Avermectins

Avermectins were first isolated at Merck Sharp & Dohme Resarch Laboratories (G. Albers-Schonberg, B. H. Arison, J.C.Chabala, A.W. Douglas, R. Eskola, M. H.Fisher, A. Lusi, H. Mrozik, J. L.Smith, and R. L. Tolman “Avermectins. Structure Determination”, J.Am.Chem. Soc. 1981, 103, 4216-4221; see also Antimicrob. Agents Chemother. 15, 361, 368, 372 (1979). Avermectins are a group of fermentation products from a strain of Streptomyces avermitilis possessing potent anthelmintic and insecticidal activities. Presently, avermectins are the active components of some insecticidal and nematocidal products used in agriculture.

Avermectins - PlantaAnalytica

Different fermentation and purification protocols afford commercial avermectin that vary in the composition of minor components. An HPLC gradient of the commercial avermectin from different sources showed that contaminants were grouped into three clusters. At the beginning of the work, the components eluting in a cluster before the main avermectin A_1a (code letter: G) were given the codes with a letter K. Two clusters of avermectins eluting after avermectin A_1a were given the codes starting with the letters L and M. Table 1 shows the composition of 5 different commercial samples of avermectin and the concentration range for various components.

The goal was to isolate about 10 mg of each avermectin present above 0.1% in the commercial product. In the initial step, the relative concentration of the avermectins was increased by 10-30 times by fractional crystallization (Table 1, colums yellow and tan). The obtained enriched mixture was then subjected to a CPC fractionation in a solvent system containing hexane, ethyl acetate, methanol and water. Table 2 shows the results of this fractionation. Each column in the Table 2 represents composition of the fraction and the concentration of the individual avermectins is expressed as percent of the fraction weight. These fractions had a much simplified composition and their final purification by preparative HPLC on C18 or C8 columns was greatly facilitated. It needs to be mentioned that while the CPC fractionation led to full recovery of the components, the yields from the preparative HPLC usually were in the range of 15 to 75 %.

Peak	HPLC RT (min)	AV-3 %	AV-4 %	AV-5 %	AV-6 %	AV-7 %	AV-3 to AV-7 % range	Mother liq. I %	Mother liq. II %
K1	22.66	0.8	1.2	1.2	1.5	0.4	0.4 - 1.2	12.5	2.6
K2	23.11	ND	ND	ND	ND	ND	0.16 - 0.6	ND	0.4
K3	25.54	0.26	0.16	0.19	0.6	0.4	0.2 - 3.5	2.2	1.9
K4	27.68	1.91	0.2	0.26	3.5	3.3	0.20 - 3.5	2.9	3.4
K5	29.15	0.24	0.26	0.26	0.2	0.2	4.2 - 5.0	1.5	1.4
K6	29.76	4.8	4.9	4.2	4.4	5	0.1 - 0.4	5.5	8.3
K7	32.51	0.25	0.1	0.12	0.29	0.4	0.13 - 0.22	4.0	4.0
K8	33.49	0.17	0.14	0.13	0.22	0.2		0.2	0.6

G (A_1a)	35.15	88.3	89.9	89.7	85.2	84.4	84.4 - 89.9	39.7	47.6

L1	39.33	ND	0.08	0.15	0.15	0.2	0 - 0.25	1.9	0.7
L2	40.01	0.8	0.7	0.78	0.78	0.7	0.7 - 0.8	1.7	2.1
L3	40.56	0.2	ND	ND	ND	0.2	0 - 0.2	1.3	1.1
L4	40.94	0.45	0.6	0.71	0.75	0.4	0.4 - 0.75	ND	0.4

M1	44.86	1.2	1.4	1.9	1.8	1.2	1.2 - 1.9	18	6.2
M2	45.79	0.6	0.27	0.26	0.23	0.6	0.12 - 0.6	1.5	3.3

Table 1

	CPC FRACTIONS
Fraction #	F-01	F-02	F-03	F-04	F05	F-06	F-07	F08	F-09	F-10	F-11	F-12	F-13	F-14	F-15
Weight (mg)	134	447	147	256	64	42	279	933	1278	598	114	149	429	130	250
Test tubes #	12	13	14	15 - 18	19-20	21 -22	23 - 27	28 - 33	34 - 35	36 - 47	48 - 58	59 -68	69-78	79 -82	83 - 96
K1												83	89	70
K2					3
K3									12	12
K4(a,b,c)					10	8				2	19			8	11
K5										7	17
K6								1	5	30
K7								5	11	16
K7a										5	5	5	5
K8						7	2

G (A_1a)							40	88	79	26

L1			6	17	22
L2					14	27	18
L3					17	31	12

M1	75	90	86	72
M2	3	4	6	6

Table 2

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