The bold

Scheme 2

values indicate C-enriched atoms from C-labeled precursors.

Recorded at 125 MHz (referenced to δ 29.84 for acetone-d ). Not

determined due to a low signal intensity.

obtained for 2 (Figure 4a and Table 4). Altogether, 1 was

thought to be biosynthesized from propionate (methylmalo-

Table 4. Incorporation of ¹³C-Labeled Precursors into A-

76356 (2)

relative enrichment

label

L-[2- C]

leucine

[1- C]

position

δ_C

propionate

acetate

glycine

179.9

42.9

74.5

48.4

41.6

24.8

22.9

22.2

14.1

160.1

110.0

54.3

0.70

0.99

0.96

0.94

1.09

1.02

1.26

1.16

0.98

0.57

1.00

0.88

0.36

0.91

0.89

2.81

1.31

1.34

1.62

1.48

1.05

0.52

1.00

0.84

4.93

1.06

0.98

1.10

1.18

1.29

1.45

1.30

1.21

0.47

1.00

0.93

0.43

0.64

4.14

0.91

0.93

1.34

1.84

1.58

1.30

0.25

1.00

0.73

0.44

1.17

1.05

1.10

1.49

1.81

2.13

2.05

1.17

2.95

1.00

1.03

Reagents and conditions: (a) IBX, EtOAc, reﬂux; (b) A, BF ·OEt ,

THF, −78 °C; (c) Ac O, pyridine, DMAP, 53% (three steps); (d)

HCl, MeOH; (e) Ac O, pyridine, Et N, DMAP, 67% (two steps).

1′

2′

3′

stereochemically unstable 6 was immediately subjected to

Brown’s crotylation

prepared from (E)-2-butene and (−)-B(Ipc) OMe, aﬀording 8

with the requisite three consecutive stereocenters as a single

isomer. The structure of 8 was determined by the X-ray

crystallographic analysis of 11, which was derivatized from 8 in

three steps, acetylation of the hydroxy group (8 → 9), removal

of the Boc group (9 → 10), and acetamide formation (10 →

25,26

using BF ·OEt and A, which was

3 2

C signal intensity of each peak in labeled 2 divided by that of the

corresponding signal in the unlabeled, normalized to the peak area of

C2′ to give an enrichment ratio for the enriched peak. The numbers

in bold type indicate C-enriched atoms from C-labeled precursors.

Recorded at 125 MHz (referenced to δ 77.0 for CDCl ).

1).

Akazaoxime (1) was synthesized from 9 in eight steps

nate), leucine, and glycine, and further modiﬁcation at the

aldoxime terminus should give nitronic acid 2.

(Scheme 3). First, the vinyl group of 9 was oxidatively cleaved

with KMnO to provide carboxylic acid 12. After the acetyl

The scarcity of 1 from the producing strain, Micromonospora

sp. AKA109, hampered us from fully evaluating its biological

activity. Thus, we decided to establish the synthetic route to 1

to obtain suﬃcient quantities for biological evaluation. The

synthetic route was planned on the basis of the biosynthetic

pathway (Scheme 1). We envisioned that 1 would be

synthesized from 4 by oxidation of the amine to aldoxime at

the ﬁnal stage. Amide 4 would be obtained from amine 5 by

condensation with Boc-glycine. The propionate part in 1 could

be stereoselectively constructed through Brown’s crotylation.

group of 12 was removed under basic conditions, the

carboxylic acid was protected as a methyl ester by the

treatment with TMSCHN . Then, removal of the Boc group in

TFA gave amine 5, which was condensed with N-Boc-glycine

by the action of EDCI·HCl and DMAP, constructing the full

carbon skeleton of 1. Prior to the ﬁnal aldoxime formation, the

methyl ester was hydrolyzed with NaOH, and the Boc group

on the glycine residue was removed with TFA. Although the

aldoxime was not obtained by the standard oxidation of 4 with

oxone or MMPP, the use of m-CPBA in EtOAc enabled the

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Article

Scheme 3

Scheme 4

Reagents and conditions: (a) KMnO , NaIO , NaHCO , t-BuOH,

H O; (b) K CO , MeOH; (c) TMSCHN , MeOH, benzene, 42%

(

three steps); (d) TFA, CH Cl ; (e) N-Boc-glycine, EDCI·HCl, Et N,

Reagents and conditions: (a) TFA, CH

; (b) N-Boc-DL-alanine/

DMAP, CH Cl , 75% (two steps); (f) aqueous NaOH, THF; (g)

N-Boc-DL-phenylalanine, EDCI·HCl, Et N, DMAP, CH Cl ; (c)

aqueous NaOH, THF; (d) TFA, CH

TFA, CH Cl ; (h) m-CPBA, Na HPO , EtOAc, 33% (three steps).

; (e) m-CPBA, Na

HPO ,

EtOAc, 35% for 17, 52% for 18 (ﬁve steps).

desired oxidation, giving rise to 1. All of the spectroscopic data

the E geometry of O-methyl nitronic acid of 2 was conﬁrmed

by the NOESY analysis and NMR chemical shift calculation of

truncated model compounds. The biosynthetic origin of their

aldoxime/nitronic acid unit was elucidated to be glycine by

precursor feeding experiments. The total synthesis of 1 was

achieved on the basis of the biosynthetic pathway in 11 steps

from N-Boc-L-leucinol (7). The synthetic route enabled us to

supply suﬃcient amounts of 1 and its unnatural analogues for

biological evaluation. Compound 1 was found to be weakly

active against Gram-positive bacterium K. rhizophila, while

compounds 4 and 18 possess antimicrobial spectra diﬀerent

from those of 1, showing selective activity against ﬁlamentous

fungi.

of synthesized 1 ([α] , IR, and H and C{ H} NMR) were

identical with those of natural 1.

Establishment of the concise and ﬂexible route of 1

motivated us to synthesize its unnatural analogues for

biological evaluation. Focusing on the eﬀect of the unique

structure of the oxidized amino acid residue on bioactivity, we

newly designed 17 and 18 as the synthetic target compounds,

which possess oxime moieties on alanine and phenylalanine

residues instead of the aldoxime of 1.

After an acid treatment of 14, the resulting amine 5 was

condensed with N-Boc-DL-alanine and N-Boc-DL-phenylalanine

to provide 19 and 22, respectively (Scheme 4). Removal of the

acetyl and Boc groups of 19 and 22, followed by m-CPBA

oxidations in EtOAc, aﬀorded 17 and 18, respectively.

The bioactivity of 1, 17, and 18, as well as synthetic

intermediate 4 for 1, was examined in antimicrobial and

cytotoxic assays (Table 5). Compounds 1 and 17 showed weak

to moderate antimicrobial activities against Gram-positive

Kocuria rhizophila, while these compounds were inactive

against other Gram-positive and -negative bacteria (Staph-

ylococcus aureus, Escherichia coli, and Rhizobium radiobacter), a

yeast (Candida albicans), and ﬁlamentous fungi Glomerella

cingulata and Trichophyton rubrum. Intriguingly, compounds 4

and 18 showed diﬀerent antimicrobial spectra with selective

activities against ﬁlamentous fungi G. cingulata and T. rubrum

with MIC values of 25−50 μg/mL. Compound 2 was weakly

active against G. cingulata with an MIC of 100 μg/mL but

inactive against T. rubrum. These results indicated that the

substituents of the C4-amino group of 1 aﬀect the

antimicrobial activity.

Enteromycin-class metabolites make up a very rare class of

natural products possessing O-methyl nitronic acid function-

ality. Our C labeling experiments suggested that this

functional group originated from glycine via an aldoxime

functionality through oxidation of the primary amino group.

Discovery of 1 validates the signiﬁcance of marine environ-

ments as yet untapped habitats for unstudied microbial species

to obtain new chemical entities.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were

measured using a JASCO P-1030 polarimeter and an ATAGO AP-

■

00 polarimeter at room temperature using the sodium D line. UV

and IR spectra were recorded on a Shimadzu UV-1800 spectropho-

tometer and a PerkinElmer Spectrum 100 instrument, respectively. H

and C{ H} NMR spectra were recorded on a Bruker AVANCE II

400 spectrometer and a Bruker ANANCE NEO 500 spectrometer at

room temperature, unless otherwise noted. Chemical shifts are

reported in parts per million on the δ scale relative to CHCl (δ 7.27

CONCLUSION

for H NMR), CDCl

(δ 77.0 for C{ H} NMR), CD

HOD (δ 3.31

■

for H NMR), CD OD (δ 49.15 for C{ H} NMR), C D H (δ 7.16

for H NMR), C D (δ 128.06 for C{ H} NMR), CD HCOCD (δ

.05 for H NMR), and CD COCD (δ 29.84 for C{ H} NMR), as

Our chemical investigation of a marine-derived Micro-

monospora AKA109 led to the isolation of a new enter-

omycin-class secondary metabolite akazaoxime (1) along with

its known congener, A-76356 (2). The absolute conﬁgurations

of 1 and 2 were determined by spectrochemical analyses, and

3 3

internal references. Structural assignments were made with additional

information from gCOSY, gHSQC, gHMBC, and gNOESY experi-

ments. High-resolution mass spectra (HRMS) were recorded by ESI

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Table 5. Antimicrobial Activity of 1 and Its Synthetic Analogues

MIC (μg/mL)

microorganism

kanamycin

amphotericin B

Kocuria rhizophila ATCC9341

S. aureus FDA209P JC-1

E. coli NIHJ JC-2

R. radiobacter NBRC14554

C. albicans NBRC0197

G. cingulata NBRC5907

T. rubrum NBRC5467

100

>100

0.39

0.78

1.25

0.39

1.25

0.78

>100

using a TOF-MS analyzer on a Bruker micrOTOF focus mass

spectrometer. An Agilent HPLC system equipped with a diode array

detector and a Shimazu HPLC system were used for analysis and

puriﬁcation. Melting points were measured on Yanaco micro melting

point apparatus and are uncorrected. The X-ray crystallographic

analysis was performed with a Bruker D8 QUEST instrument with a

PHOTON II CPAD detector and with an IμS DIAMOND

Microfocus CuKα radiation source (λ = 1.54178 Å) at 100 K.

All reactions sensitive to air or moisture were carried out in dry

solvents under an argon atmosphere, unless otherwise noted. THF

and CH Cl (dehydrated, stabilizer free) were purchased from Kanto

3/7, 4/6, 5/5, 6/4, 7/3, and 8/2 (v/v)]. Fraction 2 (3/7) was

evaporated to give 15 mg of yellow oil. Final puriﬁcation was achieved

by HPLC puriﬁcation (COSMOSIL 5C -AR-II, Nacalai Tesque, 10

mm × 250 mm) with a MeOH/0.1% HCO H solution (38/62) at a

rate of 4 mL/min. The collected fraction was evaporated under

reduced pressure, and the resulting aqueous layer was extracted with

ethyl acetate. Concentration of the organic layer gave akazaoxime (1,

8.9 mg, t = 14.1 min). Concentration of silica gel column fraction 3

(10/1) provided 0.32 g of brown oil, which was further puriﬁed by

ODS column chromatography with a step gradient of a MeCN/0.1%

HCO H solution [2/8, 3/7, 4/6, 5/5, 6/4, 7/3, and 8/2 (v/v)].

Chemical Co., Inc. All other reagents were used as supplied. Analytical

thin-layer chromatography (TLC) was performed using Merck TLC

Silica Gel 60 F254 (0.25 mm). An oil bath was used as the heat source

for reactions that required heating. Preparative thin-layer chromatog-

raphy (PTLC) was performed using Merck PLC Silica Gel 60 F254

Fraction 3 (4/6) was concentrated to give 39 mg of yellow oil, which

was subjected to HPLC puriﬁcation (COSMOSIL 5C -AR-II,

Nacalai Tesque, 10 mm × 250 mm) with a MeCN/0.1% HCO H

solution (24/76) at a rate of 4 mL/min, in a manner similar to that

described for 1, to yield A-76356 (2, 8.0 mg, t = 20.8 min).

(

0.50 mm). Flash chromatography was performed using 63−210 μm

Akazaoxime (1). Colorless amorphous; [α]

−23 (c 0.10,

−1

Silica Gel 60N (Kanto Chemical Co., Inc.).

MeOH); IR (ATR) ν_m_a_x3268, 2957, 1710, 1657, 1535 cm ; UV

The conformational search was performed with MacroModel

(MeOH) λ_m_a_x(log ε) 213 (3.56) nm; for H and C{ H} NMR data,

9,30

implemented in the Maestro 12.3 software package.

All DFT-

see Table 1; HRMS (ESI/TOF) m/z 283.1256 [M + Na] (calcd for

based calculations were performed with Gaussian 16 revision B.01.

C H N NaO , 283.1264).

Some of these computations was conducted using the Super-

Computer System (Institute for Chemical Research, Kyoto

University). Molecular structures were visualized using the Maestro

A-76356 (2). Light brown amorphous; [α] −18 (c 0.10, MeOH)

[lit. [α] −27 (c 0.70, MeOH)]; IR (ATR) ν 3324, 2955, 1710,

max

−

1640, 1538 cm ; UV (MeOH) λ (log ε) 251 (4.07) nm; for H

and C{ H} NMR data, see Table 1; HRMS (ESI/TOF) m/z

313.1364 [M + Na] (calcd for C H N NaO , 313.1370).

max

2.3 software package.

Microorganism. Micromosnospora sp. AKA109 was isolated from

a seawater sample collected at a depth of 800 m in Sagami Bay, Japan,

Preparation of PGME Amides (3a and 3b) of 1. To a solution

of 1 (1.0 mg, 3.9 μmol) in dry DMF (100 μL) and dry triethylamine

(50 μL) were added (R)-PGME (4.1 mg, 15 μmol), PyBOP (4.1 mg,

7.9 μmol), and HOBt (2.0 mg, 15 μmol) at room temperature. After

being stirred for 1 h, the reaction mixture was concentrated by

lyophilization. This crude mixture was directly puriﬁed by preparative

HPLC (COSMOSIL 5C -PAQ, Nacalai Tesque, 10 mm × 250 mm)

as previously described. The isolated strain was identiﬁed as

Micromonospora on the basis of 99.4% similarity in the 16S rRNA

gene sequence (1402 nucleotides; DDBJ accession number

LC476779) to Micromonospora rifamycinica AM105^T(accession

number LRMV01000349).

Fermentation. Strain AKA109 growing on a Bn-2 agar plate was

inoculated into 500 mL K-1 ﬂasks each containing 100 mL of seed

medium consisting of 1% soluble starch, 0.5% glucose, 0.3% NZ-case

with a gradient of a MeCN/0.1% HCO H solution (MeCN

concentration of 34% from 0 to 5 min and 34% to 100% from 5 to

(Sigma-Aldrich, Co.), 0.2% yeast extract (Kyokuto Pharmaceutical

71 min) to aﬀord (R)-PGME amide (3a, 1.2 mg, t = 26.3 min): H

Industrial, Co., Ltd.), 0.5% Tryptone (Difco Laboratories), 0.1%

NMR (400 MHz, CD OD) δ 7.47 (s, 1H, H2′), 7.40−7.32 (m, 5H,

K HPO , 0.05% MgSO ·7H O, and 0.3% CaCO (pH 7.0). The ﬂasks

-C H ), 5.50 (s, 1H, −CHPh), 4.24 (m, 1H, H4), 3.70 (s, 3H,

were shaken at 30 °C for 4 days on a rotary shaker (200 rpm). The

seed culture (3 mL) was transferred into 500 mL K-1 ﬂasks each

containing 100 mL of A-16 production medium consisting of 2.0%

-OCH ), 3.69 (overlapped, 1H, H3), 2.54 (dq, 1H, J = 6.0, 8.6 Hz,

H2), 1.61 (m, 2H, H5 and H6), 1.37 (m, 1H, H5), 1.09 (d, 3H, J =

6.9 Hz, H9), 0.94 (d, 6H, J = 6.3 Hz, H7 and H8); HRMS (ESI/

glucose, 1.0% Pharmamedia (Traders Protein), 0.5% CaCO , and

TOF) m/z 430.1943 [M + Na] (calcd for C H N NaO

20 30

1.0% Diaion HP-20 (Mitsubishi Chemical Co.) (pH 7.0). The

430.1954).

inoculated ﬂasks were placed on a rotary shaker (200 rpm) at 30 °C

(S)-PGME amide (3b, 1.4 mg, t = 25.6 min) was obtained by the

for 7 days.

reaction of 1 (1.0 mg, 3.9 μmol) and (S)-PGME in the same manner

Extraction and Isolation. At the end of the fermentation period,

as described for (R)-PGME amide (3a): H NMR (400 MHz,

00 mL of 1-butanol was added to each ﬂask, which was then agitated

CD OD) δ 7.43 (s, 1H, H2′), 7.41−7.32 (m, 5H, -C H ), 5.50 (s, 1H,

on a rotary shaker for 1 h. The mixture was centrifuged at 6000 rpm

for 10 min, and the organic layer was separated from the aqueous

layer containing the mycelium. Evaporation of the organic layer gave

approximately 3.4 g of extract from 2 L of culture. This crude material

was subjected to silica gel column chromatography with a step

−CHPh), 4.23 (m, 1H, H4), 3.70 (s, 3H, -OCH ), 3.69 (overlapped,

1H, H3), 2.53 (m, 1H, H2), 1.58 (m, 1H, H6), 1.56 (m, 1H, H5),

1.34 (m, 1H, H5), 1.17 (d, 3H, J = 6.9 Hz, H9), 0.93 (d, 6H, J = 6.5

Hz, H7 and H8); HRMS (ESI/TOF) m/z 430.1942 [M + Na]⁺

(calcd for C H N NaO 430.1954).

gradient of CHCl /MeOH [1/0, 20/1, 10/1, 4/1, 2/1, 1/1, and 0/1

Feeding Experiments. Feeding experiments were performed

(v/v)]. Concentration of fraction 4 (4/1) provided 309 mg of dark

with DL-[2- C]leucine, sodium [1- C]propionate, sodium [1- C]-

brown oil, which was further puriﬁed by ODS column chromatog-

acetate, and [1- C]glycine. Inoculation, cultivation, and puriﬁcation

raphy with a step gradient of a MeCN/0.1% HCO H solution [2/8,

were carried out in the same manner as described above. Addition of

533

J. Org. Chem. 2021, 86, 6528−6537

The Journal of Organic Chemistry

Article

¹³C-labeled precursors was initiated 48 h after inoculation and

periodically carried out every 24 h four times. After being incubated

for a further 24 h, the cultures were extracted with 1-butanol and

subjected to the same puriﬁcation protocol described above.

was ﬁltered through a short pad of Celite with Et O (100 mL).

Saturated aqueous NaHCO

the resulting solution was extracted with Et

combined organic layers were washed with brine (30 mL), dried over

Na SO , ﬁltered, and concentrated. The residue was puriﬁed by ﬂash

(30 mL) was added. After separation,

O (3 × 30 mL), and the

(

1) DL-[2- C]Leucine. After feeding of DL-[2- C]leucine (Sigma-

Aldrich, total of 80 mg, 2 mg × 10 ﬂasks × 4 days), C-labeled 1 (1.9

column chromatography on silica gel (200 g, hexane/EtOAc 10/1 to

5/1) to aﬀord crude 6, which was used in the next reaction without

further puriﬁcation.

mg) and 2 (2.9 mg) were isolated from 1 L of culture. The C{ H}

NMR spectra showed an enriched signal at δ 48.2 for 1 and δ 48.4 for

A balloon charged with trans-2-butene was connected to an ice-

cooled ﬂask at −78 °C, and trans-2-butene (∼6 mL, ∼60 mmol) was

released. After the balloon was removed, t-BuOK (3.10 g, 27.6 mmol)

in THF (15 mL) and n-BuLi (2.5 M in hexanes, 11 mL, 28 mmol)

were added. The reaction mixture was warmed to −45 °C and stirred

for 10 min. The solution was cooled to −78 °C, and then a solution of

(−)-methoxydiisopinocampheylborane (8.12 g, 25.7 mmol) in THF

(10 mL) was added. After the resulting mixture was stirred at −78 °C

(

2) Sodium [1- C]Propionate. After feeding of sodium [1- C]-

propionate (Cambridge Isotope Laboratories Inc., total of 800 mg, 20

mg × 10 ﬂasks × 4 days), C-labeled 1 (9.0 mg) and 2 (3.4 mg) were

obtained from 1 L of culture. The C{ H} NMR spectra showed an

enriched signal at δ 177.1 for 1 and δ 179.9 for 2.

(

3) Sodium [1- C]Acetate. After feeding of sodium [1- C]acetate

(

Cambridge Isotope Laboratories Inc., total of 800 mg, 20 mg × 10

ﬂasks × 4 days), C-labeled 1 (3.8 mg) and 2 (1.5 mg) were obtained

from 1 L of culture. The C{ H} NMR spectrum showed an enriched

for 30 min, BF

THF (2 × 5 mL) were added. The reaction mixture was stirred at

−78 °C for 6 h, and then a NaOH solution (3 M in H O, 10 mL) and

30% aqueous H (5 mL) were added. The mixture was warmed to

room temperature and stirred vigorously for 13 h. The resulting

solution was extracted with CH Cl (3 × 30 mL), and the combined

organic layers were washed with H O (2 × 30 mL) and brine (30

3 2

·OEt (3.5 mL, 28 mmol) and the above crude 6 in

signal at δ 75.2 for 1 and δ 74.5 for 2.

(

4) [1- C]Glycine. After feeding of [1- C]glycine (Cambridge

2 2

Isotope Laboratories Inc., total of 80 mg, 2 mg × 10 ﬂasks × 4 days),

C-labeled 1 (9.9 mg) and 2 (5.5 mg) were obtained from 1 L of

culture. The ¹³C{ H} NMR spectrum showed an enriched signal at δ

mL), dried over Na SO , ﬁltered, and concentrated to aﬀord crude 8,

162.6 for 1 and δ 160.1 for 2.

which was used in the next reaction without further puriﬁcation.

Ac O (10 mL) and DMAP (16.1 mg, 132 μmol) were added to a

Procedure for the Optimization, Energy Evaluation, and

Simulation of the NMR Spectra for Structures 2a and 2b. The

conformational search on structure 2a began upon application of

solution of crude 8 in pyridine (20 mL) at 0 °C. The reaction mixture

was warmed to room temperature and stirred for 19 h. After toluene

100000 steps of the Monte Carlo Multiple Minimum (MCMM)

method with PRCG energy minimization using the OPLS3e force

ﬁeld (gas phase) to obtain 11 conformational isomers with energies

within 35.85 kcal/mol. The next optimizations were performed at the

B3LYP/6-31+G(d,p) level of theory. Frequency calculations were

carried out at the same level of theory to conﬁrm the absence of

imaginary frequencies and to obtain thermal corrections to the Gibbs

free energies at 1 atm and 298.15 K. After single-point energies were

calculated at the B3LYP/6-311+G(d,p) level of theory with the IEF-

was added, the resulting solution was concentrated. Et

was added, and the combined organic layers were washed with a HCl

solution (1 M in H O, 2 × 30 mL), saturated aqueous NaHCO (2 ×

30 mL), and brine (30 mL), dried over Na SO , ﬁltered, and

O (30 mL)

concentrated. The residue was puriﬁed by ﬂash column chromatog-

raphy on silica gel (500 g, hexane/EtOAc 50/1 to 10/1) to aﬀord

acetate 9 (2.30 g, 7.33 mmol) in 53% yield over three steps as a

colorless oil: [α]

2950, 1704, 1519, 1366, 1248, 1169, 1025 cm ; H NMR (500 MHz,

, 336 K) δ 5.72 (m, 1H), 4.93 (br d, 1H, J = 16.9 Hz), 4.91 (dd,

−49.5 (c 1.01, CHCl ); IR (ATR) νmax 3359,

−

1 1

PCM solvation model (CHCl ), the thermal corrections at the

B3LYP/6-31+G(d,p) level were added to obtain the Gibbs free

energies. The shielding tensors of the most stable geometry were

calculated by the GIAO method at the mPW1PW91/6-311G+(2d,p)

C D

6 6

1H, J = 10.1, 1.7 Hz), 4.84 (br d, 1H, J = 5.6 Hz), 4.41 (br s, 1H),

4.19 (br s, 1H), 2.47 (m, 1H), 1.70 (s, 3H), 1.75−1.65 (m, 1H), 1.44

(s, 9H), 1.17 (m, 2H), 1.06 (d, 3H, J = 6.3 Hz), 0.95 (d, 3H, J = 6.3

level of theory with solvation of the IEF-PCM (CHCl ) model.

Because the other 10 conformers were located at signiﬁcantly higher

energies (>2.5 kcal/mol), these conformers were not subjected to the

calculation. The shielding tensors (σ) were converted the chemical

shifts (δ_c_a_l_c) using the equation δ_c_a_l_c= (intercept − σ)/−slope, where

slope and intercept are scaling factors, −1.0719 and 31.8733 for H

and −1.0420 and 186.3567 for C, respectively, reported by

Pierens. The NMR spectra of structure 2b were similarly simulated

by using seven conformers as the OPLS3e-minimized structures and

DFT-optimized structures and one conformer as the geometry for the

NMR calculation.

Hz), 0.86 (d, 3H, J = 6.7 Hz); C{ H} NMR (125 MHz, C D , 336

6 6

K) δ 169.7, 155.8, 140.6, 115.3, 78.9, 78.7, 49.5, 43.1, 40.8, 28.6, 25.1₊,

23.2, 22.3, 20.4, 17.2; HRMS (ESI/TOF) m/z 336.2144 [M + Na]

(calcd for C17

Acetamide 11. A HCl solution (3 M in H O, 0.10 mL) was added

H31NNaO , 336.2145).

to a solution of acetate 9 (100 mg, 319 μmol) in MeOH (1 mL) at

room temperature. The reaction mixture was stirred at room

temperature for 1 h, and then a HCl solution (3 M in H

mL) was added. The reaction mixture was stirred at room

temperature for 1 h, and then a HCl solution (6 M in H O, 0.4

O, 0.4

Synthetic Procedures for Synthesis of Akazaoxime (1) and

mL) was added. The reaction mixture was stirred at room

Its Analogues. Boc-L-leucinol 7. Boc O (5.9 mL, 25.6 mmol) was

temperature for 18 h, and then a HCl solution (6 M in H O, 0.4

added to a solution of a commercially available L-leucinol (2.50 g, 21.3

mmol) and Et N (4.4 mL, 32.0 mmol) in CH Cl (50 mL) at room

temperature. The reaction mixture was stirred at room temperature

mL) was added. The reaction mixture was stirred at room

temperature for 2 h, and then a HCl solution (6 M in H O, 0.4

mL) was added. The reaction mixture was stirred at room

temperature for 25 h. The reaction mixture was warmed to 50 °C

and stirred for 40 min. After the reaction mixture was cooled to room

temperature, EtOH was added. The resulting solution was

concentrated to aﬀord crude 10, which was used in the next reaction

without further puriﬁcation.

for 20 h, and then H O (110 mL) was added. After being stirred for

.5 h, the mixture was separated. The aqueous layer was extracted

with CH Cl (3 × 100 mL), and the combined organic layers were

dried over Na SO , ﬁltered, and concentrated. The residue was

puriﬁed by ﬂash column chromatography on silica gel twice (70 g,

hexane/EtOAc 10/1 to 5/1 to 3/1 to EtOAc; 100 g, hexane/EtOAc

Et N (0.10 mL, 0.73 mmol) was added to a solution of crude 10 in

0/1 to 15/1 to 10/1 to 5/1 to 2/1 to 1/2 to 1/10) to aﬀord Boc-L-

a mixture of pyridine (20 mL) and Ac O (10 mL) at 0 °C. The

leucinol 7 (3.79 g, 17.4 mmol) in 82% yield as a colorless oil. Boc-L-

leucinol 7 is a known compound, and the H NMR spectrum of

reaction mixture was warmed to room temperature and stirred for 4 h,

and then DMAP (3.0 mg, 20 μmol) was added. The reaction mixture

synthesized 7 was identical to that reported previously.

was stirred at room temperature for 15 h, and then Et N (0.20 mL,

Acetate 9. IBX (39% purity, 9.87 g, 13.7 mmol) was added to a

solution of Boc-L-leucinol 7 (3.00 g, 13.8 mmol) in EtOAc (55 mL).

The reaction mixture was heated to reﬂux temperature and stirred for

1.4 mmol) was added. The reaction mixture was warmed to 50 °C

and stirred for 2 h. After the reaction mixture was cooled to room

temperature, toluene was added. The resulting solution was

3.5 h. After being cooled to room temperature, the reaction mixture

concentrated. The residue was dissolved in Et O (30 mL), and the

534

J. Org. Chem. 2021, 86, 6528−6537

The Journal of Organic Chemistry

Article

organic layer was washed with a HCl solution (1 M in H O, 2 × 30

mg, 652 μmol) in 75% yield over two steps as a yellow oil: [α]²⁸−46

mL), NaHCO (2 × 30 mL), and brine (30 mL), dried over Na SO ,

(c 0.93, CHCl ); IR (ATR) ν 3342, 2956, 2497, 1714, 1652, 1367,

max

−

1 1

ﬁltered, and concentrated. The residue was puriﬁed by ﬂash column

chromatography on silica gel (10 g, hexane/EtOAc 10/1 to 3/1) to

aﬀord acetamide 11 (54.8 mg, 215 μmol) in 67% yield over two steps

as colorless crystals. Acetamide 11 was recrystallized by a slow

1250, 1166, 1049, 860 cm ; H NMR (500 MHz, CD OD) δ 7.20

(d, 1H, J = 9.5 Hz), 4.15 (m, 1H), 3.69 (br s, 2H), 3.66 (s, 3H),

3.68−3.62 (m, 1H), 2.51 (m, 1H), 1.66−1.51 (m, 2H), 1.46 (s, 9H),

1.34−1.28 (m, 1H), 1.12 (d, 3H, J = 7.0 Hz), 0.94−0.92 (m, 6H);

evaporation method from a mixture of Et O and hexane for the X-ray

C{ H} NMR (125 MHz, CD OD) δ 178.0, 172.54, 172.47, 158.5,

crystallographic analysis: mp 99−100 °C; [α]

CHCl ); IR (ATR) ν 3272, 2966, 2934, 2873, 1738, 1642, 1553,

366, 1234 cm ; H NMR (400 MHz, CDCl ) δ 5.62 (ddd, 1H, J =

7.1, 10.2, 9.1 Hz), 5.45 (br d, 1H, J = 9.7 Hz), 4.98 (br d, 1H, J =

7.1 Hz), 4.95 (dd, 1H, J = 10.2, 1.6 Hz), 4.74 (dd, 1H, J = 8.3, 3.0

−42 (c 0.71,

81.0, 75.9, 52.3, 45.3, 45.1, 43.01, 42.97, 28.9, 25.9, 23.7, 22.8, 14.6;

HRMS (ESI/TOF) m/z 383.2153 [M + Na]⁺(calcd for

C H N NaO , 383.2131).

max

−

1 1

Akazaoxime (1). A NaOH solution (1 M in H O, 590 μL) was

added to a solution of amide 15 (169 mg, 469 μmol) in THF (5.8

mL) at room temperature. The reaction mixture was stirred at room

Hz), 4.40−4.31 (m, 1H), 2.33 (m, 1H), 2.02 (s, 3H), 1.98 (s, 3H),

.58 (m, 1H), 1.25−1.18 (m, 2H), 1.04 (d, 3H, J = 6.8 Hz), 0.88 (d,

temperature for 3.5 h, and then a HCl solution (1 M in H O, 5 mL)

13 1

H, J = 6.3 Hz), 0.87 (d, 3H, J = 6.5 Hz); C{ H} NMR (100 MHz,

was added. The resulting solution was extracted with EtOAc (5 × 20

CDCl ) δ 170.4, 169.4, 139.7, 115.4, 78.2, 47.3, 42.1, 40.3, 24.5, 23.3,

mL), and the combined organic layers were dried over Na SO ,

3.0, 22.1, 20.8, 17.1; HRMS (ESI/TOF) m/z 278.1724 [M + Na]

ﬁltered, and concentrated to aﬀord crude carboxylic acid 16, which

was used in the next reaction without further puriﬁcation.

(

calcd for C H NNaO , 278.1727).

Methyl Ester 14. NaHCO (1.41 g, 16.8 mmol), NaIO (4.32 g,

TFA (1.0 mL) was added to a solution of crude 16 in CH Cl (4.6

0.2 mmol), and KMnO (266 mg, 1.68 mmol) were added to a

mL) at room temperature for 2 h, and then toluene was added. The

resulting solution was concentrated to aﬀord crude amine 4, which

was used in the next reaction without further puriﬁcation.

solution of acetate 9 (527 mg, 1.68 mmol) in a mixture of t-BuOH

19 mL) and H O (8.4 mL) at room temperature. The reaction

(

mixture was stirred at room temperature for 30 min, and then

Na HPO (336 mg, 2.36 mmol) and m-CPBA (∼70% purity, 285

saturated aqueous NaHSO (20 mL) was added at 0 °C. A HCl

mg, ∼1.15 mmol) were added to a solution of crude 4 in EtOAc (4.6

mL) at room temperature. The reaction mixture was stirred at room

temperature for 4 h. The resulting solution was directly puriﬁed by

ﬂash column chromatography on silica gel twice (20 g, hexane/EtOAc

solution (1 M in H O, 100 mL) was added, and the resulting solution

was extracted with EtOAc (3 × 20 mL). The combined organic layers

were dried over anhydrous Na SO , ﬁltered, and concentrated. The

residue was puriﬁed by ﬂash column chromatography on silica gel (60

g, hexane/EtOAc/AcOH 5/1/0.03 to 1/1/0.01) to aﬀord crude

carboxylic acid 12, which was used in the next reaction without

further puriﬁcation.

/1 to EtOAc/MeOH/AcOH 10/1/0.1; 20 g, hexane/EtOAc 1/1 to

EtOAc/MeOH/AcOH 10/1/0.7) to aﬀord akazaoxime (1, 22.2 mg,

5.3 mmol) as a colorless oil and the crude product (67.5 mg). The

crude product was repuriﬁed by HPLC (Mightysil RP-18GPII, 10 mm

250 mm) with a MeOH/0.1% HCO H solution (50/50) at a rate

K CO (1.66 g, 12.0 mmol) was added to a solution of crude 12 in

MeOH (11 mL) at room temperature. The reaction mixture was

stirred at room temperature for 22 h, and then a HCl solution (1 M in

of 4 mL/min (t = 7.1 min). Collected fractions were extracted with

EtOAc, and the organic layers were dried over Na SO , ﬁltered, and

H O, 10 mL) was added. The resulting solution was extracted with

concentrated to aﬀord akazaoxime (1, 18.6 mg, 71.5 mmol) as a

EtOAc (3 × 20 mL), and the combined organic layers were dried over

colorless oil. The combined yield was calculated as 33% over three

Na SO , ﬁltered, and concentrated to aﬀord crude 13, which was used

steps: [α] −31 (c 0.95, MeOH); IR (ATR) ν 3270, 2958, 2931,

max

in the next reaction without further puriﬁcation.

−

1 1

708, 1657, 1537, 1462, 1255, 1200, 1200, 1016 cm ; H NMR (500

TMSCHN (2.0 M in Et O, 1.8 mL, 3.6 mmol) was added to a

MHz, CD OD) δ 7.47 (s, 1H), 4.24 (m, 1H), 3.68 (dd, 1H, J = 9.3,

solution of crude 13 in a mixture of benzene (11 mL) and MeOH

1.1 mL) at 0 °C. The reaction mixture was warmed to room

.5 Hz), 2.41 (dq, 1H, J = 9.3, 7.0 Hz), 1.65−1.56 (m, 2H), 1.37 (m,

(

H), 1.17 (d, 3H, J = 7.0 Hz), 0.954 (d, 3H, J = 6.3 Hz), 0.947 (d,

temperature and stirred for 2 h, and then AcOH (0.2 mL) was added.

The resulting solution was concentrated. The residue was puriﬁed by

ﬂash column chromatography on silica gel (10 g, hexane/EtOAc 10/1

to 5/1) to aﬀord methyl ester 14 (127 mg, 419 μmol) in 42% yield

over three steps as a colorless oil: [α] −48.8 (c 1.02, CHCl ); IR

(

H, J = 6.3 Hz); C{ H} NMR (125 MHz, CD OD) δ 179.6, 164.9,

43.9, 75.9, 49.2, 45.4, 42.9, 26.1, 23.6, 22.8, 14.8; HRMS (ESI/TOF)

m/z 283.1269 [M + Na] (calcd for C H N NaO , 283.1264).

11 20

Analogue 17. TFA (0.12 mL) was added to a solution of methyl

ester 14 (36.6 mg, 121 μmol) in CH Cl (0.7 mL) at room

ATR) ν 3447, 3389, 2957, 2931, 2872, 1713, 1502, 1366, 1250,

max

−

temperature. The reaction mixture was stirred at room temperature

for 12 h, and then toluene was added. The resulting solution was

concentrated to aﬀord crude amine 5, which was used in the next

reaction without further puriﬁcation.

163 cm ; H NMR (500 MHz, C D , 333 K) δ 4.69 (br s, 1H),

6 6

.97 (br s, 1H), 3.59 (br d, 1H, J = 8.0 Hz), 3.34 (s, 3H), 2.69 (m,

H), 1.64 (m, 1H), 1.52 (m, 1H), 1.42 (s, 9H), 1.27−1.20 (m, 1H),

.20 (d, 3H, J = 6.6 Hz), 0.95 (d, 3H, J = 6.4 Hz), 0.88 (d, 3H, J = 6.6

Et N (50 μL, 362 μmol), EDCI·HCl (61.7 mg, 322 μmol), N-Boc-

Hz); C{ H} NMR (125 MHz, C D , 333 K) δ 176.7, 156.2, 78.8,

DL-alanine (23.5 mg, 124 μmol), and DMAP (32.6 mg, 267 μmol)

were added to a solution of crude 5 in CH Cl (1.2 mL) at 0 °C. The

5.6, 51.3, 49.8, 43.6, 42.9, 28.5, 25.2, 23.2, 22.5, 14.1; HRMS (ESI/

TOF) m/z 326.1938 [M + Na] (calcd for C H NNaO , 326.1935).

reaction mixture was stirred at room temperature for 4 h, and then

saturated aqueous NH Cl (1.0 mL) and H O (2.0 mL) were

Amide 15. TFA (0.67 mL) was added to a solution of methyl ester

4 (265 mg, 873 μmol) in CH Cl (2.5 mL) at room temperature.

successively added. The resulting solution was extracted with EtOAc

3 × 10 mL), and the combined organic layers were dried over

Na SO , ﬁltered, and concentrated. The residue was puriﬁed by ﬂash

The reaction mixture was stirred at room temperature for 12 h, and

then toluene was added. The resulting solution was concentrated to

aﬀord crude amine 5, which was used in the next reaction without

further puriﬁcation.

(

column chromatography on silica gel twice (10 g, hexane/EtOAc 3/1

to 1/1; 10 g, hexane/EtOAc 2/1 to EtOAc) to aﬀord crude amide 19,

which was used in the next reaction without further puriﬁcation.

Et N (0.36 mL, 2.6 mmol), EDCI·HCl (443 mg, 2.31 mmol), N-

Boc-glycine (183 mg, 1.04 mmol), and DMAP (32.6 mg, 267 μmol)

were added to a solution of crude 5 in CH Cl (8.7 mL) at 0 °C. The

A NaOH solution (1 M in H O, 120 μL) was added to a solution

reaction mixture was stirred for 4.5 h at room temperature, and then

saturated aqueous NH Cl (5.0 mL) and H O (3.0 mL) were

of crude 19 (33.2 mg, 88.7 μmol) in THF (1.2 mL) at room

temperature. The reaction mixture was stirred at room temperature

successively added. The resulting solution was extracted with EtOAc

5 × 10 mL), and the combined organic layers were dried over

Na SO , ﬁltered, and concentrated. The residue was puriﬁed by ﬂash

for 11 h, and then a HCl solution (1 M in H O, 1 mL) was added.

(

The resulting solution was extracted with EtOAc (5 × 10 mL), and

the combined organic layers were dried over Na SO , ﬁltered, and

column chromatography on silica gel twice (25 g, hexane/EtOAc 2/1

to 1/1; 25 g, hexane to hexane/EtOAc 1/1). to aﬀord amide 15 (235

concentrated to aﬀord crude carboxylic acid 20, which was used in the

next reaction without further puriﬁcation.

535

J. Org. Chem. 2021, 86, 6528−6537

The Journal of Organic Chemistry

https://pubs.acs.org/doi/10.1021/acs.joc.1c00358.

Article

TFA (200 μL) was added to a solution of crude 20 in CH Cl (0.9

ATCC9341, S. aureus FDA209P JC-1, E. coli NIHJ JC-2, R.

radiobacter NBRC14554, C. albicans NBRC0197, G. cingulata

NBRC5907, and T. rubrum NBRC5467.

mL) at room temperature for 2 h, and then toluene was added. The

resulting solution was concentrated to aﬀord crude amine 21, which

was used in the next reaction without further puriﬁcation.

Na HPO (68.4 mg, 482 μmol) and m-CPBA (∼70% purity, 58.9

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

■

mg, ∼239 μmol) were added to a solution of crude 21 in EtOAc (0.94

mL) at room temperature. The reaction mixture was stirred at room

temperature for 11 h. The resulting solution was directly puriﬁed by

ﬂash column chromatography on silica gel (10 g, hexane/EtOAc 2/1

to 1/1 to EtOAc to EtOAc/MeOH 10/1) and PTLC (hexane/

EtOAc/AcOH 4/1/0.1) to aﬀord analogue 17 (11.6 mg, 42.3 μmol)

UV and IR spectra of 1, one- and two-dimensional NMR

spectra of 1 and 2, HRMS spectra of 1 and 2,

spectra of labeled 1 and 2, NMR data of newly

synthesized compounds, comparison of NMR spectra

of natural and synthetic 1, Cartesian coordinates of 2a

and 2b, and crystallographic data of 11 (PDF)

in 35% yield over ﬁve steps as a colorless oil: [α] −9.34 (c 1.07,

MeOH); IR (ATR) ν 3320, 2962, 1668, 1556, 1453, 1193, 1142,

max

−

1 1

024, 801, 724 cm ; H NMR (500 MHz, CD OD) δ 7.34 (br d,

H, J = 10.1 Hz), 4.21 (m, 1H), 3.69 (br d, 1H, J = 9.5 Hz), 2.39 (m,

H), 1.98 (s, 3H), 1.63−1.53 (m, 2H), 1.36 (m, 1H), 1.15 (d, 3H, J =

.0 Hz), 0.95 (d, 3H, J = 6.6 Hz), 0.94 (d, 3H, J = 6.6 Hz); C{ H}

Accession Codes

NMR (125 MHz, CD OD) δ 179.8, 166.1, 151.2, 75.9, 49.2, 45.5,

CCDC 2058723 contains the supplementary crystallographic

data for this paper. These data can be obtained free of charge

via www.ccdc.cam.ac.uk/data_request/cif , or by emailing

data_request@ccdc.cam.ac.uk , or by contacting The Cam-

bridge Crystallographic Data Centre, 12 Union Road,

Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

−

3.3, 26.1, 23.6, 22.9, 14.8, 9.3; HRMS (ESI/TOF) m/z 273.1456 [M

−

H] (calcd for C H N O , 273.1448).

12 21 2 5

Analogue 18. TFA (0.1 mL) was added to a solution of methyl

ester 14 (24.9 mg, 82.1 μmol) in CH Cl (0.7 mL) at room

temperature. The reaction mixture was stirred at room temperature

for 13 h, and then toluene was added. The resulting solution was

concentrated to aﬀord crude amine 5, which was used in the next

reaction without further puriﬁcation.

AUTHOR INFORMATION

Corresponding Authors

■

Et N (35 μL, 246 μmol), EDCI·HCl (42.5 mg, 222 μmol), N-Boc-

DL-phenylalanine (22.4 mg, 84.6 mmol), and DMAP (3.3 mg, 27

Yasuhiro Igarashi − Biotechnology Research Center and

Department of Biotechnology, Toyama Prefectural University,

Imizu, Toyama 939-0398, Japan; orcid.org/0000-0001-

μmol) were added to a solution of crude 5 in CH Cl (0.82 mL) at 0

C. The reaction mixture was stirred for 5 h at room temperature, and

then saturated aqueous NH Cl (1.0 mL) and H O (3.0 mL) were

5114-1389 ; Email: yas@pu-toyama.ac.jp

successively added. The resulting solution was extracted with CH Cl

Daisuke Urabe − Biotechnology Research Center and

Department of Biotechnology, Toyama Prefectural University,

Imizu, Toyama 939-0398, Japan; orcid.org/0000-0002-

1999-9374 ; Email: urabe@pu-toyama.ac.jp

(

4 × 10 mL), and the combined organic layers were dried over

Na SO , ﬁltered, and concentrated. The residue was puriﬁed by ﬂash

column chromatography on silica gel twice (10 g, hexane/EtOAc 5/1

to 1/1; 10 g, hexane/EtOAc 2/1 to 1/1) to aﬀord crude amide 22,

which was used in the next reaction without further puriﬁcation.

A NaOH solution (1 M in H O, 90 μL) was added to a solution of

Authors

crude 22 (32.2 mg, 71.4 μmol) in THF (0.9 mL) at room

temperature. The reaction mixture was stirred at room temperature

Yoe Matsuyuki − Biotechnology Research Center and

Department of Biotechnology, Toyama Prefectural University,

Imizu, Toyama 939-0398, Japan

Masayuki Yamada − Biotechnology Research Center and

Department of Biotechnology, Toyama Prefectural University,

Imizu, Toyama 939-0398, Japan

Nodoka Fujihara − Biotechnology Research Center and

Department of Biotechnology, Toyama Prefectural University,

Imizu, Toyama 939-0398, Japan

for 11 h, and then a NaOH solution (1 M in H O, 90 μL) was added.

The reaction mixture was stirred at room temperature for a further 11

h, and then a HCl solution (1 M in H O, 1 mL) was added. The

resulting solution was extracted with EtOAc (5 × 5 mL), and the

combined organic layers were dried over Na SO , ﬁltered, and

concentrated to aﬀord crude carboxylic acid 23, which was used in the

next reaction without further puriﬁcation.

TFA (150 μL) was added to a solution of crude 23 in CH Cl

(

0.74 mL) at room temperature for 2 h, and then toluene was added.

Enjuro Harunari − Biotechnology Research Center and

Department of Biotechnology, Toyama Prefectural University,

Imizu, Toyama 939-0398, Japan; orcid.org/0000-0001-

8726-0865

Naoya Oku − Biotechnology Research Center and Department

of Biotechnology, Toyama Prefectural University, Imizu,

Toyama 939-0398, Japan; orcid.org/0000-0002-2171-

The resulting solution was concentrated to aﬀord crude amine 24,

which was used in the next reaction without further puriﬁcation.

Na HPO (52.0 mg, 482 μmol) and m-CPBA (∼70% purity, 48.5

mg, ∼197 μmol) were added to a solution of crude 24 in EtOAc (0.71

mL) at room temperature. The reaction mixture was stirred at room

temperature for 12.5 h. The resulting solution was directly puriﬁed by

ﬂash column chromatography on silica gel (10 g, hexane to hexane/

EtOAc 2/1 to EtOAc to EtOAc/MeOH 10/1) and PTLC (hexane/

EtOAc/AcOH 4/1/0.1) to aﬀord analogue 18 (15.0 mg, 42.8 μmol)

in 52% yield over ﬁve steps as a colorless oil: [α] −9.70 (c 1.03,

MeOH); IR (ATR) ν 3384, 1662, 1459, 1198, 1143, 850, 801, 726

cm ; H NMR (400 MHz, CD OD) δ 7.28−7.12 (m, 5H), 4.18 (m,

2168

Md. Rokon Ul Karim − Biotechnology Research Center and

Department of Biotechnology, Toyama Prefectural University,

Imizu, Toyama 939-0398, Japan; orcid.org/0000-0002-

1891-8894

Taehui Yang − Graduate School of Marine Science and

Technology, Tokyo University of Marine Science and

Technology, Tokyo 108-8477, Japan

Katsuhisa Yamada − DHC Corporation, Tokyo 106-8571,

Japan

Chiaki Imada − Graduate School of Marine Science and

Technology, Tokyo University of Marine Science and

Technology, Tokyo 108-8477, Japan

max

−

1 1

H), 3.95 (d, 1H, J = 14.2 Hz), 3.91 (d, 1H, J = 14.2 Hz), 3.66 (dd,

H, J = 9.7, 1.2 Hz), 2.31 (m, 1H) 1.58−1.45 (m, 2H), 1.31 (m, 1H),

.08 (d, 3H, J = 7.0 Hz), 0.91 (d, 3H, J = 6.6 Hz), 0.89 (d, 3H, J = 6.4

Hz); C{ H} NMR (100 MHz, CD OD) δ 179.6, 165.7, 153.5,

38.1, 130.1, 129.4, 127.4, 75.9, 49.2, 45.5, 43.1, 30.1, 26.0, 23.6, 22.8,

4.6; HRMS (ESI/TOF) m/z 349.1767 [M − H]⁻(calcd for

C H N O , 349.1769).

Bioassays. A cytotoxicity test and antibacterial assays were carried

out according to the reported procedures, with K. rhizophila

536

J. Org. Chem. 2021, 86, 6528−6537

The Journal of Organic Chemistry

(16) Zhang, Z.; Zhou, T.; Harunari, E.; Oku, N.; Igarashi, Y.

Article

Keisuke Fukaya − Biotechnology Research Center and

Department of Biotechnology, Toyama Prefectural University,

Imizu, Toyama 939-0398, Japan; orcid.org/0000-0002-

Iseolides A-C, antifungal macrolides from a coral-derived actino-

mycete of the genus Streptomyces . J. Antibiot. 2020, 73, 534−541.

(

17) Pretsch, E.; Buhlmann, P.; Aﬀolter, C. Structure Determination

148-4714

of Organic Compounds; Springer: New York, 2000.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.joc.1c00358

(18) Eshghi, H.; Alizadeh, M. H.; Davamdar, E. Regioselective

synthesis of Z -aldoximes catalyzed by H PMo O under solvent-free

12 40

conditions. J. Korean Chem. Soc. 2008 , 52 , 52 −56.

(19) Yabuuchi, T.; Kusumi, T. Phenylglycine methyl ester, a useful

Funding

tool for absolute configuration determination of various chiral

This work was supported by JSPS KAKENHI Grants

carboxylic acids. J. Org. Chem. 2000, 65, 397−404.

6K07719 to Y.I., 20K15285 to K.F., and 20H03366 to D.U.

20) Lodewyk, M. W.; Siebert, M. R.; Tantillo, D. J. Computational

Notes

prediction of H and C chemical shifts: a useful tool for natural

The authors declare no competing ﬁnancial interest.

product, mechanistic, and synthetic organic chemistry. Chem. Rev.

21) Grimblat, N.; Sarotti, A. M. Computational chemistry to the

012, 112, 1839−1862.

ACKNOWLEDGMENTS

■

rescue: modern toolboxes for the assignment of complex molecules by

C. albicans NBRC0197, G. cingulata NBRC5907, and T.

rubrum NBRC5467 were obtained from the Biotechnology

Resource Center, National Institute of Technology and

Evaluation. P388 cells were obtained from JCRB Cell Bank

under accession code JCRB0017 (lot 06252002).

GIAO NMR calculations. Chem. - Eur. J. 2016 , 22 , 12246 −12261.

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calculation of chemical shifts in commonly used solvents using density

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Worth Publishing: New York, 2000.

24) Ocejo, M.; Vicario, J. L.; Badía, D.; Carrillo, L.; Reyes, E. A

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