Natural products, with their novel and diverse structures and various biological activities, are one of the significant sources for new drug development research. However, they also have many drawbacks, such as poor biological activity, low bioavailability, nonspecificity in action, and significant toxic and side effects. Therefore, structural modification and alteration are needed to improve the properties of natural products. These modifications, including acetylation, methylation, glycosylation, and hydroxylation, can enrich the diversity of natural products while enhancing their biological activity and bioavailability to varying degrees. Among them, glycosylation modification has remarkable effects in improving solubility and stability.

Due to the complex and diverse structures of natural products, chemical glycosylation modification has always been one of the difficult problems to solve. In contrast, enzymatic glycosylation modification, which is characterized by strong selectivity, high reaction efficiency, and mild conditions, has gradually emerged as an advantageous approach in improving the solubility, stability, and biological activity of natural products, and has achieved certain research results.

Improving Solubility

Resveratrol glycoside, the main active component of Polygonum cuspidatum, has a low solubility in water, at 0.03g·L-1, requiring structural modification to enhance its bio-water solubility and increase its bioavailability. Utilizing the maltosyltransferase from Thermomonospora curvata DSM 6725 and maltotriose as a donor, glycosylation modification of resveratrol glycoside produced four glycosylated products, with the main product, α-D-maltosyl-(1,4)-resveratrol glycoside, achieving a solubility of 128.09 g/L. This represents an 8.54×103 and 1.86×103 fold increase compared to resveratrol and resveratrol glycoside, respectively. Meanwhile, the α-1,4-glycosidic bond is easily hydrolyzed by glycosidases in the human body, indicating that the metabolic process of α-D-maltosyl-(1,4)-resveratrol glycoside in vivo may be similar to resveratrol glycoside or resveratrol, enabling it to exhibit good anti-tumor and anti-atherosclerotic effects. Pterostilbene, whose core structure is resveratrol, is an extract from blueberries, grapes, and other sources, with good biological activities such as antibacterial, anti-tumor, and lipid-lowering effects. However, its solubility in water is low. Using a one-pot method to glycosylate pterostilbene results in a significant improvement in the water solubility of the product compared to pterostilbene itself.

Daidzein possesses biological activities such as treating tumors, cardiovascular diseases, and menopausal syndrome, but its extremely low water solubility limits its further drug development. Some scholars have utilized the maltosyltransferase MTase from the extreme thermophilic bacterium Thermotoga maritima and maltotriose as a donor to glycosylate daidzein, resulting in four daidzein glycosides. Among them, the solubility of the main product, daidzein 7-O-triglucoside, increased by 7.5×103 times. However, the disadvantage of non-unique products needs to be addressed and improved.

Acacetin is a dihydroxy methoxy flavonoid compound. Research has shown that acacetin possesses various biological activities, including anti-tumor and heart-protecting effects. Using brominated glucose as a glycosyl donor, glycosylation modification was performed on the 7-OH position of acacetin, resulting in the synthesis of three major target monomeric compounds. Water solubility tests were conducted on each of these target monomeric compounds, and the results indicated that the water solubility of the acacetin glycoside derivatives after glycosylation modification increased by 1000 to 18000 times.

Mangiferin is a natural organic plant polyphenol from mangoes, exhibiting extensive pharmacological activities, such as anti-inflammatory, antioxidant, anti-acute diabetes, anti-cancer, immune function regulation, and protection of heart and liver functions. It is reported that some metabolites of mangiferin may exert stronger effects, such as its phase II metabolite, mangiferin-7-O-D-glucuronide. However, the current research on the biological characteristics of mangiferin metabolite, mangiferin-7-O-D-glucuronide, is still limited, restricting further investigation of its pharmacological properties. Using natural mangiferin as the starting material, mangiferin-7-O-D-glucuronide was first synthesized through a chemical semi-synthetic method, significantly improving the water solubility of mangiferin. This provides a reference basis for the glycosylation modification of flavonoid glycosides.

Icariin, the main active ingredient of Epimedium, possesses a variety of pharmacological activities. Like most flavonoid compounds, its disadvantage is poor solubility. By using acetyl bromide glucose as a glycosyl donor to glycosylate the C7-OH of icariin and subsequently undergoing deacetylation, icariside I is obtained. Research results show that the solubility of icariside I is improved compared to icariin.

Utilizing the cyclodextrin glucosyltransferase from alkaliphilic Bacillus and water-soluble starch as a donor, site-specific glucosylation modification of naringin, neohesperidin, and hesperidin was achieved, resulting in the production of 3-α-D-glucosylnaringin, 3-α-D-glucosylneohesperidin, and 4-α-D-glucosylhesperidin. The solubility of these compounds increased by 1.0 × 103 times, 1.5 × 103 times, and 0.3 × 103 times, respectively. Additionally, the bitterness of 3-α-D-glucosylneohesperidin was reduced by 90%.

Oleanolic acid is a pentacyclic triterpenoid compound isolated from plants of the genus Swertia in the gentianaceae family, such as Prunella vulgaris and Gardenia jasminoides fruits. It exists in both free and glycoside-bound forms. It has pharmacological effects in anti-tumor, liver protection, anti-aging, and other aspects. However, due to its low solubility in water, it has not been widely used in clinical practice. Through glycosylation modification of the C3-OH group of oleanolic acid using glucose, galactose, and glucosamine as glycosyl donors, three target compounds with improved water solubility were obtained.

Increasing Stability

Anthocyanin flavonoids exist in plant cells throughout nature and are a type of water-soluble natural plant pigment with strong human biological activities, such as anti-tumor and antioxidant effects. They are widely used in the field of plant nutrition and health foods. Research results show that a single anthocyanin cannot stably exist naturally in various environments and requires chemical modification processes such as glycosylation, acyl peroxidation, and methyl peroxidation to increase its chemical stability. Using Escherichia coli heterologously expressed 3-O-glucosyltransferase (3GT) and the glycosyl donor UDP-G, glucose modification was performed on the 3-hydroxyl group of anthocyanins, resulting in anthocyanin 3-O-glucoside. Anthocyanins rapidly degrade in solutions with a pH greater than 3.0, but glycosylation modification significantly improves their stability, allowing anthocyanin 3-O-glucoside to stably exist in a pH 5.0 solution for over 96 hours.

Catechin is a type of flavonoid alcohol compound with various biological activities, such as antibacterial and antioxidant properties, which exists in nature and microbial environments. However, catechin is unstable. By using UDP-glucose glucosyltransferase (UDPG) to structurally modify the alkaline catechin, the stability of the catechin glycoside derivative is improved compared to the original drug, with an average stable existence of over 80%. The obtained glycoside derivative exhibits improved stability compared to (+)-catechin, effectively addressing the clinical trial challenges of poor chemical stability associated with various alkaline catechin products.

Rosavin is an active and indicator component of roses and Rhodiola rosea, possessing various pharmacological activities such as anti-radiation, antioxidant, and anti-tumor effects. Using fully acetylated α-D-glucose as the glycosyl donor and cinnamyl alcohol as the raw material, rosavin is synthesized through a series of reactions. Through genetic engineering and other reactions, as well as glycosylation modification, a non-natural cinnamyl alcohol diglucoside (cinnamyl alcohol) is synthesized, which exhibits improved stability compared to the original cinnamyl alcohol.

Enhancing Pharmacological Activity

Hederagenin is a type of triterpenoid compound with low solubility in water and low bioavailability, which greatly limits its application. To address this issue, structural modifications were made to hederagenin. Taking hederagenin as the lead compound, intermediate g was obtained through steps such as methylation and acetylation. Then, using glycosyl as the modifying group, structural modifications were made to intermediate g, resulting in three types of hederasaponin derivatives (h-j). Studies on the anti-tumor activity of compounds g-j showed that the hederasaponin derivatives had inhibitory effects on human bladder cancer cells. At a concentration of 1×10-6 mol·L-1, the inhibition rate of the glycoside derivatives was increased by 3 to 4 times compared to the hederagenin intermediate g.

Digitoxin is a widely used cardiotonic glycoside drug whose good antitumor activity has attracted widespread attention. Due to its high toxicity and narrow therapeutic window, structural modifications are needed. Using digitoxin as the starting material and glucose and xylose derivatives as glycosyl donors, a series of aminoglycoside derivatives of digitoxin were synthesized. Anti-tumor activity studies found that most of the glycoside derivatives exhibited an increase in activity by 1 to 10 times.

Camptothecin is an alkaloid isolated from the fruits of Camptotheca acuminata. Camptothecin possesses various anti-tumor activities and is used in the treatment of multiple malignant tumors. Glycosylation modification was performed on camptothecin to synthesize several glycoconjugates. Anti-tumor studies on the obtained camptothecin glycoside derivatives revealed that for the five tested tumor cell lines, most of the glycoside derivatives had IC50 values 2 to 10 times smaller than that of camptothecin, indicating that the modified camptothecin glycoside derivatives possessed similar or better anti-tumor activity compared to the original camptothecin.

Enhancing Target Specificity

Carbohydrate substances possess strong tumor cell-targeting specificity and recognition abilities. Therefore, the targeting performance of drug carriers can be improved after glycosylation modification, ultimately enhancing tumor treatment efficacy with lower adverse reactions. Studies have found that paclitaxel micelles modified with mannose as the glycosyl donor accumulate at higher levels in the livers of transplanted tumor mice compared to ordinary micelles. Dichondra repens, clinically used as a drug for treating chronic hepatitis, undergoes structural modification of its active ingredient, dichondrin. It was discovered that the dichondrin glycoside derivative can not only inhibit Hepatitis B virus (HBV) DNA in Hep G-2 cells, but also exhibits liver targeting properties. 10-Hydroxycamptothecin (10-HCPT), isolated from Camptotheca acuminata, possesses significant antitumor activity. Research has shown that it has an apoptotic effect on human SPC-A-1 lung cancer cell lines, and HCPT is already clinically used as an anti-lung cancer drug. However, due to its poor water solubility, sodium salt injections are required to improve solubility, which alters the structure of 10-HCPT and leads to changes in its antitumor activity. Structural modification of 10-HCPT using mannose as the glycosyl donor results in a hydroxycamptothecin glycoside derivative that exhibits significant lung targeting properties. Additionally, studies have found that using mannose as the glycosyl donor and liposomes as drug carriers, puerarin modified with these compounds can specifically target cerebral ischemic areas in rats.

According to the principles of biopharmaceutics, the oral absorption of drugs is largely limited by their solubility and dissolution rate. Therefore, introducing readily available and inexpensive sugars as glycosyl donors into the structures of medicinally valuable natural products can enhance the water solubility and improve the bioavailability of drugs, achieving the development and application of natural medicines. After structural modification of natural products, due to changes in their structures, their biological activities may also be altered, and even new biological activities may emerge. Furthermore, the utilization of chemical or biological methods to catalyze the glycosylation of natural products not only increases the diversity of drug structures but also provides feasible strategies for structural modification.

References:

[1] Lan Qing, Sun Chengxin, Wang Xianheng, Peng Junke, He Yuqi, Wang Yuhe, Zhao Changkuo. Application of Glycosylation in the Structural Modification of Natural Products [J]. Chinese Journal of New Drugs, 2022, 31(16): 1595-1601.

[2] Jin Yue, Wu Xuri, Chen Yijun. Application of Glycosyltransferases in Improving the Drug Properties of Natural Products [J]. Journal of China Pharmaceutical University, 2017, 48(05): 529-535.

About the Author

Xiaomichong, a pharmaceutical quality researcher, has been committed to pharmaceutical quality research and drug analysis method validation for a long time. Currently employed by a large domestic pharmaceutical research and development company, she is engaged in drug inspection and analysis as well as method validation.