Argadin

1. Discovery, producing organism and structure1,2,7-9)
Argadin was isolated from the culture broth of fungal strain Clonostachys FO-7314 and identified as a chitinase inhibitor. It is a cyclic pentapeptide composed of Nω-acetyl-L-arginine, D-proline, L-aspartic β-semialdehyde, L-histidine, and L-2-aminoadipic acid in which the aldehyde
carbon of aspartic β-semialdehyde is bonded to the histidyl α-amino residue. The stereochemistry of Cα and Cγ of aspartic β-semialdehyde and Cα of histidine were elucidated from the crystal
structure of the argadin-chitinase complex. Argadin is the first peptide inhibitor of glycosidase
showing nanomolar inhibition, and the argadin-chitinase complex revealed how the argadinbackbone and side chains mimic the interactions of the enzyme with chitooligosaccharides. The
first total synthesis of argadin was reported by Eggleston et al.3), and it is shown to exist as a 5:1
mixture of diastereoisomers at Cγ of aspartic β-semialdehyde.
OO

HO
H
NO

HNHN

NN
HOO

HNONHN
H

CH3NO
N
HOArgadin

Clonostachys sp. FO-7314
Bar: 10 µm

2. Physical data
White powder. C29H42N10O9; mol wt 674.72. Sol. in acidic H2O, acidic DMSO. Slightly sol. inH2O, DMSO. Insol. in MeOH, acetone, CHCl3.
3. Biological activity
1) Chitinase inhibition1,2)
IC50 (µM)aTemperature
Lucilia cuprina chitinasee
Lucilia cuprina chitinasee
Streptomyces griseus chitinase
Bacillus subtilis chitinase
Serratia marcescens chitinase Ba

20°C
37°C
37°C
37°C
37°C

ArgadinArgifinb

0.00340.15
1.9
1.9
0.0192

0.103
3.7
14.8
19.0
32.5

Riboflavinc AllosamidindN.T.
2.1
>200
>200
N.T.

S. marcescens chitinase inhibition data are reported as Ki values.
Argifin is a new compound isolated in Kitasato (see Argifin).
cRiboflavin was isolated from a Penicillium strain as a chitinase inhibitor in Kitasato4).
dAllosamidin is a representative chitinase inhibitor.
eLucilia cuprina is sheep blowfly.
ab0.0004
0.0023
0.016
0.025
0.45

2) Argadin-chitinase complex and argifin-chitinase complex2,5,6,10)
The structures of argifin and argadin in complex with Serratia marcescens chitinase B were resolved (2.0 Å resolution). These structures give an unprecedented view of how peptide based
inhibitors inactivate carbohydrate-processing enzyme. For example, the carbonyl oxygen of
the histidine in argadin occupies almost the same position as the scissile oxygen in the chitinase
B-chitooligosaccharide complex and hydrogen-bonds to the catalytic acid (Glu144), while Glu144 makes hydrogen bonds to the guanidinium group of the arginine side chain in argifin. The
structures of argifin and argadin in complex with Aspergillus fumigatus chitinase and humanbacterial-class chitinase (chitotriosidase) were also resolved5).

3) Insecticidal activity1)
Argadin was injected into American cockroach (Periplaneta americana) larvae and comparedwith mock injected controls. No mortality was observed for control cases after day 1, while 60%
and 38% mortality was observed for cockroaches injected with 20 µg and 2 µg of argadin, respectively.

4) Other biological activity1)
Argadin did not inhibit the growth of tested bacteria, yeast, or fungi at 10 µg/disc (paper discmethod). Argadin also did not inhibit the growth of P388, KB, or HL-60 cells at 25 µg/ml.

4. Total synthesis3,11)
The total synthesis of argadin was reported by two groups3,11). Below is a scheme achieved byŌmura et al.11) (See Appendix-I).
1)
OOHOH

(±) - Glycerol
polystyrene resin

OAllylO
OHC

AllylONHFmocp-TsOH2) 20% piperidine

ONHDdeH 2N

1)

ONH 2

OO

FmocN
HOO

PyBOP, DIPEA
2) 20% piperidine

1)

FmocHNN O

BocN

PyBOP, DIPEA
2) Pd(PPh 3)4, dimedone
3) 20% piperidine

DIPEA

NH

BocNOOBocNOOO
NO
NH

O

NHOO
O

N O

NH 2NH

HN
HN

2) 2% NH 2NH 2NH

HN
HNO1) HATU, DIPEA(two-cycle repetition)

NO

N O

NH

O

NHDdeONO

AllylOO

t-BuO2C

BocN

HN

OOOOH

O

NHDdeNH

H 2N

t-BuO 2CNH

NH 2HO 2COOPyBOP, DIPEA
2) 20% piperidine

OO

N O

1)

OO

t-BuO 2C

CO2t-Bu

FmocHNO

t-BuO 2C

NHFmocBocN
ONNNCH 3HN
HOOOH

NO

AllylOHBTU, DIPEA
2) 20% piperidine

BocN

OOOH1)

NHDdeNHOO(1.40 mmol/g)

HNO

AllylOONOONHOOONHCH 3
95% TFA/H2O
then HPLC separation

Argadin
4.7% overall yield
15 steps longest linear sequence5. References
1. [759] N. Arai et al., Chem. Pharm. Bull. 48, 1442-1446 (2000)
2. [805] D. R. Houston et al., Proc. Natl. Acad. Sci. USA 99, 9127-9132 (2002)
3. 		
M. J. Dixon et al., Eur. J. Org. Chem. 22, 5002-5006 (2006)
4.		
A. Turberg et al., In “Chitin and Chitosan-Chitin and Chitosan in Life Science”(Eds. by T. Uragami et al.) pp. 440-442, Kodansha Scientific (2001)
5. [891] F. V. Rao et al., Chem. Biol. 12, 65-76 (2005)
6. [947] S. Ōmura & K. Shiomi, Pure Appl. Chem. 79, 581-591 (2007)
7.			
T. Hirose, Yakugaku Zasshi 132, 1001-1010 (2012)
8.		
S. Hirono & H. Gouda, Tanpakushitsu Kakusan Koso. 54, 1590-1597 (2009)
9. [1066] T. Hirose et al., Proc. Jpn. Acad. Ser. 86, 85-102 (2010)
10. [983] H. Gouda et al., Bioorg. Med. Chem. 16, 3565-3579 (2008)
11. [1048] T. Hirose et al., J. Antibiot. 62, 495-500 (2009)