Phenylpropanoid Glycoside Analogues: Enzymatic Synthesis, Antioxidant Activity and Theoretical Study of Their Free Radical Scavenger Mechanism

Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols.苯丙素苷（Phenylpropanoid glycosides, PPGs）是一类广泛存在于多种药用植物中的天然化合物，具有优异的抗氧化能力与多样的生物学活性。由于其在植物体内的含量极低（重量百分比低于5% w/w），目前已开发出多条化学合成PPGs的路线，但此类合成方法普遍存在过程耗时冗长、产物收率偏低的缺陷。本研究首次报道了一种可用于制备天然PPGs类似物的高效替代两步生物合成路径：首先以香草醇与高香草醇为底物，在饱和乳糖溶液中利用乳酸克鲁维酵母（Kluyveromyces lactis）β-半乳糖苷酶催化的转半乳糖苷反应，合成了两种半乳糖苷，该步骤收率达30%~35%；随后以南极假丝酵母脂肪酶B（Candida antarctica Lipase B, CaL-B）为生物催化剂，将上述半乳糖结合物与饱和及不饱和羟基肉桂酸衍生物进行酯化反应，合成目标PPGs，该步骤收率为40%~60%。本研究采用2,2-二苯基-1-苦基肼自由基（2,2-diphenyl-1-picrylhydrazyl radical, DPPH•）法，评估了酚类原料、中间体及PPGs的自由基清除能力，结果显示生物合成的PPGs的自由基清除能力优于对照品抗坏血酸，且其抗氧化活性与天然PPGs相当。此外，本研究通过密度泛函理论（density functional theory, DFT）计算，阐明了PPGs的抗氧化机制为序贯质子丢失单电子转移（sequential proton loss single electron transfer, SPLET）。本研究报道的酶促合成路径，为以不同苯丙酸、糖类及酚醇为原料制备PPGs提供了一条高效且通用的途径。