*Article* **An** *O***-Demethylation Metabolite of Rabeprazole Sulfide by** *Cunninghamella blakesleeana* **3.970 Biotransformation**

**Ming Song <sup>1</sup> , Hongxiang Zhu <sup>2</sup> , Jian Wang <sup>3</sup> , Weizhuo Xu 2,\* and Wei Xu 2,\***

	- Shenyang 110016, China
	- Tel./Fax: +86-024-43520301 (Weizhuo Xu); Tel.: +86-024-43520307 (Wei Xu)

**Abstract:** To explore the potential metabolites from rabeprazole sulfide, seven strains of filamentous fungi were screened for their biotransformation abilities. Among these strains, *Cunninghamella blakesleeana* 3.970 exhibited the best result. Four different culture media were screened in order to identify the most optimal for subsequent research. Single factors such as the initial pH of culture media, culture time, inoculation volume, and media volume were individually investigated to provide the optimum biotransformation conditions. Then, an orthogonal optimization process using a five-factor, four-level L16(4<sup>5</sup> ) experiment was designed and performed. Finally, when the substrate concentration is 3 g/L, one major metabolite was detected with a transformation rate of 72.4%. Isolated by semipreparative HPLC, this metabolite was further detected by ESI-MS and NMR. The final data analysis indicated that the metabolite is *O*-demethylation rabeprazole sulfide.

**Keywords:** rabeprazole; sulfide; biotransformation; *Cunninghamella blakesleeana* 3.970; *O*-demethylation rabeprazole sulfide

**1. Introduction**

Rabeprazole is a kind of proton pump inhibitor, which has been widely used in the treatment of gastric acid secretion, and can also be applied to eradicate *Helicobacter pylori* [1]. As a benzimidazole derivative, rabeprazole has a heterocyclic molecule consisting of a pyridine and benzimidazole moiety linked by a methylsulfinyl group. This general structure guarantees the similar pharmacological properties of other proton pump inhibitors. Rabeprazole sulfide (rabeprazole thioether) is an essential intermediate for the production of rabeprazole [2,3]. Meanwhile, sulfide is also one of the metabolites produced by the rabeprazole metabolites in vivo [4–8].

Rabeprazole sulfide is an important imidazole compound, which is not only a precursor of rabeprazole synthesis, but is also one of the main metabolites of rabeprazole in vivo. As the primary metabolite of rabeprazole, rabeprazole sulfide can undergo further metabolic reactions such as demethylation and sulfuric acid binding. These demethylated metabolites often have pharmacological activity. Pharmacologically active metabolites can contribute significantly to the overall therapeutic and adverse effects of drugs. Therefore, to fully understand the mechanism of action of drugs, it is important to recognize the role of active metabolites [9–12].

Some microorganisms, especially the fungi belonging to *Cunninghamella* species, possess cytochrome P-450 mono-oxygenase systems analogous to those in mammals [13]. Hence, microbial transformation has been proposed as a complementary in vitro model for mammalian drug metabolism. *Cunnihamella* is a genus of fungi in the family *Cunninghamellaceae*, which has been implemented for in vitro biotransformations for a long time [14–20]. Various reaction types have been reported for these kinds of fungi. Previous studies have

**Citation:** Song, M.; Zhu, H.; Wang, J.; Xu, W.; Xu, W. An *O*-Demethylation Metabolite of Rabeprazole Sulfide by *Cunninghamella blakesleeana* 3.970 Biotransformation. *Catalysts* **2023**, *13*, 15. https://doi.org/10.3390/ catal13010015

Academic Editors: Zhilong Wang and Tao Pan

Received: 14 November 2022 Revised: 16 December 2022 Accepted: 17 December 2022 Published: 22 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

demonstrated their hydroxylation [21–24], epoxidation [25–27], amination [28], demethylation [29], and oxidation from sulfide to sulfoxide, which is usually the final step for the production of prazole-type molecules [30–32]. Demethylated metabolites often have some unexpected pharmacological effects. Metamizole (dipyrone) is an analgesic with antipyretic and spasmolytic properties, which has been in use for almost 100 years. The primary metabolite of metamizole, 4-methyla-

for mammalian drug metabolism. *Cunnihamella* is a genus of fungi in the family *Cunninghamellaceae*, which has been implemented for in vitro biotransformations for a long time [14–20]. Various reaction types have been reported for these kinds of fungi. Previous studies have demonstrated their hydroxylation [21–24], epoxidation [25–27], amination [28], demethylation [29], and oxidation from sulfide to sulfoxide, which is usually the final step

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 2 of 13

for the production of prazole-type molecules [30–32].

Demethylated metabolites often have some unexpected pharmacological effects. Metamizole (dipyrone) is an analgesic with antipyretic and spasmolytic properties, which has been in use for almost 100 years. The primary metabolite of metamizole, 4-methylaminoantipyrine, can be *N*-demethylated to 4-aminoantipyrine. 4-aminoantipyrine plays a role as a nonsteroidal anti-inflammatory drug, a non-narcotic analgesic, an antirheumatic drug, a peripheral nervous system drug, and an EC 1.14.99.1 (prostaglandin-endoperoxide synthase) inhibitor. It is used as a reagent for biochemical reactions, such as the production of peroxides or phenols [33,34]. Papaverine plays a role as a vasodilator agent and an antispasmodic drug. The metabolism of papaverine with *Cunninghamella echinulate* results in *O*-demethylation. 40 -*O*-demethylated papaverine, 30 -*O*-demethylated papaverine, and 6-*O*demethylated papaverine have been isolated. In silico docking studies of these metabolites using crystals of human phosphodiesterase 10a (hPDE10a) revealed that the compounds 4 0 -*O*-demethylated papaverine and 6-*O*-demethylated papaverine possess better docking scores and binding poses with favorable interactions than the native ligand papaverine [35]. minoantipyrine, can be *N*-demethylated to 4-aminoantipyrine. 4-aminoantipyrine plays a role as a non-steroidal anti-inflammatory drug, a non-narcotic analgesic, an antirheumatic drug, a peripheral nervous system drug, and an EC 1.14.99.1 (prostaglandin-endoperoxide synthase) inhibitor. It is used as a reagent for biochemical reactions, such as the production of peroxides or phenols [33,34]. Papaverine plays a role as a vasodilator agent and an antispasmodic drug. The metabolism of papaverine with *Cunninghamella echinulate* results in *O*-demethylation. 4′-*O*-demethylated papaverine, 3′-*O*-demethylated papaverine, and 6-*O*-demethylated papaverine have been isolated. In silico docking studies of these metabolites using crystals of human phosphodiesterase 10a (hPDE10a) revealed that the compounds 4′-*O*-demethylated papaverine and 6-*O*-demethylated papaverine possess better docking scores and binding poses with favorable interactions than the native ligand papaverine [35].

In this report, seven strains of filamentous fungi are investigated for their ability to metabolize the rabeprazole sulfide, in order to explore the reaction types of the whole cell transformation. Instead of hunting for the sulfoxide metabolites obtained, an *O*-demethyl rabeprazole sulfide, a new compound with potential pharmacological activity, was finally isolated and identified (Figure 1), which is the first report of the *O*-demethyl reaction in rabeprazole sulfide to date. In this report, seven strains of filamentous fungi are investigated for their ability to metabolize the rabeprazole sulfide, in order to explore the reaction types of the whole cell transformation. Instead of hunting for the sulfoxide metabolites obtained, an *O*-demethyl rabeprazole sulfide, a new compound with potential pharmacological activity, was finally isolated and identified (Figure 1), which is the first report of the *O*-demethyl reaction in rabeprazole sulfide to date.

Rabeprazole sulfide

study.

*O*-demethyl rabeprazole sulfide

**Figure 1.** Rabeprazole sulfide and its *O*-demethyl metabolite obtained by the biotransformation of *Cunninghamella blakesleeana* 3.970. **Figure 1.** Rabeprazole sulfide and its *O*-demethyl metabolite obtained by the biotransformation of *Cunninghamella blakesleeana* 3.970.

#### **2. Results and Discussion 2. Results and Discussion**

*2.1. Whole-Cell Biotransformation Results of Rabeprazole Sulfide*

*2.1. Whole-Cell Biotransformation Results of Rabeprazole Sulfide* Among the seven strains, *Cunninghamella elegans* 3.910, *Cunninghamella echimnulata* 3.967, *Cunninghamella blakesleeana* 3.970, and *Absidia coerulea* 41,050 displayed different transformation results for rabeprazole sulfide. *Cunninghamella elegans* 3.910 and *Cunninghamella blakesleeana* 3.970 demonstrated a rather significant major metabolite spectrum, whereas *Gibberella fujiluroi* 40,272, *Gibberella* sp. 2498, and *Caldariomyces fumago* 16,373 did not indicate any positive transformation result. According to the transformation TLC results (Figure 2), the fungus *Cunninghamella blakesleeana* 3.970 converted rabeprazole sulfide to rabeprazole and another, novel compound, and the conversion rate of this new Among the seven strains, *Cunninghamella elegans* 3.910, *Cunninghamella echimnulata* 3.967, *Cunninghamella blakesleeana* 3.970, and *Absidia coerulea* 41,050 displayed different transformation results for rabeprazole sulfide. *Cunninghamella elegans*3.910 and *Cunninghamella blakesleeana* 3.970 demonstrated a rather significant major metabolite spectrum, whereas *Gibberella fujiluroi* 40,272, *Gibberella* sp. 2498, and *Caldariomyces fumago* 16,373 did not indicate any positive transformation result. According to the transformation TLC results (Figure 2), the fungus *Cunninghamella blakesleeana* 3.970 converted rabeprazole sulfide to rabeprazole and another, novel compound, and the conversion rate of this new compound was high. Therefore, *Cunninghamella blakesleeana* 3.970 was selected for further study.

compound was high. Therefore, *Cunninghamella blakesleeana* 3.970 was selected for further

**Figure 2.** TLC screening results for seven strains of filamentous fungi for rabeprazole sulfide. Lanes 1 and 2, *Cunninghamella elegans* 3.910, 3 and 4, *Cunninghamella echimnulata* 3.967, 5 and 6, *Absidia coerulea* 41,050, 7 and 8, *Gibberella* sp. 2498, 9 and 10, *Caldariomyces fumago* 16,373, 11 and 12, *Gibberella fujiluroi* 40,272, 13 and 14, *Cunninghamella blakesleeana* 3.970, and 15, standard mixture of rabeprazole sulfide and rabeprazole. **Figure 2.** TLC screening results for seven strains of filamentous fungi for rabeprazole sulfide. Lanes 1 and 2, *Cunninghamella elegans* 3.910, 3 and 4, *Cunninghamella echimnulata* 3.967, 5 and 6, *Absidia coerulea* 41,050, 7 and 8, *Gibberella* sp. 2498, 9 and 10, *Caldariomyces fumago* 16,373, 11 and 12, *Gibberella fujiluroi* 40,272, 13 and 14, *Cunninghamella blakesleeana* 3.970, and 15, standard mixture of rabeprazole sulfide and rabeprazole.

#### *2.2. Optimization of Transformation Media 2.2. Optimization of Transformation Media*

In order to obtain the transformation products of rabeprazole sulfide, four microbial media were screened. The evaluation criteria were set according to both the growth of the strain and the HPLC detection diagram for the transformation solution. According to Figure S1, fewer mycelium balls were observed in medium 1 and medium 2, and the color of the mycelium ball was a little pink in medium 3, which is not its usual appearance. Only in medium 4 were the numbers of the mycelium balls ambient and the growth of mycelium good. In order to obtain the transformation products of rabeprazole sulfide, four microbial media were screened. The evaluation criteria were set according to both the growth of the strain and the HPLC detection diagram for the transformation solution. According to Figure S1, fewer mycelium balls were observed in medium 1 and medium 2, and the color of the mycelium ball was a little pink in medium 3, which is not its usual appearance. Only in medium 4 were the numbers of the mycelium balls ambient and the growth of mycelium good.

It can be seen from the HPLC results in Figure S1 that under the same substrate concentration, the ability of transformation media 1, 2, and 3 to transform rabeprazole sulfide is low, and the transformation products are relatively miscellaneous. However, transformation medium 4 had a higher ability to transform rabeprazole sulfide, and only one transformation product was produced. Therefore, based on the results in Figures S1 and S2, medium 4 was selected as the medium for subsequent fermentation. It can be seen from the HPLC results in Figure S1 that under the same substrate concentration, the ability of transformation media 1, 2, and 3 to transform rabeprazole sulfide is low, and the transformation products are relatively miscellaneous. However, transformation medium 4 had a higher ability to transform rabeprazole sulfide, and only one transformation product was produced. Therefore, based on the results in Figures S1 and S2, medium 4 was selected as the medium for subsequent fermentation.

#### *2.3. Single-Factor Evaluation for Rabeprazole Sulfide Biotransformation 2.3. Single-Factor Evaluation for Rabeprazole Sulfide Biotransformation*

## 2.3.1. Standard Curve of Rabeprazole Sulfide

rate of rabeprazole sulfide.

2.3.1. Standard Curve of Rabeprazole Sulfide Figure 3 indicates that the linear correlation coefficient R2 = 0.9995 and the correlation equation y = 18,285x + 66.794 for the concentration and peak area of rabeprazole sulfide, with a good linear correlation. This standard curve can be used to calculate the conversion Figure 3 indicates that the linear correlation coefficient R<sup>2</sup> = 0.9995 and the correlation equation y = 18,285x + 66.794 for the concentration and peak area of rabeprazole sulfide, with a good linear correlation. This standard curve can be used to calculate the conversion rate of rabeprazole sulfide. *Catalysts* **2023**, *13*, x FOR PEER REVIEW 4 of 13

> is too long, product inhibition may occur and the total transformation rate may decrease. In this section, we investigate the effect of conversion time on the production of metabo-

> **Figure 4.** Effects of culture time on metabolite conversion rate (initial pH = 6.5, media volume 50

When the transformation was performed at 28 °C, 200 rpm, it could be seen that the culture time ranged between 6 and 9 days. A culture time of 7 days resulted in the optimal

The initial pH of the media is vital for strain growth and transformation. A pH that is too low or high will cause the strains to undergo non-ambient growth and affect their metabolism capability. This is why different strains desire specific initial pH values in their media. In this section, a pH range of 5.5 to 8.0 was selected for the investigation. From Figure 5, it can be seen that both the acid and alkaline conditions are not fit for

**Figure 3.** Standard curve of rabeprazole sulfide. **Figure 3.** Standard curve of rabeprazole sulfide.

2.3.2. Evaluation of Culture Time

lites; the results are shown in Figure 4.

mL, inoculation volume 10%, substrate concentration 1 mg/mL).

biotransformation, so the best initial pH is set to 6.0.

conversion rate of 74.71%.

2.3.3. Evaluation of Initial pH

#### 2.3.2. Evaluation of Culture Time 2.3.2. Evaluation of Culture Time

**Figure 3.** Standard curve of rabeprazole sulfide.

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 4 of 13

The culture duration of whole-cell catalysis has an important effect on the reaction. If the reaction time is too short, the reaction may not be accomplished. If the reaction time is too long, product inhibition may occur and the total transformation rate may decrease. In this section, we investigate the effect of conversion time on the production of metabolites; the results are shown in Figure 4. The culture duration of whole-cell catalysis has an important effect on the reaction. If the reaction time is too short, the reaction may not be accomplished. If the reaction time is too long, product inhibition may occur and the total transformation rate may decrease. In this section, we investigate the effect of conversion time on the production of metabolites; the results are shown in Figure 4.

**Figure 4.** Effects of culture time on metabolite conversion rate (initial pH = 6.5, media volume 50 mL, inoculation volume 10%, substrate concentration 1 mg/mL). **Figure 4.** Effects of culture time on metabolite conversion rate (initial pH = 6.5, media volume 50 mL, inoculation volume 10%, substrate concentration 1 mg/mL).

When the transformation was performed at 28 °C, 200 rpm, it could be seen that the culture time ranged between 6 and 9 days. A culture time of 7 days resulted in the optimal conversion rate of 74.71%. When the transformation was performed at 28 ◦C, 200 rpm, it could be seen that the culture time ranged between 6 and 9 days. A culture time of 7 days resulted in the optimal conversion rate of 74.71%.

#### 2.3.3. Evaluation of Initial pH 2.3.3. Evaluation of Initial pH

The initial pH of the media is vital for strain growth and transformation. A pH that is too low or high will cause the strains to undergo non-ambient growth and affect their metabolism capability. This is why different strains desire specific initial pH values in their media. In this section, a pH range of 5.5 to 8.0 was selected for the investigation. From Figure 5, it can be seen that both the acid and alkaline conditions are not fit for biotransformation, so the best initial pH is set to 6.0. The initial pH of the media is vital for strain growth and transformation. A pH that is too low or high will cause the strains to undergo non-ambient growth and affect their metabolism capability. This is why different strains desire specific initial pH values in their media. In this section, a pH range of 5.5 to 8.0 was selected for the investigation. From Figure 5, it can be seen that both the acid and alkaline conditions are not fit for biotransformation, so the best initial pH is set to 6.0.

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 5 of 13

**Figure 5.** Effects of the initial pH of the media on the metabolite conversion rate (culture time 5 d, media volume 50 mL, inoculation volume 10%, substrate concentration 1 mg/mL). **Figure 5.** Effects of the initial pH of the media on the metabolite conversion rate (culture time 5 d, media volume 50 mL, inoculation volume 10%, substrate concentration 1 mg/mL). **Figure 5.** Effects of the initial pH of the media on the metabolite conversion rate (culture time 5 d, media volume 50 mL, inoculation volume 10%, substrate concentration 1 mg/mL).

#### 2.3.4. Evaluation of Media Volume 2.3.4. Evaluation of Media Volume 2.3.4. Evaluation of Media Volume

The media volume is crucial for the transformation effects, which are related to the dissolved oxygen when the conical flask volume is constant. Media volumes from 30 mL to 120 mL were selected for the investigation. From Figure 6, it can be seen that a media volume of 50 mL displayed the best transformation effect. The media volume is crucial for the transformation effects, which are related to the dissolved oxygen when the conical flask volume is constant. Media volumes from 30 mL to 120 mL were selected for the investigation. From Figure 6, it can be seen that a media volume of 50 mL displayed the best transformation effect. The media volume is crucial for the transformation effects, which are related to the dissolved oxygen when the conical flask volume is constant. Media volumes from 30 mL to 120 mL were selected for the investigation. From Figure 6, it can be seen that a media volume of 50 mL displayed the best transformation effect.

**Figure 6.** Effects of media volume on metabolite conversion rate (initial pH = 6.5, culture time 5 d, inoculation volume 10%, substrate concentration 1 mg/mL). **Figure 6.** Effects of media volume on metabolite conversion rate (initial pH = 6.5, culture time 5 d, inoculation volume 10%, substrate concentration 1 mg/mL). **Figure 6.** Effects of media volume on metabolite conversion rate (initial pH = 6.5, culture time 5 d, inoculation volume 10%, substrate concentration 1 mg/mL).

2.3.5. Evaluation of Inoculation Volume 2.3.5. Evaluation of Inoculation Volume 2.3.5. Evaluation of Inoculation Volume

Inoculation volume is a critical factor for the biotransformation process. If the inoculation volume is high, the strains will grow to the exponential phase too early and the nutrients will be depleted quickly, such that the enzymes in the strains may not sufficiently be induced and expressed, and the biotransformation may not be adequate. If the inoculation volume is low, the strains may take too long a time to grow and induce enzymes, which may decrease their metabolism capabilities. Based our previous experi-Inoculation volume is a critical factor for the biotransformation process. If the inoculation volume is high, the strains will grow to the exponential phase too early and the nutrients will be depleted quickly, such that the enzymes in the strains may not sufficiently be induced and expressed, and the biotransformation may not be adequate. If the inoculation volume is low, the strains may take too long a time to grow and induce en-Inoculation volume is a critical factor for the biotransformation process. If the inoculation volume is high, the strains will grow to the exponential phase too early and the nutrients will be depleted quickly, such that the enzymes in the strains may not sufficiently be induced and expressed, and the biotransformation may not be adequate. If the inoculation volume is low, the strains may take too long a time to grow and induce enzymes, which

ences, inoculation volumes ranging from 6% to 14% were selected for the investigation.

zymes, which may decrease their metabolism capabilities. Based our previous experiences, inoculation volumes ranging from 6% to 14% were selected for the investigation.

may decrease their metabolism capabilities. Based our previous experiences, inoculation volumes ranging from 6% to 14% were selected for the investigation.

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*Catalysts* **2023**, *13*, x FOR PEER REVIEW 6 of 13

From Figure 7, it can be seen that an inoculation volume of 10% is the best for biotransformation. From Figure 7, it can be seen that an inoculation volume of 10% is the best for biotransformation. From Figure 7, it can be seen that an inoculation volume of 10% is the best for biotransformation.

**Figure 7.** Effects of inoculation volume on metabolite conversion rate (initial pH = 6.5, culture time 5 d, media volume 50 mL, substrate concentration 1 mg/mL). **Figure 7.** Effects of inoculation volume on metabolite conversion rate (initial pH = 6.5, culture time 5 d, media volume 50 mL, substrate concentration 1 mg/mL). **Figure 7.** Effects of inoculation volume on metabolite conversion rate (initial pH = 6.5, culture time 5 d, media volume 50 mL, substrate concentration 1 mg/mL).

#### 2.3.6. Evaluation of Substrate Concentration 2.3.6. Evaluation of Substrate Concentration 2.3.6. Evaluation of Substrate Concentration

In this study, rabeprazole sulfide is not a natural substrate, which may cause severe inhibition and toxic effects for the strains. Hence, a proper substrate concentration is essential for successful biotransformation. A substrate concentration range from 1 mg/mL to 5 mg/mL was set for the investigation. In this study, rabeprazole sulfide is not a natural substrate, which may cause severe inhibition and toxic effects for the strains. Hence, a proper substrate concentration is essential for successful biotransformation. A substrate concentration range from 1 mg/mL In this study, rabeprazole sulfide is not a natural substrate, which may cause severe inhibition and toxic effects for the strains. Hence, a proper substrate concentration is essential for successful biotransformation. A substrate concentration range from 1 mg/mL to 5 mg/mL was set for the investigation.

From Figure 8, it can be seen from the decreasing conversion rate tendency that 1 mg/mL substrate concentration is the best for biotransformation. to 5 mg/mL was set for the investigation.From Figure 8, it can be seen from the decreasing conversion rate tendency that 1 mg/mL substrate concentration is the best for biotransformation. From Figure 8, it can be seen from the decreasing conversion rate tendency that 1 mg/mL substrate concentration is the best for biotransformation.

time 5 d, media volume 50 mL, substrate concentration 1 mg/mL). **Figure 8.** Effects of substrate concentration on metabolite conversion rate (initial pH = 6.5, culture time 5 d, media volume 50 mL, substrate concentration 1 mg/mL). **Figure 8.** Effects of substrate concentration on metabolite conversion rate (initial pH = 6.5, culture time 5 d, media volume 50 mL, substrate concentration 1 mg/mL).

**Figure 8.** Effects of substrate concentration on metabolite conversion rate (initial pH = 6.5, culture

According to the above single-factor investigation results, a 5-factor, 4-level, orthog-

According to the above single-factor investigation results, a 5-factor, 4-level, orthog-

onal L16(45) optimization process was designed, as shown in Table 1.

onal L16(45) optimization process was designed, as shown in Table 1.

*2.4. Orthogonal Optimization*

*2.4. Orthogonal Optimization*

#### *2.4. Orthogonal Optimization*

According to the above single-factor investigation results, a 5-factor, 4-level, orthogonal L16(4<sup>5</sup> ) optimization process was designed, as shown in Table 1.


**Table 1.** Orthogonal experimental factors and level assignments.

After the design and execution of the experiments, it could be seen that the strength of impact of the various factors on biotransformation was in the order D > B > E > C > A, i.e., the most significant influence was that of the initial pH value of the media, as shown in Table 2. When the range analysis was performed, it was found that the best combination was D2B1E3C4A4, so an initial pH of 6.0, inoculation volume of 8%, substrate concentration of 3 mg/mL, media volume of 100 mL, and culture time of 8 days were the best values. These values are consistent between the best results from the orthogonal design and those from the No.13 experimental group (total 16 groups), with a conversion rate of 72.4%. This is almost a double increase in the conversion rate compared with the unoptimized results.

**Table 2.** Orthogonal experimental results and analysis.


*2.5. Structure Identification for the Rabeprazole Sulfide Biotransformation*

The isolated metabolite was resolved in DMSO for the MS and NMR analysis. According to the ESI-MS results, showing that the *m*/*z* [M+H]<sup>+</sup> is 330.25 (Figure S3) and the *m*/*z*

[M-H]− is 328.27 (Figure S4), it could be inferred that the target metabolite has a molecular weight of 329.12, which is only a Dalton loss of 14 compared with the original rabeprazole sulfide with a molecular weight of 343.44. Hence, the loss of a methyl group is assumed.

According to the comparison of the NMR data between the substrate and metabolite, it could be found that a methyl and a carbon signal are absent in the H spectrum and C spectrum. Combined with the MS and NOESY data (Figure S7) indicating a methyl loss, it can be concluded that the target metabolite is the *O*-dimethyl rabeprazole sulfide (Figure 1). The analysis is given below.

<sup>1</sup>H-NMR (600 MHz, DMSO-*d6*) δH: 8.23 (1H, d, *J* = 5.6 Hz), 7.45 (2H, dd, *J*1 = 5.8, *J*2 = 3.2 Hz), 7.11 (2H, dd, *J*1 = 5.9, *J*2 = 3.1 Hz), 6.95 (1H, d, *J* = 5.6 Hz), 4.69 (2H, s), 4.12 (2H, t, *J* = 6.2 Hz), 3.58 (2H, t, *J* = 6.2 Hz), 2.21 (3H, s), 1.87–1.91 (2H, m).

<sup>13</sup>C-NMR (150 MHz, DMSO-*d6*) δC: 163.27, 155.16, 150.90, 148.24, 121.73, 120.20, 106.78, 65.50, 57.56, 36.74, 32.31, 10.88.

#### **3. Materials and Methods**

#### *3.1. Materials and Strains*

Rabeprazole sulfide was synthesized in the Department of Pharmaceutical Engineering, Shenyang Pharmaceutical University. Methanol and acetonitrile were purchased from Concord Technology Co., Ltd. (Tianjin, China). Peptone and yeast extract were purchased from HopeBio Co., Ltd. (Qingdao, China). All other chemical reagents were purchased from Yuwang Chemical Co., Ltd. (Shenyang, China).

*Cunninghamella blakesleeana* 3.970 and *Cunninghamella elegans* 3.910 were stored in our lab reservoir. *Cunninghamella echimnulata* 3.967, *Absidia coerulea* 41,050, *Gibberella fujiluroi* 40,272, *Gibberella* sp. 2498, and *Caldariomyces fumago* 16,373 were purchased from China Center of Industrial Culture Collection (CICC).

#### *3.2. Media and Strain Cultivations*

Slant and agar media consisted of potato (200 g), glucose (20 g), agar (20 g), and 1000 mL distilled water. Seed culture media consisted of potato starch (45 g), yeast extract (3 g), corn steep liquor (10 g), CaCO<sup>3</sup> (3 g), MgSO<sup>4</sup> (0.5 g), and FeSO<sup>4</sup> (0.05 g). Four kinds of biotransformation culture media ingredients are listed in Table 3. For all media, the pH was adjusted to 6.5 with 6 M HCl, autoclaved at 115 ◦C for 30 min, and cooled before use.

**Table 3.** Four different biotransformation media formulas.


Two loops of mycelium from a fresh growth slant were inoculated into the seed culture media and cultured at 28 ◦C, 200 rpm for 48 h. Then, 5 mL of seed culture was inoculated into the transformation media (50 mL in a 250 mL conical flask, rabeprazole sulfide as substrate) and cultured at 28 ◦C, 200 rpm for 5 days. Substrate controls were set without inoculating the fungi into the media and strain controls were set without adding the substrate into the media, with all other conditions remaining the same.

## *3.3. Thin-Layer Chromatography (TLC)*

The biotransformation results of the fungal extracts were analyzed by TLC performed on a Merck Silica gel 60 F254 plate (Merck, Darmstadt, Germany) with ethyl acetate:methanol:ammonia water (90:9:1 *v*/*v*/*v*) as the mobile phase, and a UV detection wavelength of 254 nm. A mixture of rabeprazole sulfide and rabeprazole (10 mg) was dissolved in 100 mL of isopropyl alcohol used as a standard solution. For TLC detection, 10 µL of standard solution and fungal extracts was spotted onto TLC plates.

#### *3.4. Extraction of Metabolites and HPLC Detection*

Biotransformation cultures were collected, ultrasonically treated for 30 min, and centrifugated at 3000 rpm for 10 min to retrieve the supernatant. Pellets were washed and extracted three times using ethyl acetate. The combined water phase and organic phase were extracted three times with an equal volume of ethyl acetate. The organic phase was then isolated and evaporated by reduced pressure distillation to obtain the raw metabolites. The raw metabolites were dissolved in methanol for further use.

The HPLC detection process for the metabolites was derived from the Pharmacopeia of the People's Republic of China (2020 Volume II) and optimized according to our practical work. For High-performance liquid chromatography (HPLC), a Shimadzu WondaSil C18 Superb (250 × 4.6 mm × 5 µm) column (Shimadzu Corp., Kyoto, Japan) was used. The mobile phase consisted of 0.015 mol/L Na2HPO4:acetonitrile = 60:40, the detection wavelength was 290 nm, the column temperature was 30 ◦C, the loading speed was 1 mL/min, and the loading volume was 10 µL.

#### *3.5. Isolation and Identification of Major Metabolite*

The isolation of the target metabolite was performed by semipreparative HPLC using an Elite SinoChrom ODS-BP (250 × 10 mm × 5 µm, Dalian, China) consisting of a quaternary pump, a vacuum degasser, a variable wavelength detector, and an autosampler. The mobile phase consisted of acetonitrile and water in different percentages, and the proper fractions were collected.

ESI-MS and NMR analysis were performed under standard conditions.

## *3.6. Determination of the Standard Curve*

Rabeprazole sulfide was accurately weighed, and a solution of 0.40 mg/mL was prepared, diluted step by step with methanol. Standard samples of 0.40, 0.30, 0.20, 0.15, 0.10, and 0.05 mg/mL were prepared and detected with HPLC, and a standard curve was drawn with rabeprazole sulfide concentration as the abscissa and peak area as the ordinate. This standard curve was used to calculate the conversion of rabeprazole sulfide. The total conversion rate was calculated according to the following equation:

> Conversion rate % = 1 − residue amount of raberprazole sulfide Starting amount of rabeprazole sulfide

#### **4. Conclusions and Discussion**

By screening seven filamentous fungi, rabeprazole sulfide was the most efficiently decomposed into *O*-dimethyl rabeprazole sulfide by *Cunninghamella blakesleeana* 3.970, with a conversion rate of 72.4%. This is not a common biotransformation reaction type, with only few studies having suggested the *N*-demethylation [36,37] and *O*-dimethyl reactions [38,39].

The original aim of this study was to explore the possibility of whether these fungi could exhibit some oxidase activity so that the rabeprazole sulfide could be transformed into rabeprazole, i.e., to determine whether the thioether structure could be oxidized into the sulfoxide. Despite *Cunninghamella* species having exhibited various kinds of oxidation in previous works, no oxidation metabolites were identified in this research. Several reports have shown that *Absidia* and *Gibberella* species could exhibit oxidase activity as

their P450, but positive oxidation metabolites have yet to be generated. More importantly, *Caldariomyces fumago* was selected for its strong inner chloroperoxidase expression, which exhibited multiple oxidation types, such as epoxidation [40,41], hydroxylation [42–47], halogenation [48], etc. However, none of the above reactions were identified in this rabeprazole sulfide biotransformation.

Rabeprazole sulfide is actually not a natural substrate of the fungus, which was mentioned in the previous section on the influence of substrate concentration on the conversion rate. With the increase in substrate concentration, the conversion rate decreases (Figure 8), and a too-high concentration may produce some inhibition. It is speculated that the *O*-demethylation reaction of rabeprazole sulfide is not completed in one step. It may be that rabeprazole sulfide is first converted into one or several intermediate products and then further converted into *O*-demethyl rabeprazole sulfide.

Some microorganisms can transform drugs and other xenobiotic compounds in a manner similar to that in mammals, and the utilization of microbial systems as models for mimicking and predicting the metabolism of drugs in humans and animals has received considerable attention. Liu et al. reported that the main metabolic pathways of *Cunninghamella blakesleeana* to transform verapamil are *N*-dealkylation, *O*-demethylation, and sulfate coupling, and the *O*-demethylated metabolites of verapamil have the same potency as the parent drug [49]. Xie ZY et al. reported that approximately 92.5% of pantoprazole was metabolized to six metabolites by *Cunninghamella blakesleeana* AS 3.153, one of which was 40 -*O*-demethyl-pantoprazole thioether [30]. These studies did not deeply explore the mechanism of *O*-demethylation when *Cunninghamella blakesleeana* transformed the substrate. Therefore, it is of interest to further study the mechanism of the *O*-demethylation of *Cunninghamella blakesleeana* and the pharmacological or biological characteristics of these *O*-demethylated metabolites.

In addition, further investigation should be undertaken in the hunt for fungi that may oxidize thioether to sulfoxide. Meanwhile, other rational designs for the engineered enzyme to facilitate this reaction are also welcomed.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/catal13010015/s1, Figure S1. Growth of *Cunninghamella blakesleeana* 3.970 in 4 different transformation media. Figure S2. HPLC detection results of *Cunninghamella blakesleeana* 3.970 in 4 different transformation media. Figure S3. HPLC detection results of rabeprazole sulfide and its metabolites. Figure S4. HPLC detection results of rabeprazole sulfide metabolites for MS and NMR analysis. Figure S5. MS(ESI)*m*/*z*[M+H]<sup>+</sup> diagram for the metabolite. Figure S6. MS(ESI)*m*/*z*[M+H]<sup>−</sup> diagram for the metabolite. Figure S7. <sup>1</sup>H NMR diagram for the *O*-demethyl rabeprazole sulfide. Figure S8. <sup>13</sup>C NMR diagram for the *O*-demethyl rabeprazole sulfide. Figure S9. <sup>1</sup>H NOESY diagram for the *O*-demethyl rabeprazole sulfide.

**Author Contributions:** M.S. (data curation, investigation); H.Z. (investigation, methodology); J.W. (rabeprazole and its sulfide preparation); W.X. (Weizhuo Xu) and W.X. (Wei Xu) (resources, supervision, writing—review and editing). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Data are available upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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