������ � ��

������ ���������

OH

O

O

O

HO

HO

OHOH

HO

H

Sophoraflavanone HNobiletin

O

O

OMeMeO

MeOOMe

OMeOMe

有機合成夏期セミナー 明日の有機合成化学 （2020年8月24日）

N NH

O

NH N

N

AHX

HN NH

O

NH N

NO

AOH

N NH2

O

NH

ICA

Fairy Chemicals (FCs)

ChemBioChem, 11, 1373 (2010)J. Agric. Food Chem., 58, 9956 (2010)Angew. Chem. Int. Ed., 53, 1552 (2014)

３連続窒素原子を有する６員環の天然有機化合物は， AHXとAOHが史上初

Nature 505巻, 298頁 （2014年1月16日発行）３化合物を，表題に因んでフェアリー化合物(Fairy Chemicals：FCsと略称）と命名

N NH

O

NH N

N

AHX（2-azahypoxanthine）コムラサキシメジから発見

HN NH

O

NH N

NO

AOH （2-aza-8-oxohypoxanthine）AHX代謝産物，イネから発見

N NH2

O

NH

ICA（imidazole-4-carboxamide）コムラサキシメジから発見

AHXとAOHのシバに対する活性（成長促進）

Control 0.2 mMAHX

1 mMAHX

0.2 mMAOH

1 mMAOH

ICAのシバに対する活性（成長抑制）

0 0.05 mM 1 mM0.2 mMControl 0.05 mM 1 mM0.2 mM

�������������

AICA•HCl DICA AHX

NaNO2

aq. HCl

H2Pd/C

MeOH

ICA

Xanthine oxidaseH2O

AOH

Reported Synthetic Method of Fairy Chemicals

NH

HN

NNNH

O

O

NH3 aq.

H2O

Bioorg. Med. Chem. Lett., 27, 2144 (2017)

Dropwise 15 ~ 30 min

0 °C~5 °C(Amber suspension) Filtered Filtered

rtDICA

(Amber solid)

Water 28% NH3 aq

rt rt rtdark blue solution

evap.

dark red solid

MeOH

red presipitate(red sol.)

Yellow sol.

Activated Carbon

colorless sol.

Filtered

AHX (White solid)

rt

30 min

evap.

NaNO2 in water

AICA·HCl in 2 M HCl THF

Dark blue sol.

AICA•HCl DICA AHX

NaNO2

aq. HCl

NH3 aq.

H2O

Reported Synthetic Method for AHX

Bioorg. Med. Chem. Lett., 27, 2144 (2017)

AICA•HCl DICA AHX

NaNO2

aq. HCl

H2Pd/C

MeOH

ICA

Xanthine oxidaseH2O

AOH

Synthesis of Fairy Chemicals (AHX, ICA, AOH)

Δ

MeOH

NH

HN

NNNH

O

O

N

N

N

NH

O

NH

NH

N

N

NH

O

N

AHX

N

N

N

NH2

O

N

DICA

N

N

N

NH2

O

NN

N

N

N

O

N

H

H

–H+H1, 5-hydrogen shift

suprafacial

NaNO2 in water

AICA·HCl in 2 M HCl

Dropwise 15 ~ 30 min

0 °C~5 °C(Amber suspension) Filtered Filtered

rtDICA

(Amber solid)

MeOH

Activated Carbon

colorless sol.

Filtered

AHX (White solid)

50 °C

evap.Yellow sol.

THF

DICA AHX

NaNO2

aq. HCl

Δ

MeOH

Our improved synthetic method for AHX

Kan and Kawagishi et al. Angew. Chem. Int. Ed., 53, 1552 (2014)

100� ������������

NaNO2

aq. HCl

H2Pd/C

MeOH

ICA

Xanthine oxidaseH2O

AOH

Synthesis of Fairy Chemicals (AHX, ICA, AOH)

Δ

MeOH

H2Rh/CMeOH

��#��"�$����!�AHX�&��%

��'� (Rh 309 ppb��

50����� ��� �����

NH

HN

NNNH

O

O

AICA•HCl DICA AHX

Conversion from AHX to AOH by Bacterial Cell

AOHAHXXanthine oxidase

AHX AOHBurkholderia contaminans

�����60 ������1 / 45�� �1 / 14�� �1 / 281

*S. Tokuyama, H. Kwagishi et. al Biosci. Biotechnol. Biochem. 80, 2045 (2016)

*

Xanthine oxidaseH2O

AOH

NH

HN

NNNH

O

O

XOD, ���300u/���

NaNO2

aq. HCl

Δ

MeOH

AICA•HCl DICA AHX

AICA-ribotide AICA

AHX-ribotide

AHX

AOH��� �� �

Novel Purine Metabolic Pathway

Purinemetabolism

pathway

ICA

Angew. Chem. Int. Ed., 53, 1552 (2014), Org. Lett., 20, 312 (2018)Sci. Rep., 9, 9899 (2019), Org. Lett., 21, 7841 (2019)

プリン代謝経路

Novel Pathway

PRPP

�������� �

�� �

Attempted to Ribosylation of AHX and ICA

OAcO

OAcAcO

OAc

+

OTMS

NTMSMeCH2ClCH2Cl, 70°C;

TMSOTf

OAcO

OAcAcO

OAc

+

OAcO

OAcAcO

OAc

+NH2N

O

NH

N

NH

NH

O

NN

N

NH

NH

O

N

OTMS

NTMSMeCH2ClCH2Cl, 70°C;

TMSOTf

OTMS

NTMSMeCH2ClCH2Cl, 70°C;

TMSOTf

No Reaction

Complex mixture

OAcO

OAcAcO

N

NHN

O

N

HypoxanthineInosine triacetate

AHX

ICA

K2CO3

DMF, 80 ºC85%

Removal of C2 carbon of inosine

*G. Piccialli et al., J. Chem. Soc. Perkin Trans. 1, 2079 (1997).

R = H

R = TBS

TBSCl, imidazoleDMF, 60 ﾟC, 80%

THF, 50 ºC

58%

DMF, 50 ºCquant.

*

Inosine

NaNO2

MeCN/2 M HCl0 ﾟC

66% (2 steps)

NaNO2

MeCN/2 M HCl0 ﾟC

complex mixture

NH4FDMF

R = TBS

R = H

Effect of dinitrophenyl group

OHO

OTBSTBSO

NN

O

N NN NO2

NO2

ORO

OTBSTBSO

NH

N

O

N NH2NO2

NO2

OHO

OTBSTBSO

NH2N

O

N

OHO

OTBSTBSO

NHN

O

N NN

OHO

OTBSTBSO

NH2N

O

N NH2

low yield

AHX-riboside

Preparation of AHX-riboside and ribotide

DMF, 98%

R = 2,4-dinitrophenyl

R = H

AHX-ribotide

TBAF

THF72%

1) TBAF, THF

2) H2, Pd/CMeOH29% (3 steps)

(BnO)2PN(i-Pr)21H-tetrazoleCH2Cl2, 0 ﾟC;TBHP, -78 ﾟC

BrOH

O H2, Pd/C

AcOEt

R = -CONHMeR = H

Et3N, MeOH72% (2 steps)

AcOH, MeOH

N

O

NHMe13C

NH

O

NH

Me

AcOH

100 °C74%

Synthesis of Double-13C-labeled FCs

13CNH2

OHN

13CNH

R N

R = -CONHMe

13CNOEt

O NaNO2AcOH

H2O 13CNOEt

ONOH

13CNOEt

OH2N

13C

N13C

NH2

O

N NH2R

13CNNH2

OH2N

H13C(OEt)3 + H2N

O

NHMe

N

O

NHMe13C

NH

O

NH

Me

NH3MeOH

1) Na13CN aq. NaOH

2) H2SO4 EtOH, ∆

BrOH

O 1) Na13CN aq. NaOH

2) H2SO4 EtOH, ∆

13CNOEt

O NaNO2AcOH

H2O 13CNOEt

ONOH H2, Pd/C

AcOEt 13CNOEt

OH2N

NH3MeOH

13CNNH2

OH2N13C

NH2

OHN

13CNH

R N

R = -CONHMeR = -CONHMeR = H

Et3N, MeOH72% (2 steps)

13C

N13C

NH2

O

N NH2R

AcOH, MeOH

N

O

NHMe13C

NH

O

NH

Me

H13C(OEt)3 + H2N

O

NHMe

AcOH

100 °C74%

N

O

NHMe13C

NH

O

NH

Me

Synthesis of Double-13C-labeled FCs

NHMe

NHC

NHR

O

CNNH2

OH2N

R = CONHMe

CNNH2

OHNHN

CNH

R

OMeHN

NH2

MeHN O–

CNH2

OHN

HCNH

R N

Synthesis of Double-13C-labeled FCs

13C

N13C

NH2

O

NH

13C2-DICA 13C2-ICA13C2-AICA

13C

N13C

NH2

O

NH NH2

13C2-AICA 13C2-AHX 13C2-AOH

13C

N13C

NH2

O

NH NH2

NaNO2

aq. HCl0 °C

13C

N13C

NH2

O

N NN

1) NaNO2, aq. HCl 0 °C

2) MeOH, 60 °C

60 % (2 steps)

13C

N13C

NH

O

NH N

N N13CNH

C

HN NH

O

NO

H2, Rh/C

MeOH60 % (2 steps)

Natural Oxidase

Synthesis of Double-13C-labeled AICAr

13C

N13C

NH2

O

NH NH2

13C2-AICA

13C2-AICAr

CH(OEt)3DMF

130 °C13C

N13C

NH

O

NH N

OAcO

OAc OAc

OAc

+

NO2O2N

Cl

K2CO3

DMF, 60 °C

13C

N13C

N

N

O

OAcO

OAc OAc

N

R

R = HR = Ar

13C

N13C

NH2

NH2

O

OHO

OH OH

NH2N NH2

CH3CN

60 °C52%

Me

OTMS

NTMS

TMSOTf, 70°C

ClCH2CH2Cl, 70°C;

Our Synthesized Fairy Compounds

AHX-riboside AHX-ribotide

S-ICA-ribosyl L-homocysteine

13C

N13C

NH

NH2

O

[2,5-13C2]-ICA

13C

N13C

NH

NH

O

N

[4,8-13C2]-AHX

N OHO

OHOH

13CNH2

NH2N

13C

O

N

[2,5-13C2]-AICAr

AHX

AOH

ICA

圃場実験を可能に 存在の確認

LC-MS/MSによる極微量分析法の確定 代謝物の合成

OS

OHOH

NH2N

O

N

NH2

HO2C

S-ICA-ribosyl homocysteine

+

Homocysteine unit

ICA riboside unit

homocystineMethionine

OS

OHOH

NH2N

O

N

NH2

HO2C

Retrosynthetic analysis of S-ICAr-H

Zn

AcOH/Et2O

1) SOCl2MeOH

2) Boc2OEt3N, MeOH46% (3 steps)

Li

liq. NH3THF

−78 ﾟC;H2O2

Synthesis of L-homocysteine fragment

L-Methionine L-Homocystine

*J. Picha et al. Eur. J. Med. Chem. 65, 256 (2013)

*

Et3N, DMF91%

C-S coupling of S-ICA ribosyl L-homocysteine

1) MsClEt3NCH2Cl2

2) NaIacetone67% (2 steps)

Org. Biomol. Chem, 12, 3813 (2014)

LiOH

THF/H2O

NH4F

MeOH60 ℃

Synthesis of S-ICAr-H (L-homocysteine)

TFAMeOH50 ℃

S-ICAr-H (L-homocysteine)

Synthesis of S-ICAr-M, S-AICAr-H and S-AICAr-M

N NH2

O

O

OHOH

NS

NH2

HO2C

S-ICAr-M

N NH2

O

O

OHOH

NHO

NH2

Acadesine(AICAr)

S-ICAr-H

5 steps

MeI, AgOAc

HCO2H, AcOH

S

NH2

HO2CMe N NH2

O

O

OHOH

N

S

NH2

HO2CMe

N NH2

O

O

OHOH

NS

NH2

HO2CNH2

S-adenosyl-L-methionine(SAM)

S-AICAr-H

S-AICAr-M

Our Synthesized FCs (Fairy Chemicals)

AHX-riboside AHX-ribotide

S-ICA-ribosyl L-homocysteine

13C

N13C

NH

NH2

O

[2,5-13C2]-ICA

13C

N13C

NH

NH

O

N

[4,8-13C2]-AHX

N OHO

OHOH

13CNH2

NH2N

13C

O

N

[2,5-13C2]-AICAr

AHX

AOH

ICA

圃場実験の実現 存在の確認

微量成分の検出法の確立 代謝物合成（絶対配置確定）

OS

OHOH

NH2N

O

N

NH2

HO2C