Efficient synthesis of (R)-harmonine-the toxic principle of the multicolored Asian lady beetle (Harmonia axyridis)

Article abstract of DOI:10.1039/c5ob00461f

A flexible synthetic route to (R)-harmonine ((R)-1), the toxic principle of the Asian lady beetle Harmonia axyridis (H. axyridis), via reductive olefination of the macrocyclic lactone (S)-5, is reported. High enantiomeric purity is achieved by enantioselective saponification of the lactone rac-5 with horse liver esterase. Minor modifications in the synthetic route give access to racemic and chiral harmonine (1), analogs and putative biosynthetic precursors. In addition, the antimicrobial activity of harmonine against Leishmania major (L. major) is demonstrated and provides the rationale for harmonine-based drug development against parasitic diseases. This journal is

Full text of DOI:10.1039/c5ob00461f

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Biomolecular Chemistry
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Efficient synthesis of (R)-harmonine – the toxic
principle of the multicolored Asian lady beetle
(Harmonia axyridis)†
Cite this: Org. Biomol. Chem., 2015,
13, 5139
Nadja C. Nagel,a Anita Masic,b Uta Schurigtb and Wilhelm Boland*a
A flexible synthetic route to (R)-harmonine ((R)-1), the toxic principle of the Asian lady beetle Harmonia
axyridis (H. axyridis), via reductive olefination of the macrocyclic lactone (S)-5, is reported. High enantio-
meric purity is achieved by enantioselective saponification of the lactone rac-5 with horse liver esterase.
Minor modifications in the synthetic route give access to racemic and chiral harmonine (1), analogs and
putative biosynthetic precursors. In addition, the antimicrobial activity of harmonine against Leishmania
major (L. major) is demonstrated and provides the rationale for harmonine-based drug development
against parasitic diseases.
Received 7th March 2015,
Accepted 24th March 2015
DOI: 10.1039/c5ob00461f
www.rsc.org/obc
Introduction
The multicolored Asian lady beetle H. axyridis, also known as
the harlequin ladybird, is natively distributed from southern
Siberia to southern China and from the Altai mountains to the
Pacific coast.1 This tree dwelling beetle, of the family Coccinel-
lidae, is an important predator of aphids and scale insects and
has been introduced for biological control in many countries.
Over time populations began to establish and two decades ago
H. axyridis became an invasive species in North America,
Europe and South America threatening the native lady
beetles.1–3 The invasive success arises from intra-guild preda-
tion2 and from H. axyridis’ resistance to pathogens.3 When a
lady beetle is disturbed or attacked it releases droplets of
hemolymph from the tibio-femoral joints of its legs. This be-
havior is referred to as reflex-bleeding.4,5 The repellent and
sometimes toxic properties of the hemolymph are due to some
alkaloids, which are considered to be synthesized de novo
by the beetles.6 H. axyridis produces (R)-harmonine ((R)-1)
((17R,9Z)-octadec-9-ene-1,17-diamine) as the major defense
compound (Fig. 1).7
Fig. 1 (R)-Harmonine ((R)-1) and (9Z)-18-aminooctadec-9-en-2-ol
(rac-2).
human tumor cell lines.8 Recently microsporidia from the
hemolymph of H. axyridis were shown to infect intra-guild pre-
dators.9 In this context, (R)-harmonine ((R)-1) was postulated
to protect the harlequin beetle against self-infection.10 Cur-
rently, harmonine ((R)-1) is considered a promising lead for
clinical and agricultural use (yellow biotechnology).11
Although harmonine ((R)-1) has been isolated12 and syn-
thesized previously,12–14 there is a need for rapid and efficient
syntheses of harmonine ((R)-1) and related molecules, since
the mode of action is still unknown. In particular, for bio-
assays larger quantities are needed.
L. major is the causative agent of cutaneous leishmaniasis
with an estimated annual incidence of 800 000–1.3 million
new infections worldwide.15,16 Current chemotherapy against
leishmaniasis is limited due to the continuous development of
drug resistance accompanied by severe side effects.17 There-
fore, naturally derived compounds like harmonine are
investigated to identify and develop new therapies against
leishmaniasis. The antiparasitic activity of harmonine against
P. falciparum has already been reported and prompted the
assessment of its activity against L. major.7
(R)-Harmonine ((R)-1) displays antibacterial activity against
fast-growing mycobacteria, Mycobacterium tuberculosis, and
Plasmodium falciparum (P. falciparum), demonstrates multi-
stage antimalarial activity,7 and exhibits cytotoxicity against
aMax Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knoell-
Straße 8, D-07745 Jena, Germany. E-mail: boland@ice.mpg.de
bInstitute for Molecular Infection Biology, Josef-Schneider-Str. 2/D15, D-97080
Wuerzburg, Germany
Here, we report a short and flexible synthetic route to har-
monine ((R)-1) in only a few steps starting from the readily
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob00461f
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available macrocyclic lactone (S)-5. One-pot olefination of (S)-5
directly provides the basic skeleton of harmonine ((R)-1) and
allows the synthesis of biosynthetic precursors and structural
analogs via functional group modifications. Furthermore, we
report the antiparasitic activity of harmonine against the cau-
sative agent of cutaneous leishmaniasis, L. major.18 Cultivation
in the presence of (R)-1 leads to the inhibition of parasitic
proliferation with a consequent early necrotic cell death
phenotype.
Results and discussion
According to Scheme 1 the harmonine backbone can be
obtained from the lactone (S)-5 and the phosphonium salt 4
by a Wittig-type reaction. The chiral lactone (S)-5 is available
with high ee and excellent yield by enantioselective hydro-
lysis with horse liver esterase. Furthermore, synthetic
intermediates like ((S)-3) are promising candidates for bio-
synthetic and pharmaceutical studies.19 By modifying the
chain length of the phosphonium salt or the ring size of
Scheme 2 Synthetic sequence to harmonine (rac-1) and a putative bio-
synthetic precursor rac-2. Reagents and conditions: (a) (i) DiBAlH,
MeOH, toluene, −78 °C; (ii) KHMDS, THF, −78 °C; (iii) −78 °C to rt in 1 h,
the lactone, analogs with different positions of the double 1 h rt; (b) LiAlH4, THF, rt; (c) PPh3, DPPA, DIAD, THF, rt; (d) LiAlH4, THF, rt;
(e) Mg3N2, MeOH, 80 °C; (f) LiAlH4, THF, 45 °C.
bond and molecular size become available, thus opening a
new synthetic route to harmonine-like compounds using a
unified procedure.
Following the retrosynthetic strategy the backbone of har-
monine (rac-1) was assembled by reductive olefination17 from
aldehyde which reacts in situ with the phosphorane to the
hydroxyester rac-6 ((Z)/(E) > 98/2). The isopropylester was used
to minimize side reactions during the Wittig olefination.
Reduction of rac-6 with lithium aluminum hydride afforded
the diol rac-3 in quantitative yield.23 Following the Mitsunobu
protocol the diol rac-3 was converted into the diazide rac-7
using triphenylphosphine (PPh3), diphenyl phosphorazidate
(DPPA) and diisopropyl azodicarboxylate (DIAD).24 The final
reduction of rac-7 with an excess of LiAlH4 afforded racemic
harmonine (rac-1) in quantitative yield.24 Overall, racemic har-
monine (rac-1) could be prepared in only four steps and 22%
overall yield starting from rac-5 and 4. Spectral data of rac-1
were identical to literature values.8,12
rac-5 and the ylide of (9-isopropoxy-9-oxononyl)triphenyl-
phosphonium bromide (4) in a single operation. The resulting
isopropylester rac-6 was converted into the amide rac-8 or directly
reduced to the diol rac-3 (Scheme 2). The lactone rac-5 was
obtained from commercial cyclooctanone20 and the phos-
phonium salt 4 from 9-bromononanoic acid after esterification
and reaction with triphenylphosphine.21,22
The one-pot “reductive olefination” generates from the
lactone rac-5 and DiBAlH at low temperatures an organo alu-
minium acetal that decomposes above −40 °C to a hydroxy
For biosynthetic studies19 and pharmaceutical assays7 the
(Z)-18-aminooctadec-9-en-2-ol (rac-2) was of interest. Conver-
sion of the isopropylester rac-6 into the amide rac-8 was readily
achieved using magnesium nitride in refluxing methanol
25
(Scheme 2).23 Subsequent reduction with LiAlH4 provided
the aminoalcohol rac-2 in nearly quantitative yields over
two steps.
The synthetic protocol was easily extended to chiral
(17R,9Z)-1,17-diaminooctadec-9-ene ((R)-1). The key step was a
kinetically controlled enantioselective saponification of the
lactone rac-5 which left behind unreacted (S)-lactone ((S)-5) (ee
> 99.5%) in 43% yield (Scheme 3). For hydrolysis the lactone
(rac-5) was suspended in a NaH2PO4-buffer (0.1 M, pH = 7.2)
and horse liver esterase (HLE) was added as lyophilized
powder.26 The hydrolysis started immediately and the pH was
kept constant by addition of dil. NaOH (0.5 M) to avoid spon-
taneous hydrolysis.
Scheme 1 Retrosynthesis of harmonine ((R)-1).
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Table 1 Antileishmanial activity of harmonine
IC50 (µM)a
L. major
promastigotes
L. major
amastigotes
BMDMb
SIc
(R)-Harmonine
Miltefosine
14.2d
36.2
2.4
33.0
36.5
65.5
15.2
2.0
Scheme 3 Enantioselective, enzymatic saponification of rac-5.
Reagents and conditions: (a) HLE, NaH2PO4, NaOH, rt.
a IC50: half maximal inhibitory concentration. b BMDM: bone marrow-
derived macrophages. c SI (Selectivity Index): IC50 for BMDM/IC50 for
L. major. d IC50 synthetic rac-harmonine: 13.2.
cellular and clinically relevant L. major amastigote form. When
compared to Miltefosine (1-hexadecylphosphocholine, positive
control) with an IC50 value of 36.2 µM against L. major promas-
tigotes and 33.0 µM against L. major amastigotes,29 harmonine
showed leishmanicidal activities at significantly lower concen-
trations. Miltefosine showed an IC50 value of 65.5 µM and
harmonine showed a value of 36.5 µM against BMDM. The
antileishmanial efficacy of a tested drug compared to its cyto-
toxicity against host cells is defined as the selectivity index
(SI). SIs > 20 are considered excellent, as high antileishmanial
activity and low cytotoxicity are desirable prerequisites for drug
development.30 Here, harmonine shows a very good SI of 15.2
towards L. major which is considerably higher than the SI of
2.0 of Miltefosine.
Fig. 2 Enantiomeric analysis on
a β-6TBDM column. (A) Racemic
The antileishmanial effect of harmonine as shown in
Table 1 was further investigated. The cell morphology of L.
major promastigotes was studied by means of transmitted
light microscopy upon treatment with harmonine for 6 h,
10 h, and 24 h (Fig. 3). The visual examination of promasti-
gotes incubated with harmonine showed rapid changes of the
cell morphology. After 24 h only dead cells were observed
upon harmonine-treatment whereas in the presence of
dimethyl sulfoxide (DMSO) no significant changes in the cell
morphology of the parasites were observed. Rounding of cells
upon harmonine treatment was induced after 6 h of culture
and dead cells were visible after 10 h of cultivation. DMSO-
treated control L. major promastigotes show upon 6 h and 10 h
of culture the characteristic flagellated and slender shape of
the viable and unaffected parasite (Fig. 3).
The type of cell death induced by harmonine was investi-
gated using flow cytometric approaches. The loss of membrane
integrity in necrotic cells and the translocation of phosphati-
dylserine (PS) to the outside of the cellular membrane of apop-
totic cells can be determined by Annexin V (AV, binds PS) and
PI (DNA-binding) staining.31 Double staining with AV-fluor-
escein isothiocyanate (FITC) and PI allows the discrimination
between four Leishmania cell death phenotypes as described
elsewhere: live (AV−/PI−), late necrotic/late apoptotic (AV+/PI+),
early necrotic (AV−/PI+) and early apoptotic cells (AV+/PI−).31,32
Harmonine-treatment for 24 h induced early necrotic cell
death in 31.6% and late necrotic/late apoptotic cell death in
51.2% of all the treated cells (Fig. 3). This means that after
24 h of harmonine-treatment a total of 82.8% of cells were
lactone rac-5. (B) 98% hydrolysis of (R)-lactone. (C) Isolated lactone
(S)-5 (ee ≥ 99.5%).
The progress of the reaction was monitored using a GC-MS
equipped with a chiral column (β-6TBDM) for resolution of the
enantiomers (Fig. 2).
After hydrolysis of the (R)-lactone, the reaction stopped and
both the remaining lactone (S)-5 and the hydroxyacid (R)-9
were obtained with good yield and excellent ee (S)-5 (>99.5%),
(R)-9 (>99%).
The chiral lactone (S)-5 was converted into (R)-harmonine
((R)-1) using the same sequence of reactions as shown in
Scheme 2. The spectral data of (R)-1 were identical to literature
values.8,12,13
The ee of (R)-harmonine ((R)-1) was determined by 19F NMR
using (S)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride
(MTPA chloride) for derivatization.27,28 Due to the large chemi-
cal shifts of the α-CF3 groups of the MTPA-diastereomers the
high ee of (R)-1 was reliably confirmed (ee > 97%).
For the evaluation of the antileishmanial activity of harmo-
nine, the AlamarBlue® assay with L. major promastigotes and
the Britelite™ assay with Luciferase-transgenic L. major amas-
tigotes were employed (Table 1). The cytotoxic effect of harmo-
nine against host cells was investigated in bone marrow-
derived macrophages (BMDM). The half maximal inhibitory
concentration (IC50) of harmonine was detected at 14.2 µM
against L. major promastigotes and at 2.4 µM against the intra-
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Fig. 3 Harmonine induced cell death in L. major promastigotes. L. major promastigotes were treated with 30 µM harmonine or 1% DMSO for 6 h,
10 h, and 24 h. Annexin V and PI double staining was performed for the determination of the cell death phenotype (dot-blots) and Diff-Quick stain-
ing was performed to investigate cell morphological changes via transmission light microscopy (pictures). Scale bars = 10 µm. Numbers in the quad-
rants represent cells (%) showing early necrotic, late necrotic, or apoptotic cell death.
dead. At the same time under normal growth conditions only drugs with both parasitocidal and transmission-blocking
26.6% of cells were not viable (Fig. 3). Cell death during har- activities.7
monine-treatment was observed to increase over time, as after
6 h a total of 26.2% and after 10 h a total of 40.3% of cells
were dead, as indicated by PI staining. Early necrosis is charac-
terized by PI-binding to the DNA of cells which have lost their
Experimental
Synthesis
cell membrane integrity. Harmonine was found to induce early
General methods. NMR spectra were recorded using a
necrotic cell death in L. major, as a clear population of 24.5%
Bruker AV 400 spectrometer (Bruker, Rheinstetten/Karlsruhe,
early necrotic cells was detected after 10 h of cultivation
Germany) operating at 400 MHz (1H), 100 MHz (13C) and
(Fig. 3). The increase of late necrotic/late apoptotic cells to
1
376.5 MHz (19F). Chemical shifts (δ) in H, 13C and 19F NMR
51.15% after 24 h is a consequence of an early necrotic cell
are given in ppm and are referenced to the residual solvent
death phenotype, as the rupture of the cell membrane allows
peak (CDCl3: 7.27 (1H NMR); 77.0 (13C NMR)). GC-MS analysis
was carried out using a Trace MS, 2000 Series (ThermoQuest,
Engelsbach, Germany) equipped with an Alltech DB5 column
(30 m × 0.25 m, 0.25 µm); carrier gas: helium. Enantiomers
AV to bind to PS in the dead parasite.
were separated on a Hydrodex-β-6TBDM column (25 m ×
Conclusion
0.25 m) and analyzed with an ITQ 900 (Thermo Fischer Scienti-
In conclusion a highly efficient and flexible synthetic route to fic, Bremen, Germany). HPLC-MS spectra were recorded using
chiral (R)-harmonine ((R)-1) is reported. A highly enantio- a Finnigan LTQ (Thermo Electron Company, Waltham, Massa-
selective hydrolysis of rac-5 affords both the remaining lactone chusetts, USA) MS in APCI mode and a Purospher STAR RP18
(S)-5 and the hydroxy acid (R)-9 in high yield and excellent column (250 mm × 2 mm, 5 µm). Infrared spectra were
enantiomeric purities. Reductive olefination of the lactone recorded using a Bruker Equinox 55 FTIR spectrophotometer
(S)-5 with readily available phosphonium ylides gives direct over the 700–4000 cm−1 range with a spectral resolution of
access to the backbone of harmonine ((R)-1). Subsequent func- 2 cm−1 using the transmission mode. The optical rotation was
tional group modification provides derivatives and analogs of measured with a JASCO P1030 polarimeter at 22–24 °C at a
the natural product for structure–activity studies and mechan- specific wavelength of 589 nm. High resolution mass spectra
istic analyses to unravel the mode of action of the ladybeetle (HR-MS) were either obtained using a Hewlett Packgard
alkaloid harmonine ((R)-1). The pronounced activity of harmo- 6890 GC operated with a Phenomenex ZBSHT column (30 m ×
nine ((R)-1) against mycobacteria or the malaria parasite P. fal- 0.25 m, 0.25 µm) and connected to a Masspec MS02 (Mirco-
ciparum as is reported elsewhere,7 and the activity against mass, UK) (EI, 70 eV) or a QExactive Plus MS (Thermo Fischer
L. major as is shown for the first time in the present study are Scientific, Bremen, Germany) equipped with an RP-18 column
already encouraging observations for further investigations. (150 mm × 2.1 mm, 120 Å) or via direct injection into the ESI
Readily available synthetic harmonine and analogs may source (capillary temp.: 275 °C). For preparative column
provide a base for the development of novel anti-parasitic chromatography silica gel (40–63 µm, Macherey-Nagel, Düren,
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Germany) or Lichroprep RP-18 silica gel (40–63 µm, Merck, phases were separated and the aqueous layer was extracted
Darmstadt, Germany) was used. Thin layer chromatography with Et2O (3 × 10 ml). The combined organic extracts were
was conducted on silica gel 60 F254 aluminum sheets (Merck, washed with water (30 ml) and dried over Na2SO4. The solvent
Darmstadt, Germany). The compounds were detected using was removed under reduced pressure and the residue was puri-
Hanessian’s stain, vanillin or potassium permanganate stain. fied by column chromatography on silica gel using hexane–
Except for diethyl ether all solvents purchased were of HPLC ethyl acetate (1 : 1) for elution. After removal of the solvents,
grade and used without further purification. Diethyl ether was rac-3 was obtained as a slightly yellow oil (243 mg, 0.85 mmol,
distilled prior to use. Anhydrous solvents were purchased as 98%). Rf = 0.35 (hexane–ethyl acetate 1 : 1).
such. Horse liver esterase was obtained as lyophilized powder
HRMS m/z calcd for C18H36O2 284.2715 [M]+, found
(0.5–1.0 U mg−1, Sigma Aldrich, Saint Louis, MO, USA).
284.2705; IR (thin film, cm−1) ν 3332 (br, m), 3004 (w), 2926
1
Isopropyl (9Z)-17-hydroxyoctadec-9-enoate (rac-6). A cold (s), 2854 (s), 1654 (w), 1462 (m), 1373 (m); H NMR (400 MHz,
3
solution (−78 °C) of 9-methyloxonan-2-one (rac-5) (78 mg, CDCl3) δ 5.40–5.30 (m, 2 H, H-10/H-9), 3.79 (tq, J17,18
=
0.5 mmol) in dry toluene (2.7 ml) was treated within 10 min 3J17,16 = 5.9 Hz, 1 H, H-17), 3.63 (t, J1,2 = 6.6 Hz, 2 H, H-1),
3
with DiBAlH (0.6 ml, 0.6 mmol, 1 M in hexane). Stirring was 2.06–1.95 (m, 4 H, H-11/H-8), 1.95–1.80 (br, 2 H, O–H), 1.56 (tt,
continued for an additional 40 minutes and the excess of 3J2,3
DiBAlH was quenched by addition of dry MeOH (5 µl). Next, the 1.38–1.25 (m, 18 H, H-14–H-12/H-7–H-4), 1.18 (d, J18,17
=
3J2,1 = 6.7 Hz, 2 H, H-2), 1.49–1.38 (m, 2 H, H-16),
3
=
cold solution was transferred into a cold solution (−78 °C) of 6.0 Hz, 3 H, H-18). 13C NMR (100 MHz, CDCl3) δ 129.9, 129.8
the phosphorus ylide, prepared from 9-isopropoxy-9-oxononyl (C-10/C-9), 68.2 (C-17), 63.0 (C-1), 39.3 (C-16), 32.7 (C-2), 29.7,
triphenylphosphonium bromide (4) (325 mg, 0.6 mmol) in dry 29.7, 29.5, 29.4, 29.4, 29.2, 29.1 (C-14–C-12/C-7–C-4), 27.1
THF (3.5 ml) and KHMDS (120 mg, 0.6 mmol). The reaction (C-11/C-8), 25.7 (C-15/C-3), 23.4 (C-18).
mixture was warmed to room temperature over 1 hour. Stirring
(17S,9Z)-Octadec-9-ene-1,17-diol ((S)-3). It is prepared
was continued for 1 hour and the mixture was hydrolyzed by from (17S,9Z)-17-hydroxyoctadec-9-enoate ((S)-6) (295 mg,
the addition of 5% HCl (2.6 ml). The layers were separated and 0.87 mmol). After column chromatography (S)-3 was obtained
the aqueous phase was extracted with Et2O (2 × 5 ml). The as a colorless oil (245 mg, 0.86 mmol, 98%). [α]2D2 +3.8 (c 1.00,
combined organic extracts were washed with 5% HCl (20 ml), CHCl3). All other spectral data were identical to rac-3.
a saturated NaHCO3 solution (20 ml) and water (20 ml). After
(9Z)-1,17-Diazidooctadec-9-ene (rac-7). (9Z)-Octadec-9-ene-
removal of the solvents under reduced pressure, the crude 1,17-diol (rac-3) (218 mg, 0.77 mmol) was dissolved in dry THF
product was purified by column chromatography on silica gel (13 ml) and cooled to 0 °C. PPh3 (836 mg, 3.19 mmol), DPPA
using hexane–ethyl acetate (5 : 1) for elution. Removal of the (0.97 ml, 4.51 mmol) and DIAD (0.85 ml, 4.29 mmol) were suc-
solvents yielded rac-6 as a colorless oil (49 mg, 0.13 mmol, cessively added with stirring and the reaction mixture was
26%, (Z)/(E) > 98/2; ylide preparation with n-BuLi: 29%, (Z)/(E) allowed to warm to room temperature. After 4 hours the
80/20). Rf = 0.18 (hexane–ethyl acetate 5 : 1).
solvent was removed and the reaction product was purified by
HRMS m/z calcd for C21H40O3 340.2978 [M]+, found column chromatography on silica gel using hexane–DCM
340.2985; IR (thin film, cm−1) ν 3437 (br, m), 2977 (m), 2964 (3 : 1) for elution. Removal of solvents afforded rac-7 as a color-
(m), 2928 (s), 2855 (s), 1733 (s), 1655 (w), 1464 (m), 1374 (m), less oil (195 mg, 0.58 mmol, 76%). Rf = 0.30 (hexane–
1249 (m), 1181 (m), 1145 (m), 1110 (s); 1H NMR (400 MHz, DCM 3 : 1).
3
3
CDCl3) δ 5.40–5.31 (m, 2 H, H-10/H-9), 5.01 (sp, J1′,2′ = J1′,2″
=
HRMS m/z calcd for C18H34N6Na 357.2737 [M + Na]+, found
6.2 Hz, 1 H, H-1′), 3.80 (tq, J17,18 = J17,16 = 6.1 Hz, 1 H, H-17), 357.2737; IR (thin film, cm−1) ν 3004 (w), 2928 (s), 2855 (s),
3
3
2.26 (t, J2,3 = 7.7 Hz, 2 H, H-2), 2.04–1.96 (m, 4 H, H-11/H-8), 2094 (s), 1653 (w), 1459 (m), 1378 (w), 1249 (s); 1H NMR
3
1.61 (mc, 2 H, H-3), 1.48–1.38 (m, 2 H, H-16), 1.36–1.27 (m, (400 MHz, CDCl3) δ 5.42–5.30 (m, 2 H, H-10/H-9), 3.42 (tq,
3
3
3
3
16 H, H-15–H-12/H-7–H-4), 1.23 (d, J2′,1′ = J2″,1′ = 6.3 Hz, 6 H, 3J17,18 = J17,16 = 6.5 Hz, 1 H, H-17), 3.26 (t, J1,2 = 7.0 Hz, 2 H,
H-2′/H-2″), 1.19 (d, J18,17 = 6.2 Hz, 3 H, H-18); 13C NMR H-1), 2.07–1.94 (m, 4 H, H-11/H-8), 1.61 (tt, J2,3
= =
3J2,1
3
3
(100 MHz, CDCl3) δ 173.4 (C-1), 129.8 (C-10/C-9), 68.1 (C-17), 7.0 Hz, 2 H, H-2), 1.57–1.28 (m, 20 H, H-16–H-12/H-7–H-3),
67.3 (C-1′), 39.4 (C-16), 34.7 (C-2), 29.7, 29.5, 29.2, 29.1, 1.25 (d, 3J18,17 = 6.6 Hz, 3 H, H-18). 13C NMR (100 MHz, CDCl3)
29.1, 25.7 (C-15–C-12/C-7–C-4), 27.1 (C-11/C-8), 25.0 (C-3), δ 129.9, 129.8 (C-10/C-9), 58.0 (C-17), 51.5 (C-1), 36.2 (C-16),
23.5 (C-18), 21.8 (C-2′/C-2″).
(17S,9Z)-17-Hydroxyoctadec-9-enoate ((S)-6). It is prepared (C-2), 27.2, 27.1 (C-11/C-8), 26.7 (C-3), 26.1 (C-15), 19.5 (C-18).
from (9S)-9-methyloxonan-2-one ((S)-5) (750 mg, 4.80 mmol) as (17R,9Z)-1,17-Diazidooctadec-9-ene ((R)-7). It is prepared
29.7, 29.6, 29.4, 29.3, 29.2, 29.1, 29.1 (C-14–C-12/C-7–C-4), 28.8
described. After purification, (S)-6 was obtained as a colorless from (17S,9Z)-octadec-9-ene-1,17-diol ((S)-3) (200 mg,
oil (366 mg, 1.08 mmol, 23%). [α]2D2 +2.9 (c 1.00, CHCl3). All 0.70 mmol). Purification by column chromatography afforded
other spectral data were identical to rac-6.
(9Z)-Octadec-9-ene-1,17-diol (rac-3). LiAlH4
2.62 mmol) was suspended at 0 °C in dry THF (7.5 ml) and the
(R)-7 as a colorless oil (189 mg, 0.57 mmol, 81%). [α]2D2 −19.7
mg, (c 1.06, CHCl3). All other spectral data were identical to rac-7.
(99
(9Z)-Octadec-9-ene-1,17-diamine (rac-1). LiAlH4 (177 mg,
ester rac-6 (297 mg, 0.87 mmol) was added slowly. The reaction 3.95 mmol) was suspended with stirring in dry THF (11 ml)
mixture was allowed to come to room temperature and stirred and cooled to 0 °C. A solution of (9Z)-1,17-diazidooctadec-9-
for 4.5 hours. Et2O (9 ml) and water (10 ml) were added, the ene (rac-7) (165 mg, 0.49 mmol) in dry THF (5 ml) was added
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2
dropwise and the suspension was warmed to room tempera- δ 5.68 (d, JNH,NH′ = 81.9 Hz, 2 H, N–H2), 5.40–5.27 (m, 2 H,
3
3
ture. After stirring for 4 hours, water (30 ml) and CHCl3 H-10/H-9), 3.78 (tq, J17,18 = J17,16 = 5.7 Hz, 1 H, H-17), 2.20 (t,
(30 ml) were added, the layers were separated and the aqueous 3J2,3 = 7.5 Hz, 2 H, H-2), 2.05–1.92 (m, 4 H, H-11/H-8), 1.84
3
phase was extracted with CHCl3 (6 × 30 ml). The organic (brs, 1 H, O–H), 1.62 (tt, J3,4
=
3J3,2 = 7.1 Hz, 2 H, H-3),
extracts were combined and solvents were removed under 1.51–1.37 (m, 2 H, H-16), 1.37–1.23 (m, 16 H, H-15–H-12/H-7–
3
reduced pressure. The product was partitioned between MTBE H-4), 1.17 (d, J18,17 = 6.1 Hz, 3 H, H-18). 13C NMR (100 MHz,
(80 ml) and 2% HCl (80 ml). The layers were separated, the CDCl3) δ 175.8 (C-1), 130.3, 130.3 (C-10/C-9 (E)), 129.9, 129.8
aqueous phase was carefully extracted with MTBE (3 × 20 ml) (C-10/C-9), 68.0 (C-17), 39.3 (C-16), 35.9 (C-2), 32.5, 32.4 (C-11/
and the combined organic extracts were washed with 2% HCl C-8 (E)), 29.6, 29.6, 29.5, 29.5, 29.2, 29.2, 29.0 (C-14–C-12/C-7–
(30 ml). The aqueous solutions were combined and MTBE C-4), 27.1, 27.1 (C-11/C-8), 25.7 (C-15), 25.5 (C-3), 23.4 (C-18).
(40 ml) and NH3 (25% aqueous solution, 80 ml) were added.
After separation of the two layers, the aqueous phase was 3.00 mmol) was suspended with stirring in dry THF (3 ml) and
extracted with MTBE (3 × 60 ml). The combined organic cooled to °C. (9Z)-17-Hydroxyoctadec-9-enamide (rac-8)
(9Z)-18-Aminooctadec-9-en-2-ol (rac-2). LiAlH4 (106 mg,
0
extracts were dried over Na2SO4 and the solvent was evaporated (168 mg, 0.56 mmol), dissolved in dry THF (2 ml), was added
under reduced pressure to yield rac-1 as a colorless oil dropwise. The reaction was allowed to warm to room tempera-
(139 mg, 0.49 mmol, quant.).
ture, stirred for 4 hours at room temperature and for
HRMS m/z calcd for C18H39N2 283.3108 [M + H]+, found 19.5 hours at 45 °C. After cooling to room temperature, water
283.3106; IR (thin film, cm−1) ν 3320 (m), 3004 (w), 2921 (s), (10 ml) and CHCl3 (10 ml) were added. The layers were separ-
2851 (s), 1639 (w), 1564 (m), 1468 (m), 1430 (w), 1390 (m), 1331 ated and the aqueous phase was extracted with CHCl3 (5 ×
(m); 1H NMR (400 MHz, CDCl3) δ 5.38–5.27 (m, 2 H, H-10/H-9), 10 ml). The organic extracts were combined and the solvents
3
3
2.85 (tq, J17,18
=
3J17,16 = 6.0 Hz, 1 H, H-17), 2.66 (t, J1,2
=
were removed under reduced pressure. The product was parti-
7.0 Hz, 2 H, H-1), 2.04–1.91 (m, 4 H, H-11/H-8), 1.50–1.38 (m, tioned between MTBE (80 ml) and 2% HCl (80 ml). The
6 H, H-2, N–H2), 1.36–1.21 (m, 20 H, H-16–H-12/H-7–H-3), 1.03 aqueous phase was extracted with MTBE (3 × 20 ml) and the
3
(d, J18,17 = 6.2 Hz, 3 H, H-18). 13C NMR (100 MHz, CDCl3) combined organic extracts were washed with 2% HCl (35 ml).
δ 129.8, 129.8 (C10/C-9), 46.9 (C-17), 42.1 (C-1), 40.1 (C-16), Both aqueous solutions were combined and MTBE (50 ml) and
33.7 (C-2), 29.7, 29.6, 29.6, 29.4, 29.4, 29.2, 29.2 (C-14–C-12/ NH3 (25% aqueous solution, 80 ml) were added. The layers
C-7–C-4), 27.1 (C11/C-8), 26.8 (C-3), 26.3 (C-15), 23.8 (C-18).
were separated and the aqueous phase was extracted with
(17R,9Z)-Octadec-9-ene-1,17-diamine ((R)-1) (harmonine). It MTBE (3 × 80 ml). The combined organic extracts were dried
is prepared from (17R,9Z)-1,17-diazidooctadec-9-ene ((R)-7) over Na2SO4 and the solvent was evaporated under reduced
(161 mg, 0.48 mmol). Harmonine ((R)-1) was obtained as pressure yielding rac-2 as oily, colorless wax (151 mg,
a slightly yellow oil (135 mg, 0.48 mmol, 99%, ee > 97%). [α]2D4 0.53 mmol, 95%).
−3.4 (c 1.04, C6H6). Spectroscopic data were identical to rac-1.
HRMS m/z calcd for C18H38NO 284.2948 [M + H]+, found
(9Z)-17-Hydroxyoctadec-9-enamide (rac-8). Isopropyl (9Z)-17- 284.2935; IR (thin film, cm−1) ν 3332 (br, m), 3004 (m), 2925
hydroxyoctadec-9-enoate (rac-6) (237 mg, 0.70 mmol) was dis- (s), 2853 (s), 1632 (m), 1572 (m), 1464 (m), 1372 (m), 1315 (m);
solved at 0 °C in MeOH (2.25 ml). Mg3N2 (353 mg, 3.48 mmol) 1H NMR (400 MHz, CDCl3) δ 5.38–5.28 (m, 2 H, H-10/H-9),
3
3
was quickly added in one portion, the reaction vessel was 3.75 (tq, J17,18
= =
3J17,16 = 6.1 Hz, 1 H, H-17), 2.66 (t, J1,2
thoroughly closed and the suspension was allowed to warm to 7.0 Hz, 2 H, H-1), 2.04–1.91 (m, 4 H, H-11/H-8), 1.82–1.76 (m,
room temperature. Stirring was continued for 1 hour. The reac- 3 H, N–H2, O–H), 1.48–1.35 (m, 5 H, H-16/H-15a/H-2),
tion was heated to 80 °C and stirring was continued for 1.35–1.23 (m, 17 H, H-15b/H-14–H-12/H-7–H-3), 1.15 (d,
24.5 hours. After cooling to room temperature, CHCl3 (25 ml) 3J18,17 = 6.1 Hz, 3 H, H-18). 13C-NMR (100 MHz, CDCl3) δ 130.3,
and water (25 ml) were added. The aqueous phase was neutral- 130.2 (C-10/C-9 (E)), 129.8, 129.8 (C-10/C-9), 67.7 (C-17 (E)),
ized with 3 M HCl, the layers were separated and the aqueous 67.7 (C-17), 42.1 (C-1), 39.4 (C-16), 33.6 (C-2 (E)), 33.6 (C-2),
phase was extracted with CHCl3 (2 × 25 ml). The organic layers 32.5 (C-11/C-8 (E)), 29.6, 29.6, 29.5, 29.4, 29.4, 29.2, 29.1,
were combined and the solvent was removed under reduced (C-14–C-12/C-7–C-4), 27.1 (C-11/C-8), 26.8 (C-3), 26.8 (C-3 (E)),
pressure. The residue was purified by column chromatography 25.7 (C-15), 23.4 (C-18).
on RP-18 silica gel using H2O–MeOH (1 : 9) for elution. CHCl3
was added and the solvents were removed under reduced
Enzymatic hydrolysis of lactone (rac-5)
pressure. This procedure was repeated three times with CHCl3 Racemic 9-methyloxonan-2-one (rac-5) (3.50 g, 22.4 mmol) was
and three times with benzene to remove the last traces of suspended in NaH2PO4 buffer (0.1 M, pH = 7.2, 100 ml) and
water. The residue was concentrated under high vacuum to stirred for 15 minutes. Then horse liver esterase (350 mg, lyo-
yield rac-8 as a colorless, sticky oil. (182 mg, 0.61 mmol, 87%). philized powder) was added and the pH was kept constant by
Rf = 0.43 (H2O–MeOH 1 : 9, RP-18).
dropwise addition of 0.5 M NaOH during the whole reaction
HRMS m/z calcd for C18H36NO2 298.2741 [M + H]+, found time. After 12 hours, another portion of horse liver esterase
298.2734; IR (thin film, cm−1) ν 3358 (s), 3191 (br, m), 3003 (70 mg) was added and stirring was continued for 4 hours.
(w), 2967 (w), 2924 (s), 2852 (s), 1703 (w), 1659 (s), 1633 (s), Then Celite (3.50 g) and ice (7.00 g) were added, the suspen-
1468 (m), 1423 (m), 1412 (m); 1H NMR (400 MHz, CDCl3) sion was stirred for 5 minutes and the solids were filtered off.
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The filter cake was washed with Et2O (2 × 50 ml). The layers of 1 H, H-1′), 3.40 (t, 3J9,8 = 6.9 Hz, 2 H, H-9), 2.26 (t, 3J2,3 = 7.6 Hz,
the filtrate were separated and the aqueous phase was 2 H, H-2), 1.85 (mc, 2 H, H-8), 1.61 (mc, 2 H, H-3), 1.42 (mc,
3
3
extracted with Et2O (3 × 50 ml). The combined organic extracts 2 H, H-7), 1.34–1.28 (m, 6 H, H-6–H-4), 1.22 (d, J2′,1′ = J2″,1′
=
were washed with a saturated NaHCO3 solution (100 ml) and 6.2 Hz, 6 H, H-2′/H-2″); 13C NMR (100 MHz, CDCl3) δ 173.3
dried over Na2SO4. Evaporation of the solvent yielded (9S)-9- (C-1), 67.3 (C-1′), 34.6 (C-2), 33.9 (C-9), 32.7 (C-8), 29.0, 29.0
methyloxonan-2-one ((S)-5) as a colorless liquid.
(9S)-9-Methyl-oxonan-2-one ((S)-5). Yield: 1.51 g (9.67 mmol,
86% of (S)-enantiomer, ee ≥ 99.5%). [α]2D3 +31.8 (c 1.00, THF).
(C-5/C-4), 28.5 (C-6), 28.1 (C-7), 24.9 (C-3), 21.8 (C-2′, C-2″).
(9-Isopropoxy-9-oxononyl)triphenylphosphonium bromide
(4). Isopropyl 9-bromononanoate (10) (2.89 g, 10.35 mmol)
HRMS m/z calcd for C9H16O2 156.1150 [M]+, found was dissolved in dry toluene (65 ml) and PPh3 (2.71 g,
156.1152; IR (thin film, cm−1) ν 2929 (s), 2857 (m), 1726 (s), 10.35 mmol) was added. The solution was refluxed for
1575 (w), 1464 (w), 1427 (w), 1377 (w), 1286 (m), 1254 (s); 24 hours under argon, cooled to room temperature and the
1H NMR (400 MHz, CDCl3) δ 5.07 (mc, 1 H, H-8), 2.25 (mc, 2 H, supernatant was transferred to a new flask. There again PPh3
H-2), 1.93 (mc, 1 H, H-4a), 1.82–1.74 (m, 1 H, H-7a), 1.72–1.64 (1.36 g, 5.18 mmol) was added and the solution was stirred
(m, 1 H, H-3a), 1.67–1.55 (m, 2 H, H-6a/H-3b), 1.55–1.43 (m, under reflux for 3 days. After cooling to room temperature, the
3 H, H-7b/H-5a/H-4b), 1.37–1.27 (m, 2 H, H-6b/H-5b), 1.25 (d, supernatant was discarded and both bottom layers were com-
3J9,8 = 6.5 Hz, 3 H, H-9); 13C NMR (100 MHz, CDCl3) δ 175.5 bined and purified by column chromatography on silica
(C-1), 71.5 (C-8), 35.7 (C-2), 35.1 (C-7), 29.4 (C-5), 25.0 (C-4), gel (DCM–MeOH 14 : 1). The solvents were removed under
23.9 (C-3), 21.8 (C-6), 20.7 (C-9).
reduced pressure and 4 was obtained as a sticky syrup (4.33 g,
(8R)-8-Hydroxynonanoic acid ((R)-9). The water phase was 8.00 mmol, 77%). Rf = 0.51 (DCM–MeOH 14 : 1).
covered with Et2O (100 ml) and 2 M HCl (60 ml) was added.
HRMS m/z calcd for C30H38O2P 461.2604 [M]+, found
The layers were separated and the aqueous phase was extracted 461.2601; IR (thin film, cm−1) ν 3057 (w), 2977 (w), 2855 (s),
with Et2O (3 × 100 ml). The combined organic extracts were 2802 (w), 1720 (s), 1587 (w), 1522 (w), 1485 (m), 1104 (m), 856
washed with a saturated NaCl solution (150 ml) and dried over (s), 842 (s); 1H NMR (400 MHz, CDCl3) δ 7.88–7.76 (m, 9 H,
3
Na2SO4. The solvent was removed under reduced pressure to Har-6/Har-2/Har-4), 7.74–7.67 (m, Har-5/Har-3), 4.96 (sp, J1′,2′
=
yield (8R)-8-hydroxynonanoic acid ((R)-9) as colorless wax 3J1′,2″ = 6.2 Hz, 1 H, H-1′), 3.82–3.71 (m, 2 H, H-9), 2.20 (t,
(1.52 g, 8.72 mmol, 78% of (R)-enantiomer, ee ≥ 99%). [α]2D3 J2,3 = 7.5 Hz, 2 H, H-2), 1.68–1.56 (m, 4 H, H-8/H-7), 1.53
3
3
−8.5 (c 1.00, THF).
(mc, 2 H, H-3), 1.30–1.21 (m, 6 H, H-6–H-4), 1.20 (d, J2′,1′
=
HRMS m/z calcd for C9H18O3Na 197.1148 [M + Na]+, found 3J2″,1′ = 6.2 Hz, 6 H, H-2″/H-2′); 13C NMR (100 MHz, CDCl3)
197.1146; IR (thin film, cm−1) ν 3391 (br, s), 2933 (s), 2858 (s), δ 173.3 (C-1), 135.0, 135.0 (Car-4), 133.7, 133.6 (Car-6/Car-2),
1711 (s), 1462 (m), 1410 (m), 1376 (m), 1261 (m), 1127 (m), 130.5, 130.4 (Car-5/Car-3), 118.8, 118.0 (Car-1), 67.3 (C-1′), 34.6
1103 (m); 1H NMR (400 MHz, CDCl3) δ 5.25 (br, 2 H, O–H, (C-2), 30.3, 30.2 (C-7), 28.9, 28.9, 28.7 (C-6–C-4), 24.8 (C-3),
3
3
COO–H), 3.81 (tq, J8,9 = 3J8,7 = 6.1 Hz, 1 H, H-8), 2.36 (t, J2,3
=
23.0, 22.6 (C-9), 22.6, 22.5 (C-8), 21.8 (C-2′/C-2″).
3
7.5 Hz, 2 H, H-2), 1.65 (tt, J3,4
=
3J3,2 = 7.5 Hz, 2 H, H-3),
Antileishmanial activity
1.50–1.40 (m, 3 H, H-7/H-6a), 1.40–1.30 (m, 5 H, H-6b/H-5/
3
H-4), 1.20 (d, J9,8 = 6.1 Hz, 3 H, H-9); 13C-NMR (100 MHz, The virulent L. major isolate (MHOM/IL/81/FE/BNI) and Luci-
CDCl3) δ 179.0 (C-1), 68.2 (C-8), 39.2 (C-7), 33.9 (C-2), 29.2, 29.0 ferase-transgenic (Luc-tg.) L. major were maintained by con-
(C-5/C-4), 25.5 (C-6), 24.6 (C-3), 23.5 (C-9).
tinuous passage in female BALB/c mice. L. major amastigotes
Isopropyl 9-bromononanoate (10). 9-Bromononanoic acid were isolated from lesions as described previously33,34 and pro-
(5.2 g, 21.92 mmol) was dissolved in dry 2-propanol (50 ml) mastigotes were grown in vitro in blood-agar cultures at 27 °C,
and sulfuric acid (1 ml) was added. The flask was equipped 5% CO2, and 95% humidity. AlamarBlue® assays for the
with Dean-Stark apparatus and the solution was heated to determination of antileishmanial activities against L. major
reflux for 19 hours. After the solution was cooled down to promastigotes and BMDM cytotoxicity, and Britelite™ plus
room temperature, 2-propanol was removed under reduced (PerkinElmer, Waltham, MA, USA) assays against intracellular
pressure, the residue was dissolved in Et2O (20 ml) and the Luc-tg. L. major amastigotes were performed as previously
organic solution was washed with a saturated Na2CO3 solution reported.34
(20 ml), distilled water (20 ml) and a saturated NaCl solution
(20 ml). The ethereal solution was dried over MgSO4, the
Diff-Quick staining for transmitted light microscopy
solvent was removed and the crude product was purified by After incubation for 6 h, 10 h, and 24 h in the presence of 30
column chromatography on silica gel (DCM). After removal of µM harmonine or 1% DMSO as the solvent control, L. major
the solvent, 10 was obtained as a colorless oil (5.23 g, promastigotes were harvested and centrifuged using a Cyto-
18.73 mmol, 86%). Rf = 0.70 (DCM).
spin 3 centrifuge (Shandon, Frankfurt, Germany) on micro-
HRMS m/z calcd for C12H23O2BrNa, C12H23O281BrNa scopic slides. Cytospin preparations were stained using the
301.0774, 303.0753 [M + Na]+, found 301.0776, 303.0753; IR Differential Quick stain (Diff-Quick) dye (Medion Diagnostics
(thin film, cm−1) ν 2979 (m), 2932 (s), 2857 (m), 1731 (s), 1466 AG, Duedingen, Switzerland), according to the manufacturer’s
(m), 1374 (m), 1256 (m), 1181 (m), 1145 (w), 1110 (s), 964 (w); protocol. Diff-Quik stains the leishmanial nuclei, the kineto-
3
3
1H NMR (400 MHz, CDCl3) δ 5.00 (sp, J1′,2′ = J1′,2″ = 6.2 Hz, plasts dark purple and the cytoplasm light purple allowing the
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observation of phenotypic changes within the parasite. The 11 A. Vilcinskas and H. Schmidtberg, Biol. Unserer Zeit, 2014,
stained cells were analyzed by transmitted light microscopy 44, 386.
under a 50× objective on a Nikon ECLIPSE 50i microscope 12 M. F. Braconnier, J. C. Braekman and D. Daloze, Bull. Soc.
equipped with a digital camera (Nikon, Tokyo, Japan). The Chim. Belg., 1985, 94, 605.
images were processed using NIS Elements D software (Nikon). 13 D. Enders and D. Bartzen, Liebigs Ann. Chem., 1991, 1991,
569.
Determination of the cell death phenotype by flow cytometric
analysis
14 S. Chandra Philkhana, P. Dhasaiyan, B. L. V. Prasad and
D. S. Reddy, RSC Adv., 2014, 4, 30923.
15 J. Alvar, I. D. Velez, C. Bern, M. Herrero, P. Desjeux,
J. Cano, J. Jannin and M. den Boer, PLoS One, 2012, 7,
e35671.
16 World Health Organization, 2014.
17 S. L. Croft, S. Sundar and A. H. Fairlamb, Clin. Microbiol.
Rev., 2006, 19, 111.
L. major promastigotes were either treated for 6 h, 10 h, and
24 h with 30 µM harmonine or 1% DMSO as the solvent control.
Cell staining was performed using an Annexin V-FITC Apoptosis
detection kit (Sigma-Aldrich, Saint Louis, MO, USA) according to
the manufacturer’s protocol. The stained samples were immedi-
ately analyzed by flow cytometry using a MACS Quant Analyzer
(Miltenyi Biotech, Bergisch Gladbach, Germany).
18 H. J. de Vries, S. H. Reedijk and H. D. Schallig, Am. J. Clin.
Dermatol., 2015, 16, 99–109.
19 E. Haulotte, P. Laurent and J.-C. Braekman, Eur. J. Org.
Chem., 2012, 2012, 1907.
Acknowledgements
20 J. van Buijtenen, B. A. C. van As, M. Verbruggen,
L. Roumen, J. A. J. M. Vekemans, K. Pieterse,
P. A. J. Hilbers, L. A. Hulshof, A. R. A. Palmans and
E. W. Meijer, J. Am. Chem. Soc., 2007, 129, 7393.
21 Y. Yang, M. R. Mannion, L. N. Dawe, C. M. Kraml,
R. A. Pascal and G. J. Bodwell, J. Org. Chem., 2011,
77, 57.
22 S. H. Woo, S. Frechette, E. A. Khalil, G. Bouchain,
A. Vaisburg, N. Bernstein, O. Moradei, S. Leit, M. Allan,
M. Fournel, M.-C. Trachy-Bourget, Z. Li, J. M. Besterman
and D. Delorme, J. Med. Chem., 2002, 45, 2877.
23 T. Ooi, Y. Uematsu and K. Maruoka, J. Org. Chem., 2003, 68,
4576.
We thank Dr Christian Paetz (MPI CE, Jena) for recording the
19F-NMR spectra and Sybille Lorenz (MPI CE, Jena) for HR-MS
measurements. Furthermore, the authors thank Martina
Schultheis (Institute for Molecular Infection Biology, Wuerz-
burg) for the determination of IC50 and cytotoxicity values,
and excellent technical support. This work was supported and
financed by the Max Planck Society and by a grant of the
Deutsche Forschungsgemeinschaft (DFG), Collaborative Research
Center 630 (SFB 630/TP B3), “Recognition, Preparation and Func-
tional Analysis of Agents against Infectious Diseases”.
24 L. Jiao, E. Herdtweck and T. Bach, J. Am. Chem. Soc., 2012,
134, 14563.
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