Confirmation of the absolute configuration of tenvermectin B by single-crystal X-ray diffraction analysis of its furan formate derivative

Abstract Tenvermectins A and B are a kind of 16-membered macrocyclic lactone antibiotics with potent insecticidal property, which have been obtained from two genetically engineered strains Streptomyces avermitilis MHJ1011 and S. avermitilis AVE-H39. Aiming at confirming the absolute configuration of tenvermectin B by X-ray diffraction method, 4″, 5 -di-(2-furoyloxyl) tenvermectin B (1) was prepared and a suitable crystal of 1 was obtained from MeOH. The absolute configuration of 1 was unambiguously established by single-crystal X-ray diffraction analysis. The determination of the stereochemistry of tenvermectin B could promote its chemical and biological studies as agricultural and veterinary agents. In addition, compound 1 displayed weak acaricidal and nematocidal activities against Tetranychus cinnabarinus and Bursaphelenchus xylophilus respectively. Graphical Abstract


Introduction
Tenvermectins A and B are produced by the genetically engineered strains Streptomyces avermitilis MHJ1011 and Streptomyces avermitilis AVE-H39 with better insecticidal property than ivermectin Zhang et al. 2015). As a new kind of sixteen-membered macrocyclic lactone antibiotics, tenvermectins A and B are the potential lead compounds to develop new generation of agricultural and veterinary drugs Fei et al. 2018;Zhu et al. 2021). Especially, tenvermectin B is being developed as a new insecticide.
Information on chemical structure (including configuration) for the drug substance must be submitted to support the approval of original new animal drug applications (NADAs) and abbreviated new animal drug applications (ANADAs) (FDA. 2010). Single crystal X-ray structure determination is a powerful technique for structure research, particularly in that it provides a great deal of information dealing with stereochemistry. Initially, the chemical structures of tenvermectins A and B were determined by extensive spectroscopic analysis. Their absolute configurations were established by comparison of the NMR data of the acid hydrolysates with those of 13-a-hydroxy-milbemycin A4 and methyl a-L-oleandroside . Although a series of 16membered macrolactone metabolites have been obtained from the tenvermectin-producing strain Zhang et al. 2020), single-crystal X-ray diffraction analysis of tenvermectins A and B or their derivatives have never been reported. As a kind of new insecticidal metabolite produced by combinatorial biosynthesis, confirmation of the stereochemistry of tenvermectin B by X-ray crystallography could promote the further chemical modifications and biological studies of tenvermectins and their derivatives as agricultural and veterinary agents.

Results and discussion
Confirmation of the absolute configuration of tenvermectins A and B by X-ray crystal analysis is very important for their further chemical and biological studies. However, it is difficult to obtain a suitable single crystal of tenvermectin A or tenvermectin B. Generally, a single crystal may be formed by introducing substituent group to the hydroxyl group, because the structures of milbemycins A 4 and b 1 had been detected by X-ray diffraction of their derivativesp-bromophenylurethanes (Mishima et al. 1974(Mishima et al. , 1975. Therefore, some derivatives of tenvermectin B were synthesized (Chen et al. 2020), but no crystals suitable for X-ray diffraction analysis were obtained. Fortunately, compound 1 was prepared by treatment tenvermectin B with furoyl chloride and a single crystal of 1 was successfully obtained by slow diffusion of methanol at cold storage. Its structure was determined by HR-ESIMS, NMR and X-ray diffraction analysis.
The structure of 1 was elucidated by analysis of its HR-ESIMS and NMR spectroscopic data. Its molecular formula was determined to be C 56 H 74 O 18 as deduced from the HR-ESIMS at m/z 1057.4774 [M þ Na] þ , indicating the 288 mass units higher than the corresponding parent compound, tenvermectin B. The 1 H and 13 C NMR data of 1 were assigned by the 1 H-1 H COSY, HMQC and HMBC experiments and were shown in Table S1. Comparison of the 1 H and 13 C NMR data of 1 with those of tenvermectin B  revealed that two 2-furoyl moieties were appeared in 1. The downfield chemical shifts of H-5 (d H 5.74) and H-4 00 (d H 4.88) indicated that the two 2furoyloxyl moieties were situated at C-5 and C-4 00 , respectively. The HMBC correlations from H-5 to C-33 (d C 158.0) and from H-4 00 to C-38 (d C 158.3) further confirmed the assignments. Then, the structure of 1 was established as 4 00 , 5-di-(2-furoyloxyl) tenvermectin B (Figure 1).

Experimental
See in a supplementary file online.

Conclusions
In this study, the absolute configuration of tenvermectin B was elucidated definitely by derivatizing it to the corresponding 4 00 , 5-di-(2-furoyloxyl) tenvermectin B. This result showed that the stereochemistry of tenvermectin B is 2 R, 5 R, 6 R, 7S, 12S, 13S, 17 R, 19S, 21 R, 24S, 25 R, 1 0 R, 3 0 S, 4 0 S, 5 0 S, 1 00 S, 3 00 S, 4 00 S and 5 00 S. The confirmation of the absolute configuration of tenvermectin B could promote the further chemical and biological studies of tenvermectins and their derivatives as agricultural and veterinary agents. Moreover, the derivatization approach may also be used to prepare single crystals of the analogues.

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