*2.4. Actinobacterial Producer*

To confirm and replicate the production of these metabolites, we undertook a scale up culture of *Verrucosispora* sp. SN26\_14.1. Thus, 10 L of the Actinobacterium culture were grown, and extracted through the use of amberlite XAD-16 resin, yielding 1 g of crude extract with a brownish coloration. This extract was subjected to stepwise flash chromatography using iso-octane and ethyl acetate gradients, which produced a total of ten fractions. The fractions were evaluated through HPLC to find the fractions containing Lupinacidin A (**1**) and Galvaquinone B (**2**). The chromatogram evaluation showed that only the orange colored fraction two, which was eluted with 90% iso-octane and 10% ethyl acetate, contained 78 mg of metabolites enriched with Lupinacidin A (**1**) and Galvaquinone B (**2**). The purification of Lupinacidin A (**1**) and Galvaquinone B (**2**) was achieved through HPLC using normal and reverse phase chromatography.

Lupinacidin A (**1**) was isolated as a yellow powder, with a yield of 11 mg from a 10 L culture, which suggested to be an intermediate yield compared with *Streptomyces* and *Micromonospora* producers [24,25,28]. High resolution APCI-MS gave an [M + H]+ adduct of m/z 341.1378, which results in a molecular formula of C20H20O5. The calculation of the degree of unsaturation indicated 11 degrees. 1H NMR showed the characteristic exchangeable protons of (**1**) at δ at 14.18 and 12.96 ppm, in addition to the three neighboring aromatic proton signals δ 7.26, 7.62, and 7.79 ppm that showed the expected coupling pattern for three neighboring aromatic protons in a para-ortho, ortho-meta relationship (two duplets, and one duplet of duplets). The 13C NMR experiment showed 20 carbons of which two represented ketone signals (δ 190.2 and 186.9 ppm), 12 aromatic carbons, and six aliphatic carbons (see Table 1). Two-dimensional NMR experiments, Homonuclear COrrelated SpectroscopY (COSY), Heteronuclear Single Quantum Correlation (HSQC), and Heteronuclear Multiple Bond Correlation (HMBC), helped to confirm the identity of the molecules. These data were in agreemen<sup>t</sup> with the published information [24,25,28].


**Table 1.** Spectroscopic NMR data of Lupinacidin A (**1**) and Galvaquinone B (**2**).

\*\* 1H NMR (600 MHz) Solvent: CDCl3(δ1H, mult, J in Hz), \*\*\* 13C NMR (125 MHz), Solvent: CDCl3.

Galvaquinone B (**2**) was isolated as a red powder, with a yield of 7 mg from a 10 L culture, which is an intermediate yield compared with *Streptomyces* and *Micromonospora* producers [24,28]. High resolution APCI-MS gave an [M + H]+ adduct of m/z 369.3510 and a molecular formula of C21 H20 O6. The calculation of the degree of unsaturation indicated 12 degrees. Galvaquinone B (**2**) showed characteristic exchangeable proton signals at δ 13.49, 12.50, and 12.14 ppm, respectively. Aromatic signals were similar to those found in compound (**1**), showing three neighboring aromatic protons in a para-ortho, ortho-meta relationship (two duplets, and one duplet of duplets), but with a higher frequency (see Table 1). The 13C NMR experiment showed 21 carbons of which three represented ketone signals (δ 205, 190.2 and 186.9 ppm), 12 aromatic carbons, and six aliphatic carbons (see Table 1). Two dimensional experiments (COSY, HSQC, HMBC) confirmed the identity of the molecules and were in agreemen<sup>t</sup> with the published data [24,28].

### *2.5. Phylogeny of the Producer*

To determine whether the present isolate was a new species and to evaluate its evolutionary relationship, we performed a phylogenetic evaluation of the strain based of the 16S rRNA gene sequence (Figure 5). The evaluation of the 16S gene showed a high similarity (99%) to the next related type strain, *Verrucosispora maris* DSM 45365T. However, analysis of the gyrase subunit B taxonomic marker (gyrB) showed 94.4% similarity to *Verrucosispora maris* DSM 45365T. The construction of the phylogenetic tree with the closest relatives in terms of the 16S rRNA gene sequence as well as known producers of (**1**) and (**2**) confirmed that the producer strain SN26\_14.1 belongs to the genus *Verrucosispora*, and that quite likely it represents a new species within the genus. This result represents the first report of anthraquinone production for the *Verrucosispora* genus. Interestingly, it appears that anthraquinones are more widespread metabolites in the *Actinobacteria* phylum, since compounds (**1**) and (**2**) have now been found in three actinobacterial genera, *Micromonospora*, *Streptomyces*, and *Verrucosispora* producers [24,25,28].

**Figure 5.** Phylogenetic tree based on 16S rRNA gene sequence of *Verrucosispora sp*. SN26\_14.1. The tree was calculated using a neighbor-joining statistical method and Jukes–Cantor model. • Red dots highlight Lupinacidin A (**1**) and Galvaquinone B producers (**2**).
