Chiral hydroxyl can enhance the biological activity of chiral pesticides, such as uniconazole, diniconazole, and tebuconazole. If a chiral hydroxyl group is assembled in the precursor skeleton molecule of L-pyroglutamate, the bactericidal activity of the molecule can be enhanced without affecting other functional groups. As shown in Scheme 1 , two methods exist to construct the 4S-hydroxy-L-pyroglutamate ester framework. In pathway A, the chiral hydroxyl was induced to the alpha position of the carbonyl group in L-pyroglutamic acid, as starting material via oxidation hydroxylation (Chen et al., 2022; Negi et al., 2022). However, chiral hydroxyl groups have yet to be successfully constructed after many asymmetric oxidation methods have been tried. On the other side of the strategy, the original idea may be realized by preassembling chiral hydroxyl groups onto pyrrole rings of L-hydroxyproline and then oxidative carbonylation of active methylene.

Based on the design idea, L-hydroxyproline was used as the starting material to oxidize carbonyl at the active methylene position. First, the three active functional groups of the reaction starting material must be protected; otherwise, side reactions are easy to occur. As shown in Scheme 2 , the three functional groups are successively protected by N-Boc amide, OTBS silicon ether, and p-chlorophenyl formate to obtain the critical intermediate C04. Notably, 4-chlorophenol was introduced to increase the antibacterial activity of our derivatives, inspired by Zhang’s work.

With compound C04 in hand, the key carbonyl assembly will be investigated. As shown in Table 1 , we tried to perform oxidative carbonylation via TBHP (Hossain and Shyu, 2016), DIB/TBHP (Zhao et al., 2013), and KMnO₄ (Lai and Lee, 2002). However, we did not get the desired products with the three oxidants (entries 1~3). The subject fell into the darkest, and we searched everywhere for appropriate oxidation methods. Fortunately, Yoshifuji reported a two-phase oxidation reaction using RuO₂ to convert cyclic A-amino acids to A-amino dicarboxylic acids in 1995 (Yoshifuji and Kaname, 1995). In 2001, Zhang successfully oxidized 4-hydroxyproline methyl ester into 4-hydroxypyroglutamate methyl ester using this method (Zhang et al., 2001). This method is intended to be used for oxidative carbonylation. Encouragingly, compound C04 was oxidized by RuO₂·H₂O in the solvent of EtOAc/H₂O at room temperature to generate amide C05 with excellent reactivity in 90% yield (entry 4).

Sulfonyl esters are widely used in medicine and pesticides due to their remarkable biological activities. For example, the main active substance thiothiesulfate obtained from garlic extraction and separation not only has the effect of reducing blood lipid (Ackermann et al., 2001; Rahman, 2001) but also has antitumor (Bianchini and Vainio, 2001), antiviral, and a variety of medical activities such as antibacterial, bactericidal, and viricidal (Harris et al., 2001). In the study of pesticide activity, sulfonate compounds were shown to have insecticidal, acaricidal, and bactericidal activities. In addition, sulfonate compounds also have herbicidal and plant growth-regulating effects.

Sulfonyl and sulfonamide groups are important pharmacophore groups in many drugs, and introducing these groups can effectively improve the activity of compounds. In 2011, Zhao reported curcumin benzoyl sulfonate compounds’ synthesis and acaricidal activity (Zhao, 2011). Taking curcumin as the lead, the authors produced a reaction with benzene sulfonyl chloride compounds to synthesize a series of curcumin benzoyl sulfonate derivatives and tested the acaricidal activity. The results showed that compared with curcumin itself, its acaricidal and ovicidal activities were significantly improved.

As shown in Scheme 3 , sulfonyl esterification was carried out on the chiral hydroxyl group, and different sulfonyl functional groups were introduced to enhance the efficacy. Most sulfonyl groups have a good reaction effect when introduced. Unfortunately, the synthesis of C07d showed a poor reaction effect and low yield. The analysis might be due to the presence of the N atom in the sulfonyl group, which increased the density of the electron cloud on the S atom and reduced the overall reactivity, resulting in poor reactivity.

The amide group, as an essential group in natural products, often appears in a state without other substitutions, which is conducive to the compound’s participation and thus enhances bioactivity. However, the amide group in the C07 series compounds obtained in this study is connected with the electron-pulling group, which is quite different from the natural products, and may impact the activity. In order to further explore whether the existence of electron-pulling groups on the amide N structure affects the antibacterial activity, as shown in Scheme 4 , a C07 series of compounds was successfully removed from N-Boc to obtain C08a~n under a solution of trifluoroacetic acid (TFA) in dichloromethane.

In this study, the antibacterial activity of 31 compounds against five species of fungi (Pyricularia oryzae, Fusarium graminearum, Alternaria brassicae, Valsa mali, and Alternaria alternariae) at 100 μg/ml was evaluated by an approach of inhibiting mycelium growth rate. The preliminary bioactivity test results displayed that most of the 31 target compounds had inhibitory effects on these five fungi. Among them, compounds C07l~m and C08a~n were more potent than any other against Fusarium graminearum and Valsa mali, they were superior to the commercial control drug hymexazol and equal to chlorothalonil. It is shown in Table 2 that the antibacterial activities of C07l and C07m were equal to those of C08l and C08m, but the antibacterial activities of other compounds were lower than those of C08 series compounds, the results indicated that the amide group attached to the Boc group was detrimental to the inhibitory activity. The activities of L-pyroglutamic acid derivatives with alkane chains were slightly higher than those of aromatic compounds. In addition, the introduction of naphthalene (C08l) and alkane (C08a) on the hydroxyl group could effectively enhance the inhibitory activity of Fusarium graminearum. As for the effects of substituents on aromatic compounds, available data exhibited that an electron-withdrawing group played an important role to the enhancement of antibacterial activity.

According to the above results, the structure–activity relationship (SAR) could be done. All C08 series compounds showed significant antifungal activities against Fusarium graminearum ( Table 2 ). In particular, the activity of the compounds with naphthalene was more potent than those with phenyls, and the electron-withdrawing groups on the aromatic ring facilitated the inhibitory effects of C08e~k. In addition, the introduction of 4-chlorophenol into compound C04 was beneficial to the antibacterial activity against the five fungi.

All reactions were performed in flame-dried glassware under a nitrogen atmosphere. Solvents were distilled prior to use. Reagents were used as purchased from Aladdin, Macklin, Innochem, or TLC unless otherwise noted. Chromatographic separations were performed using a silica gel, AR, 200–300 mesh. ¹H and ¹³C NMR spectra were obtained on Bruker 400 MHz NMR and JNM-ECZR 500 MHz NMR instruments using CDCl₃ and DMSO as the solvent, which were provided by the School of State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University. Optical rotations were obtained on an InsMark digital polarimeter using a sodium (589 nm, D line) lamp and are reported as follows: [ α ] λ T  ° C (c = g/100 ml, solvent). TLC analysis was visualized using UV and phosphomolybdic acid stains. High-resolution mass spectra were obtained using Q Exactive. All spectral data obtained for new compounds are reported here.

The biological reagents used were glucose, AGAR, and streptomycin. In addition, potatoes were bought from supermarkets.

The synthetic procedures for the key intermediate C06 from L-hydroxyproline are shown in Scheme 5 .

To a solution of L-hydroxyproline C01 [20.0 g, 152 mmol, [ α ] D 25 = + 89.7 ( c 0.1 , H 2 O ) ] in H₂O (76 ml) was added a solution of 10% NaOH (aqueous, 60 ml, 167 mmol). The resulting solution was stirred for 2 h followed by adding a solution of Boc₂O (36.6 g, 167 mmol) in THF (152 ml) in one portion via a syringe. The mixture was stirred for 24 h at room temperature (rt), and the reaction was quenched by addition of 10% KHSO₄ solution in water to adjust pH = 4~5, and the organic layer was separated. The aqueous layer was further extracted with EtOAc (100 ml × 3). The combined organic extracts were washed with the saturated solution of NaCl (50 ml × 1), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo to afford C02 as white solid (30.0 g, 85%) and was used directly in the next step without further purification. Mp = 117.8~118.6 °C. R _f = 0.3 (100% MeOH/CH₂Cl₂). ¹H NMR (400 MHz, DMSO-d₆ , 25°C, mixture of rotamers) δ 12.49 (br s, 1H), 5.04 (br s, 1H), 4.25~4.22 (m, 1H), 4.11 (td, J = 7.9, 2.4 Hz, 1H), 3.33~3.41 (m, 1H), 3.28~3.20 (m, 1H), 2.05~2.14 (m, 1H), 1.84~1.92 (m, 1H), 1.36 (d, J = 18.8 Hz, 9H). ¹³C NMR (100 MHz, DMSO-d₆ , 25°C, mixture of rotamers) δ 174.5, 174.0, 153.9, 153.3, 78.9, 78.9, 68.6, 67.9, 57.8, 57.5, 54.7, 54.4, 28.2, 28.0. HRMS (ESI+): m/z calculated for C₁₀H₁₇NO₅ [M+Na]⁺ 254.1106, found 254.0995.

To a solution of C02 (5.0 g, 22 mmol) in CH₂Cl₂ (45 ml) were added t-butylchlorodimethylsilane (7.2 g, 48 mmol) and imidazole (6.5 g, 95 mmol). The mixture was stirred for 10 h at room temperature, and the reaction was quenched by addition of 1 M HCl solution to adjust pH = 4~5. The organic layers were separated, and the aqueous layer was extracted with CH₂Cl₂ (50 ml × 3). The combined organic extracts were washed with the saturated solution of NaCl (50 ml × 1), dried over anhydrous NaSO₄, filtered, and concentrated in vacuo. The residue was subjected to silica gel chromatography (petroleum ether: EtOAc = 3:1) to afford C03 as colorless oil (6.7 g, 90%). R _f = 0.3 (30% EtOAc/petroleum ether). ¹H NMR (500 MHz, CDCl₃, 25°C, mixture of rotamers) δ 6.42 (br s, 1H), 4.46~4.30 (m, 2H), 3.61~3.45 (m, 1H), 3.41~3.32 (m, 1H), 2.28~2.02 (m, 2H), 1.45 (d, J = 31.4 Hz, 9H), 0.86 (s, 9H), 0.06 (d, J = 3.5 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃, 25°C, mixture of rotamers) δ 178.3, 175.3, 156.6, 154.1, 81.5, 80.7, 70.1, 69.8, 58.1, 55.0, 54.7, 39.9, 38.0, 31.2, 28.5, 28.3, 25.8, 25.77, 25.7, 18.1. HRMS (ESI+): m/z calculated for C₁₆H₃₁NO₅Si [M+Na]⁺ 368.2971, found 368. 3153.

To a solution of C03 (4.2 g, 12 mmol) in CH₂Cl₂ (40 ml) were added DCC (3.0 g, 15 mmol) and 4-chlorophenol (1.9 g, 15 mmol). The mixture was stirred for 3 h at room temperature. CH₂Cl₂ was added to dilute and filter solids. The organic layer was concentrated in vacuo. The residue was subjected to silica gel chromatography (petroleum ether: EtOAc = 10:1) to afford C04 as colorless solid (4.1 g, 74%). Mp = 56~59.8 °C. R _f = 0.3 (10% EtOAc/petroleum ether). ¹H NMR (500 MHz, CDCl₃, 25°C, mixture of rotamers) δ 7.35~7.29 (m, 2H), 7.07~7.02 (m, 2H), 4.61~4.48 (m, 1H), 4.47~4.45 (m, 1H), 3.65~3.58 (m, 1H), 3.51~3.35 (m, 1H), 2.37~2.26 (m, 1H), 2.21~2.11 (m, 1H), 1.45 (d, J = 12.7 Hz, 9H), 0.87 (s, 9H), 0.07 (s, 6H). ¹³C NMR (100 MHz, CDCl₃, 25°C, mixture of rotamers) δ 171.6, 171.5, 154.7, 153.9, 149.3, 149.1, 131.4, 131.3, 129.6, 129.5, 123.0, 122.5, 80.6, 80.4, 70.6, 69.8, 58.3, 58.0, 55.1, 54.8, 40.1, 39.0, 35.0, 28.5, 28.4, 25.8, 25.8, 25.5, 24.8, 18.1, 18.0. HRMS (ESI+): m/z calculated for C₂₂H₃₄ClNO₅Si [M+Na]⁺ 478.1894, found 478.1779.

To a solution of NaIO₄ (3.8 g, 17 mmol) in H₂O (30 ml) was added RuO₂·H₂O (0.13 g, 1 mmol) at room temperature. The resulting green yellow solution was stirred for 2 h followed by addition of C04 (2.0 g, 4 mmol) in EtOAc (15 ml) in one portion via a syringe. Additional aliquots of NaIO₄ were added to maintain a yellow-colored solution during the reaction. The mixture was stirred for 9 h at room temperature, and EtOAc (100 ml) to dilution reaction. The reaction was quenched by addition of sat. aq. Na₂S₂O₃, which immediately resulted in the precipitation of Ru black. The organic layers were separated, and the organic extract was filtered through a pad of Celite. The filtrate was then washed with the saturated solution of NaCl (50 ml × 1), dried over anhydrous Na₂SO₄, and evaporated under reduced pressure. The resulting residue was subjected to silica gel chromatography (petroleum ether: EtOAc = 10:1) to afford C05 as a white solid (1.85 g, 90%). Mp = 73.7~76.9°C. R _f = 0.33 (10% EtOAc/petroleum ether). ¹H NMR (400 MHz, CDCl₃, 25°C, mixture of rotamers) δ 7.20~7.16 (m, 2H), 6.89~6.85 (m, 2H), 4.60 (dd, J = 9.8, 1.7 Hz, 1H), 4.31 (dd, J = 10.0, 8.2 Hz, 1H), 2.36~2.30 (m, 1H), 2.19~2.11 (m, 1H), 1.34 (s, 9H), 0.72 (s, 9H), 0.05 (d, 6H). ¹³C NMR (100 MHz, CDCl₃, 25°C, mixture of rotamers) δ 171.6, 169.7, 149.7, 148.7, 131.9, 129.7, 122.4, 84.4, 69.7, 55.2, 31.8, 28.0, 25.7, 18.2. HRMS (ESI+): m/z calculated for C₂₂H₃₂ClNO₆Si [M+Na]⁺ 482.1687, found 492. 1571.

To a solution of C05 (1.8 g, 4 mmol) in THF (40 ml) at room temperature was added hydrogen fluoride (70% in HF, 3.1 ml, 24 mmol). The mixture was stirred for 4 h at room temperature, and the reaction was quenched by addition of solid NaHCO₃ filtered and concentrated in vacuo. The residue was subjected to silica gel chromatography (petroleum ether: EtOAc = 3:1) to afford C06 as white solid (1.2 g, 85%). Mp = 120~121 °C. [ α ] D 25 = + 92.3   ( c 0:1 , CHCl 3 ) . R _f = 0.4 (100% EtOAc/petroleum ether). ¹H NMR (400 MHz, CDCl₃, 25°C, mixture of rotamers) δ 7.38~7.35 (m, 2H), 7.09~7.05 (m, 2H), 4.84 (dd, J = 9.9, 1.2 Hz, 1H), 4.55 (dd, J = 10.8, 8.4 Hz, 1H), 3.05~2.97 (m, 1H), 2.60~2.55 (m, 1H), 1.54 (s, 9H). ¹³C NMR (100 MHz, CDCl₃, 25°C, mixture of rotamers) δ 200.9, 173.4, 169.2, 149.0, 148.6, 132.0, 129.8, 122.3 84.82, 68.7, 55.5, 30.6, 27.9. HRMS (ESI+): m/z calculated for C₁₆H₁₈ClNO₆ [M+Na]⁺ 378.0822, found 378.3245.

To a solution of C06 (100 mg, 0.28 mmol) in CH₂Cl₂ (1.0 ml) were added DMAP (0.03 mmol) and Et₃N (45 mg, 0.42 mmol). The resulting solution was stirred for 10 min followed by addition of sulfonyl chloride (0.42 mmol) in CH₂Cl₂ (0.5 ml) in one portion via a syringe. The reaction mixture was stirred for 1~4 h at room temperature, and the reaction was quenched by addition of sat. aq. NH₄Cl. The organic layers were separated and the aqueous layer was extracted with CH₂Cl₂ (2 ml × 3), and the combined organic extracts were washed with the saturated solution of NaCl (10 ml × 1) and dried over anhydrous Na₂SO₄. The residue was subjected to silica gel chromatography (petroleum ether: EtOAc = 10:1) to afford C07a~m.

TFA (2.2 equiv) was added to sulfonyl esters C07a~m (1.0 equiv) in CH₂Cl₂ (0.25 M), and the mixture was stirred at room temperature for 0.5~2 h. The reaction was quenched with saturated NaHCO₃ aqueous solution, and the mixture was separated through a separating funnel. The aqueous phase was extracted by CH₂Cl₂ (×3) and dried with anhydrous Na₂SO₄. After removal solvent by reduce pressure, the crude residue was purified using silica gel flash column chromatography [eluent: EtOAc/petroleum ether] to give compound C08a~n.

In vitro antifungal activity: L-pyroglutamic acid analogues were screened in vitro for their antifungal activities against five phytopathogenic fungi by poisoned food technique. Five phytopathogenic fungi, namely, Pyricularia oryzae (SD), Fusarium graminearum (XC), Alternaria brassicae (BH), Valsa mali (PF), and Alternaria alternariae (YC), were used for the assays. Potato dextrose agar (PDA) medium was prepared in the flasks and sterilized. The target compounds were dissolved in acetone before mixing with PDA, and the concentration of the test compounds in the medium was fixed at 100 μg/ml. The medium was then poured into sterilized Petri dishes. All types of fungi were incubated in PDA at 27 ± 1°C for 4 days to get new mycelia for the antifungal assays, and a mycelium disk of approximately 4 mm in diameter cut from the culture medium was picked up with a sterilized inoculation needle and inoculated in the center of the PDA Petri dishes. The inoculated Petri dishes were incubated at 27 ± 1°C for 4 days. Acetone without any compounds mixed with PDA was served as a negative control, whereas hymexazol and chlorothalonil, two commercial agricultural fungicides, were used as positive controls. For each treatment, three replicates were conducted. The radial growths of the fungal colonies were measured, and data were statistically analyzed. The inhibitory effects of the test compounds on these fungi in vitro were calculated by the following formula: Inhibition rate (%) = (C-T) × 100/(C-4 mm), where C represents the diameter of fungi growth on untreated PDA and T represents the diameter of fungi on treated PDA. Statistical analysis was processed by the SPSS 21.0 (SPSS Inc., Chicago, USA) software.