An eco-friendly enantioselective access to (R)-naringenin as inhibitor of proinflammatory cytokine release

Article abstract of DOI:10.1002/cbdv.201200227

(RS)-Naringenin is a flavanone well-known for its beneficial health-related properties, such as its anti-inflammatory activity. The preparative enantioselective chromatographic resolution of commercial (RS)-naringenin was performed on a Chiralpak AD-H column (500×50 mm i.d., d_p 20 μm) using MeOH as eluent. The developed method is in accordance with the principles of green chemistry, since the environmental impact was lowered by recycling of the eluent, and allowed the production of gram amounts of each enantiomer with high purity (chemical purity >99%, enantiomeric excess (ee) >94%). Racemic and enantiomeric naringenin were subjected to an exhaustive in vitro investigation of anti-inflammatory activity, aimed at evaluating the relevance of chirality. The assay with cultured human peripheral blood mononuclear cells (hPBMC) activated by phytohemagglutinin A revealed that (R)-naringenin was more effective in inhibiting T-cell proliferation than the (S)-enantiomer and the racemate. Moreover, (R)-naringenin significantly reduced proinflammatory cytokine levels such as those of TNF-α and, with less potency, IL-6. These results evidenced the anti-inflammatory potential of naringenin and the higher capacity of (R)-naringenin to inhibit both in vitro hPBMC proliferation and cytokine secretion at non toxic doses. Thus, (R)-naringenin is a promising candidate for in vivo investigation. Copyright

Full text of DOI:10.1002/cbdv.201200227

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1531
An Eco-Friendly Enantioselective Access to (R)-Naringenin as Inhibitor of
Proinflammatory Cytokine Release
a
a
a
b
c
by Raffaella Gaggeri ), Daniela Rossi ), Maria Daglia ), Flavio Leoni ), Maria Antonia Avanzini ),
c
d
a
Melissa Mantelli ), Markus Juza ), and Simona Collina* )
a) Department of Drug Sciences, University of Pavia, Viale Taramelli 12, I-27100 Pavia
(
phone: þ39-0382987379; fax: þ39-0382422975; e-mail: simona.collina@unipv.it)
b) Italfarmaco Research Center, Viale dei Lavoratori 54, I-20092 Cinisello Balsamo (MI)
c) Pediatric Haematology/Oncology Department, Fondazione IRCCS Policlinico San Matteo, Piazzale
Golgi 19, I-27100 Pavia
d) Analytical Development SIL, DSM Nutritional Products, Hauptstrasse 4, CH-4334 Sisseln
(
RS)-Naringenin is a flavanone well-known for its beneficial health-related properties, such as its
anti-inflammatory activity. The preparative enantioselective chromatographic resolution of commercial
(
RS)-naringenin was performed on a Chiralpak AD-H column (500ꢀ50 mm i.d., d 20 mm) using MeOH
p
as eluent. The developed method is in accordance with the principles of green chemistry, since the
environmental impact was lowered by recycling of the eluent, and allowed the production of gram
amounts of each enantiomer with high purity (chemical purity >99%, enantiomeric excess (ee) >94%).
Racemic and enantiomeric naringenin were subjected to an exhaustive in vitro investigation of anti-
inflammatory activity, aimed at evaluating the relevance of chirality. The assay with cultured human
peripheral blood mononuclear cells (hPBMC) activated by phytohemagglutinin A revealed that (R)-
naringenin was more effective in inhibiting T-cell proliferation than the (S)-enantiomer and the
racemate. Moreover, (R)-naringenin significantly reduced proinflammatory cytokine levels such as those
of TNF-a and, with less potency, IL-6. These results evidenced the anti-inflammatory potential of
naringenin and the higher capacity of (R)-naringenin to inhibit both in vitro hPBMC proliferation and
cytokine secretion at non toxic doses. Thus, (R)-naringenin is a promising candidate for in vivo
investigation.
Introduction. – The main processes of both innate and specific immunity are widely
mediated by hormones called cytokines. Among them, interleukin-6 (IL-6), tumor
necrosis factor-a (TNF-a), and interferon-g (IFN-g) are pleiotropic and exert a pivotal
role in a large spectrum of immunological phenomena. Indeed, high concentrations of
these cytokines may cause inflammation and organ injury, leading to chronic
inflammatory and autoimmune diseases, such as rheumatoid arthritis (RA), Crohnꢀs
inflammatory bowel disease, and multiple sclerosis [1][2]. Particularly, a deeper
understanding of the RA pathogenesis has provided new therapeutic opportunities
directly targeting proinflammatory cytokines, such as TNF-a and IL-6, which are the
most important mediators of the RA process [3]. Moreover, clinical evidence has
clearly shown that TNF-a blockers are also a new effective tool in the treatment of
bowel diseases and psoriasis [4]. Recently, our ethnobotanical-directed search for novel
anti-inflammatory drugs revealed that Amygdalus lycioides Spach, an Iranian
medicinal plant, showed interesting anti-inflammatory activity exerted via a TNF-a
ꢁ
2013 Verlag Helvetica Chimica Acta AG, Zꢂrich

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
blocker mechanism [5]. Interestingly, the bioassay-guided fractionation of the A.
lycioides root extract allowed identifying naringenin as the compound responsible of
the anti-TNF-a effect of the crude extract. Naringenin is a well-known natural
flavanone with a stereogenic center at C(2), mainly distributed in citrus fruits. The
beneficial, health-related properties of (RS)-naringenin have been extensively inves-
tigated [6–10].
Interestingly, the anti-inflammatory activity of (RS)-naringenin via the inhibition of
TNF-a together with interleukin-1b was reported [9], but the enantioselectivity of this
effect has not been investigated yet. This might be related to a lack of easily available,
pure naringenin enantiomers.
Both naringenin enantiomers are present in natural sources, such as orange, apple,
and tomato juices, even if in low amounts (Table) [11].
Table. Stereospecific Distribution of Naringenin in Natural Sources
Natural source
(S)-Naringenin [mg/100 ml of juice]
(R)-Naringenin [mg/100 ml of juice]
Orange juice
Grapefruit juice
Apple juice
0.036ꢁ0.001
0.031ꢁ0.059
0.031ꢁ0.001
0.759ꢁ0.029
0.017ꢁ0.001
0.077ꢁ0.002
0.018ꢁ0.001
0.693ꢁ0.022
Tomato juice
Several extraction methods have been described to obtain (S)-naringenin from
different vegetal matrices, such as A. lycioides Spach branches [12] and Typha
angustata pollen [13], but with low extraction yields. Over recent years, various
attempts towards asymmetric synthesis of flavanones have been undertaken repeatedly
[
14–21]. However, only a small number of strategies allowed synthesizing a stereo-
center at C(2), such as the synthesis of chiral flavanones by using natural chirons (chiral
synthons) [14][15], by kinetic resolution with lipase [16][17], and by using chiral
catalysts [18–21]. In contrast to the various approaches of enantioselective synthesis of
(
(
S)-naringenin [22–25], to the best of our knowledge, no enantioselective synthesis of
R)-naringenin has been performed yet. Thus, to obtain both naringenin enantiomers
in suitable amounts and purity sufficient for an exhaustive biological investigation, we
explored an enantioselective chromatographic approach. Indeed, enantioselective
chromatography is a powerful tool to obtain a single enantiomer in sufficient amount
and of appropriate quality to support biological studies, and might be more effective
than the current synthetic routes [26].

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1533
As a part of our ongoing research on new small molecules for treating inflammatory
diseases, in this work, we focused on the evaluation of the relevance of chirality in the
anti-inflammatory effect exerted by naringenin. Firstly, to have gram amounts of the
naringenin enantiomers available in purity adequate to perform biological assays,
direct chiral chromatographic resolution of commercial (RS)-naringenin at preparative
scale was performed. Then, an exhaustive in vitro evaluation of the inhibitory effect of
each enantiomer towards T-cell proliferation as well as cytokine release was carried out
in comparison with the one exerted by the racemic form.
Results and Discussion. – Preparative Enantioselective Chromatography. The
preparative method previously developed by our research group [27], with some
modifications allowing to process a larger amount of (RS)-naringenin, was used. This
led to the production of the quantity of each enantiomer necessary for an exhaustive
biological in vitro and in vivo evaluation. The enantioselective separation, previously
achieved on a Chiralpak AD-H column (150ꢀ4.6 mm i.d., d 5 mm), was scaled up by
p
using a dynamically axial compressed (DAC) column (4.1 cm i.d.) packed with 100 g of
bulk Chiralpak AD CSP (d 20 mm) and using pure MeOH as eluent, which ensured
p
short retention times (t 8.9 and 12.3 min). Since the most important prerequisite for an
R
economic and productive preparative enantiomer separation is the use of a mobile
phase consisting of a pure, low-cost solvent, together with t as short as possible, these
R
experimental conditions were considered to be proper scaled-up. For the envisaged
scale of the present project, the commercially available preparative Chiralpak AD
column (500ꢀ50 mm i.d., d 20 mm) was used by achieving an increased enantiose-
p
lectivity and recovery, with respect to the former approach. In this way, a larger amount
of racemate was processed in each cycle, reducing time and eluent consumption.
Actually, 50 mg of (RS)-naringenin were processed in a single run (20 min) at a flow
rate of 40 ml/min at room temperature (Fig. 1). The overlap of the first- and second-
eluting enantiomer made the collection of a small intermediate fraction necessary.
The production of the required amount of enantiomers was achieved by repeated
injections (100) of a MeOH solution (50 mg/ml) of (RS)-naringenin, processing a total
of 5 g of racemate. The enantiomeric excess (ee) of the obtained fractions was
determined by HPLC/PAD/CD analysis (Chiralpak AD-H column, 150ꢀ4.6 mm i.d.,
d 5 mm; eluent, 100% MeOH; flow rate, 1 ml/min; detection at 290 nm). Overall, 2.1 g
p
(ee 96%) of the first enantiomer as well as 1.8 g (ee 94%) of the second one were
obtained together with an intermediate fraction of 980 mg as a mixture of the two
enantiomers. According to our previous study [27], the absolute configuration of the
enantiomers was assigned as follows: the first eluted enantiomer, showing a negative
2
5
CD signal at 290 nm and a negative optical rotatory power ([a] ¼ ꢂ28.7, c¼0.36,
D
EtOH), was the (S)-enantiomer, while the (R)-enantiomer corresponded to the
second eluted peak and was characterized by a positive CD signal and a positive optical
2
5
rotatory power ([a] ¼ þ22.8, c ¼ 0.30, EtOH). Therefore, the developed method led
D
to recover each enantiomer with high enantiomeric purity and in a sufficient amount
for in-depth biological investigation. Moreover, the use of MeOH as mobile phase
allowed the rapid recovery of the separated enantiomers (by evaporation) and easy
recycling of the solvent, thus reducing costs. The productivity and the feasibility of the

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Fig. 1. Preparative enantiomer separation of (RS)-naringenin on a Chiralpak AD column (500ꢀ50 mm
i.d., d 20 mm)
p
entire process were evaluated in terms of time consumption (35 h) as well as solvent
consumption (85 l).
Taking into account these considerations, the method developed has proved to be a
convenient approach to obtain naringenin enantiomers. Furthermore, it is in
accordance with the principles of green chemistry, by lowering the impact on the
environment through the reduction of solvent consumption and the recycling of the
mobile phase (100% MeOH). This is an improvement compared to already published
methods using preparative chiral stationary phases (CSPs) for the enantioseparation of
racemic naringenin.
Biological Evaluation. Since the anti-inflammatory effect of (RS)-naringenin has
already been demonstrated [9], and in the view of our ongoing research for novel anti-
inflammatory agents, the effect of the naringenin enantiomers was evaluated using in
vitro cultured human peripheral blood mononuclear cells (hPBMC) and compared to
the one obtained for (RS)-naringenin. Briefly, hPBMC from three healthy donors were
stimulated with phytohemagglutinin A (PHA) in the presence of increasing doses (6–
200 mg/ml) of both enantiomers and (RS)-naringenin, and the cell proliferation was
evaluated by monitoring the thymidine incorporation. Both enantiomers and the
racemate were clearly able to inhibit PHA-stimulated T-cell proliferation in a dose-
dependent manner, being active from 200 to 12 mg/ml (Fig. 2).
It has to be outlined that in vitro inhibition due to (S)-naringenin was comparable
to that observed for (RS)-naringenin with a proliferation partially restored at 25 mg/ml.
On the other hand, at the same concentration (25 mg/ml) of (R)-naringenin, the
stimulated hPBMC proliferation resulted to be considerably reduced. Moreover, the
(
R)-enantiomer showed to be also significantly more active than the (S)-enantiomer
and the racemate at lower concentration (12 mg/ml). (R)-Naringenin appeared to be
the most potent, with an IC50 value between 6 and 12 mg/ml, whereas (S)-naringenin

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1535
Fig. 2. Antiproliferative effect of (RS)-, (S)-, and (R)-naringenin on phytohemagglutinin A-stimulated
hPBMC
showed an IC50 between 25 and 50 mg/ml. (RS)-Naringenin showed a potency in
between the two enantiomers, as expected.
To confirm that the inhibitory effect was specific and to exclude that it was due to
cell death, the cell viability of the PHA-stimulated hPBMC was evaluated by the trypan
blue exclusion method in the presence of serial doses (6–200 mg/ml) of (RS)-, (S)-, and
(
R)-naringenin. The cell viability after culture was 98%, confirming the absence of in
vitro cytotoxicity (data not shown). Moreover, neither the enantiomers nor the
racemate did induce any proliferative effect on unstimulated hPBMC, indicating that
they are devoid of any adjuvant activity.
Given that stimulated hPBMC release cytokines, the observed inhibitory effect
could be confirmed by a decrease of the cytokine amount in the supernatants. Hence,
the quantification of cytokines (TNF-a, IL-6, and IFN-g) in the supernatants obtained
under the same culture conditions as described above (with or without PHA, (RS)-
naringenin, (S)-naringenin, and (R)-naringenin at the same concentrations as
mentioned above) was carried out. Analogously to the observations in the cell-
proliferation assay, (R)-naringenin showed to be more effective in reducing cytokine
levels than the (S)-enantiomer and the racemate, being the only one able to lower both
the IL-6 and TNF-a levels. Regarding TNF-a, its levels were decreased in a dose-
dependent manner in the presence of both enantiomers and the racemate in the
concentration range of 100–6 mg/ml (Fig. 3). As mentioned above, (R)-naringenin
reduced the TNF-a release with a significantly higher activity than the (S)-enantiomer
and the racemate. Interestingly, (RS)-naringenin showed the lowest activity in
inhibiting TNF-a, whereas (S)-naringenin showed an intermediate potency (Fig. 3).
Concerning the IL-6 release, it is only affected by (R)-naringenin, which was able to
reduce IL-6 levels by ca. 50% at a concentration of 25 mg/ml, whereas (S)-naringenin as
well as the racemate showed no effect on the production of this cytokine (Fig. 4). It is
interesting to note that neither the enantiomers nor (RS)-naringenin showed any
significant inhibitory effect towards IFN-g (Fig. 4).

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Fig. 3. Effect of (RS)-, (S)-, and (R)-naringenin at different doses (6–100 mg/ml) on the in vitro TNF-a
release of phytohemagglutinin A-stimulated hPBMC
Fig. 4. Effect of (RS)-, (S)-, and (R)-naringenin at 25 mg/ml on the in vitro cytokine release of
phytohemagglutinin A-stimulated hPBMC (*, p<0.05)
Fig. 4 shows the decrease in cytokine levels detected at 25 mg/ml. Actually, the
secretion of TNF-a and IL-6 was still inhibited in the presence of 12 mg/ml of (R)-
naringenin.
These data clearly evidenced the anti-inflammatory potential of (R)-naringenin
exerted through the inhibition of IL-6 and, in particular, of TNF-a. Moreover, the
inhibition of hPBMC proliferation suggests a possible immunosuppressive action that
should be evaluated in future studies.
Conclusions. – This work reports, for the first time, the evaluation of the
enantioselectivity of the anti-inflammatory effect of naringenin. The biological results
clearly showed that (R)-naringenin exerted the higher effect than the (S)-enantiomer
at non-toxic doses, being the eutomer. Moreover, we proposed an easy and reliable
method to produce (R)-naringenin. To the best of our knowledge, there are no
enantioselective routes to (R)-naringenin to date. The gram amounts of (R)-naringenin
isolated in high enantiomeric excess will be useful to further characterize the biological
profile of this promising molecule. Indeed, the in vivo biological evaluation (in a
murine model of systemic inflammation) is currently in progress and will be reported in
due course.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1537
The results of the present study and those deriving from the in vivo assays might
represent the rationale for the use of (R)-naringenin both in pharmaceutical and
nutraceutical formulations.
Experimental Part
General. (RS)-Naringenin (¼(ꢁ)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one) was ob-
tained from SigmaꢂAldrich (I-Milan). The solvents used as eluents were of HPLC grade and were
obtained from Merck-VWR (Milan, Italy). Analytical chiral resolutions were performed with a Jasco
system (Jasco Europe, Cremella, LC, Italy) equipped with a Jasco AS-2055 plus autosampler, a PU-2089
plus pump, and a MD-2010 plus multi-wavelength detector coupled with a CD-2095 plus circular
dichroism detector. The prep. chiral separations were carried out with a Varian Chromatographic Systems
apparatus (Walnut Creek, CA, USA) consisting of two Rainin SD-1 pumps with 500 ml/min pump heads,
a 410 Varian autosampler, a 320 Varian Prostar UV-detector, and a 320 Varian fraction-collection module.
Optical rotations: Jasco DIP 1000 photoelectric polarimeter (Jasco Europe, Cremella, LC, Italy), r.t., 1-
dm cell, Na lamp.
Chiral Chromatographic Resolution. Anal. Method. The analyses were carried out using a Chiralpak
AD-H column (250ꢀ4.6 mm i.d., dp 5 mm; Daicel Industries Ltd., Japan, obtained from Chiral
Technologies Europe, F-Illkirch). Sample solns. were prepared by dissolving the analyte ((RS)-
naringenin) at 1 mg/ml in EtOH and filtering the soln. through 0.45 mm PTFE membranes. The injection
volume was 10 ml, the flow rate 1 ml/min, and the detection set at 290 nm. The HPLC analyses were
performed at 208, and the separation factor (a) and the resolution (rs) were calculated according to
international recommendations.
Prep. Method. The prep. enantioselective chromatography was carried out at r.t. using a Chiralpak
AD column (500ꢀ50 mm i.d., d 20 mm; Daicel Industries Ltd., Japan, obtained from Chiral Technologies
p
Europe, F-Illkirch) and pure MeOH as mobile phase with a flow rate of 40 ml/min. The eluate was
properly partitioned according to the UV profile (detection at 290 nm). The anal. in-process control of
the collected fractions was performed using a Chiralpak AD-H column (250 mmꢀ4.6 mm i.d., d 5 mm)
p
and a mobile phase consisting of pure MeOH (flow rate, 1 ml/min; UV detector at 290 nm). The solvent
of the obtained fractions containing the enantiomers was evaporated at 334 mbar and 408 and dried in a
vacuum oven at 0.1 mbar and 258.
In vitro Anti-Inflammatory Assay. Proliferation in Bulk Culture. The in vitro proliferation assay using
human peripheral blood mononuclear cells (hPBMC) was carried out as described in [28]. Briefly,
hPBMC were separated by Ficoll-Hypaque centrifugation from healthy donor peripheral blood and
resuspended in RPMI 1640 medium (Gibco-BRL Life Technologies) supplemented with 10% of fetal
bovine serum (FBS; Euroclone, Celbio). The hPBMC were cultured in the presence of increasing doses
(
6–200 mg/ml) of (RS)-naringenin or its enantiomers with or without stimulation with phytohemag-
glutinin A (PHA; 4 mg/ml PHA-L; Roche, D-Mannheim). After incubation for 3 d at 378 in a humidified
% CO atmosphere and 18 h before harvesting, 25 mCi/well of 3 H-thymidine (3 HTdR; Amersham
5
2
Pharmacia Biotec, I-Milan) was added to each well. The radioactivity was measured (TopCount, Packard
Instrument), and the results were expressed as stimulation index (SI¼cpm of stimulated cultures/cpm of
unstimulated cultures). The same culture conditions were used to collect supernatants for the cytokine
quantification. The collected supernatants were kept at ꢂ208 until analysis.
Cytokine Quantification in Supernatants. The concentration of IL-6, IFNg, and TNF-a in the
supernatant was quantified by ELISA using monoclonal antibody pairs (Pierce Endogen, Rockford, IL,
USA). Briefly, plates (Corning Costar) were coated with purified antibodies at the appropriate
concentrations. Standard curves were prepared with recombinant human cytokine (Pierce Endogen).
Biotin-labeled antibodies (Pierce Endogen) were added and HRP-conjugated streptavidin (Pierce
Endogen) was used to develop the reactions. The plates were read at 450 nm (Titertek Plus MS 212M),
and the results were expressed as pg/ml.
Cytotoxicity Assay. The viability of the cultured cells (under the conditions of the bulk culture) was
evaluated after 72 h of culture by adding a 0.4% soln. of trypan blue (Sigma, Italy).

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Statistical Analysis. The results obtained from the in vitro proliferation and cytokine-quantification
assays were analyzed with the non-parametric Friedman test and then by a multiple comparison test (p<
.05 was considered significant).
0
REFERENCES
1] S. K. Agarwal, J. Manag. Care Pharm. 2011, 17, S14.
[
[
2] S. Hayer, M. Tohidast-Akrad, S. Haralambous, B. Jahn-Schmid, K. Skriner, S. Trembleau, H.
Dumortier, S. Pinol-Roma, K. Redlich, G. Schett, S. Muller, G. Kollias, J. Smolen, G. Steiner, J.
Immunol. 2005, 175, 8327.
[
[
[
3] G. Kollias, Semin. Arthritis Rheumat. 2005, 34, 3.
4] P. Ornetti, H. Chevillote, A. Zerrak, J. F. Maillefert, Drugs Aging 2006, 23, 855.
5] R. Gaggeri, D. Rossi, N. Hajikarimian, E. Martino, F. Bracco, P. Grisoli, C. Dacarro, F. Leoni, G.
Mascheroni, S. Collina, O. Azzolina, Open Nat. Prod. J. 2010, 3, 20.
[
[
[
[
6] W. L. Bear, R. W. Teel, Anticancer Res. 2000, 20, 3609.
7] G. Du, L. Jin, X. Han, Z. Song, H. Zhang, W. Liang, Cancer Res. 2009, 69, 3205.
8] G. Galati, O. Sabzevari, J. X. Wilson, P. J. OꢀBrien, Toxicology 2002, 177, 91.
9] C. Bodet, V. D. La, F. Epifano, G. Grenier, J. Periodont. Res. 2008, 43, 400.
[
[
[
[
10] S. Shinkaruk, V. Lamothe, J. M. Schmitter, C. Manach, C. Morand, A. Berard, B. Bennetau, C.
Bennetau-Pelissero, Food Chem. 2010, 118, 472.
11] J. A. Yanez, C. R. Remsberg, N. D. Miranda, K. R. Vega-Villa, P. K. Andrews, N. M. Davies,
Biopharm. Drug Dispos. 2008, 29, 63.
12] R. Gaggeri, D. Rossi, M. S. Christodoulou, D. Passarella, F. Leoni, O. Azzolina, S. Collina,
Molecules 2012, 17, 1665.
13] J.-J. Lee, H. Yi, I.-S. Kim, Y. Kim, N. X. Nhiem, Y. H. Kim, C.-S. Myung, J. Ethnopharmacol. 2012,
139, 873.
[
[
14] K. J. Hodgetts, Tetrahedron Lett. 2001, 42, 3763.
15] M. Kawasaki, H. Kakuda, M. Goto, S. Kawabata, T. Kometani, Tetrahedron: Asymmetry 2003, 14,
1529.
[
16] M. Kawasaki, Y. Asano, K. Katayama, A. Inoue, C. Hiraoka, H. Kakuda, A. Tanaka, M. Goto, N.
Toyooka, T. Kometani, J. Mol. Catal. B: Enzym. 2008, 54, 93.
[
[
[
[
17] T. Todoroki, A. Saito, A. Tanaka, Biosci. Biotechnol. Biochem. 2002, 66, 1772.
18] C. Dittmer, G. Raabe, L. Hintermann, Eur. J. Org. Chem. 2007, 35, 5886.
19] M. M. Biddle, M. Lin, K. A. Scheidt, J. Am. Chem. Soc. 2007, 129, 3830.
20] H. F. Wang, H. F. Cui, Z. Chai, P. Li, C. W. Zheng, Y. Q. Yang, G. Zhao, Chem.ꢂEur. J. 2009, 15,
13299.
[21] L. Wang, X. Liu, Z. Dong, X. Fu, X. Feng, Angew. Chem., Int. Ed. 2008, 47, 8670.
[22] C. Schmidt-Danert, to Fisch & Richardson P. C., U.S. Pat. 2005/006587.
[23] M. L. Hamilton, J. C. Caulfield, J. A. Pickett, A. M. Hooper, Tetrahedron Lett. 2009, 50, 5656.
[24] L. Britsch, W. Heller, H. Grisebach, Z. Naturforsch., C 1981, 36, 742.
[25] M. L. Hagmann, W. Heller, H. Grisebach, Eur. J. Biochem. 1983, 134, 547.
[26] W. R. Leonard, D. W. Henderson, R. A. Miller, G. A. Spencer, O. S. Sudah, M. Biba, C. J. Welch,
Chirality 2007, 19, 693.
[
27] R. Gaggeri, D. Rossi, S. Collina, B. Mannucci, M. Baierl, M. Juza, J. Chromatogr., A 2011, 1218,
5414.
[
28] M. E. Bernardo, M. A. Avanzini, C. Perotti, A. M. Cometa, A. Moretta, E. Lenta, C. Del Fante, F.
Novara, A. De Silvestri, O. Zuffardi, R. Maccario, F. Locatelli, J. Cell. Physiol. 2007, 211, 121.
Received June 28, 2012