donor-two-acceptor dye design: A distinct gateway to NIR fluorescence

Article abstract of DOI:10.1021/ja308124q

The detection of chemical or biological analytes upon molecular reactions relies increasingly on fluorescence methods, and there is a demand for more sensitive, more specific, and more versatile fluorescent molecules. We have designed long wavelength fluorogenic probes with a turn-ON mechanism based on a donor-two-acceptor π-electron system that can undergo an internal charge transfer to form new fluorochromes with longer π-electron systems. Several latent donors and multiple acceptor molecules were incorporated into the probe modular structure to generate versatile dye compounds. This new library of dyes had fluorescence emission in the near-infrared (NIR) region. Computational studies reproduced the observed experimental trends well and suggest factors responsible for high fluorescence of the donor-two-acceptor active form and the low fluorescence observed from the latent form. Confocal images of HeLa cells indicate a lysosomal penetration pathway of a selected dye. The ability of these dyes to emit NIR fluorescence through a turn-ON activation mechanism makes them promising candidate probes for in vivo imaging applications.

Full text of DOI:10.1021/ja308124q

Article
pubs.acs.org/JACS
“Donor−Two-Acceptor” Dye Design: A Distinct Gateway to NIR
Fluorescence
Naama Karton-Lifshin,† Lorenzo Albertazzi,‡,§ Michael Bendikov,⊥ Phil S. Baran,∥ and Doron Shabat*,†
†School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv 69978, Israel
‡Institute for Complex Molecular Systems, Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of
Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
§NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, I-56127 Pisa, Italy.
⊥Department of Organic Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel.
∥Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: The detection of chemical or biological analytes upon
molecular reactions relies increasingly on fluorescence methods, and
there is a demand for more sensitive, more specific, and more versatile
fluorescent molecules. We have designed long wavelength fluorogenic
probes with a turn-ON mechanism based on a donor−two-acceptor π-
electron system that can undergo an internal charge transfer to form
new fluorochromes with longer π-electron systems. Several latent
donors and multiple acceptor molecules were incorporated into the
probe modular structure to generate versatile dye compounds. This new library of dyes had fluorescence emission in the near-
infrared (NIR) region. Computational studies reproduced the observed experimental trends well and suggest factors responsible
for high fluorescence of the donor−two-acceptor active form and the low fluorescence observed from the latent form. Confocal
images of HeLa cells indicate a lysosomal penetration pathway of a selected dye. The ability of these dyes to emit NIR
fluorescence through a turn-ON activation mechanism makes them promising candidate probes for in vivo imaging applications.
INTRODUCTION
■
Fluorescence probe design is a rapidly expanding area of
research in the chemical and biological sciences.1,2 The
detection of chemical or biological analytes in response to
molecular changes relies increasingly on fluorescence,3,4 and
thus, there is a demand for more sensitive, more specific, and
more versatile fluorescent molecules that can be used in vivo.5
Such molecules must have high physiological stability, high
quantum yields of fluorescence, long emission wavelengths,
large Stokes shifts, and good photostability.6−8 Most
fluorescent probes were developed for in vitro use.9,10 In
order to apply fluorescent probes for in vivo imaging, one must
consider the requirements for tissue optical penetration and the
autofluorescence of biological molecules. Near-infrared (NIR)
fluorescence has emerged as a tool for a broad spectrum of in
vivo applications including nondestructive small animal
imaging.11−15 Because tissue absorbance and autofluorescence
are significantly lower within this spectral range, NIR light can
penetrate deep into tissues and provide useful diagnostic
information16,17 with specificity that makes it an attractive and
less expensive alternative to classical radioisotope detection
methods for molecular imaging. In this respect, we have
recently developed a novel class of turn-ON NIR cyanine-based
probes.18 The probes are based on the new fluorochrome
QCy719,20 (Figure 1), which is obtained upon removal of a
specific trigger moiety by an analyte of interest. A distinctive
Figure 1. General synthesis and mechanism of activation of D2A
cyanine-like fluorescence probes.
change of the π-electron system leads to generation of a
cyanine dye with strong NIR fluorescence. The probe was used
Received: August 20, 2012
Published: November 29, 2012
© 2012 American Chemical Society
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to image endogenous hydrogen peroxide produced in an acute
inflammation model in mice.18 The synthetic strategy used to
prepare the QCy7 fluorochrome is versatile and can be
modularly utilized to obtain a novel family of fluorogenic
compounds. Here we report a new library of NIR fluorescent
molecular probes based on the distinctive donor−two-acceptor
(D2A) rational design approach.
RESULTS AND DISCUSSION
■
Design, Synthesis and Fluorescence Measurements.
The fluorescence of cyanine dyes is due to a polymethine π-
electron system with a donor−acceptor pair between two
nitrogen atoms. For instance, the fluorochrome of Cy7 (Figure
1) has one nitrogen atom with an electron pair as a donor
moiety and a second positively charged nitrogen atom as an
acceptor, bridged by a heptamethine π-electron chain. We have
demonstrated use of a distinctive molecular design to introduce
a turn-ON mechanism option in a cyanine molecule. As
illustrated in Figure 1, the condensation of phenol-dialdehyde 1
with two equivalents of a general acceptor molecule affords dye
compound 2. This dye is composed of a phenol moiety that
functions as a latent donor in conjugation with two acceptors.
Deprotonation of the phenol leads to formation of a phenolate
active donor III that is able to donate a pair of π-electrons to
either one of the conjugated acceptors (structures I and II of
compound 3). This intramolecular charge transfer (ICT)
generates a resonance species with a π-electron pattern similar
to that of Cy7 fluorochrome. On the basis of calculations
described below, structure I (Figure 1) is a preferred resonance
structure, whereas structures II and III have less resonance
contribution. The donor capability of the phenolate species III
can be masked either by a proton or by a specific protecting
group. Such a protected phenol can be used as a molecular
probe for detection or imaging of an analyte that can react with
the probe to remove the protecting group. The π-electron
arrangement of structure II can be viewed as a general D2A
system, and this dye family can be used to prepare probes with
a turn-ON option.
Figure 3. Preparation of (A) donor−two-acceptor and (B) donor−
three-acceptor dye systems.
fluorochrome. The donor moiety can be condensed with either
two identical acceptor molecules or with two different ones.
Following the described strategy, we synthesized a series of
compounds to form a library of new dye molecules with a
donor−two-acceptors or donor−three-acceptors mechanisms
of action. The structures, and the corresponding spectroscopic
data obtained for dye molecules prepared with donor 1, donor
1a, and donor 1b are respectively presented in Tables 1, 2, and
3
The UV−vis and the fluorescent spectra of the dyes from this
library support our prediction of obtaining NIR fluorescence
through a D2A design. Spectra of representative dyes from each
table are shown respectively in Figures 4, 5 and 6. The dye of
entry 6 comprises a phenol latent donor and two different
acceptors, a picolinium moiety and an indolium one (Figure 4).
Deprotonation of the phenolic dye results in formation of a
new donor−acceptor pair fluorochrome. The UV−vis spectrum
exhibits a major absorption peak with a maximum at 550 nm,
and the fluorescence spectrum indeed shows an emission peak
in the NIR region at a wavelength of 650 nm.
A variety of molecules that function as π-electron acceptors
can be incorporated in dye 2, thereby creating a family of D2A
dyes. Examples of possible acceptor-molecules with such
capability are presented in Figure 2.
The dye shown in entry 10 is composed of a phenol latent
donor and two identical acceptors based on a picolinium
moiety (Figure 5). Deprotonation of the phenolic form leads to
formation of a new donor−acceptor pair fluorochrome. The
UV−vis spectrum exhibits two absorption peaks with maxima
at 330 and 490 nm, and the fluorescence spectrum shows an
emission peak in the NIR region at a wavelength of 660 nm.
The dye shown in entry 18 is similarly composed of a phenol
latent donor and three acceptors: two picolinium moieties and
an indolium one (Figure 6). Deprotonation of the phenolic
compound leads to formation of three resonance structures of
new fluorochrome. The UV−vis spectrum exhibits an
absorption peak with maximum at 550 nm, and the
fluorescence spectrum shows an emission peak in the NIR
region at a wavelength of 680 nm.
Figure 2. Chemical structure of potential acceptor moieties.
This approach can be further extended by replacing the
donor moiety in dye 2 with two other phenol derivatives
(Figure 3). Phenol 1a can be condensed with two equivalents
of a general acceptor molecule to afford dye compound 2a,
whereas phenol 1b can be condensed with three equivalents of
a general acceptor to produce dye compound 2b. Deprotona-
tion of phenols 2a and 2b generates the corresponded
conjugated donor−acceptor pairs 4 and 5 with a new
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Table 1. Chemical Structures and Spectroscopic Data of Dyes Obtained from Donor 1
This library of fluorophores offers choice of dye compounds
with various characteristics. To demonstrate the potential
advantage of such a library, we evaluated the aqueous stability
of four selected compounds in comparison to that of the known
NIR fluorophore, Cy7. The dyes were incubated in PBS (pH
7.4), at 37 °C, and the NIR fluorescent emission of each was
monitored over 72 h (Figure 7). Three dyes had higher
aqueous stability than that of Cy7 (dyes of entries 6, 10, and
18); entry-1 dye had stability similar to that of Cy7.
Interestingly, dyes of entries 6 and 10 were highly stable; no
loss of NIR fluorescence was observed even after 72 h.
As illustrated in Figure 1, the ICT from the phenolate donor
to one of the two acceptor moieties leads to formation of a new
fluorochrome with NIR fluorescence. Importantly, the proto-
nated form of the dye (the phenol structure) does not emit
fluorescence in the NIR region (see Supporting Information).
Therefore, these dyes can be used as NIR fluorescence probes
for determination of pH in aqueous solution in the region of
the pKa value of the phenols. The types of the acceptor
moieties present in the dye molecule affect the pKa of each
phenol. To demonstrate this, the pKa’s of three dyes were
measured by monitoring their emitted fluorescence as a
function of the environmental pH (Figure 8). The phenols of
dyes from entries 18, 10 and 6 had pKa’s of 2.9, 3.9, and 5.2,
respectively.
phenolate form. In an aqueous environment, the ICT mode of
action will take place, and the dyes will emit NIR fluorescence.
Computational Study. In order to better understand the
mechanism of NIR fluorescence from the QCY7′-type
chromophore, we performed a computational study (at
B3LYP/6-31G(d) level of theory, see Supporting Information
for details) of the excitation and emission spectra of CY7′,
QCY7′, protonated QCY7′ (QCY7H′), QCY7′ with elongated
conjugation length (QCY7CC′), and QCY7′ with electron-
withdrawing groups (QCY7F2′), see Scheme 1. The results are
summarized in Table 4.
Generally, the computations reproduced well the observed
experimental trends and allowed the analysis of the factors
responsible for high fluorescence of QCY7′ and low
fluorescence of the protonated form. The source of strong
fluorescence is a conjugated backbone between the two
acceptor units. This is evident by the very strong predicted
fluorescence of the parent system (CY7′, Table 4). Once this
conjugated backbone is interrupted by an aromatic ring, as in a
protonated system (QCY7H′), fluorescence is weak. Shapes of
the frontier orbitals of CY7′ and CY7H′, given in Figure 9, also
support this explanation. Indeed, LUMO of CY7′ is located on
a conjugated oligoene backbone, while conjugation in LUMO
of CY7H′ is partially interrupted by an aromatic ring.
Additionally, the change in the orbital shape from HOMO to
LUMO is relatively small in the backbone of QCY7′ but is
significantly larger in the case of protonated QCY7H′ (Figure
The relative low pKa values of our phenolic dyes emphasize
that under physiological conditions, these dyes would be in a
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Table 2. Chemical Structures and Spectroscopic Data of Dyes Obtained from Donor 1a
Table 3. Chemical Structures and Spectroscopic Data of Dyes Obtained from Donor 1b
9). This leads to a large Stokes shift and also to minimal
fluorescence of the protonated form. Elongation of the π-
bonding system by addition of two double bonds to the
conjugated backbone (as in the QCY7CC′, Scheme 1 and
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Figure 6. Absorption and fluorescence spectra of entry-18 dye, 50 μM
in PBS, pH 7.4.
Figure 4. Absorption and fluorescence spectra of entry-6 dye, 50 μM
in PBS, pH 7.4.
Figure 7. Normalized intensity of NIR fluorescence emission for dyes
◆
■
▲
entries 1 (green -·-), 6 (blue - -), 10 (red - -), 18 (orange - -), and
□
Cy7 (- -) as a function of time (in PBS, pH 7.4, 37 °C).
parent. Elongation of the conjugated backbone in Cy7 D2A
systems is an excellent strategy to increase the fluorescence and
to shift it to the NIR region, as also shown in experimental
results given in Tables 1−3.
Confocal Fluorescent Images of Cells. Fluorogenic
probes are useful for imaging various biological functions.
Some of the newly synthesized dyes were found to have good
cell permeability and were likely to generate in vitro fluorescent
images. Figure 10 shows confocal images of HeLa cells
incubated with dye 18. The accumulation of the dye in the
lysosomal vesicles indicates cell penetration through endocy-
tosis. As expected, the dye emitted NIR fluorescence even
under the acidic conditions of the lysosome (pH ≈ 5). To
further demonstrate the utility of the fluorophore for NIR in
vitro imaging, cells were incubated with dye 18 overnight,
washed, and imaged with a confocal microscope (Figure 10).
The NIR fluorescence emitted by the dye indicates its chemical
stability inside cells.
Figure 5. Absorption and fluorescence spectra of entry-10 dye, 100
μM in PBS, pH 7.4.
Table 4) leads to even stronger fluorescence than predicted for
the parent that is also significantly shifted toward the NIR
region. Addition of an electron-withdrawing group on the
aromatic ring (as in the QCY7F2′, Scheme 1 and Table 4)
resulted in slightly weaker predicted fluorescence that is only
slightly shifted toward the NIR region relative to that of the
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Table 4. Calculated Absorption and Emission Transitions
and Their Calculated Oscillator Strength for Selected D2A
Systems Given in Scheme 1
λmax,abs (eV)
fabs
λmax,em (eV)
fem
QCY7′
CY7′
QCY7H′
QCY7CC′
QCY7F2′
2.20 eV (563 nm)
2.25 eV (550 nm)
2.65 eV (467 nm)
1.87 eV (663 nm)
2.16 eV (575 nm)
1.53
2.46
0.84
2.32
1.26
1.97 eV (629 nm)
2.08 eV (595 nm)
2.09 eV (595 nm)
1.56 eV (792 nm)
1.84 eV (672 nm)
1.02
2.15
0.13
1.17
0.68
Figure 8. Determination of pKa values for phenols of dyes shown in
■
▲
entries-6 (red -•-), -10 (black - -) and -18 (blue - -). The pKa’s of
the dyes were measured in various buffer solutions at the indicated pH.
Buffer solutions preparation conditions: pH 1- KCl + HCl 0.2 M; pH
2−5 - AcOH + NaOH 0.1 M; pH 5−8.3 - PBS 0.1 M, pH 8.8
NaHCO3 + NaOH 0.1 M. Fluorescence measurements wavelength:
entries-6 and -18 dyes λex = 550 nm, λem = 650 nm, entry-10 dye λex
=
490 nm, λem = 660 nm.
Scheme 1. Chemical Structures of Model Compounds Used
in Calculations
Figure 9. Frontier orbital pictures. (Top two): QCY7′ (LUMO and
HOMO). (Bottom two): QCY7H′ (LUMO and HOMO) calculated
at B3LYP/6-31G(d) level.
in removal of the masking group. To demonstrate that the
synthesized dyes could be incorporated into turn-ON probes,
we synthesized probe 6, a masked form of dye 10 (Figure 11).
Reaction of hydrogen peroxide with the phenyl boronic acid
protecting group caused release of the active dye.21 Whereas
probe 6 emitted fluorescence in the green region, dye 10
emitted in the NIR region. Therefore, the probe ratiometric
activity can be used to image hydrogen in cells. An NIR laser
was used to excite the sample at 820 nm to obtain two-photon
Masking of the active phenol functional group of the dye
molecules provides a convenient path to fluorescent probes
with a turn-ON mode of action. Such probes can be used to
detect an analyte of interest that reacts with the probe to result
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Figure 11. Two-photon imaging of hydrogen peroxide in living HeLa
cells. HeLa cells were treated with probe 6 for 4 h at 37 °C, washed,
and then treated with 1 mM of H2O2 or left untreated. Fluorescence
from the caged compounds (green channel) and the uncaged dyes
(red channel) was simultaneously acquired.
between the former two acceptors. The newly formed push−
pull structural element has a larger π-system than that of the
original donor−acceptor π-system and thus can emit
fluorescence in longer wavelengths. Several electron-with-
drawing groups were found to be good acceptors; however,
only a phenolate acted as an efficient donor in our analysis.
Attempts to incorporate an aniline functional group as a donor
did not result in dyes that emitted noteworthy NIR
fluorescence.
Figure 10. Confocal imaging of HeLa cells. (Images on left) Dye 18
co-localizes with the lysosome marker lysosensor, demonstrating that
the dye is localized mainly in the lysosomal vesicles. (Images on right)
Confocal images of HeLa cells incubated overnight with dye 18.
Samples were excited with a 561-nm laser, and fluorescence was
acquired in the range of 570−800 nm.
The Stokes shifts between the excitation and the emission
wavelengths of about 150−200 nm observed for most of the
new dyes are significantly larger those observed for the known
cyanines such as Cy5 and Cy7 (10−20 nm). This larger Stokes
shift is a desirable feature for fluorescence probes as it is
correlated with a greater signal-to-noise ratio. Fluorescent dyes
that absorb in wavelengths of 350−550 nm can be excited in
the NIR region with a two-photon laser.22 This option provides
the advantages of NIR excitation for chromophores that do not
absorb in the NIR range. To demonstrate the potential of dyes
in our library to undergo a two-photon excitation with an NIR
laser, dye 10 was evaluated. The dye exhibits a two-photon
excitation spectrum in wavelengths between 800 and 1000 nm
and therefore could be used to provide confocal cell images
(see Supporting Information).
Cyanine molecules are considered to be among the most
efficient NIR fluorophores. However, their chemical structure
does not readily allow functionalization to mask their
fluorescence in order to generate a turn-ON probe. Previous
efforts to design such turn-ON probes typically had limitations
of low fluorescence quantum yields, low extinction coefficients,
and complex synthesis procedures.23,24 Our approach allows us
to mask a cyanine fluorochrome with various protecting groups
and to release the active dye through the ICT mechanism. The
modular chemical structure of the probe allowed us to prepare
excitation. Cells were incubated with probe 6 for 4 h, washed,
and treated with exogenous hydrogen peroxide. Figure 11
shows confocal images of HeLa cells incubated with probe 6 in
the absence and in the presence of hydrogen peroxide. As
expected, only green fluorescence was observed from the cells
in the absence of hydrogen peroxide (the observed NIR
fluorescence was negligible). However, in the presence of
hydrogen peroxide, NIR fluorescence was observed. The
enlarged photo of several cells provides strong indication of
in vitro activation of the probe by hydrogen peroxide (see
Supporting Information).
DISCUSSION
■
The chromophores of most known fluorescent dyes have
conjugated π-electrons and a push−pull structural element. The
push component (the donor) is typically a heteroatom such as
oxygen or nitrogen, while the pull component (the acceptor)
can be any of various electron-withdrawing groups. Our dye
molecules are composed of a phenol latent donor in
conjugation with two acceptors. The latent donor is turned
ON upon formation of a phenolate ion. The unique design
enables an ICT from the activated donor to one of the two
acceptors to form a new push−pull conjugated chromophore
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a variety of masked cyanine dyes; all are equipped with the
turn-ON mode of action. Of those tested, the use of indoloum
or picolinium groups as acceptors and phenol as a donor
resulted in formation of dyes with the optimal spectroscopic
characteristics, including long emission wavelength and
quantum optical yield.
also supported by computation. Specifically, the addition of
electron-withdrawing groups on the aromatic ring and the
elongation of the π-electron system are predicted to result in
shifted emission wavelength toward the NIR region. Indeed,
such characteristics are observed empirically when using
hydrolytically stable, stronger π-electron acceptors.
Predicting the fluorescence behavior of organic compounds
solely on on the basis of chemical structure is not
straightforward. Indeed, the relationship between the molecular
structure and spectroscopic properties of the obtained dyes
cannot always be fully understood since even subtle
modifications can lead to fluorescence quenching through
CONCLUSIONS
■
In summary, we have developed a novel strategy for design of
long-wavelength fluorogenic probes. Our design is based on a
D2A π-electron system that can undergo an ICT to form a new
fluorochrome with a longer π-conjugated system. Several
different latent donors and multiple acceptor molecules were
incorporated into the probe modular structure to generate
versatile dye compounds. This strategy was demonstrated by
synthesis of a new library of dyes that had fluorescence
emission in the NIR region. Masking of the phenolate donor,
by a proton or an analyte-induced functional-group inter-
conversion, provided a convenient route for obtaining a turn-
ON probe. As with other fluorogenic dyes, our new probes
could be used to monitor various functions in cells. A
computational study supported the observed experimental
trends and allowed analysis of the factors responsible for high
fluorescence of the D2A active form and the low fluorescence
observed from the latent form as well as factors responsible for
shift of the fluorescence into NIR region. Confocal images of
HeLa cells indicated that the tested dye penetrates cells through
the lysosomal pathway. Such probes can be used as pH sensors
or for detection of specific analytes that have cleavage reactivity
for the phenolate-masking group. The ability of these dyes to
emit NIR fluorescence through a turn-ON activation
mechanism makes them promising candidate probes for in
vivo imaging applications. We anticipate that our unique
strategy will open a new door for further NIR fluorescence
probe discovery.
various pathways such as FRET (Forster resonance energy
̈
transfer), PET (photoinduced electron transfer), and more.
The D2A-conjugated chromophore approach described here is
without precedent since organic compounds are transformed
into NIR fluorophores after initial ICT.
In order to understand how and why the fluorescence
properties of the dyes are tuned by different acceptors, we have
prepared a library of analogues and analyzed their absorbances
and fluorescence spectra. In general, stronger acceptors
produced dyes with higher quantum yield and longer
fluorescence emission wavelength. For example, the indolium
group is known to be a stronger acceptor than a picolinium
one. In entry 1 (Table 1), the dye is equipped with two
indolium acceptors, whereas in entry 2, the dye has two
picolinium acceptors. Accordingly, the fluorescence emission of
entry-1 dye is observed at a longer wavelength (710 nm) than
that of entry-2 dye (630 nm). Following this trend, dye 1 had a
quantum yield of 16%, whereas the quantum yield of dye 2 is
only 0.4%. In the case of the dye in entry 5 (Table 1), where
the acceptor is the more powerful pyrilium species, the failure
to fluoresce is due to the chemical instability under aqueous
conditions. Thus, it can be concluded that, barring chemical
instability, the acceptor ability of the D2A system can be used
to qualitatively predict the fluorescence capabilities.
Within the D2A dye system, the electrophilic nature of the
ortho-acceptor is another significant factor that affects
fluorescence properties. Thus, those derivatives bearing an
indolium acceptor at the ortho position underwent a slow
spiro-cyclization to form spirocyanine III (Figure 12). The
ASSOCIATED CONTENT
■
S
* Supporting Information
Full experimental details, characterization data of all new
compounds, description of computational methods, spectro-
scopic assay conditions, and in vitro experimental conditions.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
chdoron@post.tau.ac.il
Notes
The authors declare no competing financial interest.
Figure 12. Merocyanine−spirocyanine reversal transformation.
ACKNOWLEDGMENTS
■
spiro-derivative can no longer function as a D2A fluorophore.
Although this transformation is reversible, such derivatives
exhibited faster lost of the NIR fluorescence in comparison to
their counterparts with a picolinium acceptor (see Figure 7).
To summarize, the new donor−acceptor π-electron system
reported here produces NIR fluorescence emission with good
and, as mentioned above, often predictable spectroscopic
characteristics. Collectively, the studies of how the donor and
acceptor units interact (structure−activity relationships) are
D.S. thanks the Israel Science Foundation (ISF), the Binational
Science Foundation (BSF), and the German Israeli Foundation
(GIF) for financial support. This work is supported in part by a
grant from the Israeli National Nanotechnology Initiative in the
Focal Technology Area: Nanomedicines for Personalized
Theranostics. M.B. is a member ad personam of the Lise
Meitner-Minerva Center for Computational Quantum Chem-
istry.
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