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Article

Epimeric Mixtures of Brassinosteroid Analogs: Synthesis, Plant Growth, and Germination Effects in Tomato (Lycopersicum esculentum Mill.)

by
Nitza Soto
1,
César González
1,
Marco Mellado
2,
Andrés F. Olea
3,
Yamilet Coll
4,
Katy Díaz
1,* and
Luis Espinoza
1,*
1
Departamento de Química, Universidad Técnica Federico Santa María, Av. España No. 1680, Valparaíso 2390123, Chile
2
Instituto de Química, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Av. Universidad #330, Curauma, Valparaíso 2340000, Chile
3
Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile, Santiago 8910339, Chile
4
Center for Natural Products Research, Faculty of Chemistry, University of Havana, Zapata y G, La Habana 10400, Cuba
*
Authors to whom correspondence should be addressed.
Agronomy 2020, 10(6), 808; https://doi.org/10.3390/agronomy10060808
Submission received: 13 May 2020 / Revised: 2 June 2020 / Accepted: 3 June 2020 / Published: 5 June 2020
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Abstract

:
Brassinosteroids (BRs) play an important role in the growth and development of plants. Herein, we describe the synthesis of epimeric mixtures of BR analogs with 24-norcholane type side chains, S/R configuration at C22 and A/B ring cis-type fusion. All epimeric mixtures were synthetized from hyodeoxycholic acid. The biological activity of mixtures was evaluated by using rice lamina inclination test and germination of tomato (Lycopersicum esculentum) seeds. The results show that these epimeric mixtures exhibit similar bioactivity to brassinolide in both bioassays. Thus, our results corroborate that the A/B junction has almost no effect on bioactivity and open the possibility of using epimeric mixtures instead of pure compounds. In this approach, the synthesized BR analogs maintain a good level of bioactivity, whereas the synthesis is shorter, cheaper and with higher yields. All these factors make this alternative very interesting for potential application.

1. Introduction

Brassinosteroids (BRs) are plant polyhydroxysteroids that from the pivotal discovery of brassinolide (BL) by Grove et al. [1] and have been found all around the plant kingdom [2,3]. BRs participate in a series of physiological, biochemical and response processes in plants, such as cell division and elongation, photomorphogenesis, flower developmental processes, male sterility, stomatal developmental processes and enzyme activation [4,5,6,7,8,9]. BRs are naturally found in seeds and it has also been shown that they play an important role in promotion of seed germination [10]. It was observed that the percentage of germination in Cicer arietinum, Triticum aestivum and Brassica juncea, was increased by soaking seeds in BR solutions [11,12,13,14]. So far, the applied research on BRs has been done mainly in agriculture [5]; however, there have also been some studies in forestry showing that BRs stimulate seed germination of forest species such as Robinia pseudoacacia L, Pinus tabulaeformis Carr. and Ailanthus altissima Mill. [15,16]. Rapid and synchronized germination is an important feature in commercial plant production in both agriculture and forestry. Germination or dormancy problems generate a reduction in the quality and crop yield and increase the time of the production cycle. Moreover, BRs increase resistance in plants to various kinds of biotic and abiotic stress factors, i.e., low and high temperature, drought, heat, salinity, heavy metal toxicity, and pesticides [17,18,19,20,21,22].
The chemical structures of the most common and biologically active BRs are shown in Figure 1.
Application of exogenous BRs enhances seed germination and yield of crops under stressed and [23] stress-free conditions [24,25]. Thus, BRs are recommended as safe plant growth promoters with potential applications in agriculture and horticulture [23,26]. Besides, the enhancement of crop yields and increasing plant stress tolerance obtained by exogenous BRs have prompted research into their potential applications in phytoremediation [27]. However, the high cost of BR synthesis has prevented their extensive use by farmers. To overcome this problem, an increasing number of BR analogs have been synthesized and assayed. This topic is under continuous and extensive research [28].
The effect of BRs on the growth and development of plants has been widely studied, and structure–activity relationships have been proposed [28,29,30,31,32]. Recently, the effects of the shortest side chains and a variety of their substituents have been explored [33,34,35,36]. To determine the structural effect of the side alkyl chain on growth-promotion activity, as measured by Rice Lamina Inclination Tes (RLIT) assay, we have recently reported the synthesis and growth-promoting activity of BRs’ 24-nor-5α-cholan type analogs [37,38]. One of the main results suggests that the activity of epimeric mixtures comes from independent contributions of both diastereoisomers R and S. From a practical point of view, the direct use of epimeric mixtures instead of pure compounds makes them cost effective, because important savings associated with steps of separation and purification are obtained.
Thus, in this work, BR analog type 24-norcholane, with structural variations in the side chain, and type cis (5-β) A/B ring fusion (Figure 2) were synthesized from hyodeoxycholic acid. The products of synthesis are mixtures of two diastereomers, S and R, originated by the presence of one stereocenter at C-22. These epimers differ only in the configuration of this C atom, and, therefore, it is very difficult to separate them. Thus, the effects of these epimeric mixtures on growth promotion in the RLIT and germination process of tomato seeds were evaluated.

2. Materials and Methods

2.1. Chemical

2.1.1. General Experimental Methods

All reagents were purchased from commercial suppliers and used without further purification. Melting points were measured on an SMP3 apparatus (Stuart Scientific, now Merck KGaA, Darmstadt, Germany) and are uncorrected. 1H, 13C, 13C DEPT-135, gs 2D HSQC, and gs 2D HMBC NMR spectra were recorded in CDCl3 or MeOD solutions, and are referenced to the residual peaks of CHCl3 at δ = 7.26 ppm and δ = 77.00 ppm for 1H and 13C, respectively, and CD3OD at δ = 3.30 ppm and δ = 49.00 ppm for 1H and 13C, respectively, on an Avance 400 Digital NMR spectrometer (Bruker, Rheinstetten, Germany) operating at 400.1 MHz for 1H and 100.6 MHz for 13C. Chemical shifts are reported in ppm and coupling constants (J) are given in Hz; multiplicities are reported as follows: singlet (s), doublet (d), doublet of doublets (dd), doublet of triplets (dt), triplet (t), quartet (q), multiplet (m), broad singlet (bs). IR spectra were recorded as KBr disks in an FT-IR 6700 spectrometer (Nicolet, Thermo Scientific, San Jose, CA, USA) and frequencies were reported in cm−1. Unitary mass spectra (MS) were recorded in an GCMS-QP2010 Ultra (SHIMADZU Gas Chromatograph–Mass Spectrometer, Japan RTx-5 MS column, temperature 300 °C, solvent CHCl3, CH3OH and range 7000 a 550.10 m/z, Electron Impact Ionization). For analytical TLC, silica gel 60 in a 0.25-mm layer was used and TLC spots were detected by heating after spraying with 25% H2SO4 in H2O. Chromatographic separations were carried out by a conventional column on silica gel 60 (230–400 mesh) using EtOAc–hexane gradients of increasing polarity. All organic extracts were dried over anhydrous magnesium sulfate and evaporated under reduced pressure below 40 °C.

2.1.2. Synthesis of 3α,6α-Diformyloxy-5β-Cholan-24-Oic Acid (2)

Perchloric acid (0.2 mL, 70–72% w/w) was added to a solution of hyodeoxycholic acid (1) (2.03 g, 5.18 mmol) in HCOOH (20 mL, d = 1.218 g/mL, 98–100%, 529.22 mmol). The reaction mixture was refluxed at 40 °C for 2 h. The end of the reaction was verified by TLC. Later, water (81 mL) was added and the precipitate was filtered and washed with NaHCO3 (25 mL, saturated solution) and water (50 mL). The neutralized solid was dried in vacuo and subsequently dissolved in Ac2O (30 mL), dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in dichloromethane (DCM) (10 mL) and chromatographed on silica gel with hexane/EtOAc mixtures of increasing polarity (19.8:0.2 → 0.0:20.0). Compound 2 was obtained (2.203 g, 95% yield). IR νmax (cm−1): 3422(O-H), 2939(C-H), 2869 (C-H), 1742 (C=O), 1723 (C=O). 1H NMR (CDCl3) δ (ppm): 8.04 (1H, s, HCO2-C3), 8.01 (1H, s, HCO2-C6), 5.32–5.27 (1H, dt, J = 12.0 and 5.0 Hz, H-6), 4.84–4.82 (1H, m, H-3), 2.39–2.36 (2H, m, H-22), 2.30–2.23 (2H, m, H-23), 0.99 (3H, s, H-19), 0.92 (3H, d, J = 8.0 Hz, H-21), 0.65 (3H, s, H-18). 13C NMR (CDCl3) δ (ppm): (C1) 34.92, (C2) 26.13, (C3) 73.55, (C4) 30.68, (C5) 45.38, (C6) 70.91, (C7) 26.40, (C8) 34.63, (C9) 39.85, (C10) 36.13, (C11) 20.66, (C12) 39.80, (C13) 42.89, (C14) 55.89, (C15) 28.03, (C16) 24.05, (C17) 56.02, (C18) 12.01, (C19) 23.21, (C20) 35.24, (C21) 18.22, (C22) 30.94, (C23) 31.23, (C24) 179.98, (CO2H) 160.60, (HCO2-C3), 160.51 (HCO2-C6). MS m/z (%): M+: 448 (2.5), 403 (28.7, M+ -HCO2), 341 (23.7, M+ -2 × HCO2 -OH), 281 (56.7 M+), 207 (66.4), 197 (19.5), 147 (34.8), 135 (77.2), 85 (30.7, CHCH2CH2CO2), 84 (21.7 CH=CHCH2CO2), 83 (52.3, CH2=CH=CH2CH2C≡O), 77 (21.2), 73 (100, CH2CH2CO2H).

2.1.3. Synthesis of 5β-Cholan-24-Nor-22-En-3α,6α-Diyl Diformate (3)

Pyridine (0.5 mL) and Cu(OAc)2 (0.20 g 1.10 mmol) were added to a solution of 2 (1.00 g, 2.23 mmol) in dry benzene (50 mL). Then, under reflux, Pb(OAc)4 (2.50 g 5.64 mmol) was added in four portions at hourly intervals. After addition was completed, the reaction was continued for 8 h. The end of the reaction was verified by TLC, then the mixture was filtered, and the solvent was evaporated under reduced pressure. The crude was re-dissolved in DCM (8 mL) and chromatographed on silica gel with PE/EtOAc mixtures of increasing polarity (19.8:0.2 → 15.8:4.2). Compound 3 (0.494 g, 55% yield) was obtained as a colorless solid: m.p. 86 ± 3 °C (hexane/Et2O = 1/1); IR νmax (cm−1): 3078 (C=C-H), 1726 (C=O), 1710 (C=O), 1632 (C=C), 1184 (C-O), 1197 (C-O). 1H NMR (CDCl3) δ (ppm): 8.01 (1H, s, HCO2-C3), 8.01 (1H, s, HCO2-C6), 5.67–5.58 (1H, m, H-22), 5.31 (1H, dt, J = 12.0 and 5.0 Hz, H-6), 4.88 (1H, dd, J = 17.1 and 0.8 Hz, H-23a), 4.80 (1H, dd, J = 10.0 and 1.6 Hz, H-23b), 4.90–4.78 (1H, m, H-3), 1.00 (3H, d, J = 6.6 Hz, H-21), 0.98 (3H, s, H-19), 0.66 (3H, s, H-18). 13C NMR (CDCl3) δ (ppm): (C1) 34.89, (C2) 26.32, (C3) 73.43, (C4) 31.17, (C5) 45.30, (C6) 70.79, (C7) 26.06, (C8) 34.56, (C9) 39.87, (C10) 36.07, (C11) 20.59, (C12) 39.64, (C13) 42.74, (C14) 55.46, (C15) 28.26, (C16) 24.01, (C17) 56.01, (C18) 12.11, (C19) 23.15, (C20) 41.04, (C21) 20.00, (C22) 144.88, (C23) 111.68, (HCO2-C3) 160.44, (HCO2-C6) 160.37. MS m/z (%): M+: 403 (0.16), 402 (0.46), 345 (49.2 M+ -HCO -H2C=CH2), 310 (20.5 M+ -COH, HCO2H -CH3 -H2), 256 (21.9 M+ -2 × CO2H -C2H3 -CH2 -CH3), 255 (100 M+ -C2H3 -2 × CO2H - 2× CH3), 213 (25.9), 159 (23.7), 81 (22.0).

2.1.4. Synthesis of Mixture 22(S), 23-Dihydroxy-24-Nor-5β-Cholan-3α,6α-Diyl Diformate (4a) and 22(R), 23-Dihydroxy-24-Nor-5β-Cholan-3α,6α-Diyl Diformate (4b)

NMO (0.45 g, 3.84 mmol) was added to a solution of alkene 3 (2.00 g, 4.97 mmol) in acetone (40 mL). After mixture homogenization by magnetic stirring, a solution of 4% OsO4 (0.25 g 0.98 mmol in H2O 6.25 mL) was added dropwise with stirring and then reacted for 36 h at room temperature. The end of the reaction was verified by TLC. The solvent was removed (up to 10 mL approximate volume) and water (25 mL) and Na2S2O3 × 5H2O (25 mL saturated solution) were added. The organic layer was extracted with EtOAc (2 × 25 mL), washed with water (2 × 50 mL), dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in DCM (10 mL) and chromatographed on silica gel with hexane/EtOAc mixtures of increasing polarity (19.8:0.2 → 9.8:10.2). Two fractions were obtained. Fraction I: Alkene 3 unreacted (0.580 mg). Fraction II: A mixture of 4a/4b = 5.0/1.0 (1.56 g, 72% yield).
Mixture 4a/4b: IR νmax (cm−1): 3433 (O-H), 2942 (C-H), 2868 (C-H), 1723 (C=O), 1704 (C=O). MS m/z (%): M+: 437 (0.13), 429 (22.4 M+ -4 × H2), 405 (19.5), 358 (26.0 M+ -CH3 -H2O -CHO2), 344 (27.0 M+ -H2 -2 × CHO2), 341 (48.7 M+ -2 × OH -CH3 -CHO2 -H2), 331 (19.7 M+ -C2H5O2 -CHO2), 327 (34.3), 313 (29.1 M+ -CH3 -H2O -2 × CHO2), 312 (100 M+ -2 × OH -2 × CHO2), 297 (30.1), 284 (37.3 M+ -CHO2 -H2CO2 -C2H5O2), 282 (20.8), 281 (68.2), 274 (26.4), 269 (26.0), 259 (24.1), 253 (66.8), 255 (31.4 M+ -2 × CH3 -C2H5O2 – 2 × CHO2), 254 (28.7), 229 (19.8), 228 (49.9 M+-C4H9O2 -2 × CH3 -2 × CHO2), 214 (30.0), 213 (98.0), 208 (19.8), 207 (95.8), 199 (20.0), 173 (21.8 M+ - 2 × CH3 -C4H10O2 -C4H7 -CHO2 -H2CO2), 171 (23.5), 159 (38.7), 157 (26.3), 147 (47.8), 146 (21.7), 145 (55), 143 (28.3), 135 (43.7),133 (45.7),131 (37.5), 121 (24.5), 119 (35.0), 117 (21.3), 109 (20.4), 107 (36.6), 105 (57.6), 95 (40.5), 93 (50.1), 91 (54.7), 81 (55.3), 79 (48.6), 73 (71.6).
Compound 4a: 1H NMR (CD3OD) δ (ppm): 8.05 (1H, s, HCO2-C3), 8.04 (1H, s, HCO2-C6), 5.27 (1H, dt, J = 12.0 and 4.4 Hz, H-6), 4.78–4.71 (1H, m, H-3), 3.71–3.68 (1H, m, H-22), 3.58 (1H, dd, J = 11.3 and 2.4 Hz, H-23a), 3.38 (1H, dd, J = 11.3 and 8.9 Hz, H-23b), 1.00 (3H, s, H-19), 0.92 (3H, d, J = 6.9 Hz, H-21), 0.70 (3H, s, H-18). 13C NMR (CD3OD) δ: (C1) 30.09, (C2) 28.95, (C3) 73.60, (C4) 33.92, (C5) 48.18, (C6) 76.31, (C7) 37.39, (C8) 37.48, (C9) 55.72, (C10) 38.66, (C11) 28.71, (C12) 42.50, (C13) 45.74, (C14) 58.75, (C15) 26.69, (C16) 23.20, (C17) 58.31, (C18) 13.58, (C19) 25.00, (C20) 42.58, (C21) 14.84, (C22) 76.62, (C23) 64.62, (HCO2-C3) 163.92, (HCO2-C6) 163.78.
Compound 4b: 1H NMR (CD3OD) δ (ppm): 8.05 (1H, s, HCO2-C3), 8.04 (1H, s, HCO2-C6), 5.27 (1H, dt, J = 12.0 and 4.4 Hz, H-6), 4.78–4.71 (1H, m, H-3), 3.71–3.68 (1H, m, H-22), 3.58 (1H, dd, J = 11.3 and 2.4 Hz, H-23a), 3.38 (1H, dd, J = 11.3 and 8.9 Hz, H-23b), 1.00 (3H, s, H-19), 0.87 (3H, d, J = 6.0 Hz, H-21), 0.70 (3H, s, H-18). 13C NMR (CD3OD) δ: (C1) 32.08, (C2) 28.95, (C3) 73.60, (C4) 33.92, (C5) 55.05, (C6) 76.31, (C7) 37.39, (C8) 37.48, (C9) 55.05, (C10) 40.10, (C11) 28.71, (C12) 42.50, (C13) 45.26, (C14) 58.75, (C15) 26.69, (C16) 23.20, (C17) 58.31, (C18) 13.58, (C19) 25.00, (C20) 43.44, (C21) 14.84, (C22) 76.62, (C23) 64.62, (HCO2-C3) 163.92, (HCO2-C6) 163.78.

2.1.5. Synthesis of Mixture 3α,6α, 22(S), 23-Tetrahydroxy-24-Nor-5β-Cholan (5a) and 3α,6α, 22(R), 23-Tetrahydroxy-24-Nor-5β-Cholan (5b)

An aqueous solution of K2CO3 (5% w/w, 15 mL) was added to a methanolic mixture of 4a/4b (100 mL, 1.00 g, 2.29 mmol). Then, the suspension was stirred at reflux temperature for 1 h. The end of the reaction was verified by TLC. The solvent was removed, and the dry residue was acidified with 2.5% w/w HCl (5 mL). The obtained solid was filtered and washed with 5% NaHCO3 (10 mL) and water (2 × 10 mL) and dried. Recrystallization (MeOH/Et2O = 2/1) of crude product led to epimeric mixture 5a/5b = 7.0/1.0 (0.784 g, 89.9% yield).
Mixture 5a/5b: IR νmax (cm−1): 3376 (O-H), 2937 (C-H), 2887 (C-H), 2863 (C-H). MS m/z (%): M+: 381 (0.07), 284 (24.3 M+ -4 × OH- 2 × CH3), 117 (100), 116 (25.7 M+ -C17H28O2).
Compound 5a: 1H NMR (CD3OD) δ (ppm): 4.01 (1H, dt, J = 12.0 and 4.7 Hz, H-6), 3.73–3.70 (1H, m, H-22), 3.61 (1H, dd, J = 11.4 and 2.6 Hz, H-23a), 3.51–3.50 (1H, m, H-3), 3.40 (1H, dd, J = 11.4 and 8.9 Hz, H-23b), 0.93 (3H, s, H-19), 0.94 (3H, d, J = 5.4 Hz, H-21), 0.70 (3H, s, H-18). 13C NMR (CD3OD) δ (ppm): (C1) 36.57, (C2) 30.09, (C3) 73.38, (C4) 32.13, (C5) 54.73, (C6) 69.64, (C7) 42.31, (C8) 37.25, (C9) 55.43, (C10) 37.93, (C11) 22.91, (C12) 37.79, (C13) 45.31, (C14) 58.71, (C15) 26.42, (C16) 30.99, (C17) 58.26, (C18) 13.19, (C19) 25.07, (C20) 43.03, (C21) 14.44, (C22) 76.25, (C23) 64.20.
Compound 5b: 1H NMR (CD3OD) δ (ppm): 4.01 (1H, dt, J = 12.0 and 4.7 Hz, H-6), 3.73–3.70 (1H, m, H-22), 3.61 (1H, dd, J = 11.4 and 2.6 Hz, H-23a), 3.51–5.50 (1H, m, H-3), 3.40 (1H, dd, = 11.4 and 8.9 Hz, H-23b), 0.95 (3H, s, H-19), 0.89 (3H, d, J = 6.2 Hz, H-21), 0.71 (3H, s, H-18). 13C NMR (CD3OD) δ (ppm): (C1) 36.57, (C2) 30.09, (C3) 73.38, (C4) 32.13, (C5) 45.31, (C6) 69.64, (C7) 42.48, (C8) 39.75, (C9) 54.73, (C10) 37.93, (C11) 22.91, (C12) 37.79, (C13) 45.31, (C14) 58.71, (C15) 26.26, (C16) 29.72, (C17) 58.26, (C18) 13.36, (C19) 25.07, (C20) 42.48, (C21) 13.41, (C22) 75.50, (C23) 66.56.

2.1.6. Synthesis of Mixture 22(S)-22,23-Epoxy-24-Nor-5β-Cholan-3α,6α-Diyl Diformate (6a) and 22(R)-22,23-Epoxy-24-Nor-5β-Cholan-3α,6α-Diyl Diformate (6b)

m-chloroperoxybenzoic acid (mCPBA) (77%, 0.21 g, 1.24 mmol) and NaHCO3 (2.5 mg, 0.029 mmol) were added to a solution of alkene 3 (0.5 g, 1.24 mmol) in CH2Cl2 (20 mL). The mixture was stirred for 48 h and the end of the reaction was verified by TLC. The reaction mixture was filtered to eliminate formed m-chlorobenzoic acid, and the liquid was evaporated under reduced pressure. The residue was dissolved in AcOEt (30 mL), and the solution was washed with a saturated solution of NaHCO3 (2 × 10 mL) and water (3 × 10 mL), dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in DCM (10 mL) and chromatographed on silica gel with hexane/EtOAc mixtures of increasing polarity (19.8:0.2 → 12.8:7.2). A mixture of 6a/6b = 0.39/1.0 was obtained (0.488 g, 94% yield).
Mixture 6a/6b: IR νmax (cm−1): 2954 (C-H), 2890 (C-H), 2867 (C-H), 1727 (C=O), 1712 (C=O). MS m/z (%): M+: 418 (1.8), 343 (28.9 M+ -2 × CH3 -CHO2), 342 (69.0 M+ -2 × CH3 -HCHO2), 341 (45.2), 327 (48.0), 296 (49.1), 282 (26.9), 281 (99.5), 254 (27.8), 253 (71.9 M+ -C11H18O), 227 (386), 213 (35.0), 208 (21.2 M+ -C11H18O -HCHO2), 206 (100), 185 (30.3), 171 (20.3), 159 (27.4), 157 (22,5 M+ -C18H29O), 147 (41.4), 145 (38.4), 143 (23.8 M+ -C19H31O), 135 (38.9), 133 (37.2), 131 (30.1), 121 (20.4), 119 (32.6), 117 (35.8), 107 (29.8), 105 (48.4), 95 (36.7), 93 (41.2), 91 (49.8), 82 (31.0 M+ -C12H19O3 -C7H9O4), 81 (36.5), 79 (39.6 M+ -C2H4 -C16H25O3 -HCHO2), 73 (66.3).
Compound 6a: 1H NMR (CDCl3) δ (ppm): 8.03 (1H, s, HCO2-C3), 8.01 (1H, s, HCO2-C6), 5.30 (1H, dt, J = 11.9 and 5.6 Hz, H-6), 4.86–4.80 (1H, m, H-3), 2.66 (1H, dd, J = 4.4 and 4.4 Hz, H-23β), 2.63–2.61 (1H, m, H-22), 2.40–2.39 (2H, m, H-23α), 1.00 (3H, s, H-19), 0.95 (3H, d, J = 6.6 Hz, H-21), 0.64 (3H, s, H-18). 13C NMR (CDCl3) δ (ppm): (C1) 34.92, (C2) 26.38, (C3) 73.49, (C4) 44.76, (C5) 55.63, (C6) 70.81, (C7) 39.65, (C8) 34.65, (C9) 53.78, (C10) 36.14, (C11) 20.64, (C12) 39.97, (C13) 43.08, (C14) 57.05, (C15) 24.30, (C16) 26.89, (C17) 45.36, (C18) 12.15, (C19) 23.20, (C20) 39.51, (C21) 15.60, (C22) 56.05, (C23) 49.03, (HCO2-C3) 160.51, (HCO2-C6) 160.44.
Compound 6b: 1H NMR (CDCl3) δ (ppm): 8.03 (1H, s, HCO2-C3), 8.01 (1H, s, HCO2-C6), 5.30 (1H, dt, J = 11.9 and 5.6 Hz, H-6), 4.86–4.80 (1H, m, H-3), 2.80 (1H, dd, J = 4.3 and 4.3 Hz, H-23β), 2.73–2.71 (1H, m, H-22), 2.59–2.58 (1H, m, H-23α), 1.00 (3H, s, H-19), 0.95 (3H, d, J = 6.6 Hz, H-21), 0.64 (3H, s, H-18). 13C NMR (CDCl3) δ (ppm): (C1) 34.92, (C2) 26.13, (C3) 73.49, (C4) 31.23, (C5) 53.96, (C6) 70.81, (C7) 39.65, (C8) 34.65, (C9) 53.78, (C10) 36.14, (C11) 20.61, (C12) 39.94, (C13) 43.15, (C14) 57.31, (C15) 24.16, (C16) 27.18, (C17) 45.36, (C18) 12.10, (C19) 23.20, (C20) 39.51, (C21) 16.89, (C22) 55.67, (C23) 49.03, (HCO2-CC3) 160.51, (HCO2-C6) 160.44.

2.1.7. Synthesis of Mixture 22(S)-22,23-Epoxy-24-Nor-5β-Cholan-3α,6α-Diol (7a) and 22(R)-22,23-Epoxy-24-Nor-5β-Cholan-3α,6α-Diol (7b)

An aqueous solution of K2CO3 (5% w/w, 15 mL) was added to a methanolic mixture of 6a/6b (0.70 g, 1.92 mmol, 35 mL). Then, the suspension was stirred at reflux for 1 h. The end of the reaction was verified by TLC. The solvent was removed, and the dry residue was acidified with 2.5% w/w HCl (5 mL). The obtained solid was filtered and washed with 5% NaHCO3 (10 mL) and water (2 × 10 mL) and dried. Recrystallization (MeOH/Et2O = 2/1) of crude product made it possible to obtain epimeric mixture 7a/7b = 0.5/1.0 (0.564 g, 93% yield).
Mixture 7a/7b: IR νmax (cm−1): 3394 (O-H), 2937 (C-H), 2866 (C-H). MS m/z (%): M+: 362 (0.91), 344 (60.6), 329 (49.0), 326 (43.3), 311 (35.5), 272 (26.39), 271 (23.6), 255 (25.0), 253 (30.3), 246 (38.5 M+ -3 × CH3 -OH), 232 (26.5), 231 (66.2), 229 (22.1 M+ -3 × CH3 -2 × OH), 228 (40.8 M+ -3 × CH3 -OH -H2O), 227 (23.6), 215(23.1), 214 (32.2 M+ -2 × CH3 -C5H8O -H2O -OH) 213 (100), 211 (27.7), 199 (31.8), 187 (23, 3 M+ -3 × CH3 -2 × OH -C3H6), 185 (25.8), 173 (39.0), 172 (21.4), 171 (31.4), 161 (31.0 M+ -OH -H2O -C2H4 -C9H15O), 160 (22.0), 159 (45.3), 157 (36.4), 147 (49.4), 146 (22.6), 145 (56.3), 143 (29.0), 135 (30.1), 134 (20.2), 133 (46.6), 131 (46.3), 121 (36.3), 119 (46.1), 117 (20.7), 109 (28.8 M+ -C11H18O -C4H7O2), 108 (20.4), 107 (4.9), 105 (60.8), 95 (91.0 M+ -C16H27O3), 94 (28.3), 93 (64.8), 91 (53.9), 81 (70.1), 79 (65.5).
Compound 7a: 1H NMR (CDCl3) δ (ppm): 4.05 (1H, dt, J = 11.3 and 5.3 Hz, H-6), 3.66–3.60 (1H, m, H-3), 2.66 (1H, t, J = 4.5 Hz, H-23β), 2.62–2.61 (1H, m, H-22), 2.41–2.39 (1H, m, H-23α), 0.95 (3H, d, J = 6.5 Hz, H-21), 0.90 (3H, s, H-19), 0.64 (3H, s, H-18). 13C NMR (CDCl3) δ (ppm): (C1) 35.54, (C2) 26.94, (C3) 71.54, (C4) 30.22, (C5) 53.96, (C6) 68.00, (C7) 39.69, (C8) 34.84, (C9) 38.49, (C10) 35.95, (C11) 20.68, (C12) 39.88, (C13) 43.05, (C14) 57.10, (C15) 24.42, (C16) 29.16, (C17) 48.38, (C18) 12.11, (C19) 23.44, (C20) 39.61, (C21) 15.55, (C22) 56.05, (C23) 50.00.
Compound 7b: 1H NMR (CDCl3) δ (ppm): 4.05 (1H, dt, J = 11.3 and 5.3 Hz, H-6), 3.66–3.60 (1H, m, H-3), 2.79 (1H, dd, J = 4.3 and 4.3 Hz, H-23β), 2.74–2.72 (1H, m, H-22), 2.58–2.58 (1H, m, H-23α), 0.95 (3H, d, J = 6.5 Hz, H-21), 0.90 (3H, s, H-19), 0.64 (3H, s, H-18). 13C NMR (CDCl3) δ (ppm): (C1) 34.99, (C2) 27.23, (C3) 71.54, (C4) 30.22, (C5) 55.73, (C6) 68.00, (C7) 39.78, (C8) 34.84, (C9) 38.49, (C10) 35.95, (C11) 20.68, (C12) 39.88, (C13) 43.05, (C14) 57.41, (C15) 24.29, (C16) 29.19, (C17) 48.38, (C18) 12.16, (C19) 23.44, (C20) 39.61, (C21) 17.00, (C22) 55.76, (C23) 50.00.

2.2. Biological Activity

Epimeric mixtures. Stock solutions 1 × 10−5 M were prepared by dissolving 10 mg of epimeric mixtures in ethanol (1 mL) and then diluting with sterile water. Final concentrations of 1 × 10−6 M, 1 × 10−7 M and 1 × 10−8 M were obtained by adding different aliquots to Petri plates.

2.2.1. Rice Lamina Inclination Test (RLIT)

The growth-promoting activity of BR analogs and epimeric mixtures was evaluated using the rice lamina inclination test [8]. Rice seeds (Oryza sativa) of variety Zafiro, provided by the National Agricultural Research Institute, INIA, La Platina, Chile, were used in these bioassays. The seeds were washed and soaked with sterile distilled water for 24 h, then seeded on substrate + perlite + vermiculite (in the ratio 2:1:1) inside a greenhouse maintained at a 16 h day/8 h night (22 °C) cycle. Once the plants reached the ideal size to obtain the second internode of the rice lamina, segments of the leaf of approximately 6 cm were excised; six of these segments per treatment were incubated in 50 mL of sterile distilled water for 48 h in Petri plates containing different concentrations (1 × 10−8 M, 1 × 10−7 M and 1 × 10−6 M) of testing samples (epimeric mixtures: 4a/4b, 5a/5b, 6a/6b and 7a/7b). BL was used as a positive control (APE BIO, USA), whereas pure water was used as a negative control. After incubation for 48 h at 22 °C in the dark, the magnitude of the opening angle between the leaf and the sheath was measured with a protractor. Images were taken using a Leica EZ4HD stereo microscope with camera software. Measured angle values are given as the mean of twelve measurements with standard deviations (n = 12). This test was made twice for each treatment.

2.2.2. Effect of Epimeric Mixtures on Tomato Seeds Germination (Lycopersicum esculentum Mill)

Tomato seeds (variety Cal-Ace) were obtained from SEMILLAS MUSIC® (Santiago, Chile). Seeds were treated with sodium hypochlorite 0.05% (m/V) and washed three times with deionized water. After sterilization, seeds were soaked into ethanol/water solutions of epimeric mixtures (4a/4b, 5a/5b, 6a/6b and 7a/7b) and BL at different concentrations (1 × 10−8 M, 1 × 10−7 M y 1 × 10−6 M) for 24 h. A piece of humid towel paper was placed on top of each Petri plate. Three replicates of 25 previously soaked tomato seeds each were germinated in an incubator (BIOBASE, Shandong, China) under dark, at 22 °C and 50% HR (day/night). Seeds were continuously humidified to keep HR. The number of germinated seeds was recorded every day, considering as germinated all seeds with a visible radicle tip of at least 2 mm. Mean germination time (MT) was calculated as MT = ∑ n i   t i ∑ n i   , where ni is the number of germinated seeds at time ti. Mean germination rate (MR) is simply the reciprocal of MT. After 5 days, all germinated seeds were dried in an oven at 30 °C and dry weight was determined. Results were analyzed following a reported methodology [39].

2.2.3. Statistical Analysis

The data were reported as mean values ± standard deviation (SD). One-way ANOVA and post-hoc HSD Tukey tests were used with a confidence level of 0.95. The significant differences between the positive control and each compound were calculated in each measure (germination percentage, MT, MR, and dry weight). These statistical analyses were performed using Statistical 7.0 software (Stat Soft. Inc., Tulsa, OK, USA).

3. Results and Discussion

3.1. Chemical Synthesis

Epimeric mixtures of BRs analogs with 24-norcholane side chains and cis (5β) A/B ring fusion were obtained from hyodeoxycholic acid as shown in Figure 3. Hydroxyl groups in 3α and 6α were protected following a methodology previously reported for other bile acids [40]. Formylation of hyodeoxycholic acid (1) with a HCOOH/HClO4 system produced the diformylated derivative 2 with 95% yield. The presence of both formyl functions in compound 2 was confirmed by 1H-NMR and 13C-NMR spectroscopy (see Figure S1, Supplementary Materials). In the 1H NMR spectrum, signals observed at δH = 8.04 and 8.01 ppm were assigned to H atoms of formyl group (1H, s, HCO2-C3) and (1H, s, HCO2-C6), respectively; whereas, in the 13C NMR spectrum, signals observed at δC = 160.60 and 160.51 ppm were assigned to formyl carbonyls HCO2-C3 and HCO2-C6, respectively.
Compound 2 was submitted to oxidative decarboxylation of the side chain with Pb(OAc)4/Cu(OAc)2, leading to alkene 3 with 55% yield [37,41,42,43,44]. Formation of alkene 3, with a terminal double bond between C22–C23, was confirmed by 1H-NMR through the signals observed at δH = 5.67 (1H, m), 4.88 (1H, dd, J = 17.1 and 0.8 Hz) and 4.80 (1H, dd, J = 10.0 and 1.6 Hz) ppm, which were assigned to H atoms, H-22, H-23a and H-23b, respectively, and by signals observed at δC = 144.88 and 111.68 ppm in 13C-NMR spectrum, which were assigned to carbons C23 and C22, respectively (Figure S2, Supplementary Materials).
Alkene 3 was dihydroxylated by Upjohn reaction (step III, Figure 3) using a method described for terminal alkenes in steroidal nuclei. These studies have shown that this reaction gives a mixture of diastereomers with the 22S epimer as the major product [37,45,46,47,48,49]. In this work, this reaction produced the epimeric mixture of glycols 4a/4b with 28% yield. The presence of the glycol function in the epimeric mixture was established by 1H-NMR and 13C-NMR spectroscopy: signals at δH = 3.71 (1H, m), 3.58 (1H, dd, J = 11.3 and 2.4 Hz) and 3.38 (1H, dd, J = 11.3 and 8.9 Hz) ppm were assigned to the hydrogens H-22, H-23a and H-23b of both epimers 4a/4b, respectively, whereas signals at δC = 64.62 and 76.62 ppm were assigned to hydroxylated carbon C23 and C22, respectively (see Figure S3). The diastereomeric ratio of each glycol in the mixture was established as 4a:4b = 5.0:1.0, by integration of 1H-NMR signals assigned to the C21 methyl group, which appear at δH = 0.92 and 0.87 ppm in 4a and 4b epimers, respectively.
Subsequently, saponification of epimeric mixture 4a/4b produces the tetraol mixture 5a/5b with 89.9% yield. The diastereomeric ratio of each tetraol in the mixture was 5a:5b = 7.0:1.0 (Figure S4, Supplementary Materials).
It has been reported that the epoxidation reaction on steroidal nuclei of similar structure and carrying terminal double bonds C22/C23 mainly produces epoxide 22R [45,46,47,49]. Epoxidation of alkene 3 with m-CPBA (step V, Figure 3) leads to a mixture of epimeric epoxides 6a/6b in the ratio 0.39:1.0, with 94% yield. Thus, our results are in line with previous work, and the amount of epoxide 22R is twofold larger than that of 22S. This proportion was calculated using the integrals of signals assigned to hydrogens H-22 (δ = 2.73 ppm), H-23α (δ = 2.58 ppm) and H-23β (δ = 2.80 ppm) of compound 6b (22R), and H-22 (δ = 2.62 ppm), H-23α (δ = 2.40 ppm) and H-23β (δ = 2.66 ppm) of compound 6a (22S) (see Figure S5, Supplementary Materials). For the determination of the majority epoxide, experimental and NMR comparisons spectroscopic data correlations were used. Finally, saponification of epoxides 6a/6b produces 7a/7b with 93% yield (step IV, Figure 3). The ratio of 7a/7b epimers in the mixture was calculated as 7a/7b = 0.5/1, following the same procedure described for the mixture 6a/6b. (Figure S6, Supplementary Materials)
In summary, the synthetic strategy outlined in Figure 3 allows obtaining BRs with 24-norcholane side chains and cis (5β) A/B ring fusion with good yields. Hyodeoxycholic acid was the starting material because it is a very common and accessible material that has been used in the synthesis of various BR analogs [8]. Glycol and epoxide analogs with hydroxyl or formyl groups in positions 3α and 6α were obtained as epimeric mixtures, with the diastereoisomer 22S as the major component in glycol mixtures 4a/4b and 5a/5b, whereas the opposite is obtained in epoxide mixtures 6a/6b and 7a/7b. Thus, these mixtures were used to assess the effect of different functional groups and the configuration in C22 on the bioactivity of these BR analogs.

3.2. Bioactivity of Epimeric Mixtures of Brassinosteroids

3.2.1. Rice Lamina Inclination Assay

It is well known that growth-promoting activity is one of the main features of natural BRs [50,51]. A number of different bioassays have been developed—first bean internode, root growth, and rice lamina inclination—and the obtained results have been used to establish structure–activity relationships [28,30,52]. Rice Lamina Inclination Test (RLIT) is the most widely used test due to its high specificity and sensitivity, that make it possible to detect BL concentrations as low as 0.05 ng/L [8,53]. Thus, this test has been used to determine the growth-promoting activity of epimeric mixtures using BL as positive control. The measured bending angles are listed in Table 1 as the mean ± standard deviation of two independent experiments with at least six replicates each (n = 12) for each tested concentration.
The results indicate that BL is the most active compound at all tested concentrations. However, the growth-promoting activity of epimeric mixtures is closer to that measured for BL, especially at the lowest tested concentrations. This is an interesting result because it has been shown that, in plants, BRs act mainly in this range of concentrations.
Structure–activity relationships have established that BR bioactivity is associated to the presence of some functional groups, the length of the side alkyl chain, and the configuration of the C22 atom [28,29,30,31]. Our results suggest that the presence of hydroxyl groups in the mixtures 5a/5b and 7a/7b makes no significant difference in the exhibited growth-promoting activity as compared with the mixtures where these groups were formylated. Similar conclusions can be drawn from comparison of mixtures where the hydroxyl groups in the chain were changed by an epoxide group. Besides, the greatest activity has also been attributed to BR analogs having R configurations in the C22 atom of the side alkyl chain. This effect is not apparent in these mixtures because changing the ratio of diastereoisomers has no effect on bioactivity. Finally, regarding the configuration at the A/B junction, it is worth mentioning that there are some controversial reports regarding the importance of this structural factor. As BR analogs with A/B cis junctions are not found in nature, it was assumed that this is a structural requirement for BR bioactivity. Besides, it was shown that the BL epimer with an A/B cis junction is three orders of magnitude less active in the RLIT assay than BL [54]. However, Brosa et al. have shown that BR analogs with non-natural A/B cis junctions exhibit good bioactivity [30]. Thus, our results corroborate that the A/B junction has almost no effect on bioactivity and support the idea that it depends more on the molecular spatial distribution than on the presence or absence of one specific functional group in the molecule [30,38].

3.2.2. Effect of Epimeric Mixtures on Germination of Tomato Seeds (Lycopersicum esculentum Mill.)

To assess the effect of epimeric mixtures on germination, various parameters were determined; namely, germination percentage, mean germination time (MT), mean germination rate (MR) and dry weight of the germinated plant. This study was carried out with mixtures 4a/4b, 5a/5b and BL, used as positive controls, in the concentration range 1 × 10−6–1 × 10−8 M.
The mean time taken for seed germination was calculated following the method reported by Ranal et al. [39], which requires recording the number of germinated seeds each day. At the end of germination test, seeds with normal seedlings were counted and the percentage of germination was determined for all treatments (see Figure 4).
The seedlings were then cut (considering radicle growing and some leaf primordia) and the dry weights were also determined.
The values of germination percentage and dry weight obtained for both mixtures and BL are given in Table 2.
The results indicate that all treatments produce higher germination percentages than the negative control (only added water). Besides, both mixtures were as efficient as BL in promoting tomato seed germination at all tested concentrations. In fact, there were no significant differences in the germination percentages measured for all of them, especially at the lowest tested concentration.
On the other hand, the values of dry weights clearly indicate that there are no significant differences between treatments and the negative control.
However, the data indicate that, at the lowest concentration, the largest dry weights are obtained with the epimeric mixture 4a/4b. It is worth mentioning that the radicle is longer in seedlings of seeds treated with epimeric mixtures and BL than in the negative control. These results agree with those reported for 24-epibrassinolide and have been attributed to the growth-promoting effect of BRs [14].
Finally, the MT values obtained for all treatments at different concentrations are shown in Figure 5.
The data indicate that, at the lowest tested concentration (1 × 10−8 M), all treatments accelerate the germination process and consequently decrease MT. Seeds of the negative control took a mean time of 4.5 days to germinate, whereas, in the presence of epimeric mixtures and BL, this time is reduced, becoming 3.5 days in presence of mixture 4a/4b. The magnitude of this effect follows the order 4a/4b > 5a/5b > BL. The values of MR are in the range 0.29 to 1.29 (4a/4b and negative control, respectively) and follow the same pattern as MT.
The same tendency has been found in preliminary experiments carried out in the presence of copper. This study aimed to assess the alleviating effect of these mixtures on abiotic stress produced by heavy metals, and is in progress.

4. Conclusions

In conclusion, our results suggest that the epimeric mixture 4a/4b exhibits growth-promotion activity similar to BL, and a higher germination effect than that measured for BL and other mixtures. Considering that all tested BR analogs have the non-natural cis configuration at the A/B junction, our results confirm that this junction has no effect on the bioactivity of BR analogs. In addition, the possibility of using epimeric mixtures instead of pure compounds, maintaining a good level of activity, makes this approach even more interesting for potential application. Purification and chromatographic separations are not needed and, therefore, the synthesis is shorter, cheaper and with higher yield.

Supplementary Materials

The following are available online at https://www.mdpi.com/2073-4395/10/6/808/s1, Figure S1: NMR spectra of 3α,6α-diformyloxy-5β-cholan-24-oic acid (2); Figure S2: NMR spectra of 5β-cholan-24-nor-22-en-3α,6α-diyl diformate (3); Figure S3: NMR spectra of mixture: 22(S), 23-dihydroxy-24-nor-5β-cholan-3α,6α-diyl diformate (4a) and 22(R), 23-dihydroxy-24-nor-5β-cholan-3α,6α-diyl diformate (4b); Figure S4: NMR spectra of mixture: 3α,6α, 22(S), 23-tetrahydroxy-24-nor-5β-cholan (5a) and 3α,6α, 22(R), 23-tetrahydroxy-24-nor-5β-cholan (5b); Figure S5: NMR spectra of mixture: 22(S)-22,23-epoxy-24-nor-5β-cholan-3α,6α-diyl diformate (6a); 22(R)-22,23-epoxy-24-nor-5β-cholan-3α,6α-diyl diformate (6b); Figure S6: NMR spectra of mixture: 22(S)-22,23-epoxy-24-nor-5β-cholan-3α,6α-diol (7a) and 22(R)-22,23-epoxy-24-nor-5β-cholan-3α,6α-diol (7b).

Author Contributions

Conceptualization, K.D. and L.E., methodology, N.S., C.G. and M.M.; software, M.M. and Y.C.; investigation, N.S., C.G. and K.D.; writing-original draft preparation, N.S., K.D., L.E.; writing-review and editing, A.F.O., K.D., L.E. and Y.C.; supervision, K.D. and L.E.; funding acquisition, project administration, L.E. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by FONDECYT (Fondo Nacional de Desarrollo Científico y Tecnológico) (grant N° 1160446 and N° 1191330).

Acknowledgments

N.S. thanks the support of Dirección General de Investigación y Postgrado (DGIP-USM) of Universidad Técnica Federico Santa María.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Grove, M.D.; Spencer, G.F.; Rohwedder, W.K.; Mandava, N.; Worley, J.F.; Warthen, J.D.; Steffens, G.L.; Flippenanderson, J.L.; Cook, J.C.; Brassinolide, A. Plant growth-promoting steroid isolated from brassica-napus pollen. Nature 1979, 281, 216–217. [Google Scholar] [CrossRef]
  2. Bajguz, A. Brassinosteroids-occurence and chemical structures in plants. In Brassinosteroids: A Class of Plant Hormone; Hayat, S., Ahmad, A., Eds.; Springer: Dordrecht, The Netherlands, 2016; pp. 1–27. [Google Scholar]
  3. Fujioka, S. Natural occurrence of brassinosteroids in the plant kingdom. In Brassinosteroids: Steroidal Plant Hormones; Sakurai, A., Yokota, T., Clouse, S.D., Eds.; Springer: Tokyo, Japan, 1999; pp. 21–45. [Google Scholar]
  4. Hayat, S.; Ahmad, A.; Mobin, M.; Hussain, A.; Fariduddin, Q. Photosynthetic rate, growth, and yield of mustard plants sprayed with 28-homobrassinolide. Photosynthetica 2001, 38, 469–471. [Google Scholar] [CrossRef]
  5. Choudhary, S.P.; Yu, J.-Q.; Yamaguchi-Shinozaki, K.; Shinozaki, K.; Tran, L.-S.P. Benefits of brassinosteroid crosstalk. Trends Plant Sci. 2012, 17, 594–605. [Google Scholar] [CrossRef] [PubMed]
  6. Vardhini, V.; Anjum, N. Brassinosteroids make plant life easier under abiotic stresses mainly by modulating major components of antioxidant defense system. Front. Environ. Sci. 2014, 2, 67. [Google Scholar] [CrossRef] [Green Version]
  7. Oklestkova, J.; Rarova, L.; Kvasnica, M.; Strnad, M. Brassinosteroids: Synthesis and biological activities. Phytochem. Rev. 2015, 14, 1053–1072. [Google Scholar] [CrossRef]
  8. Duran, M.I.; Gonzalez, C.; Acosta, A.; Olea, A.F.; Diaz, K.; Espinoza, L. Synthesis of five known brassinosteroid analogs from hyodeoxycholic acid and their activities as plant-growth regulators. Int. J. Mol. Sci. 2017, 18, 516. [Google Scholar] [CrossRef] [Green Version]
  9. Gomes, M.M.A. Physiological effects related to brassinosteroid application in plants. In Brassinosteroids: A Class of Plant Hormone; Hayat, S., Ahmad, A., Eds.; Springer: Dordrecht, The Netherlands, 2011; pp. 193–242. [Google Scholar]
  10. Leubner-Metzger, G. Brassinosteroids promote seed germination. In Brassinosteroids; Hayat, S., Ahmad, A., Eds.; Kluwer Academic Publisher: Amsterdam, The Netherlands, 2003. [Google Scholar]
  11. Leubner, G. Brassinosteroids Promote Seed Germination; Springer: Berlin/Heidelberg, Germany, 2003; pp. 119–128. [Google Scholar]
  12. Ali, B.; Hayat, S.; Ahmad, A. Response of germinating seeds of cicer arietinum to 28-homobrassinolide and/or potassium. Gen. Appl. Plant Physiol. 2005, 31, 55–63. [Google Scholar]
  13. Hayat, S.; Ahmad, A. 28-Homobrassinolide induced changes favoured germinability of wheat grains. Bulg. J. Plant Physiol. 2003, 29, 55–62. [Google Scholar]
  14. Sharma, P.; Bhardwaj, R. Effect of 24-epibrassinolide on seed germination, seedling growth and heavy metal uptake in Brassica junceae L. Gen. Appl. Plant Physiol. 2007, 19, 203–210. [Google Scholar]
  15. Li, K.-R.; Zhang, W.-B.; Li, H.-K. Effect of natural brassinolide on germination of Ailanthus altissima seeds. For. Stud. China 2005, 7, 12–14. [Google Scholar] [CrossRef]
  16. Li, K.; Zhang, S.; He, X. Effect of natural brassinolide on germination of Pinus tabulaeformis and Robinia pseudoacacia seeds. Sci. Silvae Sin. 2002, 38, 150–153. [Google Scholar]
  17. Özdemir, F.; Bor, M.; Demiral, T.; Türkan, İ. Effects of 24-epibrassinolide on seed germination, seedling growth, lipid peroxidation, proline content and antioxidative system of rice (Oryza sativa L.) under salinity stress. Plant Growth Regul. 2004, 42, 203–211. [Google Scholar]
  18. Yuan, L.Y.; Yuan, Y.H.; Du, J.; Sun, J.; Guo, S.R. Effects of 24-epibrassinolide on nitrogen metabolism in cucumber seedlings under Ca(NO3)(2) stress. Plant Physiol. Biochem. 2012, 61, 29–35. [Google Scholar] [CrossRef] [PubMed]
  19. Liu, J.; Gao, H.; Wang, X.; Zheng, Q.; Wang, C.; Wang, X.; Wang, Q. Effects of 24-epibrassinolide on plant growth, osmotic regulation and ion homeostasis of salt-stressed canola. Plant Biol. 2014, 16, 440–450. [Google Scholar] [CrossRef]
  20. Shu, S.; Tang, Y.Y.; Yuan, Y.H.; Sun, J.; Zhong, M.; Guo, S.R. The role of 24-epibrassinolide in the regulation of photosynthetic characteristics and nitrogen metabolism of tomato seedlings under a combined low temperature and weak light stress. Plant Physiol. Biochem. 2016, 107, 344–353. [Google Scholar] [CrossRef]
  21. Sharma, A.; Kumar, V.; Kumar, R.; Shahzad, B.; Thukral, A.K.; Bhardwaj, R. Brassinosteroid-mediated pesticide detoxification in plants: A mini-review. Cogent Food Agric. 2018, 4, 1436212. [Google Scholar] [CrossRef]
  22. Vardhini, B.V. Chapter 8-brassinosteroids are potential ameliorators of heavy metal stresses in plants. In Plant Metal Interaction; Ahmad, P., Ed.; Elsevier: Amsterdam, The Netherlands, 2016; pp. 209–237. [Google Scholar]
  23. Ali, B. Practical applications of brassinosteroids in horticulture—Some field perspectives. Sci. Hortic. 2017, 225, 15–21. [Google Scholar] [CrossRef]
  24. Ghorbani, P.; Eshghi, S.; Haghi, H. Effects of brassinosteroid (24-epibrassinolide) on yield and quality of grape (Vitis vinifera L.) ’thompson seedless’. Vitis 2017, 56, 113–117. [Google Scholar]
  25. Steber, C.M.; McCourt, P. A role for brassinosteroids in germination in arabidopsis. Plant Physiol. 2001, 125, 763–769. [Google Scholar] [CrossRef] [Green Version]
  26. Bhardwaj, R.; Sharma, I.; Kapoor, D.; Poonam; Gautam, V.; Kaur, R.; Bali, S.; Sharma, A. Brassinosteroids: improving crop productivity and abiotic stress tolerance. In Physiological Mechanisms and Adaptation Strategies in Plants Under Changing Environment; Ahmad, A., Wani, M.R., Eds.; Springer Science Business Media: New York, NY, USA, 2014; Volume 2. [Google Scholar]
  27. Barbafieri, M.; Tassi, E. Brassinosteroids for phytoremediation application. In Brassinosteroids: A Class of Plant Hormone; Hayat, S., Ahmad, A., Eds.; Springer: Dordrecht, The Netherlands, 2011; pp. 403–437. [Google Scholar]
  28. Liu, J.; Zhang, D.; Sun, X.; Ding, T.; Lei, B.; Zhang, C. Structure-activity relationship of brassinosteroids and their agricultural practical usages. Steroids 2017, 124, 1–17. [Google Scholar] [CrossRef]
  29. Takatsuto, S.; Ikekawa, N.; Morishita, T.; Abe, H. Structure activity relationship of brassinosteroids with respect to the A/B-ring functional-groups. Chem. Pharm. Bull. 1987, 35, 211–216. [Google Scholar] [CrossRef] [Green Version]
  30. Brosa, C.; Capdevila, J.M.; Zamora, I. Brassinosteroids: A new way to define the structural requirements. Tetrahedron 1996, 52, 2435–2448. [Google Scholar] [CrossRef]
  31. Back, T.; Pharis, R. Structure-activity studies of brassinosteroids and the search for novel analogues and mimetics with improved bioactivity. J. Plant Growth Regul. 2004, 22, 350–361. [Google Scholar] [CrossRef] [PubMed]
  32. Ferrer-Pertuz, K.; Espinoza, L.; Mella, J. Insights into the structural requirements of potent brassinosteroids as vegetable growth promoters using second-internode elongation as biological activity: CoMFA and CoMSIA Studies. Int. J. Mol. Sci. 2017, 18, 2734. [Google Scholar] [CrossRef] [Green Version]
  33. Černý, V.; Strnad, M.; Kamínek, M. Preparation of 2α,3α-dihydroxy-7-oxa-6-oxo-23,24-dinor-B-homo-5α-cholanic acid, its esters and amides as brassinolide analogues. Collect. Czech. Chem. Commun. 1986, 51, 687–697. [Google Scholar] [CrossRef]
  34. Uesusuki, S.; Watanabe, B.; Yamamoto, S.; Otsuki, J.; Nakagawa, Y.; Miyagawa, H. Synthesis of brassinosteroids of varying acyl side chains and evaluation of their brassinolide-like activity. Biosci. Biotechnol. Biochem. 2004, 68, 1097–1105. [Google Scholar] [CrossRef] [Green Version]
  35. Šíša, M.; Hniličková, J.; Swaczynová, J.; Kohout, L. Syntheses of new androstane brassinosteroids with 17β-ester groups-butyrates, heptafluorobutyrates, and laurates. Steroids 2005, 70, 755–762. [Google Scholar] [CrossRef]
  36. Kvasnica, M.; Oklestkova, J.; Bazgier, V.; Rárová, L.; Korinkova, P.; Mikulík, J.; Budesinsky, M.; Béres, T.; Berka, K.; Lu, Q.; et al. Design, synthesis and biological activities of new brassinosteroid analogues with a phenyl group in the side chain. Org. Biomol. Chem. 2016, 14, 8691–8701. [Google Scholar] [CrossRef]
  37. Carvajal, R.; Gonzalez, C.; Olea, A.F.; Fuentealba, M.; Espinoza, L. Synthesis of 2-deoxybrassinosteroids analogs with 24-nor, 22(S)-23-dihydroxy-type side chains from hyodeoxycholic acid. Molecules 2018, 23, 1306. [Google Scholar] [CrossRef] [Green Version]
  38. Diaz, K.; Espinoza, L.; Carvajal, R.; Conde-Gonzalez, M.; Niebla, V.; Olea, A.F.; Coll, Y. Biological activities and molecular docking of brassinosteroids 24-norcholane type analogs. Int. J. Mol. Sci. 2020, 21, 1832. [Google Scholar] [CrossRef] [Green Version]
  39. Ranal, M.A.; Santana, D.G.D.; Ferreira, W.R.; Mendes-Rodrigues, C. Calculating germination measurements and organizing spreadsheets. Braz. J. Bot. 2009, 32, 849–855. [Google Scholar] [CrossRef] [Green Version]
  40. Schteingart, C.D.; Hofmann, A.F. Synthesis of 24-nor-5 beta-cholan-23-oic acid derivatives: A convenient and efficient one-carbon degradation of the side chain of natural bile acids. J. Lipid. Res. 1988, 29, 1387–1395. [Google Scholar] [PubMed]
  41. Bacha, J.D.; Kochi, J.K. Alkenes from acids by oxidative decarboxylation. Tetrahedron 1968, 24, 2215–2216. [Google Scholar] [CrossRef]
  42. Vaidya, A.S.; Dixit, S.M.; Rao, A.S. Degradation of bile acid side chain with lead tetraacetate. Tetrahedron Lett. 1968, 50, 5173–5174. [Google Scholar] [CrossRef]
  43. Zhou, W.S.; Tian, W.S. Studies on steroidal plant-growth hormones. 2. stereoselective synthesis of (22S, 23S)-typhasterol from hyodeoxycholic acid. Acta Chim. Sin. 1985, 43, 1060–1067. [Google Scholar]
  44. Zhou, W.S.; Jiang, B.; Pan, X.F. Stereoselective synthesis of the brassinolide side-chain-novel syntheses of brassinolide and related-compounds. Tetrahedron 1990, 46, 3173–3188. [Google Scholar] [CrossRef]
  45. Ishiguro, M.; Kajikawa, A.; Haruyama, T.; Ogura, Y.; Okubayashi, M.; Morisaki, M.; Ikekawa, N. Synthetic studies of withanolides. Part 1. Synthesis of 5,6beta-epoxy-4beta-hydroxy-5beta-cholest-2-en-1-one and related compounds. J. Chem. Soc. Perkin Trans. 1 1975, 22, 2295–2302. [Google Scholar] [CrossRef]
  46. Espinoza, L.; Cortes, M. Synthesis and biological activities of two new brassinosteroids functionalized in ring C. Bol. Soc. Chil. Quim. 2002, 47, 335–347. [Google Scholar]
  47. Espinoza, L.; Cortes, M. Synthesis and biological activity of brassinosteroids analogues. Bol. Soc. Chil. Quim. 2002, 47, 511–516. [Google Scholar]
  48. Espinoza, L. Synthesis of four new brassinosteroids analogues 11-Oxo-functionalized on C Ring, with 24-nor side chain and containing 5â-cholanic acid skeleton. Org. Chem.Curr.Res. 2015, 4, 1–6. [Google Scholar]
  49. Oyarce, J.; Aitken, V.; Gonzalez, C.; Ferrer, K.; Olea, A.F.; Parella, T.; Espinoza, L. Synthesis and structural determination of new brassinosteroid 24-nor-5α-cholane type analogs. Molecules 2019, 24, 4612. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  50. Clouse, S.D.; Langford, M.; McMorris, T.C. A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development. Plant Physiol. 1996, 111, 671–678. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Khripach, V.; Zhabinskii, V.; de Groot, A. Twenty years of brassinosteroids: Steroidal plant hormones warrant better crops for the XXI century. Ann. Bot. 2000, 86, 441–447. [Google Scholar] [CrossRef] [Green Version]
  52. Takatsuto, S.; Yazawa, N.; Ikekawa, N.; Takematsu, T.; Takeuchi, Y.; Koguchi, M. Structure activity relationship of brassinosteroids. Phytochem. 1983, 22, 2437–2441. [Google Scholar] [CrossRef]
  53. Wada, K.; Marumo, S.; Ikekawa, N.; Morisaki, M.; Mori, K. Brassinolide and homobrassinolide promotion of lamina inclination of rice seedlings. Plant Cell Physiol. 1981, 22, 323–325. [Google Scholar]
  54. Seto, H.; Fujioka, S.; Koshino, H.; Suenaga, T.; Yoshida, S.; Watanabe, T.; Takatsuto, S. Epimerization at C-5 of brassinolide with sodium methoxide and the biological activity of 5-epi-brassinolide in the rice lamina inclination test. J. Chem. Soc. Perkin Trans. 1 1998, 20, 3355–3358. [Google Scholar] [CrossRef]
Agronomy 10 00808 g001 550
Figure 1. Structure of natural occurring brassinosteroids.
Figure 1. Structure of natural occurring brassinosteroids.
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Agronomy 10 00808 g002 550
Figure 2. Epimeric mixtures (diastereomer compounds with S and R stereochemistry at position C-22) of brassinosteroid analogs with 24-norcholane side chains and cis (5β) A/B ring fusion.
Figure 2. Epimeric mixtures (diastereomer compounds with S and R stereochemistry at position C-22) of brassinosteroid analogs with 24-norcholane side chains and cis (5β) A/B ring fusion.
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Agronomy 10 00808 g003 550
Figure 3. Obtaining epimeric mixtures 4a/4b, 5a/5b, 6a/6b and 7a/7b from hyodeoxycholic acid (1). Conditions. I. HCOOH/H2SO4 (Reflux); II. Pb(OAc)4, Cu(OAc)2/C6H6 (Reflux); III. OsO4, NMO/(CH3)2CO/H2O; IV. K2CO3/MeOH (Reflux), HCl 2%; V. m-CPBA/CH2Cl2/NaHCO3.
Figure 3. Obtaining epimeric mixtures 4a/4b, 5a/5b, 6a/6b and 7a/7b from hyodeoxycholic acid (1). Conditions. I. HCOOH/H2SO4 (Reflux); II. Pb(OAc)4, Cu(OAc)2/C6H6 (Reflux); III. OsO4, NMO/(CH3)2CO/H2O; IV. K2CO3/MeOH (Reflux), HCl 2%; V. m-CPBA/CH2Cl2/NaHCO3.
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Agronomy 10 00808 g004 550
Figure 4. Germination of tomato seeds after 5 days of application: (a) negative control; (b) positive control (BL); (c) epimeric mixture 4a/4b. BR concentration was 1 × 10−8 M.
Figure 4. Germination of tomato seeds after 5 days of application: (a) negative control; (b) positive control (BL); (c) epimeric mixture 4a/4b. BR concentration was 1 × 10−8 M.
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Agronomy 10 00808 g005 550
Figure 5. Effect of epimeric mixtures (4a/4b and 5a/5b) and BL on the mean germination time of tomato seeds after five days of germination. * Indicates the most significant different values as compared to the negative control.
Figure 5. Effect of epimeric mixtures (4a/4b and 5a/5b) and BL on the mean germination time of tomato seeds after five days of germination. * Indicates the most significant different values as compared to the negative control.
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Table
Table 1. Effect of epimeric mixtures 4a/4b, 5a/5b, 6a/6b and 7a/7b on the lamina inclination of rice seedlings. BL was used as the positive control.
Table 1. Effect of epimeric mixtures 4a/4b, 5a/5b, 6a/6b and 7a/7b on the lamina inclination of rice seedlings. BL was used as the positive control.
Bending Angle between Lamina and Sheaths (Degrees ± Standard Error)
Epimeric MixturesStructureConcentration (M)
1 × 10−81 × 10−71 × 10−6
4a/4b 5.0:1.0 Agronomy 10 00808 i001 Agronomy 10 00808 i002
21 ± 2.7		 Agronomy 10 00808 i003
26 ± 2.7		 Agronomy 10 00808 i004
30 ± 4.5
5a/5b 7.0:1.0 Agronomy 10 00808 i005 Agronomy 10 00808 i006
8 ± 3.5		 Agronomy 10 00808 i007
14 ± 2.2		 Agronomy 10 00808 i008
44 ± 4.2
6a/6b 0.39:1.0 Agronomy 10 00808 i009 Agronomy 10 00808 i010
20 ± 1.6		 Agronomy 10 00808 i011
20 ± 0.8		 Agronomy 10 00808 i012
21 ± 1.0
7a/7b 0.50:1.0 Agronomy 10 00808 i013 Agronomy 10 00808 i014
17 ± 2.4		 Agronomy 10 00808 i015
30 ± 1.2		 Agronomy 10 00808 i016
28 ± 2.9
BL (C+) Agronomy 10 00808 i017 Agronomy 10 00808 i018
31 ± 1.0		 Agronomy 10 00808 i019
41 ± 4.5		 Agronomy 10 00808 i020
70 ± 7.6
Water (C-) Agronomy 10 00808 i021
7 ± 4.5
Table
Table 2. Effect of epimeric mixtures 4a/4b and 5a/5b on the germination percentage of tomato seeds and dry weight of seedlings after 5 days of treatment.
Table 2. Effect of epimeric mixtures 4a/4b and 5a/5b on the germination percentage of tomato seeds and dry weight of seedlings after 5 days of treatment.
Germination, %
Dry Weight, mg
Concentration, M
Treatment1 × 10−81 × 10−71 × 10−61 × 10−81 × 10−71 × 10−6
BL98 ± 3.5100 ± 0.0100 ± 0.040.9 ± 4.949.9 ± 5.044.4 ± 2.5
4a/4b100 ± 0.0100 ± 0.0100 ± 0.045.2 ± 2.144.3 ± 3.640.1 ± 4.2
5a/5b100 ± 0.095 ± 7.198 ± 3.543.6 ± 1.939.3 ± 3.946.7 ± 4.5
H2O (C-) 90 ± 0.0 39.1 ± 4.0

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Soto, N.; González, C.; Mellado, M.; Olea, A.F.; Coll, Y.; Díaz, K.; Espinoza, L. Epimeric Mixtures of Brassinosteroid Analogs: Synthesis, Plant Growth, and Germination Effects in Tomato (Lycopersicum esculentum Mill.). Agronomy 2020, 10, 808. https://doi.org/10.3390/agronomy10060808

AMA Style

Soto N, González C, Mellado M, Olea AF, Coll Y, Díaz K, Espinoza L. Epimeric Mixtures of Brassinosteroid Analogs: Synthesis, Plant Growth, and Germination Effects in Tomato (Lycopersicum esculentum Mill.). Agronomy. 2020; 10(6):808. https://doi.org/10.3390/agronomy10060808

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Soto, Nitza, César González, Marco Mellado, Andrés F. Olea, Yamilet Coll, Katy Díaz, and Luis Espinoza. 2020. "Epimeric Mixtures of Brassinosteroid Analogs: Synthesis, Plant Growth, and Germination Effects in Tomato (Lycopersicum esculentum Mill.)" Agronomy 10, no. 6: 808. https://doi.org/10.3390/agronomy10060808

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