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

: 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 e ﬀ ect 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.


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 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' 24nor-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. 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.

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. 1 H, 13 C, 13 C 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 1 H and 13 C, respectively, and CD3OD at δ = 3.30 ppm and δ = 49.00 ppm for 1 H and 13 C, respectively, on an Avance 400 Digital NMR spectrometer (Bruker, Rheinstetten, Germany) operating at 400.1 MHz for 1 H and 100.6 MHz for 13 C. 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 EtOAchexane gradients of increasing polarity. All organic extracts were dried over anhydrous magnesium sulfate and evaporated under reduced pressure below 40 °C.

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. 1 H, 13 C, 13 C DEPT-135, gs 2D HSQC, and gs 2D HMBC NMR spectra were recorded in CDCl 3 or MeOD solutions, and are referenced to the residual peaks of CHCl 3 at δ = 7.26 ppm and δ = 77.00 ppm for 1 H and 13 C, respectively, and CD 3 OD at δ = 3.30 ppm and δ = 49.00 ppm for 1 H and 13 C, respectively, on an Avance 400 Digital NMR spectrometer (Bruker, Rheinstetten, Germany) operating at 400.1 MHz for 1 H and 100.6 MHz for 13 C. 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 CHCl 3 , CH 3 OH 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% H 2 SO 4 in H 2 O. 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 NaHCO 3 (25 mL, saturated solution) and water (50 mL). The neutralized solid was dried in vacuo and subsequently dissolved in Ac 2 O (30 mL), dried over MgSO 4 , 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  (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. An aqueous solution of K 2 CO 3 (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% NaHCO 3 (10 mL) and water (2 × 10 mL) and dried. Recrystallization (MeOH/Et 2 O = 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 ν max (cm m-chloroperoxybenzoic acid (mCPBA) (77%, 0.21 g, 1.24 mmol) and NaHCO 3 (2.5 mg, 0.029 mmol) were added to a solution of alkene 3 (0.5 g, 1.24 mmol) in CH 2 Cl 2 (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 NaHCO 3 (2 × 10 mL) and water (3 × 10 mL), dried over MgSO 4 , and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in DCM (10 mL) and chromatographed on silica gel

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.

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 n i is the number of germinated seeds at time t i . 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].

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).
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 1 H-NMR and 13 C-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 1 H-NMR signals assigned to the C21 methyl group, which appear at δH = 0.92 and 0.87 ppm in 4a and 4b epimers, respectively.
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 1 H-NMR and 13 C-NMR spectroscopy: signals at δ H = 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 δ C = 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 1 H-NMR signals assigned to the C21 methyl group, which appear at δ H = 0.92 and 0.87 ppm in 4a and 4b epimers, respectively.
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.

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]. 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. 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.

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. 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. 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.

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. 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. 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.

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. 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. 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.

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. 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. configuration in C22 on the bioactivity of these BR analogs.

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. configuration in C22 on the bioactivity of these BR analogs.

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. configuration in C22 on the bioactivity of these BR analogs.

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. configuration in C22 on the bioactivity of these BR analogs.

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. configuration in C22 on the bioactivity of these BR analogs.

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. configuration in C22 on the bioactivity of these BR analogs.

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. 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. configuration in C22 on the bioactivity of these BR analogs.

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. 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. configuration in C22 on the bioactivity of these BR analogs.

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. 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. 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.

Bending Angle between Lamina and Sheaths (Degrees ± Standard Error
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 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 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 30  Water (C-)

± 4.5
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 28 ± 2.9

± 4.5
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 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 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 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 70 ± 7.6

± 4.5
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 7 ± 4.5

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 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.

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.
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.

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.