No abstract

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙+ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages.

Engineering dual-function single polypeptide catalysts with two abiotic or biotic catalytic entities (or combinations of both) supporting cascade reactions is becoming an important area of enzyme engineering and catalysis. Herein we present the development of a PluriZyme, TR 2 E 2 , with efficient native transaminase (k cat : 69.49 � 1.77 min À 1 ) and artificial esterase (k cat : 3908-0.41 min À 1 ) activities integrated into a single scaffold, and evaluate its utility in a cascade reaction. TR 2 E 2 (pH opt : 8.0-9.5; T opt : 60-65 °C) efficiently converts methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate into 3-(R)-amino-4-(2,4,5-trifluorophenyl)butanoic acid, a crucial intermediate for the synthesis of antidiabetic drugs. The reaction proceeds through the conversion of the β-keto ester into the β-keto acid at the hydrolytic site and subsequently into the β-amino acid (e.e. > 99 %) at the transaminase site. The catalytic power of the TR 2 E 2 PluriZyme was proven with a set of β-keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions.

(–)-Epigallocatechin gallate (EGCG), the predominant catechin (≥50%) in green tea ( Camellia sinensis ), displays several bioactive properties but its stability and bioavailability are low. In this work, the properties of two α-glucosyl derivatives of EGCG (3′- and 7-O-α-D-glucopyranoside), obtained by enzymatic synthesis, were assessed. The α-glucosylation enhanced the pH and thermal stability of EGCG. The analysis of scavenging activity toward ABTS · + radicals showed that the α-glucosylation at C-7 of A-ring caused a higher loss of antioxidant activity compared with the sugar conjugation at C-3′ of B-ring. The 3′-glucoside also showed higher potential to alleviate intracellular reactive oxygen species (ROS) levels and to boost REDOX activity. The toxicity of EGCG and its monoglucosides was tested in human SH-S5Y5 neurons, RAW 264.7 macrophages, MRC5 fibroblasts, and HT-29 colon cancer cells. Interestingly, the 3′-O-α-D-glucoside increased the viability of neural cells in vitro (2.75-fold at 100 μM) in the presence of H 2 O 2 , whilst EGCG gave rise only to a 1.7-fold enhancement. In conclusion, the α-glucoside of EGCG at C-3′ has a great potential for nutraceutical, cosmetic and biomedical applications.

Glycosylation of polyphenols may increase their aqueous solubility, stability, bioavailability and pharmacological activity. Herein, we used a mutant of sucrose phosphorylase from Thermoanaerobacterium thermosaccharolyticum engineered to accept large polyphenols (variant TtSPP_R134A) to produce phloretin glucosides. The reaction was performed using 10% (v/v) acetone as cosolvent. The selective formation of a monoglucoside or a diglucoside (53% and 73% maximum conversion percentage, respectively) can be kinetically controlled. MS and 2D‐NMR determined that the monoglucoside was phloretin 4’‐O‐α‐D‐glucopyranoside and the diglucoside phloretin‐4’‐O‐[α‐D‐glucopyranosyl‐(1→3)‐O‐α‐D‐glucopyranoside], a novel compound. The molecular features that determine the specificity of this enzyme for 4’‐OH phenolic group were analysed by induced‐fit docking analysis of each putative derivative, using the crystal structure of TtSPP and changing the mutated residue. The mono‐ and diglucoside were, respectively, 71‐ and 1200‐fold more soluble in water than phloretin at room temperature. The α‐glucosylation decreased the antioxidant capacity of phloretin, measured by DPPH and ABTS assays; however, this loss was moderate and the activity could be recovered upon deglycosylation in vivo. Since phloretin attracts a great interest in dermocosmetic applications, we analyzed the percutaneous absorption of glucosides and the aglycon employing a pig skin model. Although the three compounds were detected in all skin layers (except the fluid receptor), the diglucoside was present mainly on superficial layers.

The glycosylation of plant polyphenols may modulate their solubility and bioavailability and protect these molecules from oxygen, light degradation, and during gastrointestinal transit. In this work, the synthesis of various α-glucosyl derivatives of (-)-epigallocatechin gallate, the predominant catechin in green tea, was performed in water at 50 °C by a transglycosylation reaction catalyzed by cyclodextrin glycosyltransferase from Thermoanaerobacter sp. The molecular weight of reaction products was determined by high-performance liquid chromatography coupled to mass spectrometry. Using hydrolyzed potato starch as a glucosyl donor, two main monoglucosides were obtained with conversion yields of 58 and 13%, respectively. The products were isolated and chemically characterized by combining two-dimensional nuclear magnetic resonance methods. The major derivative was epigallocatechin gallate 3'- O-α-d-glucopyranoside (1), and the minor derivative was epigallocatechin gallate 7- O-α-d-glucopyranoside (2).

No abstract

The regioselective α-glucosylation of hesperetin was achieved by a transglycosylation reaction catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. using soluble starch as glucosyl donor. By combining mass spectrometry (ESI-TOF) and 2D-NMR analysis, the main monoglucosylated derivative was fully characterized (hesperetin 7-O-α-d-glucopyranoside). In order to increase the yield of monoglucoside, several reaction parameters were optimized: Nature and percentage of cosolvent, composition of the aqueous phase, glucosyl donor, temperature, and the concentrations of hesperetin and soluble starch. Under the optimal conditions, which included the presence of 30% of bis(2-methoxyethyl) ether as cosolvent, the maximum concentration of monoglucoside was approximately 2 mM, obtained after 24 h of reaction. To our knowledge, this is the first report of direct glucosylation of hesperetin employing free enzymes instead of whole cells.