Letter pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Biocatalytic C‑Glucosylation of Coumarins Using an Engineered C‑Glycosyltransferase Dawei Chen, Ridao Chen, Kebo Xie, Tian Yue, Xiaolin Zhang, Fei Ye, and Jungui Dai* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China S Supporting Information *
ABSTRACT: The enzymatic synthesis of coumarin C-glucosides by an engineered C-glycosyltransferase, MiCGTb− GAGM, was explored in vitro and in vivo. MiCGTb− GAGM exhibited a robust C-glucosylation capability toward structurally diverse coumarin derivatives. The whole-cell bioconversion of MiCGTb−GAGM was exploited for largescale production of coumarin C-glucosides. Two C-glucosides exhibited potent SGLT2 inhibitory activities with IC50 values at 10−6 M. These findings provide cost-effective and practical synthetic strategies to generate structurally diverse and novel bioactive coumarin C-glycosides for drug discovery.
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unprecedented substrate promiscuity by protein engineering. To investigate the C-glycosylation capacity of these variants toward coumarins, 5,7-dihydroxycoumarin (1, the aglycon of dauroside D) was initially used as a sugar acceptor, and uridine diphosphate α-D-glucose (UDP-Glc) was used as a sugar donor. The recombinant proteins were overproduced as N-terminal His6-tagged fusions in Escherichia coli BL21 (DE3) and purified with Ni-affinity chromatography.11 The reactions (50 mM NaH2PO4−Na2HPO4, pH 8.0; 0.4 mM UDP-Glc; 0.2 mM 1; 100 μg of purified enzyme; 30 °C, 12 h) were analyzed by HPLC−UV/MS (high-performance liquid chromatography− UV absorption/mass spectrometry). Control reactions lacking either enzyme or UDP-Glc confirmed that the reactions were dependent upon both the enzyme and UDP-Glc. To our delight, a quadruple mutant, MiCGTb−GAGM (S60G/ V100A/T104G/I152M), exhibited higher C-glucosylation activity than the wild type toward 1 (Scheme 1; Figure S1). MS and MS2 analysis of 1a displayed the appearance of an ion peak at m/z 339 [M − H]−, which was 162 amu greater than
mong the naturally occurring C-glycosides, only a few coumarin C-glycosides are known.1,2 Dauroside D (1a, also known as mulberroside B), a 6-C-glucosyl coumarin from Haplophyllum dauricum, exhibited spasmolytic and hypotensive activities, but with mild toxicity.3 Two 8-C-glucosyl coumarins (8-C-glucosyl-6-methoxy-5,7-dihydroxy-3,4-dihydrocoumarin and 8-C-glucosyl-5-vinyl-6,7-dimethoxy-3,4-dihydrocoumarin) from Diceratella elliptica showed cytotoxic activity.4 Given the various biological activities of coumarin derivatives, especially the effects of the C-glycosyl moiety on the activity,5 several examples of chemically synthesizing coumarin C-glycosides in which the C-3 or C-4 position contains a glycosyl residue have been described.6 The chemical synthesis of coumarin 8-Cglucosides has also been reported by Mahling and Schmidt.7 However, chemical C-glycosylation remains restricted by such disadvantages as poor regio- and stereoselectivities and the tedious protection and deprotection of functional groups.8 The catalysis of enzymatic C-glycosylation by specific C-glycosyltransferases (CGTs; EC 2.4) can alleviate these disadvantages, making these enzymes powerful tools.9 In recent years, significant progress has been achieved in the enzymatic Cglycosylation of natural and unnatural compounds, including flavone,10 benzophenone and xanthone,11 and anthraquinone.12 However, the biocatalytic C-glycosylation of coumarin derivatives have not been reported. Thus, exploitation of universally applicable green approaches with catalytically promiscuous biocatalysts to generate structurally novel and diverse bioactive coumarin C-glycosides is highly desired. Herein, we report the in vitro and in vivo synthesis of structurally diverse bioactive 6- or 8-C-glucosylated coumarins using an engineered C-glycosyltransferase. We have recently characterized two benzophenone CGTs from Mangifera indica.11 In an effort to expand their substrate scope, we generated a panel of variants of the two CGTs with © XXXX American Chemical Society
Scheme 1. MiCGTb−GAGM Catalyzes the C-Glucosylation of 1
Received: February 2, 2018
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DOI: 10.1021/acs.orglett.8b00378 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters that of 1, and peaks corresponding to the characteristic fragment ions of C-glucoside, found at m/z 249 [M − H − 90]− and m/z 219 [M − H − 120]− (Figure S2).11 The product 1a was then prepared on a larger scale for structural characterization. All the MS and 1 H and 13 C NMR spectroscopic data of 1a were in good agreement with those of dauroside D,3 and a large coupling constant (J = 9.8 Hz) of the anomeric proton (H-1′) further supported the formation of the β-anomer. Therefore, MiCGTb−GAGM could regio- and stereospecifically C-glucosylate 1 in a high yield (82.2% by HPLC) and was thus selected for further investigations. This report is the first example of a CGT that catalyzes C-glucosidic bond formation with coumarin. The biochemical characteristics of this C-glucosylation catalyzed by MiCGTb−GAGM were then further investigated by using 1 and UDP-Glc as acceptor and donor substrates. MiCGTb−GAGM displayed its maximum activity at pH 8.0 and 40 °C and was divalent cation independent (Figure S3). Kinetic analysis revealed that MiCGTb−GAGM exhibited Km values of 647.1 μM and 230.4 μM for 1 and UDP-Glc, respectively, and that the corresponding kcat values were 1.6 and 0.3 min−1 (Figure S4). To explore the coumarin-substrate spectrum of MiCGTb− GAGM and investigate substrate structure−enzyme catalytic activity relationships, an acceptor library of representative natural and unnatural coumarins with different substituents at the C-3, C-4, C-5, or/and C-6 positions (Figure 1) was assessed with UDP-Glc in vitro. An initial hint to the enzyme’s broad capability for C-glucosylation was provided by HPLC−UV/MS analysis (Figure 1; Figures S5 and S6), which revealed the substantial flexibility of MiCGTb−GAGM to C-glucosylate 12 (1−4, 7, 8, 10−13, 16, and 17) of the 20 library members. Moreover, high C-glucosylation conversion rates (>80%) were observed with nine substrates (1−4, 7, 8, and 10−12). The reactions of 10 members (1−3, 7, 8, 11−13, 16, and 17) led to a single, chromatographically distinct mono-C-glucoside, which indicates the regiospecificity of MiCGTb−GAGM. In addition, MiCGTb−GAGM exhibited both C- and O-glucosylation activity toward three substrates (1, 2, and 13; Table S2). Hence, the substrate scope and synthetic potential of MiCGTb−GAGM were further expanded in the field of enzymatic C-glycosylation, which rendered MiCGTb−GAGM a promising enzyme for the production of coumarin Cglycosides that exhibit structural and bioactive diversities. In addition to UDP-glucose, we have tested the tolerance of MiCGTb−GAGM toward other five UDP-sugars (such as UDP-galactose, UDP-glucuronic acid, UDP-rhamnose, UDP-Nacetylglucosamine and UDP-xylose) with acceptor 1. However, MiCGTb−GAGM was only able to catalyze UDP-galactose with low activity (ca. 10%). To understand substrate structure−enzyme catalytic activity relationships, we subsequently performed a detailed structural comparison of the above 20 coumarins. The fact that 5,7dihydroxycoumarins (1−4, 7, 8, 10−13, 16, and 17) were converted and that neither 7-hydroxycoumarin (18 and 19) nor 6,7-dihydroxycoumarin (20) were converted to C-glucosides indicates that the 5,7-dihydroxy substituents on the coumarin framework are of stringent necessity for the C-glycosylation of MiCGTb−GAGM. Hence, the hydroxy groups at the C-5/7 positions enhanced the electron density around the attacked carbon atom, which was crucial for the C-glucosylation of MiCGTb−GAGM and improved the reactivity and regioselectivity of the glucosyl group substitution on the coumarin
Figure 1. Acceptor promiscuity of MiCGTb−GAGM. (A) Percent conversion of the C-glucosylated coumarins catalyzed by purified MiCGTb−GAGM. The members are listed based on the structural scaffolds in part B. (B) Structures of library members and the corresponding C-glucosylated coumarins. The conversion rates of Oglucosylated products (1, 2, 5, 6, 9, 13, 14, and 18−20) are shown in Table S2. *C-glucosylated coumarins were prepared, and their structures were confirmed by HR-ESI-MS and 1H and 13C NMR spectroscopic analyses (Figures S9−S33). N.D.: not detected.
skeleton.13 Moreover, MiCGTb−GAGM exhibited C-glucosylation activity on 5,7-dihydroxycoumarins with hydrophobic substituents at the C-4 position (1−4 vs 5, 6 and 14), linear substituents at the C-3 position (7 and 8 vs 9 and 14) or cyclic substituents at the C-3/4 positions (10−13). Furthermore, a double bond at the C-3/4 positions of coumarins (1, 2, and 4) had a beneficial effect on the catalytic efficiency of MiCGTb− GAGM for C-glucosylation, while the conversion rates of 3,4dihydrocoumarins (15−17) were not detected or clearly decreased. These findings facilitate chemoenzymatic syntheses of additional novel structurally diverse coumarin C-glycosides with these functional groups, which might be important for various pharmacological properties.2,14 While enzymatic reactions are routinely carried out to characterize the associated enzyme’s biochemical function, labor-intensive protein purification, the lack of substrate solubility in aqueous buffer and the cost of UDP-Glc can all be hurdles to preparative-scale biocatalysis with practical potential.15 To overcome these barriers, whole E. coli cells harboring MiCGTb−GAGM and the inherent UDP-Glc were B
DOI: 10.1021/acs.orglett.8b00378 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters used as biocatalyst with coumarins in M9 medium.16,17 All samples were analyzed by HPLC-UV/MS, which revealed that the substrate scope of MiCGTb−GAGM for C-glucosylation in vivo was identical to that of purified enzyme with only one exception (16, Figures 1 and 2). In addition, the conversion
Table 1. Preparative-Scale Coumarin C-Glucosides with Purified Enzyme or Whole Cells Containing MiCGTb− GAGM
Figure 2. Conversion rates of coumarin C-glucosides using whole cells containing MiCGTb−GAGM. The conversion of 11 was indicated at the final concentration of 0.1 mM in 1 mL mixtures, while other coumarins were indicated at 0.4 mM (Figure S7). The conversion rates of O-glucosylated products (1, 2, 4, 6−8, and 11−13) are shown in Table S2. N.D.: not detected.
no.
catalyst
C-glucosylated products
isolated yield (%)
1 2 3 4 7 8 10 10 11 12 17
enzyme enzyme whole cells whole cells whole cells enzyme enzyme whole cells whole cells whole cells whole cells
1a 2a 3a 4a/4b 7a 8a 10a/10b 10a/10b 11a 12a 17a
80.0 85.6 95.6 80.4/7.6 68.4 23.8 26.7/60.2 88.7/5.3 33.4 88.0 7.3
compounds with IC50 values of 2.2 × 10−6 and 6.3 × 10−6 M (Table S3), respectively. In summary, the potential of a uniquely permissive engineered C-glycosyltransferase MiCGTb−GAGM to function as a catalyst for the C-glucosylation of coumarins was highlighted in vitro and in vivo. MiCGTb−GAGM exhibited a robust C-glucosylation capacity toward a series of coumarin derivatives with different substitutions at the C-3 or/and C-4 positions in vitro. Moreover, MiCGTb−GAGM was exploited as a whole-cell biocatalyst to generate several coumarin Cglucosides. Two coumarin C-glucosides (10a and 11a) displayed potent inhibitory effects against SGLT2. These findings demonstrated the opportunities of performing the biocatalytic C-glycosylation of structurally diverse coumarins with endogenous NDP-sugars of host strains. The present study collectively demonstrates for the first time the significant potential of a CGT as a powerful biocatalyst for the in vitro and in vivo synthesis of additional novel bioactive natural and unnatural coumarin C-glycosides as drug leads and will enhance ongoing efforts to develop the combinatorial biosynthesis of NDP-sugars for in vivo C-glycorandomization and the synthetic biology of bioactive coumarin C-glycosides.
rates of 1a, 2a and 8a by MiCGTb−GAGM in vivo were significantly lower than that of the purified enzyme, while the yields of their respective O-glucoside were increased (Table S2). Interestingly, the ratio between 10a and 10b yields was changed from 41:59 to 93:7 when the MiCGTb−GAGM was used as a whole-cell biocatalyst in place of purified enzyme (Figures 1 and 2). These observed different yields or regioselectivity from purified enzymes and whole cells may be due to the different reaction microenvironment around proteins between in vitro and in vivo. High C-glucosylation conversion rates (>80%) were achieved with six substrates (3, 4, 7, and 10−12) with the endogenous UDP-Glc of the host strains. High yields of 7a were obtained from 0.5 to 8 h with different concentrations of 7 (Figure S8). Therefore, the success of these C-glucosylation reactions through whole-cell biotransformation establishes a cost-effective and applicable green approach for the convenient synthesis of structurally diverse and novel bioactive coumarin C-glucosides.18 To further confirm the catalytic properties of MiCGTb− GAGM and biologically assay the coumarin C-glucosides, preparative-scale reactions were performed with a purified enzyme or whole cells based on their respective yields of products. In total, we obtained 12 C-glucosylated coumarins (Table 1), 11 (2a−4a, 4b, 7a, 8a, 10a−12a, 10b, and 17a) of which were novel compounds. The isolated yields of six Cglucosides (1a−4a, 10a, and 12a) were greater than 80%. Their structures were identified with HR-ESI-MS and 1H and 13C NMR spectroscopic data (Figures S9−S33). Most of the Cglucosylated products (1a−4a, 7a, 8a, 10a−12a, and 17a) were coumarin 6-C-glucosides, and the sugar moieties were attached at the C-8 position of the coumarins (4b and 10b). All anomers of coumarin C-glucosides were in the β-configuration, which was deduced from the large coupling constants (J = 9.7−9.9 Hz) of the anomeric protons. The resulting 12 C-glucosylated coumarins were biologically evaluated for potential effects on type-2 diabetes, which was assayed using human sodiumglucose cotransporter 2 (SGLT2) inhibitory activity in vitro;19 this assay revealed that 10a and 11a exhibited potential as lead
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b00378. Experimental procedures, including protein expression, whole cells bioconversion, reactions analysis and product purification, HPLC/MS, HR-ESI-MS, and NMR characterization data and spectra of products (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Jungui Dai: 0000-0003-2989-9016 Notes
The authors declare no competing financial interest. C
DOI: 10.1021/acs.orglett.8b00378 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
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ACKNOWLEDGMENTS This work was supported by the National Science Foundation of China (Grant Nos. 81703369 and 21572277) and CAMS Innovation Fund for Medical Sciences (CIFMS-2016-I2M-3012).
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DOI: 10.1021/acs.orglett.8b00378 Org. Lett. XXXX, XXX, XXX−XXX
