*2.7. Isolation and Identification of 3-(2,4-Dihydroxyphenyl)-propionic Acid Bioconversion Product*

Due to difficulties in detecting colorless *meta*-cleavage product of catechol derivative, and since no reasonable mass spectra could be registered, we decided to transform a cleavage product into the derivative of picolinic acid by incubation with NH4Cl as described in Materials and Methods. The formation of a picolinic acid derivative was proven by HPLC-MS analysis, which showed the formation of [M−H]<sup>−</sup> ion 210.00 mass, as shown in Figure 6, corresponding to the addition of NH3 to the *meta*-cleavage product and the loss of two H2O molecules [40].

The 1H NMR spectrum of this derivative [δ 7.73 (s, 1H), 6.89 (s, 1H), 2.65 (t, *J* = 7.4 Hz, 2H), 2.38 (t, *J* = 7.4 Hz, 2H)] showed a set of two aryl protons that, from the coupling pattern (singlet + singlet), were in *meta*- or *para*-positions to each other on the aromatic ring [41], as shown in Figure S12 in the Supplementary Material. The appearance in the spectrum of two triplets with chemical shifts of 2.38 and 2.65 ppm indicated the presence of four methylene protons [41]. The 13C NMR spectrum [δ 181.51, 179.99, 171.01, 143.14, 136.74, 130.11, 115.22, 35.50, 24.00] showed two sp3 carbons with chemical shifts of 24.00 and 35.50 ppm, and three sp<sup>2</sup> carbons of carbonyl groups with chemical shifts of 171.01, 179.99, and 181.51 ppm, respectively, as shown in Figure S13 in the Supplementary Material. The carbonyl carbon atoms were the most strongly deshielded and their resonances formed a separate region at the highest frequency. Another four sp2 carbon signals were in the aromatic carbon region [41,42]. The presence of the third carbonyl group indicated the formation of *oxo*-pyridine, for which six possible theoretical structures of *oxo*-picolinic acid derivative were presumed, as shown in Figure 7. Since the 1H NMR spectrum showed a set of two singlet aryl protons in *meta*- or *para*-positions to each other, only structures **7** and **9**, as shown in Figure 7, were further analyzed. Besides, pyridine aromatic carbons are usually differentiated into two resonances at higher field (C-3/5, *meta* position) and three at lower field (C-2/6, *ortho* position; C-4, *para* position), where the electron-withdrawing effect of nitrogen is effective [41,42]. The chemical shift

of 115.22 ppm showed that the analyzed compound had relatively strongly shielded unsubstituted aromatic carbon, which should be in *meta*-position from nitrogen, in *ortho*-position from the carbonyl group, and in *meta*- or *para*-position from the carboxyl group (C-5 atom), as shown in Figure S14 in the Supplementary Material [43,44]. This led to the conclusion that structure **9**, as shown in Figure 7, 6-(2-carboxyethyl)-4-oxo-1,4-dihydropyridine-2-carboxylic acid, was formed during incubation of *meta*-cleavage product of catechol derivative with NH4Cl, as depicted in Scheme 1. These data allowed the reconstruction of the consecutive oxidation of 3-(2,4-dihydroxyphenyl)-propionic acid catalyzed by the HcdA and HcdB enzymes. Hence, 3-(2,3,5-trihydroxyphenyl)-propionic acid (**14**) was the product of oxidation of 3-(2,4-dihydroxyphenyl)-propionic acid by the HcdA hydroxylase. The molecular mass of 198.17 of 3-(2,3,5-trihydroxyphenyl)-propionic acid and capability to form *para*-quinone agreed with the UV-VIS and HPLC-MS data on the bioconversion of 3-(2,4-dihydroxyphenyl)-propionic by the HcdA hydroxylase. The formation of 3-(2,3,5-trihydroxyphenyl)-propionic acid from 3-(2,4-dihydroxyphenyl)-propionic acid was possible only through oxidative *ipso*-rearrangement, a unique reaction where *ipso*-hydroxylation (**13**) of the 3-(2,4-dihydroxyphenyl)-propionic acid takes place with a simultaneous shift of the propionic acid group to the vicinal position, as shown in Scheme 1 [45–48]. During the second step, 3-(2,3,5-trihydroxyphenyl)-propionic acid was cleaved by HcdB extradiol dioxygenase at the *meta*-position leading to the formation of (*2E*,*4E*)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid (**15**). The further imine formation and tautomerization in the presence of ammonium ions [38,49] led to 6-(2-carboxyethyl)-4-*oxo*-1,4-dihydropyridine-2-carboxylic acid (**19**), as shown in Scheme 1.

**Figure 6.** HPLC-MS analysis of 3-(2,4-dihydroxyphenyl)-propionic acid bioconversion mixture in vivo. UV 254 nm trace of picolinic acid derivative with retention time of 4.500 min (**A**), UV spectrum (**B**) and MS spectrum (**C**) of the dominant peak. The negative ions [M−H]<sup>−</sup> generated are at *m*/*z* 210.00.

**Figure 7.** Suggested structures of *oxo*-picolinic acid derivative, formed during oxidative ring cleavage of 3-(2,4-dihydroxyphenyl)-propionic acid and conversion of the ring fission product. Solid lines indicate possible positions of hydroxylation by HcdA enzyme; hollow arrays indicate the probable *oxo*-picolinic acid derivatives forming after hydroxylation at each position.

**Scheme 1.** The proposed metabolic pathway of 7-hydroxycoumarin in *Pseudomonas* sp. 7HK4 cells. Incubation of the compound **5** with NH4Cl gives picolinic acid derivative. **5**—7-hydroxycoumarin; **6**—3-(2,4-dihydroxyphenyl)-propionic acid; **13**—3-(1,2-dihydroxy-4 oxocyclohexa-2,5-dienyl)-propanoic acid; **14**—3-(2,3,5-trihydroxyphenyl)-propionic acid; **15**—(*2E*,*4E*)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid; **16**—(*E*)-2-hydroxy-4-oxopent-2-enoic acid; **17**—succinic acid; **18**—6-(2-carboxyethyl)-4-hydroxypicolinic acid; **19**—6-(2-carboxyethyl)-4 oxo-1,4-dihydropyridine-2-carboxylic acid; HcdA—3-(2,4-dihydroxyphenyl)-propionic acid 1-monooxygenase; HcdB—3-(2,3,5-trihydroxyphenyl)-propionic acid 1,2-dioxygenase; HcdC—putative (*2E*,*4E*)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid hydrolase. The dashed arrow indicates a hypothetical reaction.
