In vitro single-vessel enzymatic synthesis of novel Resvera-A glucosides

Accepted Manuscript Title: In vitro single-vessel enzymatic synthesis of novel resvera-a glucosides Author: Ju Yong Shin, Ramesh Prasad Pandey, Ha Young Jung, Luan Luong Chu, Yong Il Park, Jae Kyung Sohng PII: DOI: Reference:

S0008-6215(16)30011-8 http://dx.doi.org/doi: 10.1016/j.carres.2016.02.001 CAR 7124

To appear in:

Carbohydrate Research

Received date: Revised date: Accepted date:

8-11-2015 31-12-2015 1-2-2016

Please cite this article as: Ju Yong Shin, Ramesh Prasad Pandey, Ha Young Jung, Luan Luong Chu, Yong Il Park, Jae Kyung Sohng, In vitro single-vessel enzymatic synthesis of novel resveraa glucosides, Carbohydrate Research (2016), http://dx.doi.org/doi: 10.1016/j.carres.2016.02.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Carbohydrate Research Note

In vitro single-vessel enzymatic synthesis of novel Resvera-A glucosides Ju Yong Shin1†, Ramesh Prasad Pandey1†, Ha Young Jung1, Luan Luong Chu1, Yong Il Park2, Jae Kyung Sohng1* 1

Institute of Biomolecule Reconstruction, Department of BT-Convergent Pharmaceutical

Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 336-708, Korea. 2

Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi-do

420-743, Korea

Corresponding author: Prof. Jae Kyung Sohng Tel: +82(41)530-2246 Fax: +82(41)544-2919 Email: [email protected] †

These authors are equally contributed.

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Highlights:  An in vitro enzymatic glycosylation system is combined with UDP recycling system.  Cost-effective UDP recycling system was established to produce UDP-α-Dglucose.  Instant removal of UDP enhanced catalytic activity of GT and yield of products.  Hundred fold lower concentration of UDP-α-D-glucose used than acceptor substrate.  Two novel resvera-A glucosides were produced in practical quantities. Graphical Abstract

Abstract An in vitro enzymatic glycosylation system is developed for the efficient synthesis of glucosides of 3,5-dihydroxy-N-(4-hydroxyphenyl) benzamide (resvera-A), a chemically synthesized

molecule

resembling

resveratrol

in

structure.

Resvera-A

is

a

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pharamacophore-based designed molecule that exhibits anti-oxidant, antibacterial, antiinflammatory, and anticancer activities. In this study, an alternative cost-effective uridine diphosphate (UDP) recycling system was established to produce UDP-α-D-glucose through a two-step enzyme-catalyzed reaction using easily available cheap sources. This UDP-α-D-glucose biosynthesis system was combined with a glycosyltransferase (YjiC, from Bacillus licheniformis)-catalyzed reaction for the synthesis of glucoside derivatives of resvera-A. The side product of the glycosylation reaction, UDP, was used as a precursor for the biosynthesis of UDP-α-D-glucose, which is used by YjiC for glycosylation, thus recycling UDP. As a result, two novel molecules, resvera-A 3-O-α-Dglucoside (42.33 mg, 2.10 mM, 0.84 mg/mL) and resvera-A 4ʹ-O-α-D-glucoside (99.38 mg, 4.87 mM, 1.98 mg/mL), were synthesized within 4 h from 50 mL preparative scale reaction using only 0.1 mM of UDP-α-D-glucose, 100 folds lower concentration than the concentration of resvera-A (10 mM) used. Structures of both products were elucidated using liquid chromatography, mass spectroscopy, and nuclear magnetic resonance analysis. Keywords: Resvera-A, Glycosylation, Glycosyltransferase, Bacillus, UDP-recycling

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Resveratrol

(trans-3,4ʹ,5ʹ-trihydroxystilbene),

a

naturally

occurring

hydroxystilbene, is an essential constituent of red wine, grape juices, and other grape derived food and drinks.1,2,3 Structurally, it has two configurations, trans (E)- and cis (Z)(Fig. 1A), and its biological activities are mostly related to its trans configuration.4,5 Resveratrol exhibits biological activities against type II diabetes,6,7,8 obesity, artherosclerosis,9 Alzheimer’s disease,10 cardiovascular diseases,11 and various cancers12 due to its intrinsic free radical (reactive oxygen species (ROS) and reactive nitrogen species (RNS)) scavenging properties.13 Ultraviolet irradiation generates ROS, which is the main contributing factor of premature aging of the skin.14,15 Long-term exposure to sunlight generates excessive ROS, which overcomes the ROS defense system of skin resulting in skin damage.16 Thus, application of antioxidants to skin could counteract the harmful effects of ROS generated as a result of UV light exposure. Though scavenges ROS, upon exposure to UV light, (E)-resveratrol transforms to the (Z)-form.9,17,18 Thus, there is an ongoing search for alternative molecules to resveratrol. Resvera-A (3,5-dihydroxy-N-(4-hydroxyphenyl) benzamide), which mimics resveratrol

structurally,

was

developed

as

an

alternative

to

resveratrol

by

pharamacophore-based molecular design which mimics to resveratrol structurally (Fig. 1A). It exhibits biological activities remarkably similar to resveratrol such as antioxidant activities, whitening, wrinkle suppression, antitumor effects, and cell regeneration efficiency, cell life extension.19 However, the solubility of the molecule was compromised, which limited its application in pharmaceuticals and cosmetics. Glycosylation could be a promising approach to enhance the water solubility of the molecule. Glycosylation is a post-biosynthesis modification of natural products (NPs)

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that diversifies the structural chemistry of molecules by combining sugar moieties from nucleotide diphosphate (NDP) sugars with biological acceptor molecules with the help of glycosyltransferases (GTs). The conjugation of a bulky sugar moiety in aglycon has been found to enhance the water solubility and stability of many natural products.20-23 NDPsugars are used as the sugar donors by most of the GTs during the glycosylation reaction. However, NDP-sugars are expensive for the enzymatic synthesis of NP glycosides on an industrial scale. Moreover, the chemical synthesis of NP glycosides is tedious and hazardous, and is a multi-step process. Engineered microbial cells have been used as an alternative to the chemical and enzymatic synthesis of various NP glycosides, especially flavonoids and related compounds.24-26 However, a number of potential hurdles of in vivo production of NP glycosides, such as low production titer, difficulty of target compound production, extraction, and purification always presented challenges in the development of an efficient in vitro cofactor recycling system for easy production of NP glycosides. In an effort to reduce the cost of NDP-sugars, a single-vessel enzymatic synthesis system was developed more than a decade ago in order to produce uridine diphosphate (UDP)-D-glucose in large amounts.27 Recently, one-pot in situ UDP--D-glucose and UDP--D2-deoxyglucose generating systems were developed and coupled with the glycosylation reaction for the production of small molecule glycosides.22,28,29 In this study, we established an alternative cost-effective UDP recycling system using minimum enzyme-catalyzed reactions with easily available cheap sources for the glucosylation reaction. The concentration of UDP--D-glucose required in an in vitro glucosylation reaction is often equal or higher than substrate concentration. Thus, largescale production of NP glucosides requires the expense of high cost NDP-sugar, which 5 Page 5 of 29

ultimately increases the cost of the final product. Hence, the in vitro enzymatic synthesis of glycosylated product demands a cost-effective alternative sugar donor, which is usually expensive to buy in the commercial market. In the glucosylation reaction using UDP--D-glucose as sugar donor, glucose moiety is attached to the acceptor substrate while UDP remains as waste in the reaction mixture. As the glucosylation reaction proceeds, UDP is accumulated, which is inhibitory for the forward glucosylation reaction as it competes with UDP--D-glucose to bind in the donor binding pocket of GT. So, if we could trap this free UDP and recycle it to synthesize UDP--D-glucose, we could enhance the glucosylation forward reaction without further addition of UDP--D-glucose in the reaction mixture. In this hypothesis, two additional enzymes utilizing UDP and consequently converting it to UDP--D-glucose were introduced in the glucosylation reaction. Acetyl phosphate kinase (ACK) traps UDP and generates uridine triphosphate (UTP) by using a molecule of acetyl phosphate. UTP is further utilized by glucose 1phosphate uridylyltransferase (GalU) and produces UDP--D-glucose using a molecule of glucose 1-phosphate. Both glucose 1-phosphate and acetyl phosphate are low cost materials. This UDP recycling system22 is coupled with the Bacillus licheniformis GT, YjiC30-35-catalyzed glucosylation reaction using resvera-A as an acceptor molecule. As a result, UDP--D-glucose regeneration is complete and YjiC can efficiently synthesize NP glucosides (Fig. 1B). The recombinant proteins, ACK (~38 kDa), GalU (~38 kDa), and YjiC (~ 45 kDa) were overexpressed in E. coli BL21 (DE3) (Fig. 1C). The crude enzymes were used for one-pot reactions while purified YjiC was used for the regular reaction. YjiC was

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purified as described in the materials and methods section, and approximately 95% pure protein was quantified by Bradford’s method prior to use for reaction. To modify the structure of Resvera-A by glycosylation, we assessed the activity of purified YjiC with UDP--D-glucose as glucose donor substrate by regular glycosylation reaction as described in experimental section. HPLC analysis of the reaction mixtures revealed two new peaks at 270 nm (Fig. 2B) in comparison to control reaction (Fig. 2A). Peaks PI and PII had retention times (tRs) of 10.0 min and 10.2 min, respectively. HPLC-PDA analysis also showed that in regular reaction, the overall conversion of resvera-A was 48.8% in 180 min. Since both new peaks had lower values of tR than standard resvera-A (tR = 12.6 min), those peaks could be possible glucosylated derivatives of resver-A. To further confirm the products, we analyzed the same reaction mixture by HPLC-PDA combined with HRQTOF-ESI/MS. The exact mass analysis of each peak was determined. Both peaks had an exact mass [(PI or PII)+H+] of m/z+~ 408.1308 for the molecular formula C19H22NO9 for which the calculated mass was 408.1295, representative of monoglucoside derivatives of resvera-A (Fig. 2). To further characterize the reaction products obtained, a preparative-scale reaction was carried out in a 50 mL reaction volume. The products were purified as described in the experimental section (Fig. S1) and were subjected to 1H-NMR analysis. 1H-NMR analysis of each product and standard resvera-A is presented in Fig. 3 and Table 1. The 1

H-NMR of resvera-A and both products were compared with the previously reported 1H-

NMR of standard resvera-A.19 The spectra of standard resvera-A matches exactly with previously reported data except for minor chemical shifts.

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1

H-NMR of PI showed an absence of a spectrum for the proton of the 4ʹ-OH peak

at δ = 9.2 ppm, which is present in standard resvera-A. The peaks representing 2ʹ and 6ʹ protons and 3ʹ and 5ʹ protons were shifted a little downstream in comparison to the corresponding peaks of standard resvera-A (Fig. 3, Table 1). However, the proton spectra of the 2nd, 4th and 6th carbons are intact, as in resvera-A (Fig. S2 and S4). The presence of a doublet spectra of an anomeric proton at δ = 5.30 ppm (d, J = 4.7 Hz) confirmed the glucosylated product with alpha (α) configuration. Other spectra for glucose moiety were present between δ (5.1 to 3.0 ppm). Together, this evidence confirmed the purified PI product as resvera-A 4ʹ-O-α-D-glucoside (N-4ʹ-O-α-D-glucopyranosyloxyphenyl -3,5dihydroxybenzen carboxamide, R4ʹG). The numbers of carbons in the molecule were further confirmed by

13

C-NMR (Fig. S5). The attachment of sugar in resvera-A was

clearly seen in the spectra as there are additional carbon spectra of glucose in the region of δ = (60-80) ppm and anomeric carbon at δ =101.3 ppm. Similarly, PII was also analyzed by 1H and

13

C-NMR. Absence of the proton of the 3rd -OH group, which is

present at δ = 9.5 ppm in standard resvera-A and resvera-A 4ʹ-O-α-D-glucoside derivatives, provided evidence of conjugation of glucose moiety at the 3-OH position. Further analysis of the spectra showed a more downstream shift of the proton representing 4-H at δ = 6.62 ppm than the shift of the standard resvera-A and resvera-A 4ʹ-O-α-D-glucoside for which the shift is located at ~ δ = 6.4 ppm. Other spectra of PII matched the spectra of PI. The presence of an anomeric proton at δ = 5.33 ppm and respective glucose spectra between δ = (5.1 to 3) ppm, as in resvera-A 4ʹ-O-α-Dglucoside, confirmed PII as resvera-A 3-O-α-D-glucoside (N-4-hydroxybenzyl-3-O-α-Dglucopyranosyloxy-5-hydroxyphenyl carboxamide, R3G) (Fig. S6). The

13

C-NMR

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further confirmed the structure of resvera-A 3-O-α-D-glucoside since additional carbon spectra were present in the region of δ =(60-80) ppm for glucose along with anomeric carbon at δ =100.58 ppm (Fig. S7). Because of the short commercial supply of most NDP-sugars and their high cost, large-scale in vitro production of natural product glycosides on the industrial level is limited. To address this problem, we developed a one-pot reaction system for the efficient synthesis of small molecule glucosides. This system was designed to regenerate the cofactors in a pot, thus further supplementation of additional co-factors in order to proceed with the reaction is not necessary. Exhausted co-factors, such as UDP-α-D-glucose, are regenerated instantaneously and utilized for the further glycosylation of remaining acceptor substrate by the GT. At the first step, the UDP by-product produced after glycosylation is reutilized by ACK to convert UDP into UTP by consuming a molecule of acetyl phosphate. At the second step, GalU catalyzes the conversion of glucose 1phosphate to UDP-α-D-glucose by using UTP, a product of the ACK-catalyzed reaction. Altogether, acetyl phosphate and glucose 1-phosphate are consumed in the reaction, whereas acetate and pyrophosphate are released as by-products. Thus, in this system, the UDP-α-D-glucose donor substrate necessary for the glucosylation of resvera-A is continuously supplied until the reaction is finished. This system may be efficient for the large-scale production of many kinds of natural products by utilizing comparatively inexpensive starting substrates over UDP-α-D-glucose donors. UDP, the by-product of the GT reaction, competitively inhibits YjiC by competing with the UDP-sugar donor substrate at the donor substrate-binding site of the enzyme. Conversion of the acceptor substrate to glycoside is limited, even with the supplementation of a higher concentration

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of UDP-sugars in the single-step reactions. Thus, this approach of recycling UDP could be an efficient process for the glucosylation reaction in the forward direction. In the onepot glucosylation system, the requirement of UDP-α-D-glucose is also expected to be very low, thus lowering the cost of the reaction. To compare the efficiency of the newly developed one-pot system, the reaction was started with various concentration ratios (1:1, 1:3, 1:5) of UDP-α-D-glucose and resvera-A (1 mM UDP-α-D-glucose : 1 mM resvera-A, 1 mM UDP-α-D-glucose : 3 mM resvera-A, 1 mM UDP-α-D-glucose : 5 mM resvera-A). The reactions were preceded and samples were taken out at different time intervals (0 min, 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min). Each sample was subjected to HPLC-PDA analysis at an absorbance of 270 nm. In contrast to regular reactions, resvera-A was rapidly converted to resvera-A glucosides in the early stage of one-pot reactions, even within 30 min (Fig. 4A-C), indicating that the one-pot system supplies UDP-α-D-glucose efficiently, and the activity of YjiC GT is more effective because of the conversion of UDP to UTP, a possible competitive inhibitor in the forward reaction. The absence of UDP in the reaction may also have prevented the deglycosylation reaction, which was observed in the regular glucosylation reaction of phloretin33 and resveratrol.35 In the presence of excess amount of UDP in the reaction mixture with a glucoside derivative and YjiC, the enzyme was able to reverse the glucosylation reaction to produce aglycon and UDP-α-Dglucose from phloretin 4ʹ-O-β-D-glucoside and resveratrol 3-O-β-D-glucoside. In most of the regular glucosylation reactions with YjiC, deglucosylation of glucosides occurred because of the accumulated UDP, the byproduct of glucosylation reaction, upon longer incubation of the reaction mixture.30,33,35

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Furthermore, the reaction was started with increasing concentrations of resvera-A to higher ratios. For example, 1:10, 1:25, and 1:50 ratios of UDP-α-D-glucose and resvera-A and additional resvera-A (5 mM) were also added at an interval of 90 min with the progress of the reaction (Fig 4D-F). Within 90 min, most of the resvera-A was converted to respective glucoside derivatives. However, upon further addition of resveraA, the conversion to glucoside derivatives was halted. Finally, we tested a ratio of 1:100 of UDP-α-D-glucose to resvera-A without addition of resvera-A during the reaction (Fig. 4G). The results of this reaction showed that the recycling system demonstrates almost 87% conversion of substrate within the 3 h reaction time. Thus, the designed one-pot glycosylation system was applied for the preparative scale production of resvera-A glucosides. To investigate the formation of glycosylated products from recycling system, the preparative scale reaction was performed in 50 mL volume under a 1:100 ratio of UDP-αD-glucose and resvera-A, increasing the starting concentration of resvera-A to 10 mM in the reaction, and the yield of each product was calculated at different time points. The yield of PI and PII consistently increased at all reaction times (Fig. 4H). After 4 h of reaction, 4.87 mM (99.38 mg) of resvera-A 4ʹ-O-α-D-glucoside and 2.10 mM (42.33 mg) of resvera-A 3-O-α-D-glucoside was produced in the reaction mixture. In the recycling system, resvera-A decreased to a concentration of 3.02 mM during the reaction time. The analysis of the one-pot reaction mixture of resvera-A (Fig. 4) at different time points showed that the proportion of resvera-A 4ʹ-O-α-D-glucoside production in the reaction mixture was always higher in comparison to resvera-A 3-O-α-D-glucoside.

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One of the main purposes of this experiment was to develop an easier, inexpensive, and more efficient method for the glycosylation of natural products. The greatest advantage of the one-pot enzymatic reaction is that the catalysis of many enzymes can be performed in a single reaction with high regio- and stereo-specificity, and the many other strengths of the one-pot reaction include inexpensive materials, no intermediate purification, and high yield production. In this study, the one-pot glucosylation system included the recycling of UDP-α-D-glucose, which not only led to cost reduction of the reaction, but also enhanced the efficiency of the glycosylation reaction. Secondly, the use of uridine monophosphate kinase (UMK) in our previous study for recycling adenosine triphosphate22 was the rate-limiting step of the whole onepot glucosylation system as UMK has low stability and a low reaction catalysis rate. To eliminate this step, we developed a simple UDP-recycling system in this study. A possible disadvantage of the recycling system is the continuous decrease in the pH of the reaction mixture, as acetate is produced in one successive step, lowering the activity of the enzymes in the one-pot system. Therefore, continuous observation and pH maintenance of the reaction mixture is essential for the efficient continuation of the reaction system. The efficiency of our one-pot system was demonstrated by the production of resvera-A glucosides in gram per liter concentrations. In agreement with previous reports,28,29 the enhanced productivity was due to the immediate removal of UDP, a product inhibitor of UGTs. Furthermore, the one-pot systems maintained a continuous supply of UDP-α-D-glucose necessary for the maximum activity of the GTs. Since the newly developed one-pot glucosylation system can supply UDP-α-D-glucose continuously, the pool of UDP-α-D-glucose is consistently available until the energy

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source (acetyl phosphate and glucose-1-phosphate) is exhausted. This recycling system has the potential to replace in vivo glucosylation reactions. Our results indicated the potential of one-pot glucosylation reactions for higher yield production of resvera-A glucosides using significantly lower concentrations of UDP-α-D-glucose (0.1 mM) in the reaction mixture. As a result, we successfully produced two novel derivatives of resveraA glucosides. In a separate in vivo study, we have also tried to produce resvera-A glucosides from engineered E. coli, as with other small molecules.34 However, we were unable to detect glucosylated derivatives of resvera-A in culture supernatant as well as in cell lysate (results not shown). Resvera-A was not detected even in cell lysate. This result confirmed that resvera-A did not enter into the cell. This could be due to the charge generated in the resvera-A molecule, which has an amide functional group in its structure. The in vitro glucosylation reaction products of resvera-A and resveratrol were also drastically different in terms of the nature of glucose conjugation and the amount of product formation while using the same YjiC enzyme as catalyst. In our recent report,35 we were able to produce four different glucosylated derivatives of resveratrol including two monoglucosides, two diglucosides and a triglucoside. Though resvera-A resembles the structure of resveratrol and has same number of prominent hydroxyl groups for glucosylation as in resveratrol, YjiC was able to produce only two monoglucosides. Moreover, the glucose moieties attached to resveratrol were in the β-configuration while those sugars were in the α-configuration in resvera-A. Many important drug properties, such as pharmacokinetics, pharmacodynamics, solubility, mechanism, and potency, can be affected by glycoconjugation. Furthermore,

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sugar residues can enhance in vitro uptake through the sugar transporter (GLUT), which is over expressed in tumors, and thus may improve the oral bioavailability and efficacy of drugs.5 Therefore, we anticipate a potential enhancement in the anticancer activity of resvera-A after glucosylation.

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1. Experimental procedures 1.1 Chemicals and reagents Standard

UDP--D-glucose,

D-glucose-1-phosphate,

isopropyl--D-

thiogalactopyranoside (IPTG), and acetyl phosphate were purchased from GeneChem (Daejeon, South Korea). Resvera-A was provided by Amore Pacific Corporation (Yongin, Korea). Dimethyl sulfoxide-d6 (DMSO-d6) was purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade acetonitrile and water were purchased from Mallinckrodt Baker (Phillipsburg, NJ, USA). All other chemicals used were of high analytical grade and commercially available. 1.2 Plasmids and culture condition Escherichia coli BL21 (DE3) (Stratagene, USA) was used as the host for over-expression of proteins. Plasmids harboring genes encoding for acetate kinase (ACK-pET24ma),22 glucose 1-phosphate uridylyltransferase (GalU-pET24ma),22 and glycosyltransferase (YjiC-pET28a) were previously constructed.32-35 For easy manipulation, GalU and YjiC (G-Y) were co-expressed in a single transformant. All E. coli strains were grown at 37 C in Luria-Bertani (LB) broth or on agar plates supplemented with the appropriate amount of antibiotics (ampicillin up to 100 g/mL and kanamycin up to 50 g/mL). 1.3 Protein expression, purification, and quantification The recombinant strains were cultured in 4 mL LB medium supplemented with appropriate antibiotics for 3 h and transferred to 50 mL of LB medium using 250 mL shake flasks. The cells were grown to an OD600 value of 0.5-0.6 at 37 C. Then, protein 15 Page 15 of 29

expression was induced by addition of 0.5 mM IPTG followed by continued growth at 20 C for 18 h. The cells were harvested by centrifugation at 194 x g and 4 C, and washed twice with buffer (50 mM Tris-HCl and 10% glycerol of pH 7.5). Crude extracts were prepared by sonicating the cell suspension in 1 mM phenylmethylsulfonyl fluoride and 1 mL of 50 mM Tris-HCl (pH 7.5) containing 1 mM dithiothreitol 10% glycerol, followed by centrifugation at 13,000 x g for 30 min at 4 C for the separation of soluble and insoluble proteins. Protein expression was analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The crude enzymes were used for onepot reactions. Purified YjiC was used for initial assessment of resvera-A glucosylation by regular reaction. Soluble crude YjiC fractions were applied to a TALON metal nikel affinity resin (Takara Bio, Shiga, Japan) equilibrated with buffer containing 300 mM NaCl and 50 mM Tris-HCl (pH 7.5). The resin-bound protein was eluted using a discontinuous imidazole gradient (10, 50, 100, 200, 500 mM) prepared in the equilibration buffer. The fractions containing purified protein were analyzed by 12 % SDS-PAGE and further concentrated using Amicon® Ultra 15 mL filters (Millipore, 30 K NMWL device; Milford, MA, USA). Protein quantitation was performed by Bradford protein assay.36 1.4 Regular glucosylation reaction The 200 L regular reactions were carried out using 50 g/mL purified YjiC, 2 mM resvera-A, and other substrates in 50 mM Tris-HCl buffer (pH 7.5) with 2 mM UDP-D-glucose and 10 mM MgCl2.

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1.5 One-pot enzymatic reaction conditions The one-pot reaction mixture (500 L) for the synthesis of resvera-A glucosides contained 300 mM Tris buffer, 10 mM MgCl2, 60 mM acetyl phosphate, 10 mM glucose1-phosphate, (1-10) mM resvera-A, (0.1-1) mM UDP--D-glucose, and ~50 g/mL of each crude enzyme (ACK, GalU, and YjiC). The reaction mixture was incubated at 37 C for 3 h. For the product analyses, 10 L of reaction sample was taken from 500 L of reaction mixture and diluted in 90 L methanol at different time points for the time-dependent study. After centrifugation at 13,000 x g for 20 min, the reaction mixtures were analyzed by HPLC. For preparative-scale glycosylation, the reactions were performed in 50 mL of 10 mM (~123 mg) resvera-A and 0.1 mM (3.05 mg) UDP--D-glucose, in a shaking water bath incubator for 4 h. Remaining constituents of the preparative reaction were in similar proportion as in small scale reactions. The reactions were quenched by adding 100 mL of chilled methanol to the mixture, followed by centrifugation at 13,000 x g for 30 min. The supernatant was concentrated by evaporation in a rotary evaporator and dissolved in 1 mL methanol before purification by preparative HPLC. 1.6 Spectroscopic analyses of the reaction product Reverse-phase high performance liquid chromatography (HPLC) analysis was performed with a C18 column (YMC-Pack ODS-A (250 x 4.6 mm, 5 m) connected to a photo-diode array (PDA) at 270 nm using binary conditions of H2O and 100% acetonitrile (ACN) at a flow rate of 1 mL/min for 20 min. The ACN concentrations were as follows: 20% (0-5 17 Page 17 of 29

min), 50% (5-10 min), 70% (10-15 min), 90% (15-17 min), and 10% (17-20 min). Purification of compounds was carried out by preparative-HPLC with a C18 column (YMC-Pack ODS-AQ (250 x 20 mm I.D., 10 m) connected to a UV detector (270 nm) using a min binary program with ACN 20% (0-5 min), 40% (5-10 min), 40% (10-15 min), 90% (15-20 min), 90% (20-25 min), and 10% (25-36 min) at a flow rate of 10 mL/min. High resolution quantitative time-of-flight electrospray ionization mass spectrometry (HR QTOF-ESI/MS) was carried out in positive ion mode on ACQUITY (UPLC; Waters, Milford, MA, USA) coupled with SYNAPT G2-S (Waters). The compounds were further characterized with a 300 MHz Bruker BioSpin nuclear magnetic resonance (NMR) spectrometer (Germany) using a Cryogenic TCi probe (5 mm). All samples were prepared in either methanol (CD3OD) or dimethyl sulfoxide-d6 (DMSO-d6) (Sigma-Aldrich). One-dimensional NMR (1H –NMR and 13C-NMR) was performed to elucidate the structure of the resvera-A glucosides. 3,5-dihydroxy-N-(4-hydroxyphenyl) benzamide (Resvera-A): 1H NMR (300 MHz, DMSO-d6) δ 9.83 (s, 1H), 9.51 (s, 2H), 9.21 (s, 1H), 7.60 – 7.39 (m, 2H), 6.74 (d, J = 2.2 Hz, 2H), 6.72 – 6.67 (m, 2H), 6.38 (t, J = 2.2 Hz, 1H). δ C (75 MHz, CD3OD) 105.300, 105.585, 114.850, 123.029, 130.145, 137.123, 154.305, 158.478, 167.677. λmax: 278.7; qTOF ESI/MS (m/z+) [M+H]+observed: 246.0781; [M+H]+calculated: 246.0766. Resvera-A 4ʹ-O-α-D-glucoside (PI): 1H NMR (300 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.54 (s, 2H), 7.65 (dd, J = 9.1, 2.2 Hz, 2H), 7.07 – 6.93 (m, 2H), 6.75 (d, J = 2.2 Hz, 2H), 6.40 (t, J = 2.2 Hz, 1H), 5.30 (d, J = 4.6 Hz, 1H), 5.08 (d, J = 4.3 Hz, 1H), 5.01 (d, J = 5.1 Hz, 1H), 4.80 (d, J = 7.2 Hz, 1H), 4.57 (t, J = 5.8 Hz, 1H), 3.79 – 3.60 (m, 3H), 3.54 – 3.39 (m, 2H), 3.32 – 3.08 (m, 6H). δ C (75 MHz, CD3OD) 61.156, 70.021, 73.555,

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76.606, 76.758, 101.298, 105.366, 105.598, 116.652, 122.331, 132.995, 137.037, 154.622, 158.504, 167.675. λmax: 269.7; qTOF ESI/MS (m/z+) [2M+H]+observed: 815.2502; [2M+H]+calculated:815.2511; [M+H]+observed: 408.1297; [M+H]+calculated: 408.1295; [MGlc+H]+observed: 246.0881. Resvera-A 3-O-α-D-glucoside (PII): 1H NMR (300 MHz, DMSO-d6) δ 9.87 (s, 1H), 9.77 (s, 1H), 9.24 (s, 1H), 7.51 (dd, J = 9.0, 2.4 Hz, 2H), 6.98 (dt, J = 17.0, 1.8 Hz, 2H), 6.80 – 6.66 (m, 2H), 6.62 (t, J = 2.2 Hz, 1H), 5.33 (s, 1H), 5.20 – 4.96 (m, 1H), 4.88 (d, J = 7.2 Hz, 1H), 4.60 (s, 1H), 3.69 (d, J = 11.8 Hz, 1H), 3.57 – 3.42 (m, 2H), 3.31 – 3.09 (m, 3H). δ C (75 MHz, DMSOd6) 60.625, 69.573, 73.216, 76.530, 77.095, 100.579, 106.390, 108.402, 114.929, 122.125, 130.707, 137.172, 153.612, 158.427, 164.662. λmax: 281.7; qTOF ESI/MS (m/z+) [2M+H]+observed: 815.2514; [2M+H]+calculated:815.2511; [M+H]+observed: 408.1308; [M+H]+calculated: 408.1295; [M-Glc+H]+observed: 246.0821. Acknowledgments This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ011119012015), Rural Development Administration, Republic of Korea and supported by the Korea Foundation for the Advancement of Science & Creativity (KOFAC), and funded by the Korean Government (MOE).

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Figure legends Fig. 1. A) Structures of two different forms of resveratrol and resvera-A. B) Singlevessel multi-enzyme reaction scheme showing recycling of UDP and production of resvera-A glucosides using three different enzymes. C) SDS-PAGE analysis of recombinant proteins used in in vitro reaction. Lane M, standard protein maker; lane 1, cell lysate containing ACK (~38 kDa); lane 2, purified, YjiC (~46 kDa); lane 3, cell lysate containing GalU (~38 kDa). Fig. 2. HPLC-PDA and HRQTOF ESI/MS analysis of reaction products and standard resvera-A. A) Control reaction of resvera-A with UDP-α-D-glucose using heat treated enzymes; B) Regular glucosylation reaction of resvera-A with UDP-α-D-glucose. The maxima UV absorbance and exact mass of standard resvera-A and resvera-A glucosides is shown in inset. Fig. 3. Comparison of 1H-NMR of resvera-A and its glucoside products. A) Resvera-A; B) Resvera-A 4'-O-α-D-glucoside; C) Resvera-A 3-O-α-D-glucoside. Fig. 4. Reaction profile of UDP-α-D-glucose and resvera-A at different ratio at different time points under identical reaction conditions. Ratio of UDP-α-D-glucose and resvera-A A) 1:1; B) 1:3; C) 1:5; D) 1:10 (additional 5 mM resvera-A added at 90 min); E) 1:25 (additional 5 mM resvera-A added at 90 min); F) 1:50 (additional 5 mM resvera-A added at 90 min); G) 1:100 (no addition of resvera-A during reaction); H) Preparative reaction in 50 mL volume using 1:100 ratio of UDP-α-D-glucose (0.1 mM) and resvera-A (10 mM).

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Table 1. 1H-NMR of resvera-A and its glucoside derivatives. Proton Number

Resvera-A

N-H 5-OH or 3-OH 4′-OH 2′ and 6′-H 3′ and 5′-H 2, 6-H 4-H Anomeric Proton Sugar region

9.83 9.51 9.21 7.50 6.74 6.71 6.38 nd nd

Resvera-A 4′-Oα-D-glucoside 9.98 9.54 nd 7.65 6.99 6.75 6.40 5.30 3.0-5.5

Resvera-A 3-O-αD-glucoside 9.87 nd 9.24 7.51 6.98 6.72 6.62 5.33 3.0-5.5

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Graphical Abstract

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