Enzymatic Synthesis of Acylphloroglucinol 3-C-Glucosides from 2-O-Glucosides using a C-Glycosyltransferase from Mangifera indica

Article abstract of DOI:10.1002/chem.201600411

A green and cost-effective process for the convenient synthesis of acylphloroglucinol 3-C-glucosides from 2-O-glucosides was exploited using a novel C-glycosyltransferase (MiCGTb) from Mangifera indica. Compared with previously characterized CGTs, MiCGTb exhibited unique de-O-glucosylation promiscuity and high regioselectivity toward structurally diverse 2-O-glucosides of acylphloroglucinol and achieved high yields of C-glucosides even with a catalytic amount of uridine 5′-diphosphate (UDP). These findings demonstrate for the first time the significant potential of a single-enzyme approach to the synthesis of bioactive C-glucosides from both natural and unnatural acylphloroglucinol 2-O-glucosides. One enzyme, one product: A novel C-glycosyltransferase from Mangifera indica is reported, which is a promiscuous catalyst that synthesizes bioactive C-glucosides from natural and unnatural acylphloroglucinol 2-O-gulcosides in one-pot reactions. High yields of C-glucosides were achieved even with a catalytic amount of uridine 5′-diphosphate. This study demonstrates for the first time the significant potential of a single-enzyme approach for the C-glycodiversification.

Full text of DOI:10.1002/chem.201600411

DOI: 10.1002/chem.201600411
Communication
&
Enzyme Catalysis
Enzymatic Synthesis of Acylphloroglucinol 3-C-Glucosides from 2-
O-Glucosides using a C-Glycosyltransferase from Mangifera indica
Dawei Chen,[a] Lili Sun,[b] Ridao Chen,[a] Kebo Xie,[a] Lin Yang,[b] and Jungui Dai*[a]
ural and unnatural products.[7] However, the majority of known
Abstract: A green and cost-effective process for the con-
CGTs exhibit relatively narrow substrate selectivity and require
venient synthesis of acylphloroglucinol 3-C-glucosides
rather expensive or rare nucleotide-activated sugar donors
from 2-O-glucosides was exploited using a novel C-glyco-
(NDP-sugar), limiting their availability and scope for creating
syltransferase (MiCGTb) from Mangifera indica. Compared
structurally diverse C-glycosides with potent biological activi-
with previously characterized CGTs, MiCGTb exhibited
ties as drug leads.
unique de-O-glucosylation promiscuity and high regiose-
Significant progress has been made recently using the reac-
lectivity toward structurally diverse 2-O-glucosides of acyl-
tion reversibility of O-glycosyltransferases (OGTs) in the glycodi-
phloroglucinol and achieved high yields of C-glucosides
versfication of natural and unnatural products by performing
even with a catalytic amount of uridine 5’-diphosphate
sugar and aglycon exchange reactions in vitro.[8] Although sev-
(UDP). These findings demonstrate for the first time the
eral CGTs have been cloned and their catalytic function has
significant potential of a single-enzyme approach to the
been verified, strategies for C-glycodiversification through
synthesis of bioactive C-glucosides from both natural and
a combinatorial approach from natural and unnatural O-glyco-
unnatural acylphloroglucinol 2-O-glucosides.
sides in vitro remain scarce. To date, only two examples have
been described of synthesizing 3-C-glucoside (1a, nothofagin)
from 2-O-glucoside (1, phlorizin), using OsCGT coupled with an
OGT or an engineered dual-specific O/CGT.[9] We also recently
Sugar moieties are often essential for the physiological activity,
specificity and pharmacological properties of many natural
products.[1] The majority of natural product glycosylations in-
volve the O-glycosidic bond, which, however, is usually sensi-
tive to spontaneous or enzyme-catalyzed hydrolysis in vivo.[2]
As isosteric O-glycoside mimics, C-glycosides not only maintain
their efficacy and pharmacological properties but also exhibit
outstanding resistance to glycosidic bond cleavage.[3] For ex-
ample, 3-C-glucoside of phloretin (1a, nothofagin) exhibits
more selective and stable inhibition of human sodium-glucose
co-transporter 2 (SGLT2) activities for the treatment of type 2
diabetes than 2-O-glucoside of phloretin (1, phlorizin).[4] How-
ever, natural C-glycosides appear to be comparatively rare.[2]
Chemical C-glycosylation also remains restricted by such disad-
vantages as poor regio- and stereoselectivities and the protec-
tion and deprotection of functional groups.[3b,5] Enzymatic C-
glycosylation catalyzed by specific C-glycosyltransferases (CGTs;
EC 2.4) can alleviate these disadvantages, making these en-
zymes powerful tools.[6] In the past few years, studies on CGTs
from microbes and plants have attracted increasing interest
and achieved great progress in the C-glycosylation of both nat-
reported a benzophenone CGT that generates C-glycosides
using a simple sugar donor.[7i] However, these combinatorial
approaches to the C-glycodiversification of natural and unnatu-
ral products have limited general applicability because of the
specificity of the CGTs, low catalytic efficiency or the genera-
tion of byproducts. Therefore, the exploitation of a universally
applicable green chemistry approach for the generation of bio-
active C-glycosides from naturally abundant or easily synthe-
sized O-glycosides is highly desired. Herein, we report for the
first time an applicable green and cost-effective process for
the generation of bioactive 3-C-glucosides from 2-O-glucosides
through six pairs of acylphloroglucinol 2/4-O-glucosides using
a novel C-glycosyltransferase, MiCGTb, from Mangifera indica.
This work exposes an unexpected de-O-glucosylation promis-
cuity and high regioselectivity of MiCGTb toward structurally
diverse 2-O-glucosides of acylphloroglucinol and highlights the
first highly efficient synthesis of C-glucosides by de-O-glucosyl-
ation and C-glucosylation strategies using a single C-glycosyl-
transferase.
In our ongoing attempt to identify novel CGTs with specific
catalytic properties from M. indica leaves, as described in our
previous work,[7i] a MiCGT homologue gene with 90% identity
(Figure S1 in the Supporting Information), named MiCGTb
(GenBank: KT989668), was successfully cloned and heterolo-
gously expressed in E. coli (Figure S2 in the Supporting Infor-
mation). Similarly to MiCGT, MiCGTb also exhibited catalytic
promiscuity and regio- and stereospecificity toward numerous
natural and unnatural acceptors with uridine 5’-diphosphate
(UDP)-glucose (Table S2 in the Supporting Information). More-
over, MiCGTb likewise generates only C-glucosides with a 2,4,6-
[a] D. Chen, Dr. R. Chen, Dr. K. Xie, Prof. Dr. J. 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
1 Xian Nong Tan Street, Beijing 100050 (P.R. China)
E-mail: jgdai@imm.ac.cn
[b] L. Sun, Prof. Dr. L. Yang
College of Life and Environmental Sciences, Minzu University of China
27 Zhong Guan Cun Southern Street, Beijing 100081 (P.R. China)
Supporting information for this article can be found under http://
dx.doi.org/10.1002/chem.201600411.
Chem. Eur. J. 2016, 22, 5873 – 5877
5873
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
tri-hydroxylacetophenone-type structure. Therefore, MiCGTb
can also be validated as a benzophenone CGT due to its cata-
lytic characteristics of C-glycosylation.[7i]
established protocols of OGTs’ reversible reaction.[8b,11] This
transformation reveals that MiCGTb exhibits an unexpected
de-O-glucosylation capability toward 1 in the presence of UDP
except for its C-glucosylation activity, in which UDP is the “trig-
ger” in the de-O-glucosylation process (thus catalyzing the re-
lease of 1b and formation of UDP-glucose).[8] Although the
mechanism of aryl-C-glycosylation remains unclear, these re-
sults lend strong support to the C-glycosylation through
a direct Friedel–Crafts-like reaction.[2a,3b,6c,10]
Unexpectedly, the C-glucosylated product turned out to be
3-C-glucoside nothofagin (1a), when 2-O-glucoside phlorizin
(1) and UDP-glucose were incubated with MiCGTb at pH 6.6.
The same result was also obtained when MiCGTb and 1 were
incubated with UDP (Figure 1). However, this conversion of
1 into 1a was not observed with only MiCGT (Figure S3 in the
Supporting Information) or OsCGT.[9c] These unique results in-
spired our interest in how this enzymatic conversion of 2-O-
into 3-C-glucoside occurred: through chemical Fries-type O/C-
glucosidic bond rearrangement or an aglycone intermediate
followed by a Friedel–Crafts-like reaction.[6c,9c,10] To verify these
predictions, a mixture of 1, UDP and MiCGTb was incubated at
308C from 10 to 60 min, and the time course of the reaction
was monitored by HPLC, which clearly indicated the formation
of aglycone phloretin (1b, Figure 1B). In the control experi-
ments, no reaction was detected at all in the absence of either
MiCGTb or UDP. In light of these results, the conversion is un-
ambiguously considered to proceed via the intermediary re-
lease of 1b and the formation of UDP-glucose, followed by
regio- and stereoselective C-glucosylation. However, given that
the O-glucosylation activity toward 1b with UDP-glucose by
MiCGTb was not detected (Figure S4 in the Supporting Infor-
mation), the above reaction seems implausible based on the
The biochemical characteristics of this transformation cata-
lyzed by MiCGTb were then further investigated. The pH de-
pendence of 1b C-glucosylation showed a broad optimum
over pH 6.0–11.0, whereas the optimum conversion of 1 into
1a was in a pH range of 4.0–6.0 (Figure 2A), which might be
more suitable for the de-O-glucosylation of 1. MiCGTb dis-
played its maximum activity at 408C (Figure 2B) and was diva-
lent cation independent (Figure 2C). Moreover, high yields of
1a (>90%) were achieved even with a catalytic amount of
UDP (Figure 2D), which was more efficient than the reported
results.[9] Kinetic analysis revealed that MiCGTb exhibited kcat
values of 47.3 and 0.7 minÀ1 for 1b and 1, respectively, with
the corresponding kcat/KM values of 2.8”105 and 6.4”
103 mÀ1 minÀ1 (Figure S5 in the Supporting Information). These
results clearly demonstrate that the catalytic efficiency of di-
rectly C-glucosylation 1b appeared to be at least 40-fold
higher than that of the reaction from 1 to 1a (de-O-glucosyl-
ation 1 first, and then C-glucosylation 1b), and de-O-glucosyl-
ation was thus a rate-limiting step in the conversion of 2-O-
into 3-C-glucoside. In contrast to these results, C-glucosylation
was rate-limiting for this transformation when performed by
the dual-specific O/CGT.[9c] The higher yields of C-glucoside in
this process most likely resulted from the higher catalytic effi-
ciency of the C-glucosylation step, which was a large driving
force promoting such conversion.
Figure 1. Transformation of phlorizin (1) to nothofagin (1a) using a single C-
glycosyltransferase MiCGTb. A) The reaction catalyzed by recombinant
MiCGTb. B) HPLC chromatograms of 1 and the enzyme products 1a and 1b.
All reactions were performed at pH 6.6 and 308C. The HPLC conditions are
provided in Table S1 in the Supporting Information.
Figure 2. Yields of nothofagin (1a) catalyzed by recombinant MiCGTb with
different: A) pH (phlorizin (1): gray columns; phloretin (1b): blue columns),
B) temperature, and C) divalent cation after 2 h, and D) reaction times with
different molar ratios of phlorizin/UDP. The pH profile of 1b C-glucosylation
was tested after incubation for 10 min.
Chem. Eur. J. 2016, 22, 5873 – 5877
www.chemeurj.org
5874
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
Figure 3. Exploring the generality and regiospecificity of MiCGTb toward structurally diverse glucosides of acylphloroglucinol. A) Percent yields of 3-C-gluco-
sylated products from six pairs of acylphloroglucinol 2/4-O-glucosides (1–12) listed in descending order of yield. B) Structures of the corresponding 2/4-O-glu-
cosides in part A and 3-C-glucosylated products. All reactions were performed at pH 6.0 and 408C. The HPLC conditions are provided in Table S1.
For further exploitation of the generality and regiospecificity
of the transformation of 2-O- to 3-C-glucoside, five other pairs
of 2/4-O-glucosides of acylphloroglucinol were assayed with
MiCGTb. In the presence of UDP, MiCGTb could efficiently cata-
lyze the de-O-glucosylation and 3-C-glucosylation of the 2-O-
glucosides (3, 5 and 7; >80% yields by HPLC) that appeared
to be at least as structurally flexible as 1, while the conversion
rates of 9 and 11 were drastically lower, below 35% (Figure 3).
In contrast, the de-glucosylation activity of MiCGTb on the cor-
responding 4-O-glucosides is very low, even barely detectable
in the cases of 10 and 12. Since the C-glucosylated products
(1a, 3a, 7a, 9a, and 11 a) were prepared and structurally char-
acterized in our previous work,[7i] they were used as authentic
compounds for the identification of the C-glucosides by HPLC
(Figures S4, S6–S10 in the Supporting Information) in this
study. Scaling up the reaction of 5 with the recombinant
MiCGTb provided sufficient 5a for structure elucidation by MS,
1H and 13C NMR analysis. The appearance of parent ion peak at
m/z 371 [MÀH]À, which was 162 amu more than that of 5, and
the characteristic fragment ions of C-glucoside, [MÀHÀ90]À
and [MÀHÀ120]À, suggested that 5a was a mono-C-glucosyl-
ated product (Figure S7 in the Supporting Information). The
suggested that 5a was the 3-C-glucosylated product. The large
coupling constant (J=9.9 Hz) of the anomeric proton (H-1’’)
supported the formation of the b-anomer (Figures S11 and S12
in the Supporting Information). Thus, the structure of 5a was
identified as 3-C-b-d-glucoside. Hence, MiCGTb displayed cata-
lytic promiscuity and high regioselectivity toward more linear
acylphloroglucinol 2-O-glucosides. It is important to note that
most of these C-glucosylated products exhibited potential and
selective human SGLT2 inhibitory activities for the treatment of
type 2 diabetes in vitro.[7i]
To gain additional information on the substrate specificity
toward the nucleoside 5’-diphosphate (NDP) moiety, the nu-
cleotide specificity of MiCGTb was subsequently probed with
1 and four other commercially available NDPs under the opti-
mized conditions. To our delight, MiCGTb displayed broad sub-
strate specificity in recognizing deoxythymidine 5’-diphosphate
(dTDP), adenosine 5’-diphosphate (ADP), cytidine 5’-diphos-
phate (CDP) or guanosine 5’-diphosphate (GDP) and their cor-
responding activated glucose, albeit with a much lower con-
version rate. As shown in the representative time course of the
synthesis of 1a from 1 (Figure S13 in the Supporting Informa-
tion), the substrate specificity of MiCGTb to five different types
of NDP was as follows: 95% yields were observed with UDP in
1 h or dTDP in 6 h, respectively, but less than 20% yields were
observed with ADP, CDP, or GDP after 18 h incubation, indicat-
1
disappearance of H-3 signal in the H NMR spectrum and the
chemical shift of C-3 at d=103.6 ppm and the anomeric
carbon at d=73.6 ppm (C-1’’) in 13C NMR spectrum further
Chem. Eur. J. 2016, 22, 5873 – 5877
www.chemeurj.org
5875
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
ing that UDP is a more suitable substrate for the de-O-gluco-
sylation and C-glucosylation of MiCGTb. As expected, 1b was
likewise observed when 1 was incubated with dTDP, ADP, CDP,
or GDP, which further supported the hypothesis of the trans-
formation of 2-O- to 3-C-glucoside through the intermediate
1b. Collectively, these results offer vast combinatorial potential
for in vivo C-glycodiversification from natural and unnatural O-
glucosides with different NDPs of the host strains.[12]
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Grant Nos. 21572277 and
81573317), the PUMC Youth Fund & the Fundamental Research
Funds for the Central Universities, China (No. 3332015046).
Keywords: C-glycosides · enzyme catalysis · glycosylation ·
regioselectivity · transferases
In summary, an applicable green and cost-effective process
for the convenient synthesis of acylphloroglucinol 3-C-gluco-
sides from 2-O-glucosides using a novel C-glycosyltransferase,
MiCGTb, from M. indica was presented. Compared with previ-
ously characterized CGTs, MiCGTb exhibited unique de-O-glu-
cosylation promiscuity and high regioselectivity toward struc-
turally different acylphloroglucinol 2-O-glucosides, with high
tolerance toward five different NDP moieties, rendering it
a powerful biocatalyst for the cost-effective enzymatic synthe-
sis of bioactive C-glucosides without adding activated sugars.
The studies described herein not only indicate the significant
potential of a single-enzyme approach to the synthesis of bio-
active C-glucosides from both natural and unnatural acylphlo-
roglucinol 2-O-glucosides with catalytic amounts of NDP but
also offer strong support for C-glycosylation through a direct
Friedel–Crafts-like reaction. Given the versatility of MiCGTb for
de-O-glucosylation and C-glucosylation, further systematic
structural investigation together with site-directed mutation
are imperative for elucidating the catalytic mechanism and
substrate promiscuity and for addressing how the enzyme
governs such conversion. The unique de-O-glucosylation of
MiCGTb reported here hints more exciting and novel CGTs
from plants as alternative tools for the C-glycosylation of bio-
active natural and unnatural products and would also facilitate
further enzyme engineering for the exploitation of promiscu-
ous biocatalysts in combinatorial biosynthesis and/or metabol-
ic engineering.
[1] a) R. W. Gantt, P. Peltier-Pain, J. S. Thorson, Nat. Prod. Rep. 2011, 28,
1811–1853; b) E. C. Calvaresi, P. J. Hergenrother, Chem. Sci. 2013, 4,
ˇ
ˇ
2319–2333; c) V. Kren, T. Rezanka, FEMS Microbiol. Rev. 2008, 32, 858–
ˇ
889; d) V. Kren, L. Martínkovµ, Curr. Med. Chem. 2001, 8, 1303–1328.
[2] a) C. J. Thibodeaux, C. E. MelanÅon, H.-w. Liu, Nature 2007, 446, 1008–
1016; b) B. Blauenburg, T. Oja, K. D. Klika, M. Metsä-Ketelä, ACS Chem.
Biol. 2013, 8, 2377–2382; c) S. I. Elshahawi, K. A. Shaaban, M. K. Kharel,
J. S. Thorson, Chem. Soc. Rev. 2015, 44, 7591–7697.
[3] a) P. G. Hultin, Curr. Top. Med. Chem. 2005, 5, 1299–1331; b) T. Bililign,
B. R. Griffith, J. S. Thorson, Nat. Prod. Rep. 2005, 22, 742–760.
[4] a) J. R. L. Ehrenkranz, N. G. Lewis, C. R. Kahn, J. Roth, Diabetes/Metab.
Res. Rev. 2005, 21, 31–38; b) H. JugdØ, D. Nguy, I. Moller, J. M. Cooney,
R. G. Atkinson, FEBS J. 2008, 275, 3804–3814; c) E. C. Chao, R. R. Henry,
Nat. Rev. Drug Discovery 2010, 9, 551–559.
[5] a) S. Lemaire, I. N. Houpis, T. Xiao, J. Li, E. Digard, C. Gozlan, R. Liu, A.
Gavryushin, C. Di›ne, Y. Wang, V. Farina, P. Knochel, Org. Lett. 2012, 14,
1480–1483; b) H. Satoh, S. Manabe, Chem. Soc. Rev. 2013, 42, 4297–
4309; c) C. H. Lin, H. C. Lin, W. B. Yang, Curr. Top. Med. Chem. 2005, 5,
1431–1457; d) K. Lalitha, K. Muthusamy, Y. S. Prasad, P. K. Vemula, S. Na-
garajan, Carbohydr. Res. 2015, 402, 158–171.
ˇ
[6] a) V. Kren, J. Thiem, Chem. Soc. Rev. 1997, 26, 463–473; b) E. K. Lim,
Chem. Eur. J. 2005, 11, 5486–5494; c) C. Dürr, D. Hoffmeister, S.-E. Woh-
lert, K. Ichinose, M. Weber, U. von Mulert, J. S. Thorson, A. Bechthold,
Angew. Chem. Int. Ed. 2004, 43, 2962–2965; Angew. Chem. 2004, 116,
3022–3025.
[7] a) D. Hoffmeister, G. Dräger, K. Ichinose, J. Rohr, A. Bechthold, J. Am.
Chem. Soc. 2003, 125, 4678–4679; b) M. A. Fischbach, H. Lin, D. R. Liu,
C. T. Walsh, Proc. Natl. Acad. Sci. USA 2005, 102, 571–576; c) M. Brazier-
Hicks, K. M. Evans, M. C. Gershater, H. Puschmann, P. G. Steel, R. Ed-
wards, J. Biol. Chem. 2009, 284, 17926–17934; d) M. L. Falcone Ferreyra,
E. Rodriguez, M. I. Casas, G. Labadie, E. Grotewold, P. Casati, J. Biol.
Chem. 2013, 288, 31678–31688; e) L. Li, P. Wang, Y. Tang, J. Antibiot.
2014, 67, 65–70; f) Y. Nagatomo, S. Usui, T. Ito, A. Kato, M. Shimosaka,
G. Taguchi, Plant J. 2014, 80, 437–448; g) N. Sasaki, Y. Nishizaki, E.
Yamada, F. Tatsuzawa, T. Nakatsuka, H. Takahashi, M. Nishihara, FEBS
Lett. 2015, 589, 182–187; h) Y. Hirade, N. Kotoku, K. Terasaka, Y. Saijo-
Hamano, A. Fukumoto, H. Mizukami, FEBS Lett. 2015, 589, 1778–1786;
i) D. Chen, R. Chen, R. Wang, J. Li, K. Xie, C. Bian, L. Sun, X. Zhang, J. Liu,
L. Yang, F. Ye, X. Yu, J. Dai, Angew. Chem. Int. Ed. 2015, 54, 12678–
12682; Angew. Chem. 2015, 127, 12869–12873; j) M. Mittler, A. Becht-
hold, G. E. Schulz, J. Mol. Biol. 2007, 372, 67–76; k) H. K. Tam, J. Härle, S.
Gerhardt, J. Rohr, G. Wang, J. S. Thorson, A. Bigot, M. Lutterbeck, W.
Seiche, B. Breit, A. Bechthold, O. Einsle, Angew. Chem. Int. Ed. 2015, 54,
2811–2815; Angew. Chem. 2015, 127, 2853–2857; l) F. Wang, M. Zhou,
S. Singh, R. M. Yennamalli, C. A. Bingman, J. S. Thorson, G. N. Phillips, Jr.,
Proteins 2013, 81, 1277–1282.
[8] a) C. Zhang, C. Albermann, X. Fu, J. S. Thorson, J. Am. Chem. Soc. 2006,
128, 16420–16421; b) C. Zhang, B. R. Griffith, Q. Fu, C. Albermann, X. Fu,
I. K. Lee, L. Li, J. S. Thorson, Science 2006, 313, 1291–1294; c) P. Peltier-
Pain, K. Marchillo, M. Zhou, D. R. Andes, J. S. Thorson, Org. Lett. 2012,
14, 5086–5089; d) R. Chen, H. Zhang, G. Zhang, S. Li, G. Zhang, Y. Zhu,
J. Liu, C. Zhang, J. Am. Chem. Soc. 2013, 135, 12152–12155; e) R. W.
Gantt, P. Peltier-Pain, S. Singh, M. Zhou, J. S. Thorson, Proc. Natl. Acad.
Sci. USA 2013, 110, 7648–7653; f) K. Xie, R. Chen, J. Li, R. Wang, D.
Chen, X. Dou, J. Dai, Org. Lett. 2014, 16, 4874–4877.
Experimental Section
Chemical reagents were purchased from Sigma Aldrich (St. Louis,
MO, USA), J&K Scientific Ltd. (Beijing, China), and InnoChem Sci-
ence & Technology Co., Ltd. (Beijing, China). MiCGTb was amplified
by PCR, and the gene fragment was then inserted into plasmid
pET-28a. The resultant plasmid was then transformed into E. coli
(TransGen Biotech, China) for heterologous expression. Enzymatic
products were detected on an Agilent 1200 series HPLC system
(Agilent Technologies, Germany) coupled with a LCQ Fleet ion trap
mass spectrometer (Thermo Electron Corp., USA) equipped with an
electrospray ionization (ESI) source. Compounds were characterized
1
by H NMR spectroscopy at 400 MHz on Mercury-400 spectrometer
and 13C NMR spectroscopy at 150 MHz on Bruker AVIIIHD spec-
trometer, and HRESIMS on an Agilent Technologies 6520 Accurate
Mass Q-TOF LC/MS Spectrometer. A detailed description of the ex-
perimental procedures as well as the analytical protocols can be
found in the Supporting Information.
[9] a) L. Bungaruang, A. Gutmann, B. Nidetzky, Adv. Synth. Catal. 2013, 355,
2757–2763; b) A. Gutmann, L. Bungaruang, H. Weber, M. Leypold, R.
Breinbauer, B. Nidetzky, Green Chem. 2014, 16, 4417–4425; c) A. Gut-
Chem. Eur. J. 2016, 22, 5873 – 5877
www.chemeurj.org
5876
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
mann, C. Krump, L. Bungaruang, B. Nidetzky, Chem. Commun. 2014, 50,
5465–5468.
[12] a) G. J. Williams, J. Yang, C. Zhang, J. S. Thorson, ACS Chem. Biol. 2011,
6, 95–100; b) M. Brazier-Hicks, R. Edwards, Metab. Eng. 2013, 16, 11–20.
[10] a) E. R. Palmacci, P. H. Seeberger, Org. Lett. 2001, 3, 1547–1550; b) A.
Gutmann, B. Nidetzky, Angew. Chem. Int. Ed. 2012, 51, 12879–12883;
Angew. Chem. 2012, 124, 13051–13056; c) T. Bililign, C.-G. Hyun, J. S.
Williams, A. M. Czisny, J. S. Thorson, Chem. Biol. 2004, 11, 959–969.
[11] R. W. Gantt, P. Peltier-Pain, W. J. Cournoyer, J. S. Thorson, Nat. Chem.
Biol. 2011, 7, 685–691.
Received: January 28, 2016
Published online on March 10, 2016
Chem. Eur. J. 2016, 22, 5873 – 5877
www.chemeurj.org
5877
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim