Structure elucidation of compounds
Based on the daily TLC pattern obtained from the biotransformation process in the screening scale, it was found that 96 h after adding substrate (testosterone) is the suitable extraction time for the semi-preparative scale. The TLC profile of the medium extract showed the presence of three distinct and clear bands with Rf values of 0.33, 0.45, 0.75, respectively (Fig. 1). This means that testosterone (I) has been transformed into three significant metabolites (II - IV) by Penicillium pinophilum (Fig. 2). Accordingly, the fungi produced the three metabolites, then they were isolated by preparative layer chromatography technique and finally were identified using the spectral data. All the corresponding spectra have been represented in supplementary material (Fig. S1-S9).
17β-Hydroxyandrost-4-en-3-one (Testosterone) (substrate I): White solid; Rf in Chloroform-Methanol (8.8–1.2, v/v): 0.80; ESI-MS, m/z [M + H]+: 289; 1H NMR (CDCl3, 500 MHz): δ 0.79 (3H, s, H-18), 1.20 (3H, s, H-19), 3.65 (1H, t, J = 8.5 Hz, H-17α), 5.72 (1H, s, H-4). The chemical shift values of carbon atoms in 13C NMR (CDCl3, 125 MHz) spectrum have been presented in Table 1.
Table 1
13C NMR signals of substrate (I) and its metabolites (II-IV) (δ in ppm)
Carbon | Substrate (I) | Metabolite (II) | Metabolite (III) | Metabolite (IV) |
1 | 35.77 | 35.85 | 35.75 | 37.62 |
2 | 33.95 | 33.17 | 33.95 | 34.32 |
3 | 199.3 | 199.57 | 199.95 | 200.40 |
4 | 123.93 | 124.30 | 123.79 | 124.69 |
5 | 170.97 | 170.01 | 171.34 | 171.20 |
6 | 32.79 | 32.44 | 32.63 | 33.73 |
7 | 31.57 | 25.69 | 28.60 | 31.25 |
8 | 35.71 | 38.05 | 38.95 | 35.46 |
9 | 53.95 | 46.97 | 46.78 | 59.33 |
10 | 38.77 | 38.77 | 38.79 | 40.14 |
11 | 20.67 | 19.23 | 19.75 | 69.07 |
12 | 36.45 | 24.62 | 31.44 | 48.71 |
13 | 42.84 | 52.65 | 47.01 | 43.72 |
14 | 50.53 | 80.88 | 83.30 | 49.97 |
15 | 23.36 | 30.40 | 26.18 | 23.40 |
16 | 30.49 | 34.02 | 29.56 | 29.83 |
17 | 81.61 | 218.35 | 78.47 | 81.21 |
18 | 10.90 | 17.94 | 15.00 | 12.43 |
19 | 17.44 | 17.48 | 17.24 | 18.61 |
14α-Hydroxyandrost-4-en-3,17-dione (metabolite II): White solid; Rf in Chloroform-Methanol (8.8–1.2, v/v): 0.75; ESI-MS, m/z [M + H]+: 303; 1H NMR (CDCl3, 300 MHz): δ 1.05 (3H, s, H-18), 1.22 (3H, s, H-19), 5.75 (1H, s, H-4). The chemical shift values of carbon atoms in 13C NMR (CDCl3, 75 MHz) spectrum have been presented in Table 1.
The mass spectrum of metabolite II exhibited [M + H]+ peak at m/z 303, corresponding to the formula C19H26O3. On the 1H NMR spectrum, the lack of H-17 triplet at δ 3.65 ppm along with the significant downfield shifts of H-18 signal from 0.79 ppm to 1.05 ppm showed that there would be a change at the position of C-17. These data were consistent with the 13C NMR spectrum, which revealed that the signal at 81.61 ppm had been replaced by a signal at 218.35 ppm for C-17. Collectively, these data indicated the oxidation of the C-17β hydroxyl group to C-17 carbonyl group. Furthermore, on the 13C NMR spectrum, the large shift of C-14 signal from δ 50.53 ppm to 80.88 ppm and also the downfield shifts for C-8 (Δ 2.34 ppm), C-15 (Δ 7.04 ppm), and C-13 (Δ 9.81 ppm) confirmed the presence of a hydroxyl group at C-14α position. The data were in agreement with those reported in the literature for 14α-hydroxyandrost-4-en-3,17-dione (Faramarzi et al. 2008a; Faramarzi et al. 2008b).
14α-Hydroxytestosterone (metabolite III): White solid; Rf in Chloroform-Methanol (8.8–1.2, v/v): 0.45; ESI-MS, m/z [M + H]+: 305; 1H NMR (CDCl3, 300 MHz): δ 0.89 (3H, s, H-18), 1.19 (3H, s, H-19), 4.28 (1H, t, J = 9 Hz, H-17α), 5.70 (1H, s, H-4). The chemical shift values of carbon atoms in 13C NMR (CDCl3, 75 MHz) spectrum have been presented in Table 1.
The mass spectrum of metabolite III revealed [M + H]+ peak at m/z 305, agreeing with the formula C19H28O3. The 13C NMR spectrum exhibited a large shift for C-14 signal from 50.53 ppm to 83.30 ppm along with downfield shifts for C-8 (Δ 3.24 ppm), C-15 (Δ 2.82 ppm), and C-13 (Δ 4.17 ppm). These changes suggested that hydroxylation has taken place at C-14. Additionally, the 1H NMR spectrum displayed a large downfield shift for H-17α from 3.65 ppm to 4.28 ppm (Δ 0.63 ppm). This also confirmed that the hydroxylation has occurred at C-14. The reason for this great shift is the diaxial interaction between H-17α and C-14α hydroxyl groups. Based on the spectral features and comparing them with those reported in the literature, metabolite III was identified as 14α-Hydroxytestosterone (Yildirim et al. 2018; Faramarzi et al. 2008b).
11α-Hydroxytestosterone (metabolite IV): White solid; Rf in Chloroform-Methanol (8.8–1.2, v/v): 0.33; ESI-MS, m/z [M + H]+: 305; 1H NMR (CDCl3, 500 MHz): δ 0.82 (3H, s, H-18), 1.32 (3H, s, H-19), 3.69 (1H, t, J = 8.5 Hz, H-17α), 4.04 (1H, td, J = 10.3 Hz and J = 4.6 Hz, H-11β), 5.73 (1H, s, H-4). The chemical shift values of carbon atoms in 13C NMR (CDCl3, 75 MHz) spectrum have been presented in Table 1.
The [M + H]+ peak for the metabolite IV was appeared at m/z 305, indicating the empirical formula C19H28O3. The 1H NMR spectrum revealed a new signal as a triplet of doublet at 4.04 ppm (J = 10.3 Hz and J = 4.6 Hz) and a mild downfield shift for H-19 signal (Δ 0.12 ppm). As well, the 13C NMR spectrum exhibited a significant shift for C-11 signal from 20.67 to 69.07 (Δ 48.40 ppm) along with downfield shifts for C-12 (Δ 12.26 ppm) and C-9 (Δ 5.38) ppm. These data supported the presence of a hydroxyl group at the C-11 position. On the other hand, the splitting pattern and coupling constants for the new proton signal displayed an alpha configuration. Comparison of the spectral data with those reported in the literature supported that the metabolite IV is 11α-hydroxytestosterone (Peart et al. 2013, 2016).
Time course profile of the biotransformation process
For this study, a simple HPTLC method has been designed to measure testosterone and its metabolites during the biotransformation process (Fig. 3). The final obtained profile showed that the conversion of the substrate (testosterone) started from day one, and all the three identified products were detectable from that day onward (Fig. 4). According to this, the amount of testosterone was continuously reduced so that, at the end of the process (i.e., after 144 h), about 97.4% of testosterone was metabolized. The primary product (i.e., metabolite III) was detected in 0.017 µmol on the first day and gradually decreased to 0.011 µmol on the sixth day. The metabolite II reached the maximum level of 0.006 µmol on the fourth day and then reduced. The highest amount of metabolite IV was about 0.002 µmol on day six. However, the daily changes in the amount of metabolite III were minimal.
In-silico assessment of the metabolites
In this investigation, in-silico assessment with the ED program was performed to study the increase or decrease in computed affinities for the produced metabolites due to hydroxylation or oxidation. Based on the obtained results (Table 2), it was determined that the hydroxylation at the 11α-position of testosterone leads to increase the binding affinity of the metabolite (11α-hydroxytestosterone) to the estrogen receptors α and β. In contrast, the hydroxylation at the 14α- position reduces the binding affinity of the metabolite (14α-hydroxytestosterone) to the estrogen receptors α and β. However, both hydroxylation patterns did not affect the binding affinities of the two metabolites to the androgen, progesterone, and glucocorticoid receptors. On the other hand, the oxidation of 14α-hydroxytestosterone at the 17β-position enhanced the binding affinity of the produced metabolite (i.e., 14α-hydroxyandrostenedione) to the estrogen receptor α. However, it did not affect the binding affinity of the metabolite to the other receptors.
Table 2
Prediction of binding affinities of testosterone and its metabolites with some steroid hormone receptors
Compound | Color-coded probability binding classes (and computed binding affinity score) |
Androgen receptor | Estrogen receptor α | Estrogen receptor β | Glucocorticoid receptor | Progesterone receptor |
Testosterone | (-9.9) | (-8.4) | (-9.2) | (-9.2) | (-2.8) |
14α-Hydroxyandrostenedione | (-9.9) | (-9.0) | (-9.0) | (-8.5) | (-2.8) |
14α-Hydroxytestosterone | (-9.8) | (-8.2) | (-8.5) | (-8.8) | (-2.8) |
11α-Hydroxytestosterone | (-9.4) | (-9.2) | (-10.0) | (-8.6) | (-2.8) |