Optimised synthesis of the bacterial magic spot (P)ppGpp chemosensor pyDPA

Article abstract of DOI:10.1002/cbic.201900013

Guanosine penta-or tetraphosphate (pppGpp or ppGpp, re-spectively) is a nucleotide signalling molecule with a marked effect on bacterial physiology during stress. Its accumulation slows down cell metabolism and replication, supposedly leading to the formation of the antibiotic-tolerant persister phenotype. A specifically tailored fluorescent chemosensor, PyDPA, allows the detection of (p)ppGpp in solution with high selectivity, relative to that of other nucleotides. Herein, an optimised synthetic approach is presented that improves the overall yield from 9 to 67 % over 7 steps. The simplicity and robustness of this approach will allow groups investigating the many facets of (p)ppGpp easy access to this probe.

Full text of DOI:10.1002/cbic.201900013

Accepted Article
Title: Optimized synthesis of the bacterial magic spot (p)ppGpp
chemosensor PyDPA
Authors: Gabriele Conti, Marco Minneci, and Sara Sattin
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10.1002/cbic.201900013
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Optimized synthesis of the bacterial magic spot (p)ppGpp
chemosensor PyDPA
Gabriele Conti,[a],† Marco Minneci[a],† and prof. Sara Sattin[a],*
Abstract: (p)ppGpp is a nucleotide signalling molecule with a marked
effect on bacterial physiology during stress. Its accumulation slows
down cell metabolism and replication, supposedly leading to the
formation of the antibiotic tolerant persister phenotype. A specifically
tailored fluorescent chemosensor, PyDPA, allows to detect (p)ppGpp
in solution with high selectivity compared to other nucleotides. Here
we present an optimized synthetic approach that improves the overall
yield from 9% to 67% over seven steps. The simplicity and the
robustness of this approach will allow the groups investigating the
many facets of (p)ppGpp easy access to this probe.
Beside the well-known DNA, RNA and co-factor (NADH and FAD)
nucleotides, several nucleotide-related molecules, such as c-di-
GMP, c-AMP, Ap4A, and (p)ppGpp, are being actively
investigated for their role within second messenger signalling
systems. These molecules are produced by bacteria, along with
quorum sensing (QS) autoinducers, in response to environmental
stimuli and, in turn, lead to a variety of phenotypic changes that
allow bacteria to survive or even proliferate. In the post-antibiotic
era,[2] it is of the outmost importance to acquire a detailed
understanding of how bacteria respond to stress in order to set
out effective strategies not only to prevent the insurgence of
antibiotic resistance, but also to eradicate recurrent and chronic
infections.
Introduction
Dissecting each signalling pathway requires selective detection of
each of such signalling molecules in a milieu overpopulated with
many other, structurally similar, compounds.
Molecular-recognition and sensing of nucleotides has been an
active research field over the past decades[1] due to their
biological significance.
Figure 1. left) Structure of the nucleotide signalling molecule 1 (p)ppGpp and of its specific chemosensor PyDPA. Right) Structure of the 2:1 PyDPA : ppGpp
complex.
We decided to focus our attention in particular on guanosine tetra-
(ppGpp) or penta-phosphate (pppGpp), collectively known as
(p)ppGpp, a signalling alarmone produced in response to stress
conditions[3] (e.g. heat shock, nutrient starvation, etc.).
Discovered in 1969 by Cashel and Gallant,[4] they were initially
nicknamed magic spot I and II but, even after their structure was
elucidated (1, Fig. 1), the nickname lingered due to the complexity
of the pleiotropic effects these molecules have on bacterial
physiology.[5] Indeed, (p)ppGpp impacts transcription, translation,
and DNA replication,[3] and generates virulence factors by
interfering with QS networks.[6] Accumulation of (p)ppGpp is also
at the upstream of the stringent response, a signalling cascade
implicated with the formation of a dormant bacterial phenotype
called persisters.[7] This phenotype, transiently tolerant to
antibiotic treatment, is not only largely responsible for the
[a]
G. Conti, M.Sc; M. Minneci, M.Sc.; Prof. S. Sattin
Department of Chemistry
Università degli Studi di Milano
via Golgi, 19, 20133, Milano (Italy)
E-mail: sara.sattin@unimi.it
† These authors equally contributed to this work.
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difficulties encountered in eradicating recurrent and chronic
infections but also favours to the insurgence of resistant strains.
In the course of our research project on small molecules able to
control the onset of the persistent phenotype by interfering with
the stringent response pathway, selective detection of (p)ppGpp
has become a critical factor. Detection of (p)ppGpp in solution
over 6 steps, starting from 1-bromopyrene (Scheme 1) while a
marginal improvement of the overall yield was reported a few
years later by the same group[14] starting from 1-
pyrenecarboxaldheyde (19% over 8 steps). Here we report an
optimized synthetic sequence that overcomes the critical steps of
the original and the modified synthetic approaches, increasing the
overall yield from 9-19% to 67% over 7 steps.
3
historically relied on radiolabelled compounds (either with H or
32P)[8] or HPLC methods.[9] Only in recent years, a fluorescent
chemosensor (PyDPA, Fig. 1) – a compound bearing a binding
moiety connected and communicating with a fluorophore[10] – has
been specifically designed to bind selectively (p)ppGpp over other
nucleotides, such as ATP, GTP, UTP, TTP, cAMP and cGMP[11]
From a structural point of view, PyDPA comprises two Zn2+-
dipicolylamine (Zn2+-DPA) units, well known for their ability to bind
pyrophosphate groups in water,[12] bridged to a pyrene moiety
through an alkyloxy-phthalate. One molecule of (p)ppGpp with its
two terminal pyrophosphate groups is able to chelate two
molecules of PyDPA, forcing the proximity of the corresponding
pyrene units that is exploited for its distinctive excimer emission
(Em = 470 nm).[13]
Results and Discussion
The synthesis of Rhee et al is outlined in Scheme 1. Halogen-
lithium exchange on 1-bromopyrene (nBuLi), followed by
treatment with 1,3-dibromopropane yields the monoarylation
product 3, which is used to alkylate 5-hydroxyisophthalic acid
dimethyl ester. Ester reduction (LiAlH4) and reaction of the
resulting diol with PBr3 affords dibromide 4. Alkylation of
dipicolylamine with 4 affords the structure of the ligand, which is
finally transformed in the Zn complex by treatment with with
ZnClO4. All the steps are described to proceed in good to
excellent yields, except for the first one which proceeds with a
modest 18% yield, undermining the whole synthetic sequence.
Quantification of (p)ppGpp in solution is therefore possible with
the appropriate calibration curve up to the low micromolar range.
The synthesis of PyDPA, as originally reported in 2008 by Rhee
and co-workers,[11] was achieved with a modest yield of about 9%
Scheme 1. Reported synthesis of PyDPA according to ref[11]
In order to improve on these results, we envisaged to exploit the
reactivity of the C-Br bond of 1-bromopyrene 2 to perform a Pd-
catalysed coupling reaction with dimethyl-5-propargyloxy-
isophthalate 5 (Scheme 2), under Sonogashira conditions.
Different Pd sources (Pd(PPh3)2Cl2 or Pd(PPh3)4), in the presence
or the absence of copper salts (CuI), different solvents (e.g.
tertiary amines, THF, DMF) and a range of reaction temperatures
(from 40 to 90°C) were explored without success. Indeed, the high
activation temperatures required to activate the C-Br bond were
not compatible with the thermal instability of the alkyne 5 and
either no conversion of the starting material or alkyne
decomposition was observed.
Using the more reactive 1-iodopyrene 6,[15], allowed to obtain the
desired product 7 under mild conditions, although in modest yield
(42%), but it also introduced an additional step to the synthetic
sequence, which reduced the overall yield to 35% (Scheme 2).
Scheme 2. Pd-mediated coupling of 1-Iodopyrene 6 with isophthalate 5.
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Finally, a sequential approach was adopted to introduce the
alkynyl chain first and form the ether linkage at a later stage.
Although classical Sonogashira conditions are described as
effective for the reaction of propargylic alcohol 8 with 1-
bromopyrene 2[16], in our hands only copper-free conditions, using
Pd(PPh3)4 as catalyst in n-BuNH2 as solvent at reflux gave the
desired product 9 in excellent yield (94%, Scheme 3).[17] It is worth
noting that the same reaction conditions applied to phthalate 5
only produced alkyne decomposition and a mixture of unidentified
by-products. Catalytic reduction of the triple bond of compound 9
has been described using PtO2 (15 mol%) in THF[18] but we found
that the much cheaper Pd(C) in methanol worked just as well with
a lower catalyst loading (5 mol%) and shorter reaction times (20
minutes) with an overall dramatic decrease of the reaction cost.
Finally, reaction of alcohol 10 with HBr/AcOH under MW
irradiation as described in[19] with modifications, proceeded
smoothly affording bromide 3 in quantitative yield (Scheme 3).
Alternative conditions using PBr3 for the functional group
transformation provided lower yields and complicated the reaction
work-up. Thus, compared to the reported procedure, we obtained
the same intermediate 3 with 92% overall yield, as opposed to
18%. Two additional steps are required, but only one
chromatographic purification is involved over the three steps.
Scheme 3. Optimized approach for the preparation of 1-(3-bromopropyl)pyrene 3.
At this stage, to increase the convergence of the synthetic path
and to skip the carboxymethylesters reduction step that was
described to proceed in 68% yield (Scheme 1), triol 11 was used
for the synthesis of ether 13, exploiting the lower pKa of the
phenol moiety (Scheme 4). The triol could be obtained in almost
quantitative yield by reduction of the commercially available
dimethyl-5-hydroxy-isophthalate 12 with LiAlH4[19]. Alkylation of 11
with 3 was best performed using excess K2CO3 and 1 mol equiv
of KI in refluxing acetonitrile. Under these conditions, ether 13 was
isolated in 95% yield, after 17 h.
Scheme 4. Preparation of compound 13 via alkylation of phenol 11 with compound 3 and final steps of the synthesis of PyDPA.
The final steps of the synthesis were reproduced as previously
described with comparable yields (Scheme 4). Diol 13 was
therefore treated with PBr3 to afford dibromide 4 (89%), which
gave 14 upon reaction with excess bis(2-picolyl)amine (88%).
Treatment of 14 with Zn(ClO4)2 • 6 H2O allowed to obtain
chemosensor PyDPA¸ which was used for (p)ppGpp detection
without further purification.
Spectral data
The spectral properties of PyDPA, including absorption spectra
and fluorescence spectra in the presence of ppGpp were
consistent with the data reported in the literature for the PyDPA
chemosensor (Fig. 2).
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Synthesis of 3-Pyren-1-yl-prop-2-yn-1-ol (9): 1-Bromopyrene (3)
(100 mg, 0.35 mmol) was dissolved with Pd(PPh3)4 (13 mg, 0.0105 mmol)
in nBuNH2 (12 mL) degassed with Ar. Propargyl alcohol (8) (102 µL,
1.78 mmol) was added to the mixture and the reaction was left stirring at
reflux. After 3h the reaction was complete (TLC: 2:1 Hex:EtOAc). The
solvent was evaporated at reduced pressure and the crude was purified
by automated flash chromatography (Hex: EtOAc gradient from 92:8 to
40:60). Product 9 was obtained as a slightly yellow solid (85.1 mg, 95%).
Spectral
data
matched
those
reported
in
literature.[17]
1H-NMR (400MHz, CDCl3): δ(ppm)= 8.55 (d, 1H, H3Ar, 3J=9.2Hz), 8.25-
8.19 (m, 2H, H6Ar, H8Ar), 8.19-8.15 (m, 2H, H5Ar, H9Ar), 8.14-8.01 (m, 4H,
3
H
2Ar, H4Ar, H7Ar, H10Ar), 4.52 (d, 2H, CH2OH, J=6.5Hz), 1.82 (t, 1H. OH).
13C-NMR (100MHz, CDCl3): δ (ppm)= 132.2 (C1), 131.5 (C5a), 131.3 (C8a),
131.1 (C3a), 129.9 (C10a), 128.5 (C2), 128.4 (C9), 127.3 (C5), 126.4 (C4),
126.2 (C7), 126.9 (C5a’), 126.7 (C3a’), 125.7 (C6), 125.5 (C8), 124.5 (C10),
124.4 (C3), 92.9 (C≡CH2OH), 52.2 (CH2OH), 84.9 (CCH2OH). MS (ESI)
m/z: calculated for [C19H12ONa]+=279.08, found=279.28
Figure 2. Fluorescence emission spectra of PyDPA (20 μM in 1 mM HEPES
pH 7.5) alone and upon addition of each nucleotide and ppGpp (7 μM). Ex =
344 nm.
Synthesis of 3-(pyren-1-yl)propan-1-ol (10): Compound 9 (184 mg, 0.72
mmol) was dissolved in freshly distilled MeOH (24 mL). Pd/C 10% (38 mg,
0.04 mmol) was added and the reaction mixture was stirred under
hydrogen atmosphere (1 atm) for 20 minutes (TLC: 6:4 Hex:AcOEt). The
catalyst was removed by filtration over celite and the solvent was
evaporated at reduced pressure. Pure product 10 was obtained in 96%
Indeed, while the presence of ppGpp led to the distinctive excimer
emission band at 470nm, the presence of other nucleotides, such
as AMP, GDP or ATP only showed the monomer emission bands.
[20]
yield (179mg). Spectral data matched those reported in literature
1H-NMR (400MHz, CDCl3): δ(ppm)= 8.32 (d, 1H, H3Ar, 3J=9.4Hz), 8.19-
8.15 (dd, 2H, H6Ar, H8Ar, 3J=7.5Hz), 8.14-8.09 (dd, 2H, H2Ar, H9Ar,3J=7.9Hz),
8.06-8.01 (m, 2H, H4Ar, H5Ar), 7.99 (t, 1H, H7Ar), 7.90 (d, 1H, H10Ar), 3.80 (t,
2H, CH2OH, 3J=6.1Hz), 3.50-3.43 (m, 2H, ArCH2), 2.18-2.10 (m, 2H,
CH2CH2OH). 13C-NMR (100MHz, CDCl3): δ (ppm)= 136.3 (C1), 131.6
(C5a), 131.0 (C8a), 131.1 (C3a), 128.8 (C10a), 127.7 (C2), 127.6 (C9), 127.5
(C5), 127.4 (C4), 126.2 (C7), 125.4 (C5a’), 125.3 (C3a’), 125.1 (C6), 125.0
(C8), 124.9 (C10), 123.4 (C3), 62.6 (CH2OH), 34.7 (ArCH2), 29.8 (CH2-
CH2OH). MS (ESI) m/z: calculated for [C19H16ONa]+= 283.10; found:
282.96.
Conclusions
In conclusion, we have been able to streamline the synthesis of
the PyDPA chemosensor, overcoming the critical steps of the
original synthetic approach and increasing the overall yield from
9% to 67% from the same starting material, 1-bromopyrene. We
believe this approach will be easily reproducible and useful for the
many groups that are investigating worldwide the biological
activity of the still puzzling nucleotide signalling molecule
(p)ppGpp.
Synthesis of 1-(3-bromopropyl)pyrene (3): 33% HBr in AcOH (0.8 mL)
was added to alcohol 10 (179 mg, 0.68 mmol) in a microwave vial. The
reaction was stirred under MW irradiation at 100°C for 45’ (TLC: 7:3
Hex:AcOEt). The reaction mixture was diluted with 15 mL of EtOAc and
the organic phase was washed with 50% NaHCO3 solution (3x15mL),
water (1x15mL) and then dried over anhydrous Na2SO4. The solvent was
evaporated at reduced pressure to yield pure product 3 as a brown viscous
oil (218 mg, quant.). Spectral data matched those reported in literature.[20]
1H-NMR (400MHz, CDCl3): δ(ppm)= 8.29 (d, 1H, H3Ar, 3J=9.2Hz), 8.20-
8.15 (dd, 2H, H6Ar, H8Ar, 3J=7.6Hz), 8.15-8.10 (dd, 2H, H2Ar, H9Ar,3J=7.7Hz),
8.03 (s, 2H, H4Ar, H5Ar), 7.99 (t, 1H, H7Ar), 7.90 (d, 1H, H10Ar), 3.56-347 (m,
4H, CH2OH, ArCH2), 2.41 (qui, 2H, CH2CH2OH, 3J=6.9Hz). 13C-NMR
(100MHz, CDCl3): δ (ppm)= 134.9 (C1), 131.6 (C5a), 131.3 (C3a), 131.0
(C8a), 128.8 (C10a), 127.7 (C2), 127.6 (C9), 127.0 (C5), 126.0 (C4), 125.2
(C7), 125.1 (C6), 125.0 (C8, C5a’), 124.9 (C10), 123.4 (C3), 123.3 (C3a’), 34.7
(H2C-CH2Br), 33.6 (CH2Br), 31.8 (ArCH2). MS (ESI) m/z: calculated for
[C19H16OBr]+= 323.04; found: 323.03.
Experimental Section
Chemicals were purchased by commercial sources and used without
further purification, unless otherwise indicated. When anhydrous
conditions were required, the reactions were performed under Nitrogen or
Argon atmosphere. Anhydrous solvents were purchased from Merck.
Reactions were monitored by analytical thin-layer chromatography (TLC)
performed on Silica Gel 60 F254 plates (Merck) with UV detection (254
nm) and/or staining with ammonium molybdate acid solution, potassium
permanganate alkaline solution. Silica gel 60 (40-63 µm) (Merck) was used
for flash column chromatography. NMR experiments were recorded on a
Bruker AVANCE-400 MHz instrument at 298 K. Chemical shifts (δ) are
reported in ppm. The 1H and 13C NMR resonances of compounds were
assigned with the assistance of COSY and HSQC experiments. Multiplicity
are assigned as s (singlet), d (doublet), t (triplet), q (quartet), quint
(quintet), m (multiplet). Mass spectra were recorded on Apex II ICR FTMS
(ESI ionization-HRMS), Waters Micromass Q-TOF (ESI ionization-HRMS)
or ThermoFischer LCQ apparatus (ESI ionization). Compound 11 was
prepared as described in literature[19]
Synthesis of (5-(3-(pyren-1-yl)propoxy)-1,3-phenylene)dimethanol
(13): Compounds 3 (100 mg, 0.31 mmol), 11 (57.2 mg, 0.37 mmol), oven-
dried K2CO3 (128 mg, 0.93 mmol) and KI (56 mg, 0.34 mmol) were
dissolved in 2 mL of dry CH3CN (0.15 M). The reaction mixture was stirred
at reflux under microwave irradiation for 17h (TLC: 95:5 CH2Cl2:MeOH).
The solvent was evaporated at reduced pressure and the resulting brown
solid was dissolved in 15 mL of EtOAc. The solution was washed with
water (3 x5 mL) and brine (1 x 15 mL). The organic phase was dried over
anhydrous MgSO4 and the solvent was evaporated at reduced pressure.
The crude was purified by automated flash chromatography (95:5
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CH2Cl2:MeOH) affording pure product 13 in 95% yield (116.3 mg). Spectral
Keywords: (p)ppGpp • persisters • alarmone • fluorescent
data matched those previously reported.[11] 1H-NMR (400MHz, CDCl3):
chemosensor • PyDPA
δ(ppm)= 8.32 (d, 1H, H3Ar, 3J=9.6Hz), 8.17 (d, 2H, H6Ar, H8Ar 3J=7.6Hz),
,
8.13-8.07 (dd, 2H, H2Ar, H9Ar, 3J=7.7Hz), 8.03 (s, 2H, H4Ar, H5Ar), 7.99 (t,
[1]
[2]
[3]
[4]
[5]
Y. Zhou, Z. Xu, J. Yoon, Chem Soc Rev 2011, 40, 2222-2235.
WHO, Antimicrobial Resistance: Global Report on Surveillance, 2014
K. Potrykus, M. Cashel, Annu Rev Microbiol 2008, 62, 35-51.
M. Cashel, J. Gallant, Nature 1969, 221, 838-841.
a) Z. D. Dalebroux, M. S. Swanson, Nat Rev Microbiol 2012, 10, 203-
212; b) Z. D. Dalebroux, S. L. Svensson, E. C. Gaynor, M. S. Swanson,
Microbiol. Mol. Biol. Rev. 2010, 74, 171-199.
1H, H7Ar), 7.90 (d, 1H, H10Ar), 6.95 (s, 1H, Hp-Ph), 6.87 (s, 2H, Ho-Ph), 4.67
3
(s, 4H, CH2OH), 4.07 (t, 2H, CH2OPh, J=6.1Hz), 3.56 (t, 2H, ArCH2,
3J=7.4Hz), 2.35 (quint, 2H, ArCH2CH2). 13C-NMR (100MHz, CDCl3): δ
(ppm)= 159.7 (Cq-OCH2), 142.9 (2x Cq), 135.9 (C1), 131.6 (C5a), 131.0
(C8a), 130.1 (C3a), 128.9 (C10a), 127.6 (C2), 127.5 (C9), 127.4 (C5), 126.8
(C4), 126.0 (C7), 125.3 (C5a’), 125.1 (C3a’), 125.0 (C6, C8), 124.9 (C10),
123.5 (C3), 117.6 (CHp-Ph), 112.4 (2xCHo-Ph), 67.1 (CH2OPh), 65.3
(2xCH2OH), 31.3 (ArCH2CH2), 29.9 (ArCH2). MS (ESI) m/z: calculated for
[C27H24O3Na]+= 419.16; found: 419.64.
[6]
[7]
[8]
B. M. Fontaine, Y. Duggal, E. E. Weinert, ACS Infect. Dis. 2018.
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M. Fischer, T. P. Zimmerman, S. A. Short, Anal Biochem 1982, 121,
135-139.
Chemosensor PyDPA. 1H-NMR (400MHz, CD3CN): δ(ppm)= 8.76-8.72
[10] D. Wu, A. C. Sedgwick, T. Gunnlaugsson, E. U. Akkaya, J. Yoon, T. D.
James, Chem Soc Rev 2017, 46, 7105-7123.
(d, 4H, CH-o-Py, 3J=5.3Hz), 8.37 (d, 1H, H3Ar 3J=9.2Hz), 8.21-8.09 (m,
,
3H, H6Ar, H8Ar, H9Ar), 8.07-7.90 (m, 9H, H2Ar, H4Ar, H5Ar, H7Ar, H10Ar, CH-p-
[11] H.-W. Rhee, C.-R. Lee, S.-H. Cho, M.-R. Song, M. Cashel, H. E. Choy,
Y.-J. Seok, J.-I. Hong, J. Am. Chem. Soc. 2008, 130, 784-785.
[12] a) D. H. Lee, S. Y. Kim, J. I. Hong, Angew. Chem. Int. Ed. 2004, 43,
4777-4780; b) D. A. Jose, S. Mishra, A. Ghosh, A. Shrivastav, S. K.
Mishra, A. Das, Org Lett 2007, 9, 1979-1982; c) P. Das, N. B. Chandar,
S. Chourey, H. Agarwalla, B. Ganguly, A. Das, Inorg. Chem. 2013, 52,
11034-11041.
[13] a) H. K. Cho, D. H. Lee, J. I. Hong, Chem Commun 2005, 1690-1692;
b) S. Nishizawa, Y. Kato, N. Teramae, J. Am. Chem. Soc. 1999, 121,
9463-9464.
3
3
Py), 7.67 (t, 4H, CH-m-Py, J=6.3Hz), 7.33 (d, 4H, CH-m’-Py, J=7.8Hz),
6.73 (s, 2H, Ho-Ph), 6.71 (s, 1H, Hp-Ph), 4.16 (d, 4H, PyCH2N, Jgem=16Hz),
4.02 (t, 2H, ArCH2CH2CH2O, 3J=5.5Hz), 3.82 (s, 4H, NCH2Ph,), 3.64 (d,
4H, PyCH2N), 3.61 (t, 2H, ArCH2CH2CH2O, 3J=7.3Hz), 2.36 (quint, 2H,
ArCH2CH2CH2O, 3J=6.9Hz). 13C-NMR (100MHz, CD3CN): δ(ppm)= 160.4
(Cq-OCH2), 155.4 (Cq Py), 149.1 (Co Py), 143.0 (Cp Py), 137.3 (2x Cq Ph),
134.3 (C1), 132.3 (C5a), 131.8 (C8a), 130.9 (C3a), 129.8 (C10a), 129.0 (C2),
128.5 (C9), 128.2 (C5), 127.7 (C4), 127.4 (2xCHo-Ph), 127.2 (C7), 126.4
(CH-m-Py), 126.1 (C10), 126.0 (CH-m’-Py), 126.0 (C6), 125.8 (C8), 125.7
(C5a’), 125.5 (C3a’), 124.7 (C3), 119.0 (CHp-Ph), 68.0 (ArCH2CH2CH2OPh),
[14] J. Oh, J. I. Hong, Org Lett 2013, 15, 1210-1213.
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M. Klapper, K. Mullen, U. Kaiser, A. Golzhauser, A. Turchanin, Acs
Nano 2013, 7, 6489-6497.
56.9 (PhCH2N), 55.8 (PyCH2N), 31.9 (ArCH2CH2CH2OPh), 29.8
(ArCH2CH2CH2OPh). m/z: calculated for [C51H46N6OZn2 + 3ClO4‾]+
=
4+
[M4++3ClO4‾]+ : 1187.07; found: 1187.28.
[17] P. Sarathi Addy, B. Saha, N. D. Pradeep Singh, A. K. Das, J. T. Bush,
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This project has received funding from the European Research
Council (ERC) under the European Union’s Horizon 2020
research and innovation programme (grant agreement No
758108)”. We thank Alessio Maria Caramiello for technical
assistance.
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