Templated Chromophore Assembly by Dynamic Covalent Bonds

Article abstract of DOI:10.1002/anie.201507186

Through the simultaneous use of three orthogonal dynamic covalent reactions, namely disulfide, boronate, and acyl hydrazone formation, we conceived a facile and versatile protocol to spatially organize tailored chromophores, which absorb in the blue, red, and yellow regions, on a preprogrammed α-helix peptide. This approach allowed the assembly of the dyes in the desired ratio and spacing, as dictated by both the relative positioning and distribution of the recognition units on the peptide scaffold. Steady-state UV/Vis absorption and emission studies suggest an energy transfer from the yellow and red donors to the blue acceptor. A molecular dynamics simulation supports the experimental findings that the helical structure is maintained after the assembly and the three dyes are confined in defined conformational spaces.

Full text of DOI:10.1002/anie.201507186

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201507186
German Edition: DOI: 10.1002/ange.201507186
Self-Assembly
Templated Chromophore Assembly by Dynamic Covalent Bonds
Lou Rocard, Andrey Berezin, Federica De Leo, and Davide Bonifazi*
Abstract: Through the simultaneous use of three orthogonal
dynamic covalent reactions, namely disulfide, boronate, and
acyl hydrazone formation, we conceived a facile and versatile
protocol to spatially organize tailored chromophores, which
absorb in the blue, red, and yellow regions, on a prepro-
grammed a-helix peptide. This approach allowed the assembly
of the dyes in the desired ratio and spacing, as dictated by both
the relative positioning and distribution of the recognition units
on the peptide scaffold. Steady-state UV/Vis absorption and
emission studies suggest an energy transfer from the yellow and
red donors to the blue acceptor. A molecular dynamics
simulation supports the experimental findings that the helical
structure is maintained after the assembly and the three dyes
are confined in defined conformational spaces.
N
atural photosynthesis is initiated by the absorption of
sunlight by a large number of light-harvesting pigments that
funnel the excitation energy to a reaction center where energy
conversion takes place. The efficiency of this process relies on
a particular organization and ratio of selected chromophores
that enables maximum light absorption and energy conver-
sion.[1]
One of the main challenges in mimicking natural antenna
systems is to develop high-yielding and easy-to-implement
protocols that can lead to the rational assembly of chromo-
phores into spatially organized architectures. To be efficient,
these should feature well-defined distances, orientations, and
an exact ratio of chromophoric donors to acceptors.[2] In this
respect, two approaches are followed: a sequential and
a templated route (Figure 1). Following the sequential
approach, covalent or noncovalent dendrimers,[3] macrocy-
cles,[4] supramolecular polymers,[5] nanostructures[6] and func-
tional surfaces[7] have been prepared starting from pro-
grammed monomeric chromophores. However, these systems
are limited by the synthetic accessibility and versatility to
tailor different colors, as they are often restricted to a limited
number of dyes. On the other hand, natural macromolecules
such as peptides,[8] nucleic acids,[9] and artificial polymers[10]
have been successfully used as platforms to template the
organization of chromophores (Figure 1). In this respect,
Figure 1. The sequential and templated routes for engineering multi-
chromophoric architectures.
helical peptide scaffolds appear to be suitable templates as
they can be easily programmed with a multitude of receptor
sites at given positions to spatially organize chromophores in
a specific orientation.[11] We have followed the templated
route (Figure 1), and report herein the first simultaneous
assembly of a multichromophoric architecture that consists of
three tailored chromophores linked through dynamic cova-
lent bonds to an amphiphilic a-helix peptide featuring
complementary receptor sites positioned every two pitches.
This arrangement imposes a distance of 1.08 nm and a parallel
orientation of the dyes in the final architecture.
In the search of recognition processes occurring simulta-
neously and orthogonally,[12] dynamic covalent bonds are
certainly excellent candidates because of their reversible or
irreversible nature.[13] The use of three dynamic covalent bonds
in concert has been very recently reported,[14] but their
exploitation for the simultaneous[15] assembly of multicompo-
nent architectures is unknown. With the aim of developing
a fast multicomponent assembly protocol, we have prepared
model disulfide–pyridine 1, bicyclic diol 2, and hydrazide 3
derivatives and studied their chemoselectivity in simultaneous
disulfide exchange, boronate, and acyl hydrazone formation,
respectively (Scheme 1). Whereas for the disulfide exchange
reaction a classical disulfide–pyridine functionality undergoing
fast interchange with phenyl thiol has been used,[16] 1,2-cis-diol
7-azanorbornane and 2,6-difluorobenzaldehyde functionalities
were selected to accelerate the formation of the linkages and at
[*] L. Rocard, Dr. A. Berezin, Dr. F. De Leo, Prof. Dr. D. Bonifazi
Namur Research College (NARC) and Department of Chemistry
University of Namur (UNamur)
Rue de Bruxelles 61, Namur 5000 (Belgium)
E-mail: davide.bonifazi@unamur.be
Prof. Dr. D. Bonifazi
Department of Pharmaceutical and Chemical Sciences and INSTM
UdR Trieste, University of Trieste
Piazzale Europa 1, Trieste 34127 (Italy)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002//ani.201507186.
Angew. Chem. Int. Ed. 2015, 54, 15739 –15743
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
15739

.
Angewandte
Communications
reactive thiophenol-derived blue NDI (B-NDI) 22, red PDI
(R-PDI) 27 exposing a 2,6-difluoro-l-phenyaldehyde moiety,
and phenyl boronic acid derived yellow Py (Y-Py) 30
derivatives were synthesized.
Following a synthetic methodology recently developed by
us,[23] AB-type B-NDI 22 was prepared in high yields starting
from easily accessible core-substituted monoanhydride 18
(Scheme 2). Sequential condensation reactions in the pres-
ence of amine 19 produced monoimide monoanhydride 20.
This compound was transformed into thiol-derived chromo-
phore B-NDI 22 through a condensation and substitution
reactions in the presence of amines 21 and iPrNH2, respec-
tively, followed by HgII-assisted cleavage of the thioether
group. PDI derivative 27 was synthesized starting from
cyclohexyl monoimide 23, which was subsequently reacted
with amine 19 to afford the diimide intermediate, sequentially
hydrolyzed into 24. Final condensation with aniline 25
followed by Suzuki-type cross-coupling with 26 afforded dye
27. The synthesis of yellow chromophore 30 consisted in
a Sonogashira cross-coupling between ethynylpyrene 28 and
29, followed by an acid-catalyzed hydrolysis of the boronic
Scheme 1. Simultaneous disulfide interchange, boronate, and acyl
hydrazone formation. a) m-Phenylene diamine (m-PDA), THF, RT, 1 h.
the same time to produce thermodynamically stable boronic
esters[17] and hydrazones, respectively. The azanorbornane
function was used to anchor a side tether, used for peptide
linkage, on the N atom. When operating the three reactions
simultaneously (Scheme 1), full conversion of 1,2-cis bicyclo-
diol 2 into tert-butylphenylboronate 8, disulfide
pyridine 1 into disulfide naphthalene 7, and
hydrazide 3 into acyl hydrazone 9 was achieved
in the presence of a catalytic amount of m-
PDA,[18] as observed by 1H NMR spectroscopic
and ESI-TOF mass spectrometric analyses of
the crude material (Figure 2). Purification of
the crude material allowed the isolation of the
expected products, ultimately confirming their
quantitative conversion (see the Supporting
Information).
Following these findings, a programmed a-
helix
peptide,
Ac-QLA-X(disulfide)-
QLAQLA-X(hydrazide)-QLAQLA-X(diol)-
QLA-CONH2, bearing the receptor sites on
a modified glycine every (i, i+ 7) residues, was
prepared. The amphiphilic trimeric QLA
sequence was selected as the main motif. The
presence of the hydrophilic glutamine each (i,
i+3) and (i, i+4) brings solubility and helix
stability by H-bonding to the alanine/leucine-
based motif, two residues well known for their
strong tendency to form helices.[19] Three
modified glycines were thus synthesized from
commercially available amino acid Fmoc-Gly-
(Propargyl)-OH through Cu-catalyzed azide–
alkyne cycloaddition (Scheme 2 and the Sup-
porting Information). Peptide 16 was synthe-
sized following Fmoc-based solid-phase syn-
thesis that, after disulfide interchange with 2,2’-
dipyridyl disulfide (DPS), was transformed
into peptide 17 (Scheme 2). In parallel, ethynyl
pyrene,[20] perylene diimide (PDI),[21] and
naphthalene diimide (NDI)[22] have been
selected for their complementary UV/Vis
absorption profiles that, covering the blue,
yellow and red regions, allow selective excita-
1
Figure 2. A) Expanded region of H NMR spectrum (CDCl3 , 258C) of 1) physical mixture
of 1, 2, and 3; 2) one-pot reaction; 3) disulfide naphthalene 7; 4) boronate 8; 5) acyl
tions in the three spectral zones. In this respect, hydrazone 9. B) ESI-TOF mass spectrum of the crude material.
15740 www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 15739 –15743

Angewandte
Chemie
chromism of the R-PDI bands is
observed, suggesting the forma-
tion of H-type complexes.[21a]
The energy transfer (ET) pro-
cess for the triad was investi-
gated using steady-state fluores-
cence spectroscopy. Excitation
measurements
(Figure 3C)
clearly indicated that all dyes
contribute to the emission of the
lowest-energy acceptor unit, B-
NDI (lem = 640 nm), thus con-
firming the ET from R-PDI to
B-NDI and from Y-Py to B-
NDI either by direct or stepwise
ET cascade. The quantum yield
of the acceptor unit B-NDI
(QYB-NDI) within YRB peptide
by direct excitation (lexc
=
609 nm) was determined to be
27%. Upon selective excitation
of the donor units R-PDI (lexc
495 nm) and Y-Py (lexc
=
=
398 nm), QYB-NDI were evalu-
ated to be 1.5% and 1.4%,
respectively. By calculating the
ratio of the QYB-NDI upon indi-
rect and direct excitation, the
energy-transfer efficiency FET
from the donor to the acceptor
was estimated to be 5% and
5.6% for the Y-Py!B-NDI and
R-PDI!B-NDI sensitization,
respectively. To validate our
hypothesis, FET was determined
upon comparison of the absorp-
tion and excitation spectra, both
normalized at the maximum
absorption wavelength of blue
Scheme 2. Synthetic approach for preparing YRB peptide 31. a) Fmoc-Gly(Propargyl)-OH, CuSO4·5H2O,
sodium ascorbate, DMF/H2O (4:1), RT, 3 h 76–89%; b) Rink amide MBHA resin, solid-phase peptide
synthesis (SPPS; see the Supporting Information); c) 2,2’-DPS, DIEA, DMF, RT, 1 h; d) m-PDA, DMF, RT,
4 h; e) 19, DIEA, dioxane, 1018C, 2 h, 92%; f) TFA, 738C, 24 h, 91%; g) 21, Et3N, benzoic acid, DMF,
1108C, 6 h, 72%; h) iPrNH2, 1108C, 48 h, 91%; i) 1)Hg(ClO4)2, RT, 30 min, 2)H2S, RT, 10 min À72%;
j) 19, Im, toluene/DMF (6:1), 1108C, 16 h, 67%; k) 1) KOH, iPrOH, 808C, 3 h, 2) AcOH, RT, 15 min;
l) 25, Im, toluene/DMF (6:1), 1108C, 16 h À78% over 2 steps; m) 26, K2CO3, [Pd(PPh3)4], dioxane/H2O
(7:1), 808C, 20 h, 95%; n) 28, CuI, [Pd(PPh3)2Cl2], NEt3, THF, 408C, 6 h, 62%; o) 2m aq. HCl/THF (1:2),
RT, 40 h, 85%. Fmoc=9-fluorenylmethyloxycarbonyl; DIEA=N,N-diisopropylethylamine; TFA=trifluoro-
acetic acid; Im=imidazole.
chromophore
unit
B-NDI
(lmax = 612 nm), by using the
equation FET = ExD/AD, where
ExD and AD are fluorescence
intensity and absorption of the
donor in the normalized excita-
tion and absorption spectra,
respectively. The efficiencies
were estimated to be 5% and
ester. The three chromophores were then simultaneously
assembled into peptide 31 by simply mixing the dyes with 17
at room temperature in the presence of m-PDA in anhydrous
DMF (Scheme 2). Separation by gel permeation chromatog-
raphy (GPC) afforded colored YRB-peptide 31 as pure
material, whose structure was confirmed by mass spectrom-
etry (see the Supporting Information).
The triad absorbs throughout the entire visible spectrum
with distinct peaks at 376, 398, 495, 527, and 612 nm,
characteristic of the Y-Py, R-PDI, and B-NDI chromophoric
units, respectively (Figure 3A). Notably, a significant hypo-
5.4% for Y-Py!B-NDI and R-PDI!B-NDI, respectively,
which are in agreement with previous experimental measure-
ments. To shed further light on the secondary structure of the
YRB peptide, CD studies were conducted to unravel the
conformational behavior before and after the chromophore
assembly. Whereas the measurements in DMF could not be
performed (lcut-off = 268 nm), CD spectra recorded in a solu-
tion of H2O/CF3CH2OH (TFE; 2:1; lcut-off = 190 nm) clearly
showed the typical peptide-centered peaks (positive band at
192 nm and two negative at 208 and 222 nm) fingerprinting an
a-helix secondary structure (see the Supporting Information).
Angew. Chem. Int. Ed. 2015, 54, 15739 –15743
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 15741

.
Angewandte
Communications
QYB-NDI by direct excitation of the acceptor unit (lexc
620 nm) collapsed to 6% (see the Supporting Information).
Upon excitation of the R-PDI (lexc = 495 nm) and Y-Py (lexc
=
=
398 nm) donor units, the QYB-NDI values were determined to
be 0.3% and 0.4%, respectively. As demonstrated by the
significant bathochromic shift of NDI-centered absorption
band, in this solvent, strong inter- and intramolecular are
possibly occurring and favor a significant quenching of the
Figure 3. A) Normalized absorption (solid lines) and fluorescence
(dashed lines) spectra of B-NDI 22, R-PDI 27, and Y-Py 30 in DMF;
B) Absorption spectra of B-NDI 22 (blue), R-PDI 27 (red), Y-Py 30
(yellow), their arithmetic sum (a, 22+27+30) and YRB peptide
(black) in DMF; C) Absorption (solid line, 2 mm) and emission (long-
dashed line, 2 mm, lexc =612 nm) and excitation profile (lem =640 nm)
in DMF (short-dashed line, 2 mm) for YRB peptide.
The a-helix content was estimated to be 82% by the
CONTINLL algorithm.[23] Switching the solvent to neat
TFE or a H2O/TFE solution (1:1 and 1:2) did not enhance
the inherent helicity of the secondary structure, thus implying
that the selected sequence is an ideal helical scaffold. Notably,
as both peptides 31 and 17 possess the same inherent helicity,
it suggests that the presence of large p-conjugated dyes does
not significantly perturb the secondary structure.
On the other hand, the lack of any Cotton effect in the
absorption region of the dyes implies the absence of any chiral
chromophoric organization, most likely because of the
significant distance from the helical template. In TFE, the
Figure 4. MD simulation (55 ns) of YRB peptide (explicit solvent).
A) Side and B) top views of 500 overlaid frames. C) Most stable
conformation identified at 53 ns (Etot =À1001 kcalmolÀ1).
15742 www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 15739 –15743

Angewandte
Chemie
[4] a) R. Takahashi, Y. Kobuke, J. Am. Chem. Soc. 2003, 125, 2372 –
radiative emission. Further time-resolved photophysical stud-
ies under different conditions are currently under investiga-
tion to unravel all possible deactivation pathways, including
those arising from aggregation and photoinduced charge
transfer phenomena.
MD simulation analysis for the YRB-peptide (Figure 4),
clearly showed that the majority of the residues are in the a-
helix or 3–10 helix (see the Ramachandran plot in the
Supporting Information). The chromophoric units show a dif-
ferent spatial occupancy owing to the conformational flexibility
of the linkers and the geometrical constrains imposed by the
helical template. As shown in Figure 4, the chromophores
occupy three different conformational spaces in which red,
blue, and yellow regions are easily discerned. Notably, in the
most stable conformation, the red and blue units are facing
each other through H-type complexes, thus supporting the
hypochromism observed in the UV/Vis spectra.
In summary, we have engineered the first orthogonal
multireaction system consisting of simultaneous disulfide
exchange, boronate, and acyl hydrazone formation. Exploi-
tation of this methodology allowed the simultaneous organ-
ization of tailored blue, red, and yellow chromophores on
a preprogrammed a-helix peptide. The energy transfer from
the primary donor Y-Py to the acceptor B-NDI, and from the
intermediate donor R-PDI to B-NDI was estimated to be
around 5% and 5.5%, respectively. The proof-of-concept put
forward in this paper will allow the bottom-up approach of
multichromophoric architectures exhibiting any desired ab-
sorbed or emitted colors, enabling unlimited surfing through
the color coordinate diagram.
2373; b) P. Parkinson, C. E. I. Knappke, N. Kamonsutthipaijit, K.
Sirithip, J. D. Matichak, H. L. Anderson, L. M. Herz, J. Am.
Chem. Soc. 2014, 136, 8217 – 8220.
[5] R. Abbel, C. Grenier, M. J. Pouderoijen, J. W. Stouwdam,
P. E. L. G. Leclere, R. P. Sijbesma, E. W. Meijer, A. P. H. J.
Schenning, J. Am. Chem. Soc. 2009, 131, 833 – 843.
[6] a) T. E. Kaiser, H. Wang, V. Stepanenko, F. Würthner, Angew.
Chem. Int. Ed. 2007, 46, 5541 – 5544; Angew. Chem. 2007, 119,
5637 – 5640; b) A. Lohr, F. Würthner, Angew. Chem. Int. Ed.
2008, 47, 1232 – 1236; Angew. Chem. 2008, 120, 1252 – 1256; c) A.
Ajayaghosh, V. K. Praveen, C. Vijayakumar, S. J. George,
Angew. Chem. Int. Ed. 2007, 46, 6260 – 6265; Angew. Chem.
2007, 119, 6376 – 6381; d) A. Ajayaghosh, C. Vijayakumar, V. K.
Praveen, S. S. Babu, R. Varghese, J. Am. Chem. Soc. 2006, 128,
7174 – 7175.
[7] a) N. Sakai, M. Lista, O. Kel, S. I. Sakurai, D. Emery, J. Mareda,
E. Vauthey, S. Matile, J. Am. Chem. Soc. 2011, 133, 15224 –
15227; b) G. Sforazzini, E. Orentas, A. Bolag, N. Sakai, S.
Matile, J. Am. Chem. Soc. 2013, 135, 12082 – 12090.
[8] a) R. A. Miller, A. D. Presley, M. B. Francis, J. Am. Chem. Soc.
2007, 129, 3104 – 3109; b) M. Endo, M. Fujitsuka, T. Majima,
Chem. Eur. J. 2007, 13, 8660 – 8666.
[9] a) P. K. Dutta, R. Varghese, J. Nangreave, S. Lin, H. Yan, Y. Liu,
J. Am. Chem. Soc. 2011, 133, 11985 – 11993; b) J. G. Woller, J. K.
Hannestad, B. Albinsson, J. Am. Chem. Soc. 2013, 135, 2759 –
2768.
[10] Z. M. Hudson, D. J. Lunn, M. A. Winnik, I. Manners, Nat.
Commun. 2014, 5, 3372.
[11] U. Lewandowska, W. Zajaczkowski, L. Chen, F. Bouilli›re, D.
Wang, K. Koynov, W. Pisula, K. Müllen, H. Wennemers, Angew.
Chem. Int. Ed. 2014, 53, 12537 – 12541; Angew. Chem. 2014, 126,
12745 – 12749.
[12] a) Z. Rodriguez-Docampo, S. Otto, Chem. Commun. 2008,
5301 – 5303; b) R. J. Sarma, S. Otto, J. R. Nitschke, Chem. Eur.
J. 2007, 13, 9542 – 9546.
[13] S. J. Rowan, S. J. Cantrill, G. R. L. Cousins, J. K. M. Sanders, J. F.
Stoddart, Angew. Chem. Int. Ed. 2002, 41, 898 – 952; Angew.
Chem. 2002, 114, 938 – 993.
Acknowledgements
[14] a) K.-D. Zhang, S. Matile, Angew. Chem. Int. Ed. 2015, 54, 8980 –
8983; Angew. Chem. 2015, 127, 9108 – 9111; b) K.-D. Zhang, N.
Sakai, S. Matile, Org. Biomol. Chem. 2015, 13, 8687 – 8694.
[15] A. Wilson, G. Gasparini, S. Matile, Chem. Soc. Rev. 2014, 43,
1948 – 1962.
[16] G. T. Hermanson in Bioconjugate Techniques, 3rd ed. (Ed.: G. T.
Hermanson), Academic Press, Boston, 2013, pp. 229 – 258.
[17] C. D. Roy, H. C. Brown, J. Organomet. Chem. 2007, 692, 784 –
790.
All the authors gratefully acknowledge the EU through the
ERC Starting Grant “COLORLANDS” project. We thank A.
Sciutto for the help in the preparation of some intermediates,
Prof. T. Verbiest (KU Leuven) for access to the circular
dichroism spectrometer and for measurements, Dr. A. Stopin
for the SEM measurements, and A. De Leo for his contribu-
tion to the artwork.
[18] M. Rashidian, M. M. Mahmoodi, R. Shah, J. K. Dozier, C. R.
Wagner, M. D. Distefano, Bioconjugate Chem. 2013, 24, 333 –
342.
Keywords: dynamic covalent chemistry · energy transfer ·
peptides · self-assembly · supramolecular chemistry
[19] a) J. M. Scholtz, R. L. Baldwin, Annu. Rev. Biophys. Biomol.
Struct. 1992, 21, 95 – 118; b) J. M. Scholtz, E. J. York, J. M.
Stewart, R. L. Baldwin, J. Am. Chem. Soc. 1991, 113, 5102 – 5104.
[20] R. Varghese, H. A. Wagenknecht, Chem. Eur. J. 2009, 15, 9307 –
9310.
[21] a) F. Würthner, Chem. Commun. 2004, 1564 – 1579; b) F. Würth-
ner, C. R. Saha-Moeller, B. Fimmel, S. Ogi, P. Leowanawat, D.
Schmidt, Chem. Rev. 2015, DOI: 10.1021/acs.chemrev.5b00188.
[22] N. Sakai, J. Mareda, E. Vauthey, S. Matile, Chem. Commun.
2010, 46, 4225 – 4237.
How to cite: Angew. Chem. Int. Ed. 2015, 54, 15739–15743
Angew. Chem. 2015, 127, 15965–15969
[1] a) G. D. Scholes, G. R. Fleming, A. Olaya-Castro, R. Van Gron-
delle, Nat. Chem. 2011, 3, 763 – 774; b) R. Croce, H. Van Amer-
ongen, Nat. Chem. Biol. 2014, 10, 492 – 501.
[2] L. Maggini, D. Bonifazi, Chem. Soc. Rev. 2012, 41, 211 – 241.
[3] a) M. S. Choi, T. Yamazaki, I. Yamazaki, T. Aida, Angew. Chem.
Int. Ed. 2004, 43, 150 – 158; Angew. Chem. 2004, 116, 152 – 160;
b) S. Hecht, J. M. J. FrØchet, Angew. Chem. Int. Ed. 2001, 40, 74 –
91; Angew. Chem. 2001, 113, 76 – 94; c) U. Hahn, M. Gorka, F.
Vogtle, V. Vicinelli, P. Ceroni, M. Maestri, V. Balzani, Angew.
Chem. Int. Ed. 2002, 41, 3595 – 3598; Angew. Chem. 2002, 114,
3747 – 3750; d) V. Balzani, G. Bergamini, P. Ceroni, E. Marchi,
New J. Chem. 2011, 35, 1944 – 1954.
[23] A. A. Berezin, A. Sciutto, N. Demitri, D. Bonifazi, Org. Lett.
2015, 17, 1870 – 1873.
Received: August 2, 2015
Published online: December 7, 2015
Angew. Chem. Int. Ed. 2015, 54, 15739 –15743
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 15743