Argifin

1. Discovery, producing organism and structure1-4,14,18)
Argifin was isolated from the culture broth of Clonostachys grammicosporopsis FTD-0668
and found to be a chitinase inhibitor. It is a cyclic pentapeptide of cyclo(Nω-(N-methylcarbamoyl)-L-arginyl-N-methyl-L-phenylalanyl-β-L-aspartyl-β-L-aspartyl-D-alanyl). The argifin-chitinase complex was resolved with the argadin-chitinase complex (see Argadin). The first total
synthesis was achieved by Eggleston et al.5)

OHOO

HNCH3OHNNOHOONH ONHNHONHNHNH

CH3CH3O

ArgifinGliocladium sp. FTD-0668(Clonostachys grammicosporopsis FTD-0668)
Bar: 20 µm

2. Physical data
White powder. C29H41N9O10; mol wt 675.70. Sol. in H2O. Insol. in MeOH, acetone, CHCl3.
3. Biological activity
1) Chitinase inhibition2,4,6,7) (see Argadin)
2) Argifin-chitinase complex4,6,7) (see Argadin)3) Insecticidal activity2)
Argifin (20 µg) was injected into cockroach larvae in three separate trials and compared to mockinjected controls. Mortality was assessed at 1, 5, and 20/23 days post injection. Three separate
trials repeatedly showed the efficacy of argifin against larval stages of Periplaneta americana(American cockroach) and Blattella germanica (German cockroach). Argifin showed 73% mortality in cockroaches, while the mortality rate of the control was only 12% in Periplaneta americana. Most of the dead cockroach larvae showed no signs of molting. Cockroach larvae killedduring molting showed new cuticle formation below the partially opened old cuticle. Furthermore, they were unable to leave the old exuva leading to their death shortly after sclerotizationof the new cuticle.
4) Other biological activity2)
Argifin 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 or HL-60 cells at 25 µg/ml.

4. Total synthesis5, 8-10,19,20)
The total synthesis of argifin was reported by two groups. The following scheme is Ōmura’ssolution phase tag approach10). (See Appendix-I)
C18H 37
C18H 37

OOHO

C18H 37O1) Fmoc-Asp(OAlly)-OH
DCC, DMAP100% (recrystallization)O2) 5% piperidine
100% (recrystallization)

H 2N2) 5% piperidine
100% (recrystallization)

1) Fmoc-Asp-Ot-Bu
PyBop, i-Pr 2NEt
97% (recrystallization)
2) Pd(PPh 3)4, dimedone
92% (chromatography)

AllylO

NO

O
TAGaO

O

t-BuO

1) PyBop, i-Pr 2NEt
O
85% (chromatography)
t-BuO2) 2% NH 2NH 2
100% (recrystallization)
O
BnHN
3)
NNO

N

NHBoc

N

i-Pr 2NEt
85% (recrystallization)

TAGa ONH

O

5% piperidine

Recrystallizationwith HOBt in MeOH

CH 3

BocNHOOTAGaO
CH 3NHO

NN
H

CH 3N
BnO

NNHO
TAGaOO

HN

H 3N

N O
N N

1) Pd(OH) 2, H 2

CH 3O

NHOONHCH 3O

AllylOOTAGa

CH 3ONH

DdeONO1) Fmoc-D -Ala-OH,
PyBop,i-Pr 2NE
100% (recrystallization)

t-BuO
N
H

H
NO

H 2N

2) 5% piperidine
100% (recrystallization)

NH

AllylO2) 5% piperidine
100% (recrystallization)

CH 3O

HNO

Dde

CH 3NOOOHNONHOOFmoc NHHOO
TAGa

H 3C

NHNH 2OTAGaHO-TAGa

1) Fmoc-Orn(Dde)-OH,PyBrop, i-Pr 2NEt
100% (recrystallization)

1) Fmoc-N-Me-Phe-OH,PyBop, i-Pr 2NEt
100% (recrystallization)

AllylO

DdeCH 3OCH 3O
NH

CO2HOONHNHNON
H

DdeCH 3O

Argifin2) 50% TFA in DCM(Overall yield : 44% in 16 steps)
68% for 2 steps(chromatography)
10 times recrystallization and 4 times
flash chromatography

5. Computer-aided rational molecular design from argifin11-15)
Efficient total synthesis of argifin could be applied to enable synthesis of analogs. In addition,
the 3-D structure of argifin, in complex with chitinase B from Serratia marcescens (SmChiB),
was resolved by X-ray analysis (see Argadin)11). Rational molecular design of derivatives basedon the structure of argifin led to production of novel derivatives, which showed great potential
for inhibitory activity against SmChiB12-15).
6. The active framework of argifin and use of in situ click chemistry14,16,17)
The studies of argifin and its analogs by X-ray crystallography with various chitinases revealed
that there are at least four conserved hydrogen-bond interactions between the N ω-methylcarbamoyl-L-arginine moiety and the polar groups arrayed in the hydrolytic pocket of the family 18chitinases. The remarkable fidelity of the hydrogen-bonding network between the chitinases
and the argifin ligand implicates its critical role in revealing the micromolar to nanomolar range
of inhibition. Ōmura’s group thus focused on the design and simplification to azide-bearing
N ω-methylcarbamoyl-L-arginine substrate, as a smaller analogs of macrocyclic peptide naturalproduct argifin, and the use of target-guided synthesis (TGS) depending on the 1,3-dipolar cycloaddition between the azide-molecules and the acetylenes, which is named as “in situ clickchemistry” (Figure), for the screening of novel and more potent chitinase inhibitors16).

An illustration of in situ click chemistry.
As shown in Figure for in situ click chemistry, at first, azide or alkyne building blocks are incubated in the presence of target protein. The protein binds initially the building blocks with thehighest affinity. The enforced propinquity of the azide and alkyne accelerates triazole formationas a covalent bond to link two building blocks. The newly-generated triazole compound showshigher affinity compared with corresponding monovalent building blocks.
The in situ click chemistry experiments were performed in parallel in 96-well microtiter plates toexplore the chitinase-accelerated reaction, using a mixture of SmChiA, B, and C. Utilization of
the mixed SmChi has the advantage of accelerating the identification of novel inhibitors against
each isozyme of chitinase through a one-off screening. Although a singular isozyme or multiple
isozymes of chitinase may participate in the formation of triazoles under this particular screening
condition, the identification of the actual templating isozyme or isozymes can be determined ina follow-up assay using separate isozymes. Consequently, an azide (100 µM) and 71 structurally-diverse alkynes (300 µM) were incubated in the presence of SmChis (Scheme). The inhibitory activities of syn-triazole displayed high inhibitory activity against SmChiB (IC50 value of 22nM), which is approximately 30-fold stronger than that of azide ligand (approximately 300-foldpotency compared with natural argifin)16).

N3

IC 50 = 580 nM against SmChiBONHNHBnNNHOONHNHBnazide (100 µM)+NONhit alkyne (300 µM)

CH 3N NN

Serratia marcescens chitinases(A, B and C1 ) (9.6 mUnit/mL)
ON

10% MeOH/aq. buffer (pH 7.0)
37 ˚C, 20 hO

NH ONHNO

NH

N
H

N
(on 96 wells plate)

syn-triazole
IC 50 = 22 nM against SmChiB

SmChi templated in situ click chemistry protocol and the guided triazole analog.

NHCH 3Ōmura’s group also has determined the crystal structures of complexes of SmChiB with azideligand (click precursor) and syn-triazole (generated in situ), together with the 3-component complex of [SmChiB]–[azide-ligand]–[mimic of alkyne-bearing quinoline-oxime fragment], respectively. The results of their X-ray analysis demonstrated that the assembly of azide ligand andalkyne-bearing quinolone-oxime within SmChiB is responsible for yielding a very potent syn-triazole inhibitor via an in situ click chemistry reaction, as visualized by X-ray crystallography.17)Ōmura’s strategy employed an azide substituent appended to an active domain excised, as it
were, from the more complex natural macrocyclic peptide argifin. The SmChi, which in this casewas specifically SmChiB, served as both mold and template for triazole formation between aunique pair of azide and alkyne fragments. In the process of in situ click chemistry, the highly
exergonic nature of triazole formation makes the process completely irreversible, and thereby
locks in unique information, a kind of embedded message of the encounter. More practically, it
allowed us to discover a lead template for the discovery of a selective chitinase inhibitor directed
toward the functions of SmChi, without the need for lengthy and costly analog syntheses.

7. References
1. [747] K. Shiomi et al., Tetrahedron Lett. 41, 2141-2143 (2000)
2. [754] S. Ōmura et al., J. Antibiot. 53, 603-608 (2000)
3. [755] N. Arai, et al., J. Antibiot. 53, 609-614 (2000)
4. [805] D. R. Houston et al., Proc. Natl. Acad. Sci. USA 99, 9127-9132 (2002)5.		
I. Eggleston et al., Bioorg. Med. Chem. Lett. 15, 4717-4721 (2005)
6. [891] F. V. Rao et al., Chem. Biol. 12, 65-76 (2005)
7. [947] S. Ōmura & K. Shiomi, Pure Appl. Chem. 79, 581-591 (2007)
8.			
I. M. Eggleston et al., Org. Biomol. Chem. 7, 259-268 (2009)
9. [1034]		 T. Sunazuka et al., Bioorg. Med. Chem. 17, 2751-2758 (2009)
10. [1107]		T. Hirose et al., Tetrahedron 67, 6633-6643 (2011)
11. [983] H. Gouda et al., Bioorg. Med. Chem. 16, 3565-3579 (2008)
12. [1052] H. Gouda et al., Bioorg. Med. Chem. Lett. 19, 2630-2633 (2009)
13. [1055] H. Gouda et al., Bioorg. Med. Chem. 17, 6270-6278 (2009)
14. [1066] T. Hirose et al., Proc. Jpn. Acad., Ser. B86, 85-102 (2010)
15. [1079] H. Gouda et al., Bioorg. Med. Chem. 18, 5835-5844 (2010)
16. [1031] T. Hirose et al., J. Antibiot. 62, 277-282 (2009)17. [1157] T. Hirose et al., Proc. Natl. Acad. Sci. USA 110, 15892-15897 (2013)18.			
T. Hirose, Yakugaku Zasshi 132, 1001-1010 (2012)19.		
M. J. Dixon et al., Org. Biomol. Chem. 7, 259-268 (2009)20.		
A. Andersen et al., Chem. Biol. 15, 295-301 (2008)