A Fluorogenic Screening for Enantio- and Diastereoselectivity of 2-Deoxy- d -ribose-5-phosphate Aldolases

Article abstract of DOI:10.1055/s-0035-1560723

A highly sensitive, fluorescence-based selectivity screening system for 2-deoxy-d-ribose-5-phosphate aldolases was realized by installing short, straightforward syntheses to fluorophore-coupled carbohydrates as d-ribose, l-ribose, and d-xylose. The substr

Full text of DOI:10.1055/s-0035-1560723

SYNLETT
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
© Georg Thieme Verlag Stuttgart · New York
2016, 27, 11–16
letter
11
C. Bisterfeld et al.
Letter
Synlett
A Fluorogenic Screening for Enantio- and Diastereoselectivity of
2-Deoxy-D-ribose-5-phosphate Aldolases
Carolin Bisterfelda,1
Irene Küberl, née Kullartza,1
Markus Dicka
Jörg Pietruszka*a,b
a Institute of Bioorganic Chemistry, Heinrich-Heine-University at
Forschungszentrum Jülich, and Bioeconomy Science Center (BioSC),
52426 Jülich, Germany
b Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszen-
trum Jülich GmbH, 52425 Jülich, Germany
j.pietruszka@fz-juelich.de
Dedicated to Prof. Steven V. Ley on the occasion of his 70th birthday
Received: 03.09.2015
Accepted after revision: 25.09.201
Published online: 13.10.2015
O
OH
O
O
DOI: 10.1055/s-0035-1560723; Art ID: st-2015-d0693-l
R
R
O
DERA
Abstract A highly sensitive, fluorescence-based selectivity screening
system for 2-deoxy-D-ribose-5-phosphate aldolases was realized by in-
stalling short, straightforward syntheses to fluorophore-coupled carbo-
hydrates as D-ribose, L-ribose, and D-xylose. The substrates allow the si-
DERA
OH
spontaneous
O
OH OH
O
multaneous
determination
of
enantioselectivity
and
diastereoselectivity of DERA-catalyzed aldol reactions.
R
OH
R
1
Key words 2-deoxy-D-ribose-5-phosphate
aldolase,
fluorogenic
Scheme 1 DERA-catalyzed aldol reaction towards β-hydroxy-γ-lactols 1
screening, enantioselectivity, diastereoselectivity, extremophilic aldo-
lases
proaches or protein engineering, are used to find new pos-
sible enzymatic catalysts.8 Here, a fast and reliable high-
throughput screening system is essential for the identifica-
tion of positive hits.
Fluorogenic screenings are often favored over other
methods, due to their high sensitivity, which allows low
substrate and enzyme concentrations.9 In 1998 Reymond
and coworkers reported a fluorogenic screening for aldo-
lases, based on a retroaldol reaction of umbelliferone-cou-
pled aldols.10 As activity assay for DERA, Greenberg used
2004 4-methylumbelliferone-coupled D-ribose,11 whereas
Fei et al. applied a green fluorescence coumarin deriva-
tive.12
Here we present a fluorescence screening system adapt-
ed from Reymond et al. and Greenberg et al., which we ex-
panded to a enantio- and diastereoselectivity assay by af-
fording efficient and straightforward syntheses to the fluo-
rogenic substrates 5 and 6. Based on the natural DERA
reaction (Scheme 2) the enantio- and diastereoselectivity
was assayed using 5-O-(4′-methylumbelliferyl)-2-deoxy-
pentoses, such as 2-deoxy-D-ribose (D-5), 2-deoxy-L-ribose
(L-5), and 2-deoxy-D-xylose (6). The aldose form 7 could be
cleaved, depending on the configuration of the sugar moi-
Enantiomerically pure aldol compounds represent a
highly desirable class of building blocks in organic synthe-
sis. However, the enantioselective synthesis by convention-
al methods often remains a complex challenge.2 Besides dif-
ferent organic synthetic methods,3 aldolases offer excellent
stereo- and chemoselectivity under mild reaction condi-
tions.4 A particularly promising representative is the 2-de-
oxy-D-ribose-5-phophate aldolase (DERA), due to its unique
ability to utilize an aldehyde (as a natural substrate) as the
nucleophile, giving access to a double aldol reaction result-
ing in β-hydroxy-γ-lactols 1 (Scheme 1).5 Applying this en-
zyme, various complex natural products have been synthe-
sized with good yields and high enantioselectivity without
harsh reaction conditions.6 A prominent example is the key
building block for the pharmaceutical blockbuster atorvas-
tatin synthesized by Jennewein et al.7
For synthetic purposes, it is desirable to produce all pos-
sible stereoisomers of a given compound. Unfortunately, in
the case of DERA, there is no natural stereocomplementary
enzyme known. To complete the synthetic toolbox, en-
zymes with an inverted enantio- and diastereoselectivity
are highly desired. Different methods, like metagenome ap-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, 11–16

12
C. Bisterfeld et al.
Letter
Synlett
OH
OH
O
O
DERA
2–O3PO
2–O3PO
H
OH
O
+
HO
O
O
O
O
OH
O
OH
HO
2
3
4
D-11
L-11
HO
D-5
L-5
Scheme 2 DERA-catalyzed in vivo retro-aldol reaction of 2-deoxy-D-ri-
bose-5-phosphate (2) to glyceraldehyde-3-phosphate (3) and acetalde-
hyde (4)
1) K2CO3, 4-MeUmb,
DMF, 75 °C, 16 h
2) DOWEX 50WX,
MeCN–H2O, r.t., 2 d
(D) 92%
(L) quant.
0.1% MeOH–HCl,
r.t., 12 min
(D) 88%
(L) 87%
ety, to the corresponding glyceraldehyde derivative 8. This
compound 8 decomposes spontaneously by β-elimination
to aldehyde 9 and the fluorophore 10, which can be quanti-
fied even at low concentrations (Scheme 3).
p-TsCl, pyridine,
–5 °C to r.t., 16 h
O
O
O
O
The enantiocomplementary screening substrates D-5
and L-5 were synthesized in a short sequence from 2-de-
oxy-D-ribose (D-11) and 2-deoxy-L-ribose (L-11), respec-
tively (Scheme 4). To stabilize the cyclic form of the fura-
noses, the anomeric hydroxyl group was methylated using
methanolic hydrochloride acid, carefully monitoring the re-
action to avoid undesired overmethylation of the primary
and secondary hydroxy group.13 The methylated D- and L-
ribose 12 were isolated in good to excellent yields (92%–
quant.).14 To install a good leaving group at the 5-hydroxy
position, a standard tosylation, using p-tosyl chloride in
pyridine, was applied yielding the compounds D-13
(65%) and L-13 (68%).15 Substitution with the fluorophore
4-methylumbelliferone (10) was performed in DMF using
K2CO3 as base.11 A following demethylation, applying an
acidic cation exchange resin, provided the screening sub-
strates in good yields (D-5, 88%; L-5, 87%) over two steps.16
In order to analyze the diastereoselectivity of different
DERA, it became necessary to synthesize 4-methylumbelli-
ferone-coupled D-xylose substrate 6 (Scheme 5), since 2-de-
TsO
HO
HO
(D) 65%
(L) 68%
HO
D-12
L-12
D-13
L-13
Scheme 4 Three-step synthesis towards the enantiomeric fluorogenic
substrates D-5 and L-5
oxy-D-xylose is very expensive ($1250/gram, Carbosynth).
Hence, it was synthesized based on 2-deoxy-D-ribose by in-
verting the configuration at the 3-position of the carbohy-
drate moiety. Analogously, the tosyl group was substituted
with 4-methylumbelliferone, but without subsequent de-
methylation, to uphold the cyclic form of the furanose. The
product 14 was isolated with a good yield of 80%.17 A reac-
tion with the Dess–Martin periodinane (DMP)18 in di-
chloromethane led to oxidation of the unprotected 3-hy-
droxy group, yielding 82% of compound 15.19 After reduc-
tion with NaBH4 36% of the desired diastereomer was
(A)
(B)
OH
O
O
O
O
O
OH
OH
OH
OH
O
O
O
O
O
O
O
O
O
O
O
D-5
OH
HO
HO
HO
HO
5, 6
7
O
DERA
H
O
O
O
O
L-5
O
spontaneous
pH 7
O
+
O
O
O
O
O
O–
OH
OH
10
9
8
6
λ
em = 460 nm
ex = 340 nm
λ
Scheme 3 (A) Synthesized screening substrates D-5, L-5, and 6; (B) DERA-catalyzed screening reaction
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, 11–16

13
C. Bisterfeld et al.
Letter
Synlett
work very efficiently at low temperatures – resulting in an
increased activity and a broader substrate spectrum (due to
the high flexibility of the proteins).27
DMF, K2CO3,
4-MeUmb
82%
O
O
TsO
HO
O
O
O
O
O
HO
D-13
14
DMP, CH2Cl2
80%
NaBH4,
EtOH–H2O
36%
O
UmbO
HO
O
O
O
UmbO
O
16
15
MeCN–H2O,
DOWEX 50WX
58%
Umb =
O
O
O
O
O
O
OH
HO
6
Scheme 5 Synthesis of the 2-deoxy-D-xylose fluorogenic substrate 6
by inverting the configuration of the 2-deoxy-D-ribose moiety
isolated.20 Final demethylation with DOWEX 50WX yielded
the fluorogenic D-xylose product 6 in 58%.21
To afford a high throughput, the screening was realized
in 96-well microtiter plates. The fluorescence was mea-
sured, applying an excitation wavelength of 340 nm and
measuring an emission wavelength of 460 nm. The fluoro-
genic substrates D-5, L-5, and 6 were dissolved in acetoni-
trile–water to form stable stock solutions, which were used
for the measurement in triethanolamine buffer (0.1 M, pH
7). To determine the volume activity of various DERA a cali-
bration curve using 4-methylumbelliferone (10) was re-
corded.22 The proteins were provided by their gene expres-
sion in E. coli BL21 (DE3) and inserted for the screening as
cell-free crude extract.23
Applying this screening system, we tested DERA from E.
coli and five additional aldolases from psychrophilic (Col-
wellia psychrerythraea, Shewanella halifaxensis), mesophilic
(Rhodococcus erythropolis),24 and hyperthermophilic (Pyro-
baculum aerophilum, Thermotoga maritima)25 organisms for
their enantio- and diastereoselectivity (Figure 1). Extremo-
philic enzymes are interesting targets for various applica-
tions in biotechnology: While (hyper)thermophilic en-
zymes show high stability even under harsh reaction condi-
tions,26 their psychrophilic homologues have adapted to
Figure 1 Activity of psychrophilic, mesophilic, and hyperthermophilic
DERA enzymes for the 4-methylumbelliferone-coupled D-ribose D-5, L-
ribose L-5,-and D-xylose 6 substrate. Error bars represent the standard
deviation of triplet measurements. As control cells, transformed with an
empty vector were used.
The screening results show a higher volume activity of
the DERA from psychrophilic organisms compared to the
usually used E. coli DERA at a similar expression level (see
Supporting Information). It was found that the hyperther-
mophilic variants show nearly no conversion of the sub-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, 11–16

14
C. Bisterfeld et al.
Letter
Synlett
strates. As the expression level is low, but higher than in the
negative control, these results indicate a low activity for the
used substrates. Interestingly, under these screening condi-
tions DERA from C. psychrerythraea showed an even higher
activity for the L-ribose substrate L-5 than for the D-ribose
compound D-5. This corresponds to the theory that psy-
chrophilic enzymes are more flexible than their meso- or
(hyper)thermophilic homologues. Furthermore this en-
zyme might be a promising candidate to accept a broader
substrate scope as the E. coli DERA.28
In summary, we showed short and straightforward syn-
theses to obtain useful fluorophore-coupled screening sub-
strates, which can be applied in a reliable, fast, and highly
sensitive microtiter-plate screening system. We expanded
this fluorescence-based screening from an activity assay to
a complex screening for enantio- as well as diastereoselec-
tivity. This enables us to test a high number of DERA en-
zymes to find possible new catalysts for application in or-
ganic synthesis.
(4) (a) Clapés, P.; Garrabou, X. Adv. Synth. Catal. 2011, 353, 2263.
(b) Brovetto, M.; Gamenara, D.; Saenz Méndez, P.; Seoane, G. A.
Chem. Phys. Lett. 2011, 111, 4346. (c) Dean, S. M.; Greenberg, W.
A.; Wong, C.-H. Adv. Synth. Catal. 2007, 349, 1308. (d) Fessner,
W.-D.; Helaine, V. Curr. Med. Chem. 2001, 12, 574.
(5) (a) Liu, J.; Hsu, C.-C.; Wong, C.-H. Tetrahedron Lett. 2004, 45,
2439. (b) Gijsen, H. J. M.; Wong, C.-H. J. Am. Chem. Soc. 1994,
116, 8422. (c) Liu, J.; Wong, C.-H. Angew. Chem. 2002, 114, 1462.
(d) Patel, J. M. J. Mol. Catal. B 2009, 61, 123.
(6) (a) Clapés, P.; Fessner, W.-D.; Sprenger, G. A.; Samland, A. K.
Curr. Opin. Chem. Biol. 2010, 14, 154. (b) Wolberg, M.; Dassen, B.
H. N.; Schürmann, M.; Jennewein, S.; Wubbolts, M. G.;
Schoemaker, H. E.; Mink, D. Adv. Synth. Catal. 2008, 350, 1751.
(7) Jennewein, S.; Schürmann, M.; Wolberg, M.; Hilker, I.; Luiten,
R.; Wubbolts, M.; Mink, D. Biotechnol. J. 2006, 1, 537.
(8) (a) Pricer, W. E.; Horecker, B. L. J. Biochem. Biophys. Methods
1960, 235, 1292. (b) Sgarrella, F.; Del Corso, A.; Tozzi, M. G.;
Camici, M. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol.
1992, 1118, 130. (c) Han, T. K.; Zhu, Z.; Dao, M. L. Curr. Microbiol.
2004, 48, 230. (d) Kim, Y.-M.; Chang, Y.-H.; Choi, N.-S.; Kim, Y.;
Song, J. J.; Kim, J. S. Protein Expression Purif. 2009, 68, 196.
(e) Jiao, X.-C.; Pan, J.; Xu, G.-C.; Kong, X.-D.; Chen, Q.; Zhang, Z.-
J.; Xu, J.-H. Catal. Sci. Technol. 2015, 5, 4048.
(9) (a) Wahler, D.; Reymond, J.-L. Curr. Med. Chem. 2001, 12, 535.
(b) Rogers, M. V. Drug Discovery Today 1997, 2, 156. (c) Liebl,
W.; Angelov, A.; Juergensen, J.; Chow, J.; Loeschcke, A.; Drepper,
T.; Classen, T.; Pietruszka, J.; Ehrenreich, A.; Streit, W. R.; Jaeger,
K.-E. Appl. Microbial. Biotechnol. 2014, 98, 8099.
(10) Jourdain, N.; Carlón, R. P.; Reymond, J.-L. Tetrahedron Lett. 1998,
39, 9415.
(11) Greenberg, W. A.; Varvak, A.; Hanson, S. R.; Wong, K.; Huang, H.;
Chen, P.; Burk, M. J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 5788.
(12) Fei, H.; Xu, G.; Wu, J.-P.; Yang, L.-R. Biochem. Biophys. Res.
Commun. 2015, 460, 826.
(13) Deriaz, R. E.; Overend, W. G.; Stacey, M.; Wiggins, L. F. J. Chem.
Soc. 1949, 2836.
(14) (3S,4R)-4-(Hydroxymethyl)-1-methoxytetrahydrofuran-3-ol
(D-12) and (3R,4S)-4-(Hydroxymethyl)-1-methoxytetrahy-
drofuran-3-ol (L-12)
Acknowledgment
We gratefully acknowledge the Ministry of Innovation, Science, and
Research of the German federal state of North Rhine-Westphalia
(NRW) and the Heinrich Heine University Düsseldorf (HHU; scholar-
ship within the CLIB-Graduate Cluster Industrial Biotechnology for
I.K., iGRASPseed-Graduate Cluster for M.D., and technology platform
‘ExpressO’), the Deutsche Forschungsgemeinschaft, and the For-
schungszentrum Jülich GmbH for the support of our projects. The sci-
entific activities of the Bioeconomy Science Center were financially
supported by the Ministry of Innovation, Science and Resesarch with-
in the framework of the NRW-Strategieprojekt BioSC (No. 313/323-
400-002 13). We thank Vera Ophoven for supporting experiments
and Dr. Thomas Classen for helpful discussions.
2-Deoxy-D-ribose (D-11, 0.5 g, 3.7 mmol) were dissolved in
methanolic HCl (10 mL, 0.1%) and stirred at r.t. for 12 min. The
reaction was controlled by rotary power and after full conver-
sion neutralized with Ag2CO3. The mixture was filtrated, and
the solvent was evaporated. The crude product was purified by
Kugelrohr distillation; 506 mg (92%, dr A/B = 0.6:0.4) D-12 were
isolated as a colorless oil. Analogously 100 mg (0.75 mmol) 2-
desoxy-L-ribose (L-11) were converted into 116 mg (0.78 mmol,
quantitative yield, dr A/B = 0.6:0.4) L-12. Rf = 0.06 (PE–EtOAc,
40:60). IR (film): ν = 3383, 2918, 1444, 1206, 1050 cm–1. GC–MS
(EI, +, 70 eV): m/z (%) = 147 (1) [M + H]+, 117 (100) [C5H9O3]+, 88
(52) [C5H10O]+, 71 (26) [C4H8O]+. 1H NMR (600 MHz, CDCl3): δ =
2.00 (m, 1 H, 2b-HA), 2.08–2.14 (m, 3 H, 2b-HB, 2a-HA, 3-OHA),
2.26 (ddd, J = 13.9, 6.9, 2.0 Hz, 1 H, 2a-HB), 2.50 (d, J = 5.7 Hz,
1 H, 3-OHB), 2.78 (m, 1 H, 5-OHB), 2.90 (d, J = 10.23 Hz, 1 H, 5-
OHA), 3.38 (mc, 6 H, OMeA, OMeB), 3.58–3.73 (m, 4 H, 5a-HA, 5a-
HB, 5b-HA, 5b-HB), 4.05 (ddd, J = 3.5 Hz, 1 H, 4-HB), 4.10–4.17 (m,
2 H, 4-HA, 3-HA), 4.51 (ddd, J = 8.4, 6.9, 3.5 Hz, 1 H, 3-HB), 5.10
(dd, J = 4.5, 0.2 Hz, 1 H, 1-HA), 5.11 (dd, J = 5.7, 2.0 Hz, 1 H, 1-HB)
ppm. 13C NMR (151 MHz, CDCl3): δ = 41.5 (C-2A), 42.5 (C-2B),
54.9 (OMeA), 55.4 (OMeB), 63.1 (C-5A), 63.5 (C-5B), 72.1 (C-3B),
72.8 (C-3A), 87.4 (C-4A), 87.5 (C-4B), 105.5 (C-1A), 105.6 (C-1B)
Supporting Information
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1560723.
S
u
p
p
ortiInfogrmoaitn
S
u
p
p
ortioInfgrmoaitn
References and Notes
(1) Authors contributed equally.
(2) (a) Hu, S.; Zhang, L.; Li, J.; Luo, S.; Cheng, J.-P. Eur. J. Med. Chem.
2011, 3347. (b) Hayashi, Y.; Itoh, T.; Aratake, S.; Ishikawa, H.
Angew. Chem. 2008, 120, 2112. (c) Denmark, S. E.; Stavenger, R.
A. Acc. Chem. Res. 2000, 33, 432. (d) List, B.; Lerner, R. A.; Barbas,
C. F. III. J. Am. Chem. Soc. 2000, 122, 2395. (e) Mukaiyama, T. In
Organic Reactions; John Wiley and Sons: New York, 2004.
(3) (a) Masamune, S.; Choy, W.; Petersen, J. S.; Sita, L. R. Angew.
Chem. Int. Ed. 1985, 24, 1. (b) Notz, W.; Tanaka, F.; Barbas, C. F.
III. Acc. Chem. Res. 2004, 37, 580. (c) Paterson, I.; Goodman, J. M.;
Anne Lister, M.; Schumann, R. C.; McClure, C. K.; Norcross, R. D.
Tetrahedron 1990, 46, 4663. (d) Machajewski, T. D.; Wong, C.-H.
Angew. Chem. Int. Ed. 2000, 39, 1352.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, 11–16

15
C. Bisterfeld et al.
Letter
Synlett
ppm. HRMS (ESI, +): m/z (%) calcd for C6H12O4Na [M + Na]+:
171.06333; found: 171.06275. D-12: [α]D +39.4 (c 1.0, CHCl3);
lit.13 [α]D +38.4 (c 0.6, CH3COOH). L-12: [α]D –40 (c 0.93, CHCl3).
(15) [(3S,4R)-3-Hydroxy-1-methoxytetrahydrofuran-4-yl]methyl
p-Tosylate (D-13) and [(3R,4S)-3-Hydroxy-1-methoxy-tetra-
hydrofuran-4-yl]methyl p-Tosylate (L-13)
H, 5′a-HB), 4.26 (dd, J = 5.3, 4.9 Hz, 1 H, 5′b-HB), 4.42 (ddd, J =
5.9, 1.5, 1.4 Hz, 1 H, 3′-HA), 4.47 (ddd, J = 4.6, 4.5, 2.1 Hz, 1 H, 3′-
HB), 4.59 (ddd, J = 4.8, 4.7, 1.5 Hz, 1 H, 4′-HA), 4.64 (ddd, J = 5.3,
4.9, 4.6 Hz, 1 H, 4′-HB), 5.66 (dd, J = 4.6, 0.2 Hz, 1 H, 1′-HA), 5.67
(dd, J = 5.6, 2.5 Hz, 1 H, 1′-HB), 6.14 (d, J = 1.5 Hz, 2 H, 3-HA,B),
6.79 (d, J = 2.6 Hz, 2 H, 8-HA,B), 6.85 (dd, J = 8.8, 2.6 Hz, 1 H, 6-
HA), 6.89 (dd, J = 8.8, 2.6 Hz, 1 H, 6-HB), 7.48 (d, J = 8.8 Hz, 2 H, 5-
HA,B) ppm. 13C NMR (151 MHz, CDCl3): δ = 18.8 (Me), 41.6 (C-2′),
68.8 (C-5′), 73.5 (C-4′), 85.4 (C-3′), 99.8 (C-1′), 101.8 (C-8), 112.4
(C-3), 112.6 (C-6), 114.1 (C-4a), 125.8 (C-5), 152.7 (C-8a), 155.3
(C-7), 161.4 (C-2), 161.6 (C-4) ppm. HRMS (ESI, +): m/z (%) calcd
for C15H17O6 [M + H]+: 293.10251; found: 293.10190. D-5
[α]D +30 (c 0.6, CHCl3). L-5 [α]D –42.4 (c 0.23, CHCl3).
Under an argon atmosphere D-12 (4.20 g, 28.4 mmol) was dis-
solved in pyridine (100 mL) and cooled to –5 °C. A solution of
p-TsCl (5.22 g, 27.4 mmol, 1.00 equiv) in dry CH2Cl2 (40 mL)
was added dropwise. The temperature of the reaction was
maintained between –5 °C and 0 °C. The reaction was stirred for
1 h at 0 °C, warmed up to r.t. and stirred overnight. The conver-
sion was controlled by TLC. Ice-cold H2O (130 mL) and EtOAc
were added, and the reaction was extracted with a sat. CuSO4
solution. The aqueous phases were combined and extracted
with CH2Cl2. The combined organic phases were dried over
MgSO4 and filtered. The solvent was evaporated, and the crude
product was purified by column chromatography (PE–EtOAc,
70:30); 5.60 g (18.5 mmol, 65%, dr A/B = 0.7:0.3) of D-13 were
isolated as a colorless oil. Analogously 1.00 g (8.75 mmol) of L-
12 were converted into 1.40 g (4.63 mmol, 68%, dr A/B = 0.7:0.3)
of the colorless oil L-13. Rf = 1.9 (PE–EtOAc, 40:60). IR (film): ν =
3454, 2928, 1598, 1444, 1180, 1096, 665 cm–1. GC–MS (EI, +, 70
eV): m/z (%) = 301 (1) [M – H]+, 271 (1) [M – OCH3]+, 91 (71)
[C7H8]+. 1H NMR (600 MHz, CDCl3): δ = 2.03–2.10 (m, 3 H, 2a-HB,
2b-HA, 2b-HB), 2.21 (ddd, J = 13.4, 6.8, 1.7 Hz, 1 H, 2a-HA), 2.45
(s, 6 H, MeA, MeB), 3.23 (s, 3 H, OMeB), 3.34 (s, 3 H, OMeA), 4.02–
4.13 (m, 6 H, 5-HA, 5-HB, 4-HA, 4-HB), 4.20 (ddd, J = 4.06, 4.02, 1.7
Hz, 1 H, 3-HA), 4.43 (ddd, J = 6.6, 6.5 Hz, 3.4 Hz, 1 H, 3-HB), 5.01–
5.04 (m, 2 H, 1-HA, 1-HB), 7.33–7.37 (m, 4 H, 3′-HA, 3′-HB), 7.76–
7.83 (d, J = 8.3 Hz, 4 H, 2′-HA, 2′-HB) ppm. 13C NMR (151 MHz,
CDCl3): δ = 21.7 (Me), 41.0 (C-2A/B), 41.4 (C-2A/B), 55.0 (OMeA/B),
55.1 (OMeA/B), 69.4 (C-1′A/B), 70.1 (C-1′A/B), 72.7 (C-3A/B), 72.8 (C-
(17) 7-[(3S,4R)-3-Hydroxy-1-methoxytetrahydrofuran-4-
yl]methoxy-4-methyl-2H-chromen-2-one (14)
Compound D-13 (10 g, 33.1 mmol) was dissolved in DMF (25
mL). K2CO3 (2.5 g) and 4-methylumbelliferone (7.58 g, 43 mmol,
1.3 equiv) were added. The reaction was stirred for 16 h at 75
°C, then quenched with H2O, extracted with EtOAc, and washed
with 0.1 M NaOH. The organic phase was dried over MgSO4, and
the solvent was evaporated. The crude product was purified by
column chromatography (n-pentane–EtOAc, 50:50), yielding
8.33 g (27.2 mmol, 82%, dr A/B = 0.4:0.6) of 14 as a colorless oil.
Rf = 0.33 (n-pentane–EtOAc, 20:80). IR (film): ν = 3586, 3322,
3093, 2923, 2838, 1709, 1613, 1389, 1369, 1294, 1150, 1069,
1027, 971, 838 cm–1. GC–MS (EI, +, 70 eV): m/z (%) = 133 (10)
[C5H9O4]+, 147 (8) [C6H11O4]+. 1H NMR (600 MHz, CDCl3): δ =
2.15 (ddd, J = 13.4, 6.4, 5.4 Hz, 1 H, 2′a-HA), 2.22 (ddd, J = 13.8,
6.4, 4.6 Hz, 2 H, 2′a-HB, 2′b-HB), 2.32 (ddd, J = 13.4, 6.4, 1.8 Hz,
1 H, 2′b-HA), 2.39 (d, J = 1.3 Hz, 3 H, MeA,B), 3.33 (s, 3 H, OMeA),
3.42 (s, 3 H, OMeB), 4.03 (dd, J = 10.0, 4.5 Hz, 1 H, 5′a-HB), 4.07–
4.13 (m, 3 H, 5′a-HA, 5′b-HA, 5′b-HB), 4.26 (ddd, J = 6.4, 6.4, 4.4
Hz, 1 H, 3′-HA), 4.30 (ddd, J = 6.4, 1.6, 1.5 Hz, 1 H, 3′-HB), 4.43
(ddd, J = 4.6, 4.5, 1.6 Hz, 1 H, 4′-HB), 4.57 (ddd, J = 6.7, 6.6, 4.4 Hz,
1 H, 4′-HA), 5.13 (dd, J = 5.4, 1.8 Hz, 1 H, 1′-HA), 5.16 (dd, J = 4.6,
0.2 Hz, 1 H, 1′-HB), 6.13 (d, J = 1.3 Hz, 2 H, 3-HA,B), 6.81 (d, J = 2.5
Hz, 1 H, 8-HB), 6.84 (d, J = 2.5 Hz, 1 H, 8-HA), 6.86 (dd, J = 8.8, 2.5
Hz, 1 H, 6-HB), 6.89 (dd, J = 8.8, 2.5 Hz, 1 H, 6-HA), 7.48 (d, J = 8.8
Hz, 1 H, 5-HB), 7.49 (d, J = 8.8 Hz, 1 H, 5-HA) ppm. 13C NMR (151
MHz, CDCl3): δ = 18.8 (Me), 41.2/41.7 (C-2′), 55.2/55.3 (OMe),
68.8/69.9 (C-5′), 73.0/73.3 (C-4′), 83.8/85.4 (C-3′), 101.7/101.8
(C-1′), 105.5/105.8 (C-8), 112.3/112.4 (C-3), 112.7 (C-6),
114.0/114.1 (C-4a), 125.7 (C-5), 152.6/152.7 (C-8a), 155.2/155.3
(C-7), 161.3/161.4 (C-2), 161.7/161.8 (C-4) ppm. HRMS (ESI, +):
3A/B), 83.0 (C-5/C-4), 84.6 (C-5/C-4), 106.4 (C-1A/B), 106.7 (C-
1
A/B), 128.0 (C-2′), 130.0 (C-3′), 145.1 (C-4′) ppm. HRMS (ESI, +):
m/z (%) calcd for C13H18O6SNa [M + Na]+: 325.07218; found:
325.07163. D-13 [α]D +30 (c 0.6, CHCl3). L-13 [α]D –31.5 (c 0.9,
CHCl3).
(16) 7-{[(3S,4R)-1,3-Dihydroxytetrahydrofuran-4-yl]methoxy}-4-
methyl-2H-chromen-2-one (D-5) and 7-{[(3R,4S)-1,3-Dihy-
droxytetrahydrofuran-4-yl]methoxy}-4-methyl-2H-
chromen-2-one (L-5)
Compound D-13 (7.00 g, 23.2 mmol) was dissolved in DMF
(70 mL). K2CO3 (7.00 g, 50.6 mmol) and 4-methylumbelliferone
(5.30 g, 30.0 mmol, 1.3 equiv) were added. The reaction was
stirred for 16 h at 75 °C, then quenched with H2O (70 mL),
extracted with EtOAc and washed with 0.1 M NaOH. The
organic phase was dried over MgSO4 and filtered. The solvent
was evaporated. The crude product was dissolved in MeCN–H2O
(150 mL, 1:3), mixed with DOWEX 50WX 8-100 (4.5 g), stirred
for 1.5 h, and stored for 2 d at r.t. Resulting MeOH was evapo-
rated. The solution was filtered, and the solvent was evaporated.
The crude product was purified by column chromatography
(gradient PE–EtOAc, 70:30 → 40:60 → 10:90 → 0:100) yielding
6.40 g (21.9 mmol, 88%, dr A/B 0.8:0.2) D-5. Analogously 115 mg
(0.38 mmol) of L-13 was converted into 97 mg (0.33 mmol, 87%,
dr A/B 0.8:0.2) L-5. Rf = 0.09 (n-pentane–EtOAc, 20:80). IR (film):
ν = 3450, 2928, 1736, 1611, 1366, 1216 cm–1. GC–MS (EI, +, 70
eV): m/z (%) = 293 (100) [M + H]+, 246 (10) [C14H14O4]+. 1H NMR
(600 MHz, CDCl3): δ = 2.17–2.21 (m, 1 H, 2′a-HB), 2.24 (ddd, J =
13.9, 5.9, 4.6 Hz, 1 H, 2′a-HA), 2.32–2.37 (m, 2 H, 2′b-HA,B), 2.39
(d, J = 1.5 Hz, 6 H, MeA,B), 3.99 (dd, J = 10.0, 4.7 Hz, 1 H, 5′a-HA),
4.06 (dd, J = 10.0, 4.8 Hz, 1 H, 5′b-HA), 4.18 (dd, J = 5.3, 5.3 Hz, 1
m/z (%) calcd for
307.11762. [α]D +29.8 (c 1.0, CDCl3).
C16H20O6 [M +
H]+: 307.11761; found:
(18) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277.
(19) 7-[(4R)-1-Methoxy-3-oxotetrahydrofuran-4-yl]methoxy-4-
methyl-2H-chromen-2-one (15)
To a solution of DMP (10.4 g, 24.5 mmol, 1.5 equiv) in CH2Cl2
(300 mL) compound 14 (5 g, 16.3 mmol) dissolved in CH2Cl2 (25
mL) were added. The reaction was stirred for 2 h at r.t. before it
was quenched with 1 M Na2S2O3 solution (100 mL). Sat. NaHCO3
solution (100 mL) was added, and the reaction was stirred until
both phases were clear. The mixture was diluted with CH2Cl2
and H2O, the phases were separated, and the aqueous layer was
extracted with CH2Cl2. The organic phases were combined,
dried over MgSO4, and the solvent was evaporated. The crude
product was purified by column chromatography (PE–EtOAc,
60:40) yielding 3.97 g (13.1 mmol, 80%, dr A/B 0.7:0.3) of
product 15. Rf = 0.49 (PE–EtOAc, 50:50). IR (film): ν = 3079,
2939, 2359, 1752, 1707, 1610, 1391, 1268, 1087, 1063, 1016,
982, 953, 833, 806 cm–1. GC–MS (EI, +, 70 eV): m/z (%) = 58 (100)
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, 11–16

16
C. Bisterfeld et al.
Letter
Synlett
[C3H6O]+. 1H NMR (600 MHz, CDCl3): δ = 2.38 (d, J = 1.19 Hz, 3 H,
MeB), 2.39 (d, J = 1.28 Hz, 3 H, MeB), 2.50 (dd, J = 18.21, 0.32 Hz,
1 H, 2′a-HB), 2.52 (dd, J = 18.14, 1.12 Hz, 1 H, 2′a-HA), 2.74 (dd,
J = 18.21, 5.61 Hz, 1 H, 2′b-HB), 2.83 (dd, J = 18.14, 5.58 Hz, 1 H,
2′b-HA), 3.42 (s, 3 H, OMeA), 3.48 (s, 3 H, OMeB), 4.15 (dd, J =
10.3, 7.27 Hz, 1 H, 4′-HA), 4.26–4.34 (m, 4 H, 5′a-HA, 5′’a-HB, 5′’b-
HB, 4′-HB), 4.44 (dd, J = 7.27, 3.17 Hz, 1 H, 5′b-HA), 5.40 (dd, J =
5.58, 1.12 Hz, 1 H, 1′-HA), 5.43 (dd, J = 5.61, 0.32 Hz, 1 H, 1′-HB),
6.14 (mc, 2 H, 3-HA,B), 6.83 (d, J = 2.88 Hz, 1 H, 8-HB), 6.84 (d, J =
2.42 Hz, 1 H, 8-HA), 6.84 (dd, J = 8.67, 2.88 Hz, 1 H, 6-HB), 6.90
(dd, J = 8.84, J = 2.42, 1 H, 6-HA), 7.48 (d, J = 8.67 Hz, 1 H, 5-HB),
7.50 (d, J = 8.84 Hz, 1 H, 5-HA) ppm. 13C NMR (151 MHz, CDCl3):
δ = 18.7 (Me), 43.6 (C-2′A), 43.7 (C-2′B), 55.2 (OMeA), 55.3
(OMeB), 67.2 (C-4′B), 69.5 (C-4′A), 76.1 (C-5′B), 78.0 (C-5′A), 101.6
(C-8B), 101.9 (C-8A), 101.9 (C-1′B), 102.7 (C-1′A), 112.3 (C-3A),
112.4 (C-3A), 112.5 (C-4aA,B), 114.1 (C-6A), 114.2 (C-6B), 125.6
(C-5B), 125.7 (C-5A), 152.5 (C-4A,B), 155.1 (C-8aB), 155.2 (C-8aA),
161.2 (C-7A,B), 161.3 (C-2B), 161.4 (C-2A), 210.6 (C-3′A), 210.8 (C-
3′B) ppm. HRMS (ESI, +): m/z (%) calcd for C16H18O6 [M + H]+:
305.10196; found: 305.10199. [α]D +44.5 (c 1.1, MeCN).
solid. Rf = 0.06 (PE–EtOAc, 50:50). IR (film): ν = 3356, 1705,
1609, 1364, 1207 cm–1. GC–MS (EI, +, 70 eV): m/z (%) = 293
(100) [M + H]+, 246 (12) [C14H14O4]+. 1H NMR (600 MHz, CDCl3):
δ = 2.19 (ddd, J = 13.8, 4.6, 4.6 Hz, 1 H, 2′a-HA), 2.23–2.32 (m, 3
H, 2′b-HA, 2′a-HB, 2′b-HB), 2.40 (d, J = 1.3 Hz, 6 H, MeA,B), 4.28
(dd, J = 10.0 Hz, J = 6.2 Hz, 1 H, 5′a-HB), 4.30–4.34 (m, 3 H, 5′a-
HB, 5′a-HA, 5′b-HA), 4.35 (dd, J = 6.9, 3.9 Hz, 1 H, 3′-HB), 4.42 (d, J
= 8.6, 4.6 Hz, 1 H, 3′-HA),4.50–4.54 (m, 1 H, 4′-HA), 4.69 (ddd, J =
6.2, 3.9, 3.0 Hz, 1 H, 4′-HB), 5.61 (dd, J = 4.6, 0.3 Hz, 1 H, 1′-HA),
5.78 (dd, J = 5.4, 3.3 Hz, 1 H, 1′-HB), 6.14 (q, J = 1.3 Hz, 2 H, 3-
HA,B), 6.89 (d, J = 2.5 Hz, 2 H, 8-HA,B), 6.91 (dd, J = 8.7, 2.5 Hz, 2 H,
6-HA,B), 7.50 (d, J = 8.7 Hz, 2 H, 5-HA,B) ppm. 13C NMR (151 MHz,
CDCl3): δ = 18.8 (Me), 42.1 (C-2′), 68.8 (C-5′), 71.9 (C-4′), 82.3
(C-3′), 99.4 (C-1′), 102.0 (C-8), 112.4 (C-3), 112.5 (C-6), 114.1 (C-
4a), 125.8 (C-5), 152.7 (C-8a), 155.3 (C-7), 161.4 (C-2), 161.8 (C-
4) ppm. HRMS (ESI, +): m/z (%) calcd for C16H20O6 [M + H]+:
293.10196; found: 293.10218. [α]D +11.5 (c 0.2, MeCN).
(22) Protocol of the Fluorogenic Assay
Triethanolamine buffer (120 μL, 0.1 M, pH 7.0), the specific sub-
strate (10 μL of a 10 mM solution), and BSA (10 μL of a solution
40 mg/mL in H2O) were pipetted in a 96-well microtiterplate.
Cell-free crude lysate (60 μL) was added and then directly mea-
sured with a GENios Microtiterplate Reader (Tecan, Switzer-
land) for 1–2 h, applying an excitation wavelength of 340 nm
and an emission wavelength of 460 nm with a band-width of 5
nm. For calibration a solution of 4-methylumbelliferone in
MeCN was prepared and measured in triethanolamine buffer
(using 0–2.5 nmol 4-methylumbelliferone). The calibration
curve is shown in the Supporting Information.
(20) 7-[(3R,4R)-3-Hydroxy-1-methoxytetrahydrofuran-4-
yl]methoxy-4-methyl-2H-chromen-2-one (16)
Compound 15 (3.97 g 13.1 mmol) was dissolved in EtOH–H2O
(1:1) and cooled to 0 °C. NaBH4 (1.23 g, 32.7 mmol, 2.5 equiv)
was added, and the reaction was stirred for 1 h at r.t. The reac-
tion was quenched with a sat. NH4Cl solution and extracted
with CHCl3. The organic phases were washed with H2O, dried
over MgSO4, and the solvent was evaporated. The diastereomers
(dr 1:1) could be separated by column chromatography (PE–
EtOAc, 50:50); 1.45 g (4.73 mmol, 36%) of the desired diastereo-
mer 16 were isolated. Rf = 0.17 (PE–EtOAc, 50:50). IR (film): ν =
3395, 3060, 2946, 1716, 1609, 1370, 1208 cm–1. GC–MS (EI, +,
70 eV): m/z (%) = 133 (19) [(C5H9O4)+], 147 (8) [(C6H11O4)+].
1H NMR (600 MHz, CDCl3): δ = 2.13–2.21 (m, 2 H, 2′-H), 2.37 (d,
J = 1.2 Hz, 3 H, Me), 2.96 (d, J = 10.9 Hz, 1 H, 3′-OH), 3.38 (s, 3 H,
OMe), 4.19 (dd, J = 9.7, 6.8 Hz, 1 H, 5′a-H), 4.34 (ddd, J = 6.8, 4.1
Hz, 1 H, 4′-H), 4.36–4.41 (m, 2 H, 5′b-H, 3′-H), 5.11 (dd, J = 4.0
Hz, 1 H, 1′-H), 6.10 (q, J = 1.2 Hz, 1 H, 3-H), 6.87 (d, J = 2.5 Hz, 1
H, 8-H), 6.90 (dd, J = 8.8, 2.5 Hz, 1 H, 6-H), 7.48 (d, J = 8.8 Hz, 1 H,
5-H) ppm. 13C NMR (151 MHz, CDCl3): δ = 18.8 (Me), 41.5 (C-2′),
55.3 (OMe), 69.0 (C-5′), 71.6 (C-3′), 82.5 (C-4′), 101.9 (C-8),
105.5 (C-1′), 112.2 (C-3), 112.6 (C-6), 113.9 (C-4a), 125.7 (C-5),
152.6 (C-4), 155.3 (C-8a), 161.4 (C-7), 161.9 (C-2) ppm. HRMS
(ESI, +): m/z (%) calcd for C16H20O6 [M + H]+: 307.11761; found:
307.11769. [α]D –67.4 (c 1.0, CHCl3).
(23) Protein Expression
Genes, coding for DERA (de °C) from P. aerophilum, T. maritima
and C. psychrerythraea were ordered as synthetic genes from
GenScript, followed by cloning into the pET-21a(+)-vector. For S.
halifaxensis, the corresponding gene could be isolated from
genomic DNA, whereas de °C genes from E. coli and R. erythrop-
olis were isolated previously.24 The proteins were expressed in
E. coli BL21(DE3) strain, using TB medium. Expression was
started by applying 0.1 mM IPTG, and cells were harvested after
16 h (24 h for psychrophilic enzymes) incubation at 25 °C (18
°C); 1 g cells were resuspended in potassium phosphate (KPi)
buffer (5 mL, 20 mM, pH 7). The cells were disrupted via sonifi-
cation, and after centrifugation (15 min at 12000 × g) the super-
natant was taken for kinetic measurements. A SDS-gel of all
used proteins is shown in the Supporting Information.
(24) Kullartz, I.; Pietruszka, J. J. Bioltechnol. 2012, 161, 174.
(25) Sakuraba, H.; Yoneda, K.; Yoshihara, K.; Satoh, K.; Kawakami, R.;
Uto, Y.; Tsuge, H.; Takahashi, K.; Hori, H.; Ohshima, T. Appl. Envi-
ron. Microbiol. 2007, 73, 7427.
(26) Egorova, K.; Antranikian, G. Curr. Opin. Microbiol. 2005, 8, 649.
(27) Feller, G. Scientifica 2013, 28.
(28) (a) Chen, L.; Dumas, D. P.; Wong, C. H. J. Am. Chem. Soc. 1992,
114, 741. (b) DeSantis, G.; Liu, J.; Clark, D. P.; Heine, A.; Wilson, I.
A.; Wong, C.-H. Bioorg. Med. Chem. 2003, 11, 43.
(21) 7-{[(3R,4R)-1,3-Dihydroxytetrahydrofuran-4-yl]methoxy}-4-
methyl-2H-chromen-2-one (6)
Compound 15 (980 mg, 3.2 mmol) was dissolved in MeCN–H2O
(3:1). DOWEX 50WX 8-100 (130 mg) was added, and the reac-
tion was stirred for 1.5 h at r.t. Resulting MeOH was evaporated,
and the mixture was stored at r.t. for 2 d. The solution was fil-
tered, and the solvent was evaporated. The crude product was
purified by column chromatography (PE–EtOAc, 70:30) yielding
544 mg (1.86 mmol, 58%, dr A/B 0.7:0.3) of product 6 as a white
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, 11–16