Purification and identification of two antifungal cyclic dipeptides from Bacillus cereus subsp. thuringiensis associated with a rhabditid entomopathogenic nematode especially against Fusarium oxysporum

Figures & data

Figure 1. Structure of DKPs: (A) cyclo(D-Pro-L-Met) and (B) cyclo-(D-Pro-D-Tyr).

Figure 2. HPLC profile of FDAA derivatives of the acid hydrolysates of DKPs: (A) cyclo(D-Pro-L-Met) and (B) cyclo-(D-Pro-D-Tyr).

Table 1. HPLC reversed-phase retention times of the standard L and D-amino acid and CDPs derivatives.

Amino acid derivatives	Retention time (min)
L-Pro	17.451
D-Pro	18.394
L-Met	22.225
D-Met	24.404
L-Tyr	20.514
D-Tyr	33.084

Figure 3. HPLC chromatogram of diketopiperazines on a reversed-phase C18 column (LC-20AD). Samples of 15 µl were injected to a column (250 mm × 4.6 mm × 5 mm), eluted with 100% methanol (A) cyclo(D-Pro-L-Met), retention time is 2.711 min. The calculated purity is 98% based on the peak area (B) cyclo-(D-Pro-D-Tyr), retention time is 2.820 min. The calculated purity is 92% based on the peak area.

Figure 4. DSC curves of diketopiperazines. Temperatures corresponding to the onset of transition and midpoint of the transition region and enthalpy (ΔH) were recorded by means of the built-in software: (A) cyclo(D-Pro-L-Met) and (B) cyclo-(D-Pro-D-Tyr).

Table 2. NMR data for DKPs in CDCl₃ (δ in ppm, J in Hz).

	DKP 1		DKP 2
Carbon No.	^1H	^13C	^1H	^13C
1	4.24 (t, J = 5.6)	54.5	4.24 (dd, J = 3.0, 10.0)	56.3
OH			6.88 (s)
NH	7.27 (s)		5.92 (s)
2		165.4		165.2
3	4.17 (t, J = 7.5)	59.0	4.1 (t, J = 7.5)	59.1
4		170.4		169.7
5	2.39	27.9	2.03, 2.34	28.3
6	2.06	21.9	1.93	22.5
7	3.59	45.7	3.58, 3.65	45.4
8	2.03	28.0	2.77 (dd, J = 10.0, 14.5), 3.49 (dd, J = 4.0, 14.5)	35.9
9	2.76 (td, J = 2.5, 7.5)	29.1		126.9
10	2.16	15.2	7.06 (d, J = 8.0, H10,10′)	130.3
11			6.79 (d, J = 8.5, H11,11′)	116.1
12				155.6
Ph				165.2

Table 3. MIC of diketopiperazines against test fungi.

		MIC (µg/ml)
Test organisms	DKP 1	DKP 2	Bavistin	Amphotericin B
A. flavus	32	64	100	–
C. albicans	64	32	–	64
F. oxysporum	4	2	32	–
R. solani	4	8	32	–
P. expansum	16	8	64	–

The values represents mean of three replications.

“–” denotes not tested.

Table 4. MIC and MBC of diketopiperazines against test bacteria.

	MIC (µg/ml)
	DKP 1		DKP 2		Cefotaxime		Ciprofloxacin
Test organisms	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
B. subtilis	32	64	32	64	100	100	5	5
S. aureus	16	16	64	64	250	250	5	5
E. coli	125	250	16	32	100	250	5	5
P. aeruginosa	32	64	16	16	500	1000	10	10

The values represent mean of three replications.

Table 5. Antimicrobial activity of diketopiperazines.

	Zone of inhibition (diameter in mm)
Test organisms	DKP 1	DKP 2	Ciprofloxacin	Bavastin	Amphotericin B
A. flavus	18 ± 1	20 ± 0	*	24 ± 1.52	*
C. albicans	11 ± 1	17 ± 0.57	*	*	24 ± 1.73
F. oxysporum	34 ± 1.52	32 ± 0	*	16 ± 0	*
R. solani	31 ± 1	32 ± 0.57	*	19 ± 1.52	*
P. expansum	20 ± 0.57	30 ± 0.57	*	24 ± 1.15	*
B. subtilis	20 ± 1	21 ± 0	31 ± 0.57	*	*
S. aureus	18 ± 0	23 ± 0.57	31 ± 0	*	*
E. coli	15 ± 1	25 ± 0.57	28 ± 1.52	*	*
P. aeruginosa	19 ± 1.52	23 ± 0	25 ± 0.57	*	*

(*) not tested.