The Study of Regioselective Acylation of Geniposide by Using Whole-Cell Biocatalysts in Organic Solvents

Abstract

Geniposide, the predominant bioactive constituent identified in the traditional Chinese medicine herb Gardenia jasminoides, demonstrates clinically significant pharmacological properties. However, the clinical application of geniposide is significantly limited by its insufficient lipophilicity and consequent compromised oral bioavailability. To enhance the lipophilicity and bioavailability of geniposide, a novel whole-cell-mediated catalytic approach was developed for the first time. Aspergillus oryzae whole cells exhibited the highest catalytic activity among microbial strains screened for geniposide decanoylation in the organic solvents. The optimal reaction conditions were identified as follows: acetonitrile served as the reaction solvent, with a substrate molar ratio of 15:1, a whole-cell dosage of 20 mg/mL, and the reaction temperature maintained at 50 °C. Under these optimized conditions, the initial reaction rate was 6.1 mmol/L·h, the conversion reached 99%, and the regioselectivity exceeded 99%. In addition, nine geniposide esters were successfully synthesized, exhibiting outstanding conversion efficiency and high regioselectivities. The pronounced regioselectivity exhibited by Aspergillus oryzae cells toward the 6′-hydroxy group of the glycoside ring in geniposide can be attributed to the lower steric hindrance at this position relative to other hydroxyl moieties, which may enter into the enzyme’s active site more easily to attack the acyl-enzyme intermediate.

1. Introduction

As the sustainable synthesis of natural products increasingly becomes a central focus in organic chemistry, chiral pool approaches provide significant utility, partly owing to the inherent availability of synthetic chirons that enable efficient access to structurally complex natural products [1]. Geniposide, a natural iridoid glycoside compound, serves as the key active ingredient in the traditional Chinese medicine gardeniae (Figure 1). It exhibits a variety of pharmacological activities, such as antitumor, hypotensive, hypoglycemic, hypolipidemic, hepatoprotective, choleretic, and neuroprotective effects, and is regarded as a drug or lead compound for the prevention and management of different diseases [2,3,4,5,6,7,8,9]. The molecular structure of geniposide encompasses multiple hydroxyl groups, which render it highly water-soluble yet poorly lipid-soluble. As a result, it is challenging for geniposide to traverse the cell membrane, leading to low bioavailability and restricting its pharmacological effects. Lipophilic modification has received widespread attention as a promising strategy. Studies have indicated that the geniposide derivatives obtained through acylation modification display superior pharmacological functions compared to geniposide [10]. For instance, the geniposide derivative by acylation modification has enhanced lipophilicity and increased cell membrane permeability, which can more effectively decrease serum uric acid (SUA) levels in hyperuricemic mice by inhibiting the xanthine oxidase (XOD) activity [11]. Geniposide 4-isopentyl ester (GENI) can lower the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in yeast cells, thereby demonstrating an antiaging effect [12]. Additionally, 6′-O-lauroyl geniposide demonstrates superior anti-inflammatory activity compared to geniposide [13].

Biosynthetic strategies have successfully integrated biocatalysts, such as enzymes and whole-cell catalysts, into chemical transformations, effectively minimizing the environmental impact while promoting sustainable practices. The enzymatic method for acylating polyhydroxy compounds offers the advantages of operational simplicity, high regioselectivity, and environmental friendliness. In the acylation of xylose laurate using lipase Novozym 435, the cost is relatively high owing to the complex preparation procedures for the enzyme [13]. Whole-cell catalysts can be obtained simply by the cultivation of microbial cells, circumventing the complex preparation steps of pure enzymes and reducing costs. Additionally, the cells can offer a natural, protective milieu for the enzymes, preventing their deactivation [14,15,16]. Research has demonstrated that the utilization of whole-cell catalysts can achieve the synthesis of glycoside ester derivatives in non-aqueous systems with high catalytic activity [16,17,18]. Xu et al. [19] carried out the acylation modification of phenolic glycosides by employing whole-cell biocatalysts and discovered that these catalysts manifested high catalytic activity in non-aqueous systems, with substrate conversion rates consistently exceeding 90%. In summary, in terms of efficiency and cost-effectiveness, whole-cell biocatalysis exhibits significant advantages over enzymatic methods and has emerged as a research focus in biocatalysis.

Currently, the synthesis of the geniposide ester derivatives through biocatalysis mainly involves enzymatic methods. For example, Yang et al. [13] synthesized geniposide laurate using immobilized lipase TL IM. In contrast to enzymatic catalysis, whole-cell catalysis exhibits substantial untapped research potential and broader application prospects. Nevertheless, there have been no reports specifically focusing on the acylation modification of geniposide using microbial whole-cell catalysis. Therefore, this study explored a new method for the acylation of geniposide with aliphatic acyl donors in non-aqueous media using whole-cell catalysis (Figure 1). Initially, geniposide decanoylation was employed as a model reaction to screen for whole-cell catalysts capable of efficiently catalyzing this transformation. The influences of several key variables on geniposide decanoylation were further analyzed, including the type of organic solvent, whole-cell catalyst dosage, and substrate molar ratio. Additionally, this study investigated the recognition patterns of acyl donors by Aspergillus oryzae whole cells in the acylation of geniposide. This research proposes a novel technique for the structural modification of geniposide based on whole-cell catalysts and provides theoretical and technical support for the further development and application of geniposide ester derivatives.

2. Results and Discussion

2.1. Catalytic Behaviors of Various Microbial Whole Cells in the Acylation of Geniposide

In this study, several different lipase-producing strains were selected. Following fermentation and subsequent freeze-drying processes, whole-cell catalysts were developed.

Table 1. Regioselective decanoylation of geniposide catalyzed by whole cells.

Strains	V0 (mmol/L·h)	Conversion Rate (%)	6′-Regioselectivity (%)
Rhizopus oryzae GDM 3.406	Not Detected	Not Detected	Not Detected
Aspergillus oryzae GDM 3.446	3.3 ± 0.2	86.3 ± 0.6	>99
Pseudomonas aeruginosa GDM 1.443	1.5 ± 0.0	56.4 ± 0.6	>99
Pseudomonas stutzeri GDM 1.446	Not Detected	Not Detected	Not Detected
Pseudomonas fluorescens GDM 1.209	Not Detected	Not Detected	Not Detected

Reaction conditions: 15 mM geniposide, 90 mM vinyl decanoate, 15 mg/mL catalyst preparation, 2 mL anhydrous acetonitrile, 40 °C, 200 rpm.

demonstrates that both Aspergillus oryzae whole cells and Pseudomonas aeruginosa whole cells are capable of catalyzing geniposide decanoylation, with initial reaction rates of 3.3 mmol/L·h and 1.5 mmol/L·h, respectively, and substrate conversion rates of 86% and 56%, respectively. No catalytic activities were detected in the other three lipase-producing strains. Previous studies have also shown that different microorganisms produce lipases with notable variations in type, activity, and catalytic efficiency under identical culture medium conditions [20]. It is worth noting that A. oryzae and P. aeruginosa, as whole-cell catalysts, exhibit high selectivity on the 6′-hydroxy group of the sugar ring of geniposide. Similarly, P. aeruginosa also exhibited high regioselectivity toward the 6′-hydroxyl group on the sugar ring during the acylation of glycoside compounds such as arbutin and helicid [21]. It has been reported that immobilized lipozyme TL IM exhibited high regioselectivity toward the primary hydroxyl group at the 6′ position of geniposide [12]. This regioselectivity can be ascribed to the reduced steric hindrance of the 6′-hydroxy group in the glycoside ring of geniposide relative to other hydroxyl groups, enabling it to more readily access the enzyme’s active site and react with the acyl-enzyme intermediate, thereby favoring acylation at this position. Taking into account both the conversion and the initial reaction rate, A. oryzae was selected as the whole-cell catalyst for subsequent experiments.

2.2. Effects of Organic Solvents on Decanoylation of Geniposide Catalyzed by A. oryzae Whole Cell

In the biotransformation process mediated by whole-cell catalysts, the properties of the reaction medium significantly influence the activity and stability of the whole-cell biocatalyst, substrate solubility, and cell membrane permeability [22]. Geniposide is a polyhydroxy compound, which has a higher solubility in strongly polar solvents and a lower solubility in low-polar solvents. Strongly polar organic solvents can extract the essential water from enzyme molecules, leading to catalyst deactivation. Therefore, selecting appropriate organic solvents is a crucial factor affecting the whole-cell catalyzed reaction.

Table 2. Effects of organic solvents on geniposide decanoylation catalyzed by A. oryzae whole cells.

Solvents	V0 (mmol/L/h)	Conversion Rate (%)	6′-Regioselectivity (%)
Acetone	1.8 ± 0.8	63.4 ± 0.6	>99
Acetonitrile	3.3 ± 0.2	86.3 ± 0.6	>99
tert-Butanol	Not Detected	6.1 ± 0.7	>99
Tetrahydrofuran	1.5 ± 0.3	13. 3 ± 0.9	>99
Dimethylformamide (DMF)	Not Detected	Not Detected	Not Detected
2-Methyltetrahydrofuran	1.7 ± 0.4	26.2 ± 1.1	>99
Dimethyl sulfoxide (DMSO)	Not Detected	Not Detected	Not Detected

Reaction conditions: 15 mM geniposide, 90 mM vinyl decanoate, 15 mg/mL A. oryzae cells, 2 mL anhydrous solvent, 40 °C, 200 rpm.

presents the effects of various pure solvents on the catalytic performance of A. oryzae cells during the geniposide acylation. A. oryzae whole cells showed no bioactivity in strong polar solvents, including N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). This phenomenon may be attributed to the ability of these solvents to deprive essential water of enzyme molecules, resulting in enzyme deactivation. When acetonitrile was used as the reaction medium, the catalytic activity of A. oryzae whole cells was highest, with the substrate conversion rate reaching 86% and the initial reaction rate attaining 3.3 mmol/L·h. In contrast, when acetone served as the reaction medium, the catalytic activity was moderate, resulting in a substrate conversion rate of 63% and an initial reaction rate of 1.8 mmol/L·h. However, when 2-methyltetrahydrofuran, tert-butanol, and tetrahydrofuran were used as reaction media, the catalytic activities were significantly lower, with substrate conversion rates of 26%, 6%, and 13%, respectively. Additionally, the reaction medium exerted no significant effect on the regioselectivity of geniposide decanoylation catalyzed by whole cells, with geniposide 6′-decanate being the sole product.

2.3. Influence of Several Crucial Parameters on the Decanoylation of Geniposide

To optimize the synthesis efficiency of geniposide 6′-decanoate by A. oryzae whole cells, critical parameters, including the substrate molar ratio, catalyst loading, and temperature, were systematically investigated. Concurrent with the enzymatic acylation of glycosides catalyzed with vinyl esters, the hydrolysis of the acyl donors proceeds as a competing side reaction. Consequently, a stoichiometric excess of acyl donors is typically required to drive the reaction toward completion. As illustrated in Figure 2a, the substrate molar ratio significantly influenced the initial reaction velocity and conversion of geniposide decanoylation catalyzed by A. oryzae whole cells. When the molar ratio of vinyl decanoate to geniposide was increased from 3:1 to 15:1, the substrate conversion increased from 67% to 99%. Upon further increasing the molar proportion of vinyl decanoate relative to geniposide, the substrate conversion efficiency remained largely unchanged, while the reaction rate demonstrated minimal variability. In previous studies by our research group [23], it was found that in the synthesis of salidroside esters with A. oryzae, increasing the molar ratio of vinyl caprylate to salidroside from 5:1 to 25:1 led to a significant increase in both the initial reaction rate and the substrate conversion (initial rate increased from 7.42 mmol/L·h to 15.36 mmol/L·h; substrate conversion increased from 78.6% to 99.0%). Additionally, the regioselectivity is not significantly impacted by the substrate’s molar ratio. An initial reaction rate of 4.4 mmol/L·h and a substrate conversion rate of 99% were achieved with a molar ratio of 15:1 for vinyl decanoate to geniposide, indicating that this ratio was optimal. The amount of catalyst is a critical determinant of biotransformation efficiency in the whole-cell-catalyzed decanoylation of geniposide.

As illustrated in Figure 2b, both the initial reaction rate and geniposide conversion increased with the incremental elevation of the A. oryzae dosage. The initial reaction velocity exhibited a progressive enhancement, escalating from 1.5 to 4.6 mmol/L·h as the biocatalyst dosage was increased incrementally from 5 to 20 mg/mL. Notably, further increasing the amount of the biocatalyst had a negligible effect on improving the catalytic performance. Therefore, 20 mg/mL was selected as the optimal concentration of the A. oryzae whole-cell catalyst.

Temperature not only influences thermodynamic and kinetic stability but also contributes to biocatalyst deactivation. As depicted in Figure 2c, the whole-cell catalysts demonstrated catalytic activity across the temperature range of 30–55 °C. The conversion rate peaked at 99% at 50 °C, while the initial reaction rates increased from 2.8 to 6.1 mmol/L·h with temperature elevation from 30 °C to 50 °C. Yang et al. [23] found that increasing the temperature enhanced catalyst activity and accelerated the reaction rate within a specific temperature range during the whole-cell catalytic synthesis of salidroside esters. However, when the temperature surpassed the critical threshold, both the reaction rate and substrate conversion rate exhibited a marked decline, likely attributable to reduced intracellular enzyme activity at elevated temperatures.

In conclusion, the optimal reaction conditions were determined to be as follows: acetonitrile as the reaction solvent, a substrate molar ratio (vinyl decanoate:geniposide) of 15:1, a whole-cell dosage of 20 mg/mL, and a reaction temperature of 50 °C. Under the optimized conditions, the reaction exhibited consistently excellent regioselectivity throughout all experimental trials.

2.4. Product Structure Characterization

To facilitate the structural characterization of the synthesized geniposide derivatives, the reaction was scaled up. The products were isolated and purified using flash column chromatography and were further characterized by HPLC and NMR analyses. The retention times of the geniposide ester derivatives determined by HPLC were as follows: 2.4 min for geniposide 6′-propionate, 2.5 min for geniposide 6′-butyrate, 3.0 min for geniposide 6′-hexanoate, 3.3 min for geniposide 6′-octanoate, 4.6 min for geniposide 6′-decanoate, 4.4 min for geniposide 6′-undecenoate, 5.3 min for geniposide 6′-laurate, 8.5 min for geniposide 6′-myristate, and 15.0 min for geniposide 6′-palmitate. NMR spectroscopic data (¹H and ¹³C) along with corresponding spectra are available in the Supplementary Materials. Figure 3 presents the model compound geniposide decanoate, indicating the positions of carbon and hydrogen atoms as they correspond to the nuclear magnetic resonance (NMR) data. The ¹³C NMR data of geniposide decanoate are presented in

Table 3. ¹³C NMR chemical shifts of geniposide 6′-decanoate.

Carbon Numbers	Geniposide (δC)	Geniposide 6′-O-Decanoate (δC)
Base moiety
1	95.71	96.29
2	45.85	45.58
3	144.05	144.33
4	125.42	125.59
5	37.94	38.16
6	34.40	34.69
7	110.89	110.95
8	151.50	151.47
9	166.84	166.78
10	50.94	50.92
11	59.28	59.32
Sugar moiety
1′	98.58	98.87
2′	73.26	73.16
3′	77.20	76.38
4′	69.96	70.20
5′	76.60	73.92
6′	60.95	63.27
Acyl moiety
1″		172.64
2″		33.49
3″		22.09
4″		24.43
5″		28.47
6″		28.85
7″		28.65
8″		31.28
9″		13.86
10″		9.88

. Comparative analysis of the ¹³C NMR spectra between geniposide and its decanoate derivative demonstrated ten distinct carbon signals at 172.64, 33.49, 22.09, 24.43, 28.47, 28.85, 28.65, 31.28, 13.86, and 9.88 ppm in the latter, corresponding to the complete carbon framework of the decanoyl moiety (Table 3). Moreover, in the geniposide decanoate, the carbon signal at C_6′ of the sugar moiety exhibited a downfield shift of 2.32 ppm (from 60.95 ppm to 63.27 ppm), whereas the neighboring carbon atom at C_5′ showed an upfield shift of 2.68 ppm (from 76.60 ppm to 73.92 ppm). This result indicates that the acylation occurs at the C_6′ position of the geniposide glycosyl moiety. It has been reported that during the lauric acid acylation of geniposide catalyzed by lipase TL IM, the acylation site was also identified as the C_6′ position of the geniposide sugar ring [13].

2.5. Reaction Process of Geniposide Decanoylation and Biocatalyst Reusability of Whole Cells

To further investigate the reaction process of whole-cell catalysis, the decanoylation of geniposide mediated by A. oryzae cells was conducted under optimized reaction parameters (Figure 4a). The substrate conversion increased significantly during the initial 18 h period of the reaction, rising from 20.3% to 95%, followed by a more gradual increase, likely attributed to either the reduced geniposide concentration or the partial inactivation of the whole-cell biocatalyst. A 99% peak substrate conversion rate was achieved after 24 h of reaction time.

Biocatalyst reusability analysis indicated that the whole-cell biocatalyst of A. oryzae preserved 82% of its original catalytic capacity through two consecutive reaction cycles, with progressive stabilization maintaining 41% activity after six operational cycles (Figure 4b). This phenomenon can likely be ascribed to the whole cell establishing a natural protective microenvironment for intracellular enzymes, thereby reducing their inactivation within the cell.

2.6. Regioselective Acylation of Geniposide with Various Aliphatic Acyl Donors

The cell-bound enzymes from various microbial sources have different substrate recognition characteristics. For instance, in the synthesis of arbutin esters, whole cells of Candida parapsilosis catalyzed the reaction, producing both mono- and di-esters, while the other biocatalysts tested, including Rhizopus oryzae, Rhizomucor miehei, and Pseudomonas putida, exhibited high 6′-regioselectivity (>99.0%), producing only mono esters [20]. The acylation of geniposide with various fatty acid vinyl esters catalyzed by A. oryzae cells was investigated in anhydrous acetonitrile (

Table 4. Effect of various acyl donors on regioselective acylation of geniposide catalyzed by A. oryzae cells.

Entry	Acyl Donor	V0 (mmol/L·h)	Time (h)	Conversion (%)	6′-Regioselectivity (%)
1	Vinyl propionate (C3)	3.4 ± 0.4	24	88.4 ± 0.9	>99
2	Vinyl butyrate (C4)	4.2 ± 0.6	24	87.8 ± 0.4	>99
3	Vinyl hexanoate (C6)	5.7 ± 0.2	24	93.2 ± 0.5	>99
4	Vinyl caprylate (C8)	5.9 ± 0.3	24	99	>99
5	Vinyl decanoate (C10)	6.1 ± 0.3	24	99	>99
6	Vinyl 10-undecenoate (C11)	6.7 ± 0.6	24	99	>99
7	Vinyl laurate (C12)	6.2 ± 0.7	24	99	>99
8	Vinyl myristate (C14)	5.5 ± 0.2	24	99	>99
9	Vinyl palmitate (C16)	4.2 ± 0.3	24	97.3 ± 0.2	>99

Reaction conditions: 15 mM geniposide, 225 mM acyl donor, 20 mg/mL A. oryzae cells, 2 mL anhydrous acetonitrile, 50 °C, 200 rpm.

). Notably, the reactions catalyzed by A. oryzae whole cells exclusively produced geniposide 6′-esters, as verified by comprehensive ¹H NMR and ¹³C NMR spectroscopic analysis in the Supplementary Materials. This result is consistent with findings using whole-cell catalysts derived from Pseudomonas stutzeri, which demonstrated mono-acylated regioselectivity at the C_6′ position of its glycoside moieties during the acylation of esculin [24].

Under optimal reaction conditions (substrate molar ratio of 15:1, whole-cell dosage of 20 mg/mL, and temperature of 50 °C), nine geniposide esters were successfully synthesized (Table 4). As illustrated in Table 4, A. oryzae cells demonstrated remarkable catalytic efficiency, achieving 88–99% conversion at 24 h in the geniposide acylation when employing acyl donors with different chain lengths. Moreover, the initial reaction rate rose from 3.4 mmol/L·h to 6.7 mmol/L·h as the chain length of vinyl esters increased from C3 to C11. However, as the chain length further increased from C12 to C16, the initial reaction rate exhibited a decreasing trend, likely attributable to the increased steric hindrance of the longer-chain acyl donors. Similarly, the conversion exhibited a consistent trend. Specifically, acyl donors with medium chain lengths demonstrated a higher initial reaction rate and conversion compared to those with shorter or longer chain lengths. For instance, as the acyl donor chain length increased from C3 to C10, the initial reaction rate markedly rose from 3.4 mmol/L·h to 6.1 mmol/L·h, and the conversions improved from 88% to 99%. Conversely, when the chain length was extended to C16, the initial reaction rate decreased to 4.2 mmol/L·h, while the substrate conversion reached 97% after 24 h. The results clearly indicated that the whole cells of A. oryzae exhibited enhanced specificity toward medium-chain acyl donors in geniposide acylation. This phenomenon can be ascribed to the enhanced affinity between medium-chain-length acyl groups and the hydrophobic acyl binding pocket within intracellular acylase, resulting in stronger interactions. Previous investigations have indicated that biocatalysts derived from various origins exhibited heterogeneous catalytic behaviors when interacting with various acyl donors. For example, A. oryzae cells exhibited higher specificity toward the medium-chain acyl donors during the synthesis of salidroside esters [23], while Candida parapsilosis cells demonstrated the greatest efficiency with short-chain acyl donor (vinyl acetate) in the synthesis of arbutin esters [20]. It has been reported that Thermomyces lanuginosus lipase exhibited superior catalytic activity toward medium-length acyl donors relative to short-chain ones during the acylation of 6-azauridine with fatty acid vinyl esters [25]. This phenomenon can be attributed to the hydrophobic, crevice-like binding site possessed by Thermomyces lanuginosus lipase [26].

3. Materials and Methods

3.1. Microorganisms and Materials

The microbial strains provided by the Guangdong Institute of Microbiology comprised Pseudomonas fluorescens GDM 1.209, Pseudomonas stutzeri GDM 1.446, Pseudomonas aeruginosa GDM 1.443, Rhizopus oryzae GDM 3.406 and Aspergillus oryzae GDM 3.446. Geniposide (purity 98%) was purchased from Nantong Feiyu Biotechnology Co., Ltd. (Nantong, China). The fatty acid vinyl esters serving as acyl donors were sourced from TCI (Shanghai) Chemicals and Industrial Materials Development Co., Ltd. (Shanghai, China). Among these compounds, vinyl 10-undecenoate exhibited a purity of 92%, whereas vinyl propionate and vinyl butyrate both exhibited purities of 98%. The remaining esters (vinyl hexanoate, vinyl caprylate, vinyl decanoate, vinyl laurate, vinyl myristate and vinyl palmitate) had a purity of 99%. All other reagents, including acetone, tetrahydrofuran, tert-Butanol, acetonitrile, 2-methyltetrahydrofuran, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), sucrose, peptone, beef extract, MgSO₄·7H₂O, K₂HPO₄, NaCl, (NH₄)₂SO₄, and CaCl₂, were acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and exhibited the maximum achievable purity.

3.2. Preparation of Whole-Cell Catalysts

The bacterial and fungal culture methods were based on the previously established experimental protocols of the research group [15]. The bacterial strains were initially cultured in the medium comprising 0.1% sucrose, 1% peptone, 1% beef extract, 0.02% MgSO₄·7H₂O, 0.5% K₂HPO₄, and 0.5% NaCl under controlled conditions of 30 °C and 180 rpm for 24 h. The activated bacterial seed culture (2% v/v) was inoculated into 200 mL of fermentation broth contained in a 500 mL Erlenmeyer flask and incubated for 48 h under controlled conditions of 30 °C and 180 rpm. The fermentation medium was formulated with 0.1% soybean oil, 0.2% peptone, 0.02% MgSO₄·7H₂O, and 0.5% (NH₄)₂SO₄. The fungal strains underwent pre-cultivation on potato dextrose agar (PDA) plates under controlled conditions maintained at 28 °C for 72 h. Subsequently, the activated spore suspension was aseptically introduced into a 200 mL fermentation medium (500 mL Erlenmeyer flask) formulated with 0.6% soybean oil, 0.7% peptone, 0.5% (NH₄)₂SO₄, and 0.02% CaCl₂, which underwent at 30 °C and 180 rpm for 48 h. Bacterial and fungal cells were collected using centrifugation and filtration, respectively. The harvested cells were then subjected to freeze-drying at −50 °C for 24 h before being pulverized into a fine powder, resulting in the preparation of the whole-cell catalyst.

3.3. General Procedure for Acylation of Geniposide by Whole Cells

The acylation reaction was performed in a 10 mL glass reaction flask equipped with a lid under shaking conditions at 200 rpm. The reaction system comprised 2 mL of organic solvent, geniposide (15 mM), a defined amount of fatty acid vinyl esters, and the freeze-dried whole-cell biocatalyst. Control experiments were conducted in the absence of the whole-cell biocatalyst. At predetermined time intervals (10 min, 30 min, 1 h, 3 h, 6 h, 9 h, 12 h, 18 h, 24 h, 30 h, 36 h), aliquots (20 µL) were withdrawn from the reaction mixtures, followed by dilution with the corresponding mobile phase. Subsequently, the samples were subjected to analysis via high-performance liquid chromatography (HPLC). The conversion (C) was quantified by determining the fraction of geniposide consumed with respect to its initial concentration. The initial reaction rate (V₀) was determined by assessing the reduction in geniposide concentrations during the initial 10 min reaction phase. Regioselectivity was quantified by calculating the ratio of the target product’s chromatographic peak area to the aggregate peak areas of all detected products. Each experimental procedure was repeated three times.

3.4. Biocatalyst Reusability of the Whole-Cell

The reaction was carried out at 50 °C with shaking at 200 rpm for 12 h in a 2 mL solution of anhydrous acetonitrile containing 15 mM geniposide, 225 mM vinyl decanoate, and 20 mg/mL of A. oryzae whole-cell catalyst. Following each batch of the synthetic reaction, the whole-cell biocatalysts were separated through filtration, washed with anhydrous acetonitrile, and subsequently employed in the subsequent fresh reaction. This process was repeated six times to evaluate the reusability of the whole cell catalyst.

3.5. HPLC Analysis

The analysis of all samples was conducted using high-performance liquid chromatography (HPLC) equipped with a Shimadzu LC-200C pump and a diode array detector from Shimadzu (Kyoto, Japan), with a detection wavelength of 238 nm. A Zorbax SB-C18 column (5 μm particle size, 4.6 mm × 250 mm length) supplied by Agilent Technologies Industries Co., Ltd. (Santa Clara, CA, USA) was utilized in the process. The mobile phase was composed of acetonitrile and water containing 0.1% formic acid, with a flow rate of 1.0 mL/min. The volumetric ratios of acetonitrile to aqueous 0.1% formic acid solution for the geniposide ester derivatives were established as follows: 60:40 for geniposide 6′-propionate, geniposide 6′-butyrate, and geniposide 6′-hexanoate; 70:30 for geniposide 6′-caprylate, geniposide 6′-decanoate, and geniposide 6′-undecenoate; and 80:20 for geniposide 6′-laurate, geniposide 6′-myristate, and geniposide 6′-palmitate.

3.6. Separation and Structure Identification of the Products

The target compound was successfully isolated by collecting the supernatant, performing vacuum concentration, and subsequently purifying it via silica gel column chromatography. The structural analysis of the ester derivatives was investigated utilizing ¹³C NMR spectroscopy at a frequency of 100 MHz and ¹H NMR at 400 MHz, with DMSO-d₆ as the solvent. The data were processed using MestReNova 15.0.0 software, and the chemical shifts were expressed in δ (ppm) shift. NMR spectroscopy were provided in the Supplementary Materials.

4. Conclusions

In this study, we developed a novel biocatalytic method for the convenient and efficient synthesis of geniposide ester derivatives by leveraging whole-cell catalysis technology. A range of 6′-ester derivatives of geniposide were successfully synthesized by utilizing A. oryzae whole-cell biocatalysts, which exhibited high regioselectivity and excellent conversions. Moreover, the chain length of aliphatic acyl donors utilized in the acylation process demonstrated a significant influence on the catalytic activity of the A. oryzae whole-cell biocatalysts. The catalytic system developed in this study exhibited remarkable substrate compatibility, enabling the efficient synthesis of nine geniposide aliphatic esters with substrate conversions ranging from 88% to 99%. Subsequent studies will systematically investigate the lipophilicity and biological activity of geniposide ester derivatives, thereby providing deeper insights into their pharmacological properties and drug development potential.