A new β-class milbemycin derivative from a mutant of genetically engineered strain Streptomyces avermitilis AVE-H39

Abstract A new β-class milbemycin, 13α-hydroxy milbemycin β 6 (1), was isolated from the fermentation broth of a mutant of genetically engineered strain Streptomyces avermitilis AVE-H39. Its structure and absolute configuration were elucidated by extensive spectroscopic methods and confirmed by single crystal X-ray diffraction. Graphical Abstract


Introduction
Species of the genus Streptomyces are of major pharmaceutical interest because they synthesize a variety of bioactive secondary metabolites (Ikeda et al. 2003). Avermectin and the related milbemycin, which are produced by Streptomyces, have been widely used commercially in animal health, agriculture and human infections (Ikeda et al. 1999;Wang et al. 2010). However, with the increasing resistance to avermectin and milbemycin, it brings about the keen needs for the discovery of novel products with improved bioactivities (Wang and Wu 2007 ;Geurden et al. 2015). Recent advances in combinatorial biosynthesis brought strategies like gene replacement, recombination and heterologous expression to find more novel bioactive compounds from Streptomyces ( Goss et al. 2012;Olano et al. 2010;Zhang et al. 2020). To search for more bioactive compounds, a genetically engineered strain Streptomyces avermitilis AVE-H39 was generated by combinatorial biosynthesis strategy and a series of novel macrocyclic lactones have been obtained from this strain (Zhang et al. 2015;Qi et al. 2020;Wang et al. 2020). In order to explore the diversity of its secondary metabolism, we treated the spores of S. avermitilis AVE-H39 with N-methyl-N'-nitroso-N-nitrosoguanidine (NTG) (Deli c et al. 1970;Srikrai and Robbers 1983). Then, a mutant strain AVE-H39C10 with several differences in the HPLC profiles of metabolites with respect to the parental strain was obtained. In further work to investigate the metabolites of this mutant strain, a new b-class milbemycin derivative (1) was isolated from its fermentation broth. Herein, we described the fermentation, isolation, structural elucidation and nematocidal activity of the new compound.

Structure determination
Compound 1 (Figure 1) was isolated as colorless prism crystal with a specific rotation of þ23 (c 0.09, EtOH) and UV(EtOH) k max nm (log e): 235 (4.22). Its molecular formula was established as C 32 H 46 O 7 from the HR-ESIMS ion at m/z 543.3336 [M þ H] þ (calcd 543.3316), requiring 9 degrees of unsaturation. The IR absorption bands at 3529, 1733 and 1672 cm À1 suggested the presence of hydroxyl and carbonyl groups. Analysis of the 1 H NMR spectrum (Table S1) revealed the presence of one downfield signal at d H 6.59 (1H, brs), three olefinic methyls at d H 1.74 (6H, brs), 1.53 (3H, brs), two aliphatic doublet methyls at d H 1.09 (3H, d, J ¼ 6.2 Hz), 0.79 (3H, d, J ¼ 5.7 Hz) and one aliphatic triplet methyl at d H 0.91 (3H, t, J ¼ 6.6 Hz). The 13 C NMR spectrum (Table S1) of 1 showed 32 carbon resonances in total, which were assigned by the assistance of the DEPT spectrum to two carbonyl groups [d C 197.6 and 170.2], three sp 2 quaternary carbons, five sp 2 methines, a ketal-type carbon [d C 97.3], an oxygen-bearing sp 3 tertiary carbon [d C 80.2], seven sp 3 methines (four oxygenated) and seven methylenes in addition to the six methyl groups commented above. The complete assignment of all 1 H Figure 1. The structure of compound 1. and 13 C NMR spectroscopic data of 1 was subsequently accomplished by the 1 H-1 H COSY, HSQC and HMBC experiments. Comparison of the 1 H and 13 C NMR data of 1 with those of milbemycin b 12 (Nonaka et al. 2000) revealed their similarity. The differences between them were that a carbonyl, a hydroxyl group and an ethyl group were situated at C-5, C-13 and C-25, respectively, in 1. The observed HMBC correlations ( Figure S1) of H 3 -26/C-3, C-4 and C-5, H 3 -28 and H 3 -29/C-13 (d C 77.9) and H 3 -32/C-25 supported the structural assignment of 1. From a biogenetic point of view, the relative configuration of 1 was assigned by analogy with that of 25-ethyl ivermectin (Zhang et al. 2015).

Crystal Data of 1
13a-hydroxy milbemycin b 6 (1) was crystallized from CHCl 3 : MeOH (1:1) at room temperature. The X-ray crystallographic data were obtained on a Bruker APEX-II CCD with a graphite monochromated Cu Ka radiation, k ¼ 1.54178 Å at 296 K. Crystal data: C 32 H 46 O 7 , M ¼ 542.69, tetragonal, space group P43 21 2; unit cell dimensions were determined to be a ¼ 14.4672 (2)   was solved by direct methods (SHELXS97) and refined by using full-matrix leastsquares on F 2 . In the structure refinements, non-hydrogen atoms were placed on the geometrically ideal positions using the 'ride on' method. Hydrogen atoms bonded to oxygen were located by the structure factors with isotropic temperature factors. The final R1 values were 0.0399 (I > 2r(I)). The final wR2 values were 0.1026 (I > 2r(I)). The final R1 values were 0.0458 (all data). The final wR2 values were 0.1071 (all data). The absolute configuration was determined by the Flack's parameter determined as 0.07(9). Crystal data of 1 were deposited with the Cambridge Crystallographic Data Centre (CCDC 2087769).

Conclusion
As a continuation of our efforts to discover more novel sixteen-membered macrolides, 13a-hydroxy milbemycin b 6 (1) was obtained from the fermentation broth of a mutant strain S. avermitilis AVE-H39C10. Its structure and absolute configuration were elucidated by extensive spectroscopic methods and confirmed by single crystal X-ray diffraction. Although compound 1 showed weak nematicidal activity against Bursaphelenchus xylophilus, it may shed new insight into the biosynthesis of milbemycin metabolites.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This research was financially supported by the Key Research and Development Programs of Zhejiang Province (Grant No: 2020C02028).