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doi.org/10.26434/chemrxiv.12562310.v1
What Is Best Strategy for Water Soluble Fluorescence Dyes? – a CaseStudy Using Long Fluorescence Lifetime DAOTA DyesNiels Bisballe, Bo W. Laursen
Submitted date: 25/06/2020 • Posted date: 29/06/2020Licence: CC BY-NC-ND 4.0Citation information: Bisballe, Niels; Laursen, Bo W. (2020): What Is Best Strategy for Water SolubleFluorescence Dyes? – a Case Study Using Long Fluorescence Lifetime DAOTA Dyes. ChemRxiv. Preprint.https://doi.org/10.26434/chemrxiv.12562310.v1
The applications of organic fluorophores in biological sciences rely heavily on their properties in aqueoussolution. The lipophilic nature of virtually all such chromophores provides several challenges to adapt them tobiologically relevant conditions. In this work we investigate three different strategies for achievingwater-solubility of the diazaoxatriangulenium (DAOTA+) chromophore: hydrophilic counter ions, aromaticsulfonation of the chromophore core, and attachment of cationic or zwitterionic side chains. The longfluorescence lifetime (FLT, τf » 20 ns) of DAOTA+ makes it a sensitive probe for changes in the rate ofnon-radiative deactivation and for aggregation leading to multi exponential decay profiles. Direct sulfonation ofthe chromophore, as applied in several Alexa dyes, does indeed increase solubility drastically, but at the costof greatly reduced quantum yields (QY) due to enhanced non-radiative deactivation processes. Theintroduction of either cationic (4) or zwitterionic side chains (5), however, brings the FLT (τf = 18 ns) and QY(φf = 0.56) of the dye to the same level as the parent chromophore in acetonitrile. For these derivativestime-resolved fluorescence spectroscopy also reveals a high resistance to aggregation and non-specificbinding in a high loading of bovine serum albumin (BSA). The results clearly show that addition of chargedflexible side chains is preferable to direct sulfonation of the chromophore core.
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What is best strategy for water soluble fluorescence dyes? –
A case study using long fluorescence lifetime DAOTA dyes
Niels Bisballea and Bo W. Laursen*a
Address:
a Nano-Science Center & Department of Chemistry, University of Copenhagen,
Universitetsparken 5, DK-2100, Copenhagen Ø, Denmark.
* Corresponding author e-mail:
2
Abstract
The applications of organic fluorophores in biological sciences rely heavily on their properties in
aqueous solution. The lipophilic nature of virtually all such chromophores provides several
challenges to adapt them to biologically relevant conditions. In this work we investigate three
different strategies for achieving water-solubility of the diazaoxatriangulenium (DAOTA+)
chromophore: hydrophilic counter ions, aromatic sulfonation of the chromophore core, and
attachment of cationic or zwitterionic side chains. The long fluorescence lifetime (FLT, τf 20 ns)
of DAOTA+ makes it a sensitive probe for changes in the rate of non-radiative deactivation and
for aggregation leading to multi exponential decay profiles. Direct sulfonation of the chromophore,
as applied in several Alexa dyes, does indeed increase solubility drastically, but at the cost of
greatly reduced quantum yields (QY) due to enhanced non-radiative deactivation processes. The
introduction of either cationic (4) or zwitterionic side chains (5), however, brings the FLT (τf = 18
ns) and QY (φf = 0.56) of the dye to the same level as the parent chromophore in acetonitrile. For
these derivatives time-resolved fluorescence spectroscopy also reveals a high resistance to
aggregation and non-specific binding in a high loading of bovine serum albumin (BSA). The
results clearly show that addition of charged flexible side chains is preferable to direct sulfonation
of the chromophore core.
3
Introduction
Molecular behavior of a solute in a given solvent is a critical property in several chemical sciences,
ranging from the distribution of drugs in the human body to the self-assembly of nanostructures.1,
2 This is indeed also the case in the field of bio-imaging and for assays relying on fluorescent dyes,
where enhanced water solubility of dyes leads to improved signal to background ratios and
elimination of artifacts.3-5
Organic chromophores in general consist of extended π-conjugated systems responsible for
absorption and emission of ultra-violet (UV) to near-infrared (NIR) electromagnetic radiation.
This inherently introduces a hydrophobic structural element to the fluorescent molecule, favoring
dissolution in organic solvents, while inducing aggregation due to poor solvation in aqueous
environments.6, 7 In bio-imaging the latter is seldom desirable, but notable exceptions include the
probing of membranes and staining lipophilic compartments.8, 9 Dye aggregation may lead to
unwanted changes in absorption and emission spectra, reduced fluorescence intensity and
lifetime.10-13 But even when aggregates are avoided e.g. by high dilution, moderate water-solubility
may still lead to problems arising from hydrophobic interactions with other amphiphilic solutes
such as biomacromolecules. This may lead to nonspecific binding and can in turn also alter optical
properties.14-17 Thus, for general application of fluorescence dyes, high water-solubility is a key
feature since it is expected to reduce both dye-dye interactions (aggregation and self-quenching)
as well as nonspecific interactions with biomolecules.
A testament to the importance of good solvation of fluorescent probes in aqueous media is the
pioneering work of Haugland leading to the highly successful commercial Alexa Fluor® dyes
(Thermo Fisher).18, 19 The Alexa Fluor® dyes are traditional organic chromophores, mainly
xanthenes and cyanines, modified with anionic sulfonate groups to drastically increase their water
solubility and thus performance in bioimaging. The same strategy has since been employed for
several other chromophore types and by other commercial brands.
While many different chromophore modifications have been applied for increasing water-
solubility, including anionic sulfonates and phosphonates, zwitterionic sulfobetaines and neutral
PEG chains, there is presently no general consensus about the preferred or optimal modifications
of fluorescence dyes to increase water-solubility and minimize artefacts in bio-imaging.
Rhodamine and borondipyrromethene (BODIPY) chromophores are popular in bioimaging and
4
enhancing their water-solubility is a desirable improvement. A key feature of these dyes is their
high brightness (ε ∙ φf). They also feature moderate fluorescence lifetimes (FLTs) in the range of
3-5 ns and have seen application in time resolved fluorescence spectroscopy.20-22 Inspired by the
Alexa Fluor® dyes, Kolmakov et al. (2010) have synthesized a red emitting, water-soluble
rhodamine dye by allylic sulfonation.23 They have successfully demonstrated the use of the dye in
advanced super-resolution fluorescence imaging techniques. Since, Kolmakov et al. (2012) have
also assessed the solubilizing effects of phosphonate and hydroxyl groups in the same position.24
Investigations into solubilizing the BODIPY chromophore are more varied. Early examples carry
sulfonate groups directly on the chromophore.25, 26 Later investigations have focused on
introducing water solubility through functionalized side chains to keep the electron distribution of
the chromophore intact. The solubilizing groups are typically comprised of sulfonates,
phosphonates and sulfobetaines.27-29
Another successful approach to water solubility has been post-synthetic functionalization of dyes
with a sulfonated peptide linker. The linker can be introduced through well-known couplings like
amidation and the click-reaction, which many commercially available dyes are already set up for.30-
32 This strategy, however, seems to work best for dyes that already contain hydrophilic elements.
The peptide linker may feature further functional groups suitable to generate bioconjugates.33
We have in recent years been working with synthesis and applications of various aza-/oxa-
triangulenium dyes (Chart 1), in particular the ADOTA+ and DAOTA+ derivatives. These
fluorophores display especially long intrinsic FLT in combination with good quantum yield (QY),
making them uniquely suited for time-gated imaging34, fluorescence lifetime imaging microscopy
(FLIM)35, 36 and fluorescence polarization assays37, 38. The long FLT arises from the combination
of moderate radiative rate (kf) and very low rates of non-radiative deactivation (knr).39 The major
challenge in designing and modifying long FLT dyes is to keep the rate of non-radiative
deactivation very low. which is achieved through exceptional structural rigidity.
In this work we investigate design strategies to obtain highly water soluble triangulenium dyes.
Firstly, such modifications have not been made before and would greatly increase the scope of
these unique long FLT dyes. Secondly, the long FLT of these dyes provide a highly sensitive test
case to compare various strategies for enhancing water solubility. This sensitivity arises from the
intrinsically lower rate of fluorescence, which ensures that any additional non-radiative
5
contributions to deactivation of the excited state will have a much greater impact than in standard
fluorophore systems with a shorter lifetime. We will also use fluorescence lifetime measurements
as a convenient tool to probe the degree of aggregation and/or association with bio molecules as
any of such events will lead to inhomogeneity in the population of dyes and thus deviations from
simple single exponential fluorescence decay rates.
The DAOTA+ chromophore (Chart 1) was chosen as the starting point for this study. It features a
long FLT (τf > 20 ns in MeCN and DCM) and a high quantum yield (φf = 0.58 in MeCN, 0.80 in
DCM).39
Chart 1. Structures of diazaoxatriangulenium (DAOTA+),
azadioxatriangulenium (ADOTA+), dimethoxyquinacridinium
(DMQA+), carbon-bridged diazatriangulenium (CDATA+) and benzo-
fused diazatriangulenium (BDATA+).
The DAOTA+ chromophore is promising due to high photo stability40, 41 and resistance to
quenching from amino acids.42 However, it suffers from low water solubility, which reduces its
brightness and FLT in aqueous solutions. The chromophore is closely structurally related to other
reported cationic triangulenium fluorophores such as: ADOTA+ 43, DMQA+ 44, CDATA+ 45 and
6
BDATA+ 46 dyes (Chart 1), but also to other much studied cationic dyes including helicenes47, 48,
acridines49, 50 and rhodamines51, 52. Thus, we expect the here derived guidelines to be generally
applicable to a large range of cationic fluorescent dyes.
Results and discussion
Strategies for chromophore modifications and synthesis
With a series of six new DAOTA+ dyes (Chart 2) we will evaluate various strategies for increased
water-solubility: 1) Introduction of hydrophilic counter ions. 2) Direct sulfonation of the
chromophore system. 3) Addition of charged side chains. Traditionally, triangulenium dyes have
been synthesized with the highly lipophilic tetrafluoroborate (BF4-) and hexafluorophosphate (PF6
-
) anions.
Chart 2. Structures of the six water-soluble DAOTA+ dyes 1-6, along with their QYs (φf) and FLTs (τf) in pure water. a Only the FLT corresponding to the freely solvated dye of a bi-exponential decay is shown (see Table 2 for details).
7
This strategy was developed with facile synthetic workup in mind, exploiting the lack of
dissociation of these salts in aqueous media.53 To obtain water-soluble derivatives, the anion of
Pr2DAOTA+ was exchanged for the hydrophilic and biologically prevalent chloride ion. This was
conveniently achieved using an anion exchange resin. This simple modification resulted in the
highly water-soluble (>10 mM) dye, 1 (Chart 2), from the practically insoluble BF4- salt in
quantitative yield (Scheme S1).
As mentioned in the introduction direct sulfonation of the chromophore core has been successfully
applied in several cases, including Alexa 488 and Alexa 532 to name a few,16 as well as the
aforementioned BODIPY dyes. Functionalization directly on the DAOTA+ chromophore can
roughly be broken down to occur in three different regions: the positions neighboring the O-bridge
(positions 3 and 5, Chart 1), positions neighboring the N-bridges (positions 1, 7, 9 and 11), and
positions para to the carbenium center (positions 2, 6 and 10). Delgado et al. (2018) have shown
large effects of simple functional groups attached directly to the 9-position of DAOTA+ via
electrophilic aromatic substitution, with a clear connection between electron density
(donating/withdrawing groups) and spectral properties.54 This is in line with observations from the
DAOTA+ precursor, DMQA+.55 We suspect that substitution neighboring the O-bridge is
preferable to positions neighboring the N-bridge, since steric interference with the side-chain in
the latter case is expected to reduce planarity. Despite EWGs on the 6-position enhancing
fluorescence of the helical DMQA+ 55, in planar systems functionalization next to N-bridges has a
negative impact on fluorescence properties (QY and FLT) as seen for DAOTA+, and in our
previous observations with chlorination of triazatriangulenium salts. 54, 56
Selective core sulfonation on the 3- and 5-positions of DAOTA+ was achieved by overnight
reaction in concentrated sulfuric acid (Scheme S1). A trace amount of the mono-sulfonated product
(2) could also be obtained in this way, with the primary product containing sulfonates in both
positions (3, 88 % yield). The mono-sulfonated product is likely to be a result of desulfonation
during aqueous workup. Both products are soluble in water. The selectivity for the 3- and 5-
positions is result of the strongly acidic conditions which favors electrophilic substitution in
positions most remote to the nitrogen bridges, as reported by Duwald et al. (2017) for the DMQA+
system (Chart 1).57
8
Modifying the dye with solvating groups on the side chains is expected to have little influence on
the photophysics. We have several examples of DAOTA+ modified in these positions, where the
emissive properties intrinsic to the chromophore remain largely intact.35, 39, 42 The side-chain
modified dyes were synthesized via the traditional pathway for triangulenium dyes where side
chains are introduced by substitution with primary amines during formation of the nitrogen bridges
in the ring system.53 By this classical pathway a DAOTA+ dye carrying two 3-
dimethylaminopropyl side chains (9, Scheme S2) was obtained as a key intermediate. Methylation
of 9 with iodomethane gave tri-cationic 4 after anion exchange. Zwitterionic side chains were
conveniently obtained by alkylation of 9 with 1,3-propanesultone, as previously reported for the
CalFluor chromophores58, to give 5 after anion exchange.
Finally, we were interested in combining the above strategies. By sulfonating the PF6- salt of 4
under conditions similar to the synthesis of 3, 6 was obtained after anion exchange (Scheme S2).
Full experimental details and characterizations of all new compounds are given in Supporting
Information.
Photophysical properties of water-soluble triangulenium dyes
With this diverse set of water-soluble triangulenium dyes (1-6) in hand, we first investigated the
effects of the modifications on the spectral features. As expected for the simple anion exchange
absorption and emission are nearly identical for 1 and Pr2DAOTA+ (Table 1). Absorption spectra
of 2 and 3, containing one and two sulfonic acid groups respectively, were surprisingly similar.
These modifications introduce a redshift in both absorption (S0 → S1) and emission maxima
compared to those of 1 (Table 1, Figure 1). The (S0 → S2) absorption maximum on the other hand
is blueshifted. The ionic side chains of 4 and 5 resulted in a blueshift shift in both (S0 → S1)
absorption and emission maxima. The (S0 → S1) absorption band of both 4 and 5 are narrower than
observed for 1 and Pr2DAOTA+ (Figure 1). For comparison, the absorption spectrum of 5 is similar
in width and shape to the parent chromophore, Pr2DAOTA+ in MeCN, while 1 has less pronounced
features (Figure S13).
9
Table 1 Steady state absorption and emission values for 1-6 (Chart 1) in water and Pr2DAOTA+ in acetonitrile for
comparison. Full absorption and emission spectra as well as excitation specta for 1-6 can be found in the ESI
(Figures S1-S6).
Solvent λmax (abs)
[nm]
S0 → S1
λmax (abs)
[nm]
S0 → S2
λmax (em)
[nm]
S1 → S0
Stokes shift
[cm-1]
φfa
Pr2DAOTA+ b, c MeCN 557 449 590 1004 0.58
1 H2O 558 449 587 885 0.31
2 H2O 564 440 594 895 0.27
3 H2O 565 435 594 864 0.28
4 H2O 549 448 576 854 0.56
5 H2O 548 449 576 887 0.56
6 H2O 558 439 584 798 0.36
a Quantum yields are determined relative to Rhodamine 6G in ethanol (φf = 0.95). Details on their calculation can be found in the ESI. b Data for
Pr2DAOTA+ taken from Bogh et al. (2017)39. c Full absorption spectrum can be found in Figure S13.
Figure 1. (A) Normalized overlay of absorption spectra of the DAOTA+ chromophore without modifications (1),
with a sulfonated core (3, similar to 2), with ionic side chains (5, similar to 4) and both of the latter modifications
combined (6). (B) Normalized overlay of emission spectra, analogous to A.
A narrower absorption band is an indication of a more homogeneous solvation, validating the
strategy of placing the functional groups on the side chains. Combining the approaches of
sulfonating directly on the chromophore and having quaternary ammonium groups on the side
chains (6) results in spectra clearly relatable to each individual modification: the (S0 → S1)
absorption redshift of the sulfonic acid groups, as observed for 2 and 3, and blue shift of the
400 450 500 550 600
0,0
0,2
0,4
0,6
0,8
1,0
No
rma
lize
d a
bso
rban
ce
Wavelength [nm]
1
3
5
6
A
550 600 650 700 750
0,0
0,2
0,4
0,6
0,8
1,0
Norm
aliz
ed e
mis
sio
n
Wavelength [nm]
1
3
5
6
B
10
charged side chains, as observed for 4 and 5, counteract. The resulting absorption maximum is
similar to that of 1, which contains neither modification. The blue shift of the (S0 → S2) absorption
observed for 2 and 3, and attributed to the sulfonic acid groups, persists in 6 as this transition is
unaffected by the ammonium side chains.
The spectral shifts can be explained by the electronic effects of the modifications on the electronic
transitions (Figure 2). The (S0 → S1) transition is dominated by the N-bridges donating electron
density to the formal cation center. The positively charged ammonium groups of the side chains
in 4 and 5 poses an electrostatic influence on the N-bridges impeding this process, leading to a
blueshift of the transition. Similarly, the sulfonates neighboring the O-bridge reduce its donating
capabilities in the (S0 → S2) transition. The (S0
→ S1) redshift from the sulfonates is surprising
considering its position,59 and we speculate that it might be a secondary effect of the S2 blueshift
reducing the mixing of S1 and S2 and thus increasing their separation.
Figure 2. Substituent effects on the electronic
transition dipoles for the first (red) and second (blue)
excited state illustrated for 6.43 The electron donating
capabilities of the heteroatom bridges is reduced in
both cases, leading to more energetic transitions.
Despite the similar spectral properties of 1 and Pr2DAOTA+, the QY (Table 1) is almost halved
for the former in aqueous solution. This can in part be attributed to poorer solvation of the cation
in water compared to acetonitrile. A broadening of both absorption and emission bands were
observed with increased dye concentration when measuring the QY, suggesting aggregation
(Figures S23-S24).
11
However, an intrinsic quenching of fluorescence observed for dyes dissolved in water must also
be considered to be a significant contribution. The extent of this effect can be determined by
comparing the fluorescence in water and deuterium oxide and is discussed later in this work.
Direct sulfonation of the π-system lead to a decreased QY. When comparing the absorption and
emission spectra from the QY titration of 2, a slight broadening of the bands is seen with increased
concentration which could indicate aggregation (Figures S26-S27). No broadening is observed for
the double sulfonated 3 (Figures S29-S30), but the QY was still as low as 1.
We were pleased to find that the QYs of 4 and 5 in water (Table 1) are comparable to that of
Pr2DAOTA+ in MeCN. Introducing solubilizing groups on the side chains of DAOTA+ promises
to be a viable strategy in translating the desired photophysical properties to aqueous solution. The
QY of 6 lies between the values resulting from the individual modifications (3 and 4 respectively).
This confirms that introducing the sulfonic acid groups has a negative impact on the QY.
Time resolved fluorescence spectroscopy in water
In order to explain the differences in QY of the synthesized dyes and to develop insight into the
dyes’ molecular behavior in aqueous solution, we turned to time-resolved fluorescence
spectroscopy. Measuring the FLT and looking at the decay profiles allows us to distinguish well-
solvated dyes displaying a mono-exponential fluorescence decay due to the homogeneity of the
sample from inhomogeneous samples containing subpopulations of aggregated dyes and thus
displaying multi exponential decay profiles. We found that in pure water only the dyes 3-5 display
mono-exponential fluorescence decays (Table 2) and can be considered fully and homogeneously
solvated.
The bi-exponential decay of 1 in water indicates poor solvation and the formation of aggregates,
which show a longer lifetime component. The quantum yield of 1 is almost half (Table 1)
compared to the best dyes (4 and 5) while the dominant FLT component only is reduced by 20%.
This means that the observed aggregates must have very low quantum yields and/or non-emissive
aggregates are also present.
12
Table 2. Comparison of fluorescence lifetimes of 1-6 (Chart 1) in water, PBS and PBS with the addition of BSA (600
µM). Full decay profiles can be found in the ESI (Figures S14-S19).
H2O PBS PBS + BSA
τf1 [ns] τf2 [ns]a τavg
[ns]b
τf1 [ns] τf2 [ns]a τavg
[ns]b
τf1 [ns] τf2 [ns]a τf3 [ns]a, c τavg
[ns]b
1 15.2 22.6 (19 %) 16.6 14.9 21.6 (26 %) 16.6 10.5 18.3 (60 %) 1.3 (1 %) 15.0
2 13.5 24.1 (12 %) 14.8 11.7 16.5 (54 %) 14.3 12.3 23.2 (51 %) 1.6 (2 %) 17.7
3 12.5 - - 11.5 20.2 (7 %) 12.1 11.9 25.0 (59 %) 1.8 (1 %) 19.5
4 18.3 - - 18.1 - - 18.6 - 7.7 (3 %) 18.2
5 18.6 - - 18.3 - - 18.5 - 6.3 (2 %) 18.3
6 14.6 20.5 (7 %) 15.0 14.2 - - 14.1 - 2.4 (1 %) 14.1
a Secondary fluorescence lifetime components and their weighted intensities in parenthesis. b Intensity weighted average fluorescence lifetimes,
where multiexponential fluorescence decay is observed. c Interpreted as the fluorescence lifetime component of dye bound to BSA at quenching
sites.
Two-fold sulfonation of the chromophore (3) resulted in mono-exponential fluorescence decay
reveals a homogeneous solvation of the chromophore. However, a large simultaneous drop in both
FLT (12.5 ns) and QY (0.28) (Table 1) show that the sulfonation indeed accelerates non-radiative
deactivation. The negative impact on FLY and QY from core sulfonation is confirmed by the mono
sulfonated derivative 2, which furthermore exerts a bi-exponential decay, indicating remaining
problems with aggregation of this overall neutral species.
The mono-exponential decays of 4 and 5 both show comparable FLTs above 18 ns. Along with
the measured QYs (Table 1), these results indicate that the dyes are well-solvated in water as a
result of the charged side chains. Remarkably, both the FLTs and QYs of these dyes are
comparable to Pr2DAOTA+ in MeCN solution.39
The fluorescence decay of 6 features a small secondary component in pure water. We speculate
that the charge complementarity of the two positive and two negative groups in each end of the
molecule may lead to head-to-tail aggregates. The lower FLT compared to 1 must, as discussed for
2 and 3, be attributed to the introduction of sulfonic acids groups on the chromophore core.
13
Solvation of dyes in buffers and protein solutions
To draw parallels to biological systems, time-resolved fluorescence of the dyes in phosphate
buffered saline (PBS) was also investigated (Table 2). The obtained decays were mostly similar to
the ones observed in pure water. For 3, a biexponential decay was observed. The increased salt
concentration of the solution could lower the solvation of the dye, thus inducing aggregation to
some extent.60 Inversely, a mono-exponential decay for 6 in PBS could be due to the breaking up
of the proposed head-to-tail aggregates due to the additional ionic strength stabilizing the solvation
of the highly charged dye. It should be noted, however, that for either dye these are relatively small
effects.
The fluorescence lifetime measurements were repeated with the addition of 600 µM BSA to
introduce sites for non-specific binding, relatable to biologically relevant media.61 For all dyes an
additional short-lived component was observed, leading to an overall decrease in the average FLT.
The short-lived component is more prevalent for the dyes that already display compromised
solvation in PBS. We tentatively assign the short lived component to dye bound to BSA at a
quenching site. Especially the core sulfonated anionic dye, 3, shows to be severely compromised
at high protein concentrations. This observation is noteworthy since sulfonation and overall
negative charge is the preferred modification for many commercial fluorescence dyes.
The well-solvated and bright chromophores, 4 and 5 are only slightly affected by BSA. The
population of freely dissolved chromophores in these cases does not seem to be affected by the
presence of BSA at retaining the long FLT of 18 ns, while a small fraction of the molecules bound
to BSA display a 6-8 ns lifetime. The interaction between dye molecules and BSA must therefore
be static on the nanosecond timescale. Any significant presence of one or more “dark” interactions
with the protein can be ruled out based on the relative fluorescence intensities, which show a
reduction of 6-10 % upon addition of BSA (Figures S10-S11).
Intrinsic water quenching
Aside from solubility and solvation, fluorescence of organic chromophores in water is further
complicated by a quenching effect intrinsic to the solvent. This has been illustrated several times
by enhanced fluorescence intensity and FLT in D2O.62-65 The exact mechanism by which this
14
quenching occurs and what parameters affect it is still to be fully understood and it is thus difficult
to predict the extent of the effect.
Measuring the FLT in D2O for the dyes 1-6 (Table 3) showed a significant increase compared to
H2O. A 20 % enhancement is seen for 4 and 5 already displaying long FLTs in pure water. This
effectively surpasses its parent’s performance in MeCN and brings the fluorophore on par with
Pr2DAOTA+ in DCM39, in which a similar solvent quenching effect is not expected to take place.
A 30 % enhancement is seen in the remaining cases. Where bi- exponential decays are observed,
both fluorescence decay components see an increase, suggesting that even dye aggregates are
affected by the quenching effect of water.
Table 3. Fluorescence lifetimes of 1-6 (Chart 1) in deuterium
oxide and the relative increase compared to water (Table 2).
τf1 [ns] τf2 [ns] a τavg [ns] b τ(D2O)/τ(H2O)
1 19.3 27.6 (22 %) 21.1 1.27c
2 16.8 23.6 (32 %) 19.0 1.28c
3 16.6 - - 1.33
4 21.9 - - 1.20
5 22.1 - - 1.19
6 19.2 - - 1.28c a Secondary fluorescence lifetime components and their weighted intensities
in parenthesis. b Amplitude weighted average fluorescence lifetimes, where
multiexponential fluorescence decay is observed. c Value calculated based
on the intensity weighted average fluorescence lifetime for bi-exponential
decays.
Rates of the excited state processes
With the steady state and time-resolved fluorescence results in hand we can now look at how the
structural differences of the dyes affect the associated excited state deactivation processes.
Calculating the rate constants (Table 4) from the measured FLTs and QYs reveals that 4 and 5
featuring the hydrophilic side chains have a significantly higher radiative rate kf than the remaining
derivatives. This effect we assign to the electrostatic impact of the positively charged ammonium
group on the nitrogen donor groups in the DAOTA+ chromophore, which also did result in a
blueshift and narrowing of the absorption and emission bands.
15
Most importantly, 4 and 5 display very low rates of non-radiative deactivation knr (Table 4),
showing that this design is ideal for solubilizing DAOTA+ in water. This becomes even more clear
when considering that almost 40 % of the non-radiative deactivation observed for these dyes in
water seems to be solvent specific quenching, as can be seen by comparing the rates in water and
D2O (Table 4). Interestingly, it seems that the aromatic sulfonates attached directly to the
chromophore core opens up additional paths of non-radiative deactivation increasing knr by a factor
of two. This is most clearly seen by comparison between the derivatives well solvated in D2O
where effects of aggregation and specific water quenching are not contributing. Here the core
sulfonated compounds 3 and 6 display knr twice that of 4 and 5.
Table 4. Radiative and non-radiative rate constants for the depopulation
of the excited state of 1-6 in water and deuterium oxide.
kf
[107 s-1]
knr (H2O)
[107 s-1]
knr (D2O) a
[107 s-1]
Δknr b
[107 s-1]
Pr2DAOTA+ c, d 2.6 2.1 - -
1e 2.0 4.5 3.1 1.4
2e 2.0 5.4 4.0 1.5
3 2.2 5.8 3.8 2.0
4 3.1 2.4 1.5 0.9
5 3.0 2.4 1.5 0.9
6e 2.5 4.4 2.7 1.6
a The rate constant of fluorescence is assumed to be the same in water and deuterium
oxide. b Effectively the quenching contribution of water, kQ·[Q]. c In acetonitrile. d Data
from Bogh et al. (2017).39 e Rate constants calculated from dominant component of
biexponential decay fits.
Conclusions
Six new water-soluble DAOTA+-derivatives were obtained through combinations of three
different strategies for modifying the chromophore. Exchanging the lipophilic anion for chloride
(1) lead to a substantial gain in water solubility, but solvation and aggregation of the cationic
chromophore remain unaltered. While twofold direct sulfonation of the DAOTA+ chromophore
(3) does enhance solvation in pure water, aggregation and non-specific binding are still a concern
in biologically relevant environments. Despite further effort to modify DAOTA+ in this regard (6),
16
core modifications remain problematic as the introduction of additional non-radiative deactivation
pathways compromises fluorescence lifetime and quantum yield. Excellent photophysical
properties in water resulted from introducing charged side chains in derivatives 4 and 5. This
design strategy resulted in dyes with long FLT of 18 ns and high quantum yields of 56 %, to our
knowledge, unmatched by any other water-soluble small molecule fluorophores emitting beyond
550 nm. We managed to utilize the FLT response as a tool to investigate how different solvation
strategies influence molecular events and interactions. These results help in finding the optimal
design for a bioconjugable and water-soluble FLT probe.
In first instance these results suggest that charged and zwiterionic side chains most likely also are
a preferable design strategy for organic fluorophores in general over traditional core sulfonation.
The potential usefulness of this strategy extends beyond the field of dyestuff. Tuning solvation
through different functionalizations is useful in several fields, e.g. in the science of nanostructures,
where controlled self-assembly in various solvents is of interest. Long FLT probes hold potential
as tools for investigating such solvation effects. We see the DAOTA+ chromophore as a good
candidate for further investigation of such properties, in particular due to the sensitivity and
resolution of time-resolved fluorescence spectroscopy as demonstrated. These dyes should also be
considered in the investigation of fluorescence quenching effects, since time-resolved
measurements are much more sensitive and convenient than corresponding intensity-based
experiments. Further, they provide detailed information on effects causing changes in fluorescence
response. Through combinations of these approaches, valuable information on the time scales of
the investigated events can be had, allowing for discrimination between static and dynamic
molecular events.
Conflicts of interest
The authors declare the following competing financial interests: Bo W. Laursen is
associated with the company KU-dyes, which produces and sells fluorescent dyes
(including triangulenium dyes).
17
Acknowledgements
The work was supported by the Danish Council of Independent Research (DFF-6111-00483).
References
1. G. L. Perlovich, A. O. Surov and A. Bauer-Brandl, J Pharm Biomed Anal, 2007, 45, 679-
687.
2. K. Ariga, M. Nishikawa, T. Mori, J. Takeya, L. K. Shrestha and J. P. Hill, Sci Technol Adv
Mater, 2019, 20, 51-95.
3. E. Oliveira, E. Bertolo, C. Nunez, V. Pilla, H. M. Santos, J. Fernandez-Lodeiro, A.
Fernandez-Lodeiro, J. Djafari, J. L. Capelo and C. Lodeiro, Chemistryopen, 2018, 7, 9-52.
4. A. N. Butkevich, V. N. Belov, K. Kolmakov, V. V. Sokolov, H. Shojaei, S. C. Sidenstein,
D. Kamin, J. Matthias, R. Vlijm, J. Engelhardt and S. W. Hell, Chemistry, 2017, 23, 12114-12119.
5. X. Wang, Y. Z. Hu, A. Chen, Y. Wu, R. Aggeler, Q. Low, H. C. Kang and K. R. Gee,
Chem Commun (Camb), 2016, 52, 4022-4024.
6. C. A. Hunter and J. K. M. Sanders, J Am Chem Soc, 1990, 112, 5525-5534.
7. D. Chandler, Nature, 2005, 437, 640-647.
8. J. Li, N. Kwon, Y. Jeong, S. Lee, G. Kim and J. Yoon, ACS Appl Mater Interfaces, 2018,
10, 12150-12154.
9. M. Collot, T. K. Fam, P. Ashokkumar, O. Faklaris, T. Galli, L. Danglot and A. S.
Klymchenko, J Am Chem Soc, 2018, 140, 5401-5411.
10. N. J. Hestand and F. C. Spano, Acc Chem Res, 2017, 50, 341-350.
11. K. Tani, C. Ito, Y. Hanawa, M. Uchida, K. Otaguro, H. Horiuchi and H. Hiratsuka, J Phys
Chem B, 2008, 112, 836-844.
12. A. B. Descalzo, P. Ashokkumar, Z. Shen and K. Rurack, Chemphotochem, 2019, 4, 120-
131.
18
13. R. L. Halterman, J. L. Moore and W. T. Yip, J Fluoresc, 2011, 21, 1467-1478.
14. L. C. Zanetti-Domingues, C. J. Tynan, D. J. Rolfe, D. T. Clarke and M. Martin-Fernandez,
Plos One, 2013, 8, e74200.
15. C. Sanchez-Rico, L. Voith von Voithenberg, L. Warner, D. C. Lamb and M. Sattler,
Chemistry, 2017, 23, 14267-14277.
16. L. D. Hughes, R. J. Rawle and S. G. Boxer, Plos One, 2014, 9, e87649.
17. Y. S. Zeng, R. C. Gao, T. W. Wu, C. Cho and K. T. Tan, Bioconjug Chem, 2016, 27, 1872-
1879.
18. N. Panchuk-Voloshina, R. P. Haugland, J. Bishop-Stewart, M. K. Bhalgat, P. J. Millard, F.
Mao, W. Y. Leung and R. P. Haugland, J Histochem Cytochem, 1999, 47, 1179-1188.
19. F. Mao, W. Y. Leung and R. P. Haugland, 1999, WO9915517.
20. W. Lin and T. Chen, Anal Biochem, 2013, 443, 252-260.
21. S. Daddi Oubekka, R. Briandet, M. P. Fontaine-Aupart and K. Steenkeste, Antimicrob
Agents Chemother, 2012, 56, 3349-3358.
22. K. Suhling, J. Levitt and P. H. Chung, Methods Mol Biol, 2014, 1076, 503-519.
23. K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Muller, C. Eggeling and S.
W. Hell, Chemistry, 2010, 16, 158-166.
24. K. Kolmakov, C. A. Wurm, R. Hennig, E. Rapp, S. Jakobs, V. N. Belov and S. W. Hell,
Chemistry, 2012, 18, 12986-12998.
25. H. J. Wories, J. H. Koek, G. Lodder, J. Lugtenburg, R. Fokkens, O. Driessen and G. R.
Mohn, Recl Trav Chim Pay B, 1985, 104, 288-291.
26. L. Li, J. Han, B. Nguyen and K. Burgess, J Org Chem, 2008, 73, 1963-1970.
27. S. L. Niu, G. Ulrich, R. Ziessel, A. Kiss, P. Y. Renard and A. Romieu, Org Lett, 2009, 11,
2049-2052.
28. T. Bura and R. Ziessel, Org Lett, 2011, 13, 3072-3075.
19
29. S. L. Niu, C. Massif, G. Ulrich, P. Y. Renard, A. Romieu and R. Ziessel, Chemistry, 2012,
18, 7229-7242.
30. A. Romieu, D. Tavernier-Lohr, S. Pellet-Rostaing, M. Lemaire and P.-Y. Renard,
Tetrahedron Letters, 2010, 51, 3304-3308.
31. A. Romieu, T. Bruckdorfer, G. Clave, V. Grandclaude, C. Massif and P. Y. Renard, Org
Biomol Chem, 2011, 9, 5337-5342.
32. C. Massif, S. Dautrey, A. Haefele, R. Ziessel, P. Y. Renard and A. Romieu, Org Biomol
Chem, 2012, 10, 4330-4336.
33. K. Kolmakov, V. N. Belov, C. A. Wurm, B. Harke, M. Leutenegger, C. Eggeling and S.
W. Hell, European Journal of Organic Chemistry, 2010, 2010, 3593-3610.
34. R. M. Rich, D. L. Stankowska, B. P. Maliwal, T. J. Sorensen, B. W. Laursen, R. R.
Krishnamoorthy, Z. Gryczynski, J. Borejdo, I. Gryczynski and R. Fudala, Anal Bioanal Chem,
2013, 405, 2065-2075.
35. B. P. Maliwal, R. Fudala, S. Raut, R. Kokate, T. J. Sorensen, B. W. Laursen, Z. Gryczynski
and I. Gryczynski, Plos One, 2013, 8, e63043.
36. A. Shivalingam, M. A. Izquierdo, A. L. Marois, A. Vysniauskas, K. Suhling, M. K.
Kuimova and R. Vilar, Nat Commun, 2015, 6, 8178.
37. T. J. Sørensen, E. Thyrhaug, M. Szabelski, R. Luchowski, I. Gryczynski, Z. Gryczynski
and B. W. Laursen, Methods Appl Fluores, 2013, 1, 025001.
38. S. A. Bogh, I. Bora, M. Rosenberg, E. Thyrhaug, B. W. Laursen and T. J. Sorensen,
Methods Appl Fluoresc, 2015, 3, 045001.
39. S. A. Bogh, M. Simmermacher, M. Westberg, M. Bregnhoj, M. Rosenberg, L. De Vico,
M. Veiga, B. W. Laursen, P. R. Ogilby, S. P. A. Sauer and T. J. Sorensen, Acs Omega, 2017, 2,
193-203.
40. T. J. Sørensen, M. Rosenberg, C. G. Frankær and B. W. Laursen, Advanced Materials
Technologies, 2018, 4, 1800561.
20
41. I. Dalfen, R. I. Dmitriev, G. Holst, I. Klimant and S. M. Borisov, Anal Chem, 2019, 91,
808-816.
42. I. Bora, S. A. Bogh, M. Rosenberg, M. Santella, T. J. Sorensen and B. W. Laursen, Org
Biomol Chem, 2016, 14, 1091-1101.
43. E. Thyrhaug, T. J. Sorensen, I. Gryczynski, Z. Gryczynski and B. W. Laursen, J Phys Chem
A, 2013, 117, 2160-2168.
44. C. Herse, D. Bas, F. C. Krebs, T. Burgi, J. Weber, T. Wesolowski, B. W. Laursen and J.
Lacour, Angew Chem Int Ed Engl, 2003, 42, 3162-3166.
45. M. Rosenberg, K. R. Rostgaard, Z. Liao, A. O. Madsen, K. L. Martinez, T. Vosch and B.
W. Laursen, Chem Sci, 2018, 9, 3122-3130.
46. M. Rosenberg, M. Santella, S. A. Bogh, A. V. Munoz, H. O. B. Andersen, O. Hammerich,
I. Bora, K. Lincke and B. W. Laursen, J Org Chem, 2019, 84, 2556-2567.
47. O. Kel, P. Sherin, N. Mehanna, B. Laleu, J. Lacour and E. Vauthey, Photochem Photobiol
Sci, 2012, 11, 623-631.
48. J. Guin, C. Besnard and J. Lacour, Org Lett, 2010, 12, 1748-1751.
49. Y. K. Yang and J. Tae, Org Lett, 2006, 8, 5721-5723.
50. A. Joshi-Pangu, F. Levesque, H. G. Roth, S. F. Oliver, L. C. Campeau, D. Nicewicz and
D. A. DiRocco, J Org Chem, 2016, 81, 7244-7249.
51. J. B. Grimm, A. K. Muthusamy, Y. Liang, T. A. Brown, W. C. Lemon, R. Patel, R. Lu, J.
J. Macklin, P. J. Keller, N. Ji and L. D. Lavis, Nat Methods, 2017, 14, 987-994.
52. M. Beija, C. A. Afonso and J. M. Martinho, Chem Soc Rev, 2009, 38, 2410-2433.
53. B. W. Laursen and F. C. Krebs, Chem-Eur J, 2001, 7, 1773-1783.
54. I. H. Delgado, S. Pascal, C. Besnard, S. Voci, L. Bouffier, N. Sojic and J. Lacour,
Chemistry, 2018, 24, 10186-10195.
55. I. H. Delgado, S. Pascal, A. Wallabregue, R. Duwald, C. Besnard, L. Guenee, C. Nancoz,
E. Vauthey, R. C. Tovar, J. L. Lunkley, G. Muller and J. Lacour, Chem Sci, 2016, 7, 4685-4693.
21
56. X.-M. Hu, Q. Chen, Z.-Y. Sui, Z.-Q. Zhao, N. Bovet, B. W. Laursen and B.-H. Han, Rsc
Adv, 2015, 5, 90135-90143.
57. R. Duwald, S. Pascal, J. Bosson, S. Grass, C. Besnard, T. Burgi and J. Lacour, Chemistry,
2017, 23, 13596-13601.
58. P. Shieh, V. T. Dien, B. J. Beahm, J. M. Castellano, T. Wyss-Coray and C. R. Bertozzi, J
Am Chem Soc, 2015, 137, 7145-7151.
59. E. Thyrhaug, T. J. Sorensen, I. Gryczynski, Z. Gryczynski and B. W. Laursen, J Phys Chem
A, 2013, 117, 2160-2168.
60. A. K. Chibisov, H. Görner and T. D. Slavnova, Chem Phys Lett, 2004, 390, 240-245.
61. G. L. Trainor, Expert Opin Drug Discov, 2007, 2, 51-64.
62. P. Taborsky, J. Kucera, J. Jurica and O. Pes, J Chromatogr B Analyt Technol Biomed Life
Sci, 2018, 1092, 7-14.
63. K. Klehs, C. Spahn, U. Endesfelder, S. F. Lee, A. Furstenberg and M. Heilemann,
Chemphyschem, 2014, 15, 637-641.
64. J. Olmsted, 3rd and D. R. Kearns, Biochemistry, 1977, 16, 3647-3654.
65. J. Kucera, P. Lubal, S. Lis and P. Taborsky, Talanta, 2018, 184, 364-368.
download fileview on ChemRxivPreprint.pdf (603.42 KiB)
S1
Electronic Supplementary Information
What is best strategy for water soluble fluorescence dyes? – A
case study using long fluorescence lifetime DAOTA dyes
Niels Bisballea, Bo W. Laursena*
a Nano-Science Center & Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-
2100, Copenhagen Ø, Denmark.
Table of Contents Experimental details ..................................................................................................................................... S2
Synthetic procedures .................................................................................................................................... S3
Steady state spectra of 1 to 6 ..................................................................................................................... S11
Time resolved fluorescence data for 1 to 6 ................................................................................................ S18
Determination of quantum yields for 1 to 6 .............................................................................................. S24
NMR spectra of 1 to 6 and 8 to 12 ............................................................................................................. S41
References .................................................................................................................................................. S59
S2
Experimental details Spectroscopy. All measurements were performed on dye solutions in a 10 mm quartz cuvette. Fluorescence
spectra were recorded of solutions with an absorbance below 0.1 in respect to the S0 ->S1 transition. UV/Vis
absorption spectra were recorded on an Agilent Cary 300 UV-Vis Spectrophotometer. Absorption was
measured using a single beam setup and subtracting a spectrum of the pure solvent as the background. Data
points were acquired for each 1 nm. Emission and excitation spectra were recorded on an Agilent Cary Eclipse
Fluorescence Spectrophotometer. Fluorescence lifetimes were measured by Time-correlated single photon
counting (TCSPC) on a FluoTime 300 instrument (PicoQuant, Berlin, Germany) using a PMA-182 detector
(PicoQuant) with a spectral range of 185-820 nm. Samples were excited using pulsed laser excitation at 507
nm (LDH-P-C-510, PicoQuant) and the emission monochromator was set to 590 nm. All decays were recorded
at a sample temperature of 22°C. Decay data was fitted to an exponential function (mono-, bi- or tri-
exponential) using the FluoFit software package (PicoQuant) by reconvolution with a resulting χ2 < 1.1. The
instrument response function was recorded using a dilute Ludox solution with the emission monochromator
set to the excitation wavelength.
Quantum yields of fluorescence were measured relative to Rhodamine 6G in ethanol (φ = 0.95 ± 0.015)1
according to standard protocol2 and IUPAC recommendations3. Absorption spectra were recorded as
described above with a spectral bandwidth of 2.0 nm. Emission spectra were recorded on a FluoTime 300
instrument with a sample holder temperature of 22°C. Samples were excited using a continuous-wave xenon
arc lamp (PicoQuant) at 500 nm with a spectral bandwidth of 2.0 nm. Emission was recorded using a PMA-
182 detector from 505-800 nm with a spectral bandwidth of 1.5 nm. Data points were collected with a 1 nm
interval. Emission spectra were corrected for detector sensitivity using the manufacturer-supplied correction
file and for excitation power by recording lamp intensity with a photodiode for each point. A background
spectrum recorded using pure solvent was subtracted. For each compound a linear fit was obtained by
plotting the integrated emission spectra as a function of the fraction of absorbed light, f, at 500 nm (eq. 12).
𝑓 = 1 − 10−𝐴(𝜆𝑒𝑥)
Quantum yields of fluorescence were calculated from the using eq. 2 (modified from ref. 2) by using the slope
of the linear fit obtained above (Δ), where n is the refractive index of the solvent. Values for the refractive
indices were used for the sodium D-line at 298.15K (nD, ethanol = 1.3593, nD, water = 1.3326).4
Φ𝑓 = Φ𝑓,𝑟𝑒𝑓 ×𝑛2
𝑛𝑟𝑒𝑓2 ×
Δ
Δ𝑟𝑒𝑓
Dyes and solvents. A commercially available source of Rhodamine 6G was used without further purification.
Absolute ethanol (≥ 99.98%) was acquired from VWR International. Water was purified to have a conductivity
below 0.056 µS cm-1. Phosphate buffered saline (PBS) was prepared by dissolving NaCl (4.00 g), KCl (101 mg),
Na2HPO4 (710 mg) and KH2PO4 (120 mg) in approximately 400 mL of purified water. pH was measured to be
7.36 and the solution was diluted further to 500 mL in a volumetric flask with purified water. The solution of
bovine serum albumin (BSA) in PBS was prepared by gentle dispersion of BSA (801 mg, ≥ 96%, lyophilized,
Sigma Aldrich) in about 15 mL of PBS in a 20 mL volumetric flask. The dispersion was left standing overnight
to allow foaming to cease, after which more PBS was added for a total volume of 20 mL. Fluorescence lifetime
measurements in BSA-PBS were performed on the day the solution had been prepared.
S3
Synthetic procedures
Scheme S1. Synthesis of 1-3 from PrDAOTA+ BF4-. (a) Amberlite© IRA-400(Cl) ion exchange resin,
MeCN/H2O; (b) H2SO4, rt, 1 h; (c) H2SO4, rt, 16 h.
S4
Scheme S2. Synthesis of 4-6 starting from the common triangulenium precursor 7. (a) i. 3-(Dimethylamino)-
1-propylamin, NMP, rt, 30 min; ii. 110°C, 20 min; iii. KPF6 (aq); (b) Pyridine hydrochloride, 200°C, 1.5 h; (c)
1,3-propanesultone, DIPEA, MeCN, rt, 16 h, darkness; (d) Iodomethane, K2CO3, MeCN, rt, 16 h; (e) H2SO4, rt,
16 h; (f) Amberlite© IRA-400(Cl) ion exchange resin, MeCN/H2O.
S5
General remarks. Compound 7 and Pr2DAOTA+ BF4- were prepared according to literature procedures.5, 6
Other starting materials and reagents were obtained through commercial suppliers and used as received
without further purification, unless otherwise noted. Acetonitrile was dried over 4 Å molecular sieves for at
least 48 hours prior to use, where the dry solvent was required. C18 RP silica with a pore size of 53-80 Å and
a particle size of 35-70 µm was obtained from Carl Roth Gmbh (Karlsruhe, Germany). Anion exchange was
performed using Amberlite© IRA-400(Cl) resin (CAS: 9002-24-8) obtained from Alfa Aesar Gmbh (Karlsruhe,
Germany). 1H-NMR, 13C-NMR and 19F-NMR spectra were acquired on 500 MHz instruments by Bruker.
Chemical shifts for 1H-NMR and 13C-NMR spectra are reported relative to TMS, referenced to the solvent
residual peaks; CDCl3 (1H = 7.26, 13C = 77.16), CD3CN (1H = 1.94, 13C = 1.32), CD3OD (1H = 3.31, 13C = 49.00),
D2O (1H = 4.79), except for 13C-NMR in D2O, in which signals are referenced to the (CH3)3Si- signal of the
internal DSS standard. 19F-NMR chemical shifts are referenced to neat trifluoroacetic acid as an external
reference (δ = -77.87 ppm relative to CFCl3)7 in a separate flame-sealed lock tube placed coaxially inside the
NMR tube during sample acquisition. For 19F-NMR spectra the FID was enhanced by exponential
multiplication with a line broadening factor of 8.0 Hz to reduce noise caused by FID truncation. Peak
separation (705 – 708 Hz) remains well above spectral resolution (4.23 Hz). HRMS was recorded on an ESP-
MALDI-FT-ICR instrument equipped with a 7 T magnet (the instrument was calibrated using sodium
trifluoroacetate cluster ions prior to acquiring the spectra) or a MicrOTOF-QII-system using ESP (calibrated
using formic acid). Elemental analysis was done at the University of Copenhagen, Department of Chemistry,
Elemental Analysis Laboratory, Universitetsparken 5, Copenhagen DK-2100, Denmark.
General procedure 1 – Anion exchange of triangulenium dyes: A column of Amberlite IRA-400 (Cl) ion
exchange resin (60 mm in height x 10 mm in diameter) was rinsed thoroughly with water (300 mL), before
being equilibrated with the specified eluent (100 mL). The hexafluorophosphate salt of the triangulenium
dye was dissolved in a minimal amount of the eluent and passed through column. The colored eluate was
collected and the solvents were removed under reduced pressure, followed by drying under vacuum (<
1mbar) for at least 12 h to give the corresponding chloride salt.
General procedure 2 – Aromatic sulfonation of triangulenium dyes: A small vial was charged with the
corresponding triangulenium dye and a magnetic stir bar. Concentrated sulfuric acid (98 %) was added and
gentle fuming and bright orange luminescence was briefly observed. The vial was sealed with a screw cap
and was kept at room temperature for the indicated amount of time under moderate stirring. The deep red
reaction solution was quenched by adding it to a slurry of 15 g of ice in 5 mL of water. The magenta red
solution was neutralized by careful addition of 1 M K2CO3 (18 times the volume of sulfuric acid used), resulting
in CO2 (g) formation. The solvent was carefully evaporated under reduced pressure to leave the dye on a base
of potassium sulfate. A plug of C18-functionalized silica gel (40 mm in height x 40 mm in diameter) in
water/acetonitrile 19:1 was prepared and at no point allowed to run dry of eluent. The mixture of dye and
inorganic salts was dissolved in a minimal amount of the eluent and gently loaded onto the plug without
disturbing the surface. The salts were eluted through the plug with 100 mL of the loading eluent. The eluent
was switched to water/acetonitrile 1:1 to elute the product off the plug. The eluate containing the dye was
concentrated under reduced pressure, and the product was purified by reversed-phase chromatography on
C18-functionalized silica (water-acetonitrile-formic acid) to give the pure dye.
S6
8,12-Dipropyl-8,12-dihydro-3a2H-benzo[ij]xantheno[1,9,8-cdef][2,7]naphthyridin-3a2-ylium chloride (1):
The product was obtained through the general procedure 1, starting from Pr2DAOTA+ BF4- (50 mg, 0.11
mmol). Water-acetonitrile 1:1 was used as the eluent and gave the pure product as a dark red solid (45 mg,
>99 %). 1H-NMR (500 MHz, CD3OD): δ 8.27 (t, J = 8.6 Hz, 1H), 8.08 (t, J = 8.5 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H),
7.52 (d, J = 8.6 Hz, 2H), 7.29 (d, J = 8.2 Hz, 2H), 4.49 (m, 4H), 1.97 (m[h], J = 7.5 Hz, 4H), 1.21 (t, J = 7.4 Hz, 6H). 13C-NMR (126 MHz, CD3OD): δ 154.0 (-), 142.3 (-), 141.1 (-), 141.1 (-), 140.9 (+), 139.7 (+), 112.9 (-), 110.3 (+),
109.6 (+), 108.9 (-), 107.0 (+), 50.3 (-), 20.2 (-), 11.1 (+). HRMS (ESP): Calcd for C25H23N2O+ [M+]: 367.1810.
Found: 367.1804. Elem. Anal. (Found: C, 73.25; H, 6.4; N, 6.5. C25H23ClN2O requires: C, 74.5; H, 5.75; N, 6.95
%). Rf (Water:Acetonitrile:Formic acid, 60:40:0.1) = 0.07.
8,12-Dipropyl-8,12-dihydrobenzo[ij]xantheno[1,9,8-cdef][2,7]naphthyridin-3a2-ylium-3-sulfonate (2): The
product was obtained through the general procedure 2, reacting Pr2DAOTA+ BF4- (52 mg, 0.11 mmol) in 500
µL of sulfuric acid for 1 h. Purification by reversed-phase column chromatography was achieved with an
eluent of water-acetonitrile-formic acid in a 60:40:0.1 ratio to give the product as a magenta solid (1.4 mg, 3
%). A substantial impurity could still be detected by NMR, giving rise to a single at δ 8.48, presumably being
a formate salt. Due to the low quantities of product and the insignificance of the salt in fluorescence
spectroscopy, no further effort was made to remove it. 1H-NMR (500 MHz, D2O): δ 8.30 (d, J = 9.1 Hz, 1H),
7.86 (t, J = 8.4 Hz, 2H), 7.38 (d, J = 9.2 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 7.10 (d, J = 8.6
Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H), 4.14-4.03 (m, 2H), 3.98-3.86 (m, 2H), 1.77-1.67 (m, 2H), 1.59-1.48 (m, 2H),
1.05 (t, J = 7.3 Hz, 3H), 0.98 (t, J = 7.4 Hz, 3H). Rf (Water:Acetonitrile:Formic acid, 60:40:0.1) = 0.53.
Potassium 8,12-dipropyl-8,12-dihydrobenzo[ij]xantheno[1,9,8-cdef][2,7]naphthyridin-3a2-ylium-3,5-
disulfonate (3): The product was obtained through the general procedure 2, reacting Pr2DAOTA+ BF4- (51 mg,
S7
0.11 mmol) in 500 µL of sulfuric acid for 16 h. Purification by reversed-phase chromatography was achieved
with an eluent of water-acetonitrile-formic acid in a 80:20:0.1 ratio to give the product as a magenta red solid
(56 mg, 88%). 1H-NMR (500 MHz, D2O): δ 8.45 (d, J = 9.1 Hz, 2H), 7.89 (t, J = 8.5 Hz, 1H), 7.45 (d, J = 9.3 Hz,
2H), 7.11 (d, J = 8.6 Hz, 2H), 4.20 (s br, 4H), 1.76-1.63 (m, 4H), 1.02 (t, J = 7.3 Hz, 6H). 13C-NMR (126 MHz,
D2O): δ 150.7 (-), 143.0 (-), 142.8 (+), 142.0 (-), 141.4 (-), 140.4 (+), 126.3 (-), 113.6 (-), 112.0 (+), 109.2 (-),
108.9 (+), 51.7 (-), 21.2 (-), 12.7 (+). HRMS (ESP): Calcd for C25H21N2O7S2- [M-]: 525.0790. Found: 525.0811.
Elem. Anal. (Found: C, 52.15; H, 3.6; N, 4.7; S, 10.35. C25H21KN2O7S2 requires: C, 53.2; H, 3.75; N, 4.95; S, 11.35
%). Rf (Water:Acetonitrile:Formic acid, 60:40:0.1) = 0.28.
8,12-Bis(3-(trimethylammonio)propyl)-8,12-dihydro-3a2H-benzo[ij]xantheno[1,9,8-
cdef][2,7]naphthyridin-3a2-ylium chloride (4): The product was obtained through the general procedure 1,
starting from 11 (50 mg, 54 µmol). Water-acetonitrile 1:1 was used as the eluent and gave the pure product
as a dark red solid (30 mg, 93 %). 1H-NMR (500 MHz, CD3OD): δ 8.38 (t, J = 8.6 Hz, 1H), 8.20 (t, J = 8.5 Hz, 2H),
7.81 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H), 4.71 (t, J = 7.9, 4H), 3.82 (m, 4H), 3.22
(s, 18H), 2.46 (p, J = 8.9 Hz, 4H). 13C-NMR (126 MHz, CD3OD): δ 154.3 (-), 142.4 (-), 141.8 (-), 141.5 (+), 141.1
(-), 140.3 (+), 113.0 (-), 110.3 (+), 110.1 (+), 109.2 (-), 107.4 (+), 64.1 (-), 53.9 (+), 53.9 (+), 53.8 (+), 45.5 (-),
20.9 (-). HRMS (ESP): Calcd for C31H39Cl2N4O+ [M3++2Cl-]: 553.2501. Found: 553.2511. Elem. Anal. (Found: C,
46.5; H, 6.4; N, 6.55. C31H39Cl3N4O requires: C, 63.1; H, 6.65; N, 9.5 %)i. Rf (Water:Acetonitrile:Formic acid,
60:40:0.1) = 0.22.
3,3'-((Benzo[ij]xantheno[1,9,8-cdef][2,7]naphthyridine-3a2-ylium-8,12-diylbis(propane-3,1-
diyl))bis(dimethylammoniumdiyl))bis(propane-1-sulfonate) chloride (5): The product was obtained through
the general procedure 1, starting from 10 (50 mg, 59 µmol). Water-acetonitrile 1:1 was used as the eluent
and gave the pure product as a dark red solid (42 mg, 97 %). 1H-NMR (500 MHz, D2O): δ 8.25 (t, J = 8.5 Hz,
1H), 8.07 (t, J = 8.4 Hz, 2H), 7.41 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.2 Hz, 2H), 4.36 (s br,
4H), 3.57 (s br, 4H), 3.45 (t, J = 8.6 Hz, 4H), 3.11 (s, 12H), 2.84 (t, J = 7.0 Hz, 4H), 2.29 (s br, 4H), 2.08 (s br, 4H). 13C-NMR (126 MHz, D2O): δ 154.7 (-), 143.0 (+), 142.6 (-), 142.0 (+), 141.9 (-), 141.3 (-), 113.1 (-), 111.9 (+),
i There appears to be an issue with incomplete combustion of 4, presumably due to the tricationic character. The same relative deviation from the calculated values is observed for the corresponding PF6
- salt (11).
S8
111.1 (+), 109.3 (-), 108.5 (+), 64.4 (-), 62.3 (-), 53.9 (+), 49.6 (-), 46.1 (-), 21.7 (-), 20.9 (-). HRMS (ESP): Calcd
for C35H45N4O7S2+ [M+]: 697.2730. Found: 697.2739. Elem. Anal. (Found: C, 55.05; H, 6.45; N, 7.15; S, 7.7.
C35H45ClN4O7S2 requires: C, 57.35; H, 6.2; N, 7.65; S, 8.75 %). Rf (Water:Acetonitrile:Formic acid, 60:40:0.1) =
0.36.
8,12-Bis(3-(trimethylammonio)propyl)-8,12-dihydrobenzo[ij]xantheno[1,9,8-cdef][2,7]naphthyridin-3a2-
ylium-3,5-disulfonate chloride (6): The product was obtained through the general procedure 1, starting from
12 (12 mg, 15 µmol). Water-acetonitrile 1:1 was used as the eluent and gave 8 mg of the pure product as a
dark red solid (77 %, 12 µmol). 1H-NMR (500 MHz, D2O): δ 8.49 (d, J = 9.0 Hz, 2H), 8.02 (t, J = 8.5 Hz, 1H), 7.46
(d, J = 9.2 Hz, 2H), 7.26 (d, J = 8.7 Hz, 2H), 4.61 (s br, 4H), 3.71 – 3.61 (m, 4H), 3.15 (s, 18H), 2.27 (m, 4H).
HRMS (ESP): Calcd for C31H37N4O7S2+ [M+]: 641.2104. Found: 641.0811. Rf (Water:Acetonitrile:Formic acid,
60:40:0.1) = 0.21.
5,9-Bis(3-(dimethylamino)propyl)-1,13-dimethoxy-5,9-dihydro-13bH-quinolino[2,3,4-kl]acridin-13b-ylium
hexafluorophosphate (8): A solution of 7 (2.00 g, 3.92 mmol) in NMP (10 mL) was added dropwise to a
solution of 3-(dimethylamino)-1-propylamine (1.75 mL, 13.9 mmol) in NMP (5 mL) over 15 min. After another
15 min the resulting red solution was heated to 110°C for 20 min. The dark green solution was allowed to
cool to rt and was crashed out in 500 mL of a 9:1 mixture of 0.2 M KPF6 and 1 M NaOH. The formed precipitate
was isolated by filtration. The product was dissolved in a minimal amount of acetonitrile and poured into
Et2O (750 mL) under vigorous stirring. The precipitate was isolated by filtration and washed with Et2O (3 x 50
mL). The solid was recrystallized from a hot mixture of 2-propanol and acetonitrile (9:1, 100 mL/g). The
product was obtained as 710 mg of dark crystals (56 %). 1H-NMR (500 MHz, CD3CN): δ 8.20 (t, J = 8.5 Hz, 1H),
7.91 (dd, J = 8.7, 8.0 Hz, 2H), 7.66 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.9 Hz, 2H), 6.92 (d, J = 8.0 Hz, 2H), 4.81-4.73
(m, 2H), 4.62-4.52 (m, 2H), 3.73 (s, 6H), 2.50 (t, J = 6.3 Hz, 4H), 2.28 (s, 12H), 2.21-2.07 (m, 4H). 13C-NMR (126
MHz, CD3CN): δ 160.6 (-), 143.5 (-), 143.2 (-), 140.0 (-), 137.9 (+), 137.4 (+), 120.4 (-), 113.9 (-), 108.5 (+), 105.9
(+), 103.9 (+), 57.2 (-), 56.6 (+), 45.9 (+), 25.2 (-). 19F-NMR (470 MHz, CD3CN): δ -72.26 (d, 1JPF = 705 Hz, PF6-).
HRMS (ESP): Calcd for C31H39N4O2+ [M+]: 499.3073. Found: 499.3066. Elem. Anal. (Found: C, 57.15; H, 6.2; N,
8.55. C31H39F6N4O2P requires: C, 57.75; H, 6.1; N, 8.7 %).
S9
8,12-Bis(3-(dimethylamino)propyl)-8,12-dihydro-3a2H-benzo[ij]xantheno[1,9,8-cdef][2,7]naphthyridin-
3a2-ylium hexafluorophosphate (9): 8 (1.20 g, 1.86 mmol) was added to molten pyridine hydrochloride (20
g) at 200°C and stirred gently for 1.5 h, while the dark green solution turned magenta. The reaction mixture
was allowed to cool to room temperature, before 0.2 M KPF6 (100 mL) was added to dissolve the pyridine
hydrochloride and precipitate the product. The magenta precipitate was isolated by filtration, before being
reprecipitated in 0.2 M KPF6 (100 mL, basified with 10 mL 1M NaOH) from a minimal amount of acetonitrile
and filtered again. The isolated magenta solid was reprecipitated in Et2O (250 mL) from acetonitrile and
isolated by filtration. The solid was washed with Et2O (3 x 50 mL). The product was recrystallized from hot
MeOH (25 mL/g) and washed with cold MeOH (10 mL) and Et2O (3 x 25 mL) to give 583 mg (52 %, 0.974
mmol) of red crystals. 1H-NMR (500 MHz, CD3CN): δ 8.23 (t, J = 8.6 Hz, 1H), 8.05 (t, J = 8.5 Hz, 2H), 7.64 (d, J
= 8.8 Hz, 2H), 7.55 (d, J = 8.7 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 4.53 (t, J = 7.7 Hz, 4H), 2.46 (t, J = 6.3 Hz, 4H),
2.28 (s, 12H), 2.03 (m, 4H). 13C-NMR (126 MHz, CD3CN): δ 153.6 (-), 142.1 (-), 141.0 (-), 140.8 (-), 140.6 (+),
139.4 (+), 112.7 (-), 110.3 (+), 109.3 (+), 108.7 (-), 106.8 (+), 56.8 (-), 47.2 (-), 45.8 (+), 24.6 (-). 19F-NMR (470
MHz, CD3CN): δ -72.91 (d, 1JPF = 707 Hz, PF6-). HRMS (ESP): Calcd for C29H33N4O+ [M+]: 453.2654. Found:
453.2650. Elem. Anal. (Found: C, 57.45; H, 5.45; N, 9.1. C29H33F6N4OP requires: C, 58.2; H, 5.55; N, 9.35 %).
3,3'-((Benzo[ij]xantheno[1,9,8-cdef][2,7]naphthyridine-3a2-ylium-8,12-diylbis(propane-3,1-
diyl))bis(dimethylammoniumdiyl))bis(propane-1-sulfonate) hexafluorophosphate (10): 9 (99 mg, 0.166
mmol) and 1,3-propanesultone (203 mg, 1.66 mmol) were dissolved in dry acetonitrile (1.7 mL) under argon
in a dry round-bottomed flask. Diisopropylethylamine (0.29 mL, 1.66 mmol) was added and the flask was
wrapped in aluminium foil to shield the reaction from light. The dark red reaction was stirred at room
temperature for 17 hours, before being poured into Et2O (100 mL) under vigorous stirring. The magenta
precipitate was isolated by filtration and washed with Et2O (3 x 25 mL). The product was recrystallized from
hot water (6 mL). The crystals were dried to give 104 mg (74 %, 0.123 mmol). 1H-NMR (500 MHz, D2O): δ 8.25
(t, J = 8.5 Hz, 1H), 8.08 (t, J = 8.4 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 7.17 (d, J = 8.2 Hz,
2H), 4.39 (s br, 4H), 3.56 (s br, 4H), 3.44 (t, J = 8.5 Hz, 4H), 3.10 (s, 12 H), 2.84 (t, J = 7.0 Hz, 4H), 2.30 (s br,
4H), 2.08 (s br, 4H). 13C-NMR (126 MHz, D2O): δ 154.7 (-), 143.0 (+), 142.9 (-), 142.0 (+), 142.0 (-), 141.3 (-),
113.2 (-), 111.9 (+), 111.1 (+), 109.4 (-), 108.5 (+), 64.5 (-), 62.3 (-), 53.9 (+), 49.6 (-), 46.1 (-), 21.7 (-), 20.9 (-). 19F-NMR (470 MHz, D2O): δ -72.14 (d, 1JPF = 708 Hz, PF6
-). HRMS (ESP): Calcd for C35H45N4O7S2+ [M+]: 697.2730.
S10
Found: 697.2746. Elem. Anal. (Found: C, 47.45; H, 5.65; N, 6.2; S, 6.9. C35H45F6N4O7PS2 requires: C, 49.9; H,
5.4; N, 6.65; S, 7.6 %). Rf (Water:Acetonitrile:Formic acid, 60:40:0.1) = 0.31.
8,12-Bis(3-(trimethylammonio)propyl)-8,12-dihydro-3a2H-benzo[ij]xantheno[1,9,8-
cdef][2,7]naphthyridin-3a2-ylium hexafluorophosphate (11): Iodomethane (20 µL, 321 µmol) was added to
a dark red solution of 9 (100 mg, 0.167 mmol) and K2CO3 (46 mg, 0.334 mmol) in dry acetonitrile (1.7 mL)
under argon. After 16.5 hours of mild stirring at room temperature, the bright red reaction solution was
crashed out in 0.2 M KPF6 (100 mL). The red precipitate was isolated by filtration and washed with 0.2 M KPF6
(3 x 25 mL). The precipitate was reprecipitated in Et2O (200 mL) from a minimal volume of acetonitrile under
vigorous stirring. The red precipitate was isolated by filtration and washed with Et2O (3 x 25 mL). This gave
140 mg (91 %, 0.152 mmol) of an amorphous red solid. 1H-NMR (500 MHz, CD3CN): δ 8.32 (t, J = 8.6 Hz, 1H),
8.17 (t, J = 8.5 Hz, 2H), 7.67 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H), 4.59 (t, J = 7.9
Hz, 4H), 3.64 (m, 4H), 3.09 (s, 18H), 2.37 (p, J = 8.0 Hz, 4H). 13C-NMR (126 MHz, CD3CN): δ 153.9 (-), 142.0 (-),
141.8 (-), 141.0 (+), 140.8 (-), 139.9 (+), 112.8 (-), 110.2 (+), 109.9 (+), 109.0 (-), 107.2 (+), 63.8 (-), 54.3 (+),
54.3 (+), 54.2 (+), 45.3 (-), 20.7 (-). 19F-NMR (470 MHz, CD3CN): δ -72.19 (d, 1JPF = 706 Hz, PF6-). HRMS (ESP):
Calcd for C31H39F12N4OP2+ [M3++2PF6
-]: 773.2407. Found: 773.2411. Elem. Anal. (Found: C, 29.15; H, 3.75; N,
4.05. C31H39F18N4OP3 requires: C, 40.55; H, 4.3; N, 6.1 %).
8,12-Bis(3-(trimethylammonio)propyl)-8,12-dihydrobenzo[ij]xantheno[1,9,8-cdef][2,7]naphthyridin-3a2-
ylium-3,5-disulfonate hexafluorophosphate (12): The product was obtained through the general procedure
2, reacting 11 (24 mg, 26 µmol) in 250 µL of sulfuric acid for 17 hours. Purification by reversed-phase
chromatography was achieved with an eluent of water-acetonitrile-formic acid in an 80:20:0.1 ratio to give
product as a magenta red solid (18 mg, 88 %). 1H-NMR (500 MHz, D2O): δ 8.53 (d, J = 9.1 Hz, 2H), 8.11-8.04
(m, 1H), 7.52 (d, J = 9.3 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 4.67-4.60 (, 4H), 3.73 – 3.66 (m, 4H), 3.16 (s, J = 1.8
Hz, 18H), 2.35-2.26 (s, 4H). 19F-NMR (470 MHz, D2O): δ -72.24 (d, 1JPF = 705 Hz, PF6-). HRMS (ESP): Calcd for
C31H37N4O7S2+ [M+]: 641.2104. Found: 641. 0811. Rf (Water:Acetonitrile:Formic acid, 60:40:0.1) = 0.19.
S11
Steady state spectra of 1 to 6
Figure S1. Normalized absorption, emission and excitation spectra for 1 (5 µM) in water. Wavelengths in
legend give the excitation wavelength for emission spectra and emission wavelength for excitation spectra.
Figure S2. Normalized absorption, emission and excitation spectra for 2 (5 µM) in water. Wavelengths in
legend give the excitation wavelength for emission spectra and emission wavelength for excitation spectra.
300 400 500 600 700
0,0
0,2
0,4
0,6
0,8
1,0
No
rma
lize
d a
bso
rban
ce,
em
issio
n
Wavelength [nm]
Absorption
Ems 530nm
Ems 507nm
Ems 485nm
Exc 587nm
Exc 650nm
300 400 500 600 700
0,0
0,2
0,4
0,6
0,8
1,0
No
rma
lize
d a
bso
rban
ce,
em
issio
n
Wavelength [nm]
Absorption
Ems 530nm
Ems 507nm
Ems 485nm
Exc 594nm
Exc 650nm
S12
Figure S3. Normalized absorption, emission and excitation spectra for 3 (5 µM) in water. Wavelengths in
legend give the excitation wavelength for emission spectra and emission wavelength for excitation spectra.
Figure S4. Normalized absorption, emission and excitation spectra for 4 (5 µM) in water. Wavelengths in
legend give the excitation wavelength for emission spectra and emission wavelength for excitation spectra.
300 400 500 600 700
0,0
0,2
0,4
0,6
0,8
1,0
Norm
aliz
ed a
bsorb
ance, em
issio
n
Wavelength [nm]
Absorption
Ems 530nm
Ems 507nm
Ems 485nm
Exc 594nm
Exc 650nm
300 400 500 600 700
0,0
0,2
0,4
0,6
0,8
1,0
No
rma
lize
d a
bso
rban
ce,
em
issio
n
Wavelength [nm]
Absorption
Ems 530nm
Ems 507nm
Ems 485nm
Exc 577nm
Exc 650nm
S13
Figure S5. Normalized absorption, emission and excitation spectra for 5 (5 µM) in water. Wavelengths in
legend give the excitation wavelength for emission spectra and emission wavelength for excitation spectra.
Figure S6. Normalized absorption, emission and excitation spectra for 6 (5 µM) in water. Wavelengths in
legend give the excitation wavelength for emission spectra and emission wavelength for excitation spectra.
300 400 500 600 700
0,0
0,2
0,4
0,6
0,8
1,0
Norm
aliz
ed a
bsorb
ance, em
issio
n
Wavelength [nm]
Absorption
Ems 530nm
Ems 507nm
Ems 485nm
Exc 577nm
Exc 650nm
300 400 500 600 700
0,0
0,2
0,4
0,6
0,8
1,0
No
rma
lize
d a
bso
rban
ce,
em
issio
n
Wavlength [nm]
Absorption
Ems 530nm
Ems 507nm
Ems 485nm
Exc 586nm
Exc 650nm
S14
Figure S7. Normalized absorption and emission spectra for 1 (5 µM) in PBS and with the addition of 600 µM
BSA. Emission spectra were recorded with excitation at 520 nm. The normalization of the emission
spectrum with the addition of BSA (red dash) is corrected for intensity to show the effect of BSA.
Figure S8. Normalized absorption and emission spectra for 2 (5 µM) in PBS and with the addition of 600 µM
BSA. Emission spectra were recorded with excitation at 520 nm. The normalization of the emission
spectrum with the addition of BSA (red dash) is corrected for intensity to show the effect of BSA.
400 500 600 700 800
0,0
0,2
0,4
0,6
0,8
1,0
No
rma
lize
d a
bso
rban
ce,
em
issio
n
Wavelength (nm)
Abs PBS
Abs PBS+BSA
Ems PBS
Ems PBS+BSA
400 500 600 700 800
0,0
0,2
0,4
0,6
0,8
1,0
No
rma
lize
d a
bso
rban
ce,
em
issio
n
Wavelength (nm)
Abs PBS
Abs PBS+BSA
Ems PBS
Ems PBS+BSA
S15
Figure S9. Normalized absorption and emission spectra for 3 (5 µM) in PBS and with the addition of 600 µM
BSA. Emission spectra were recorded with excitation at 520 nm. The normalization of the emission
spectrum with the addition of BSA (red dash) is corrected for intensity to show the effect of BSA.
Figure S10. Normalized absorption and emission spectra for 4 (5 µM) in PBS and with the addition of 600
µM BSA. Emission spectra were recorded with excitation at 520 nm. The normalization of the emission
spectrum with the addition of BSA (red dash) is corrected for intensity to show the effect of BSA.
400 500 600 700 800
0,0
0,2
0,4
0,6
0,8
1,0
No
rma
lize
d a
bso
rban
ce,
em
issio
n
Wavelength (nm)
Abs PBS
Abs PBS+BSA
Ems PBS
Ems PBS+BSA
400 500 600 700 800
0,0
0,2
0,4
0,6
0,8
1,0
No
rma
lize
d a
bso
rban
ce,
em
issio
n
Wavelength (nm)
Abs PBS
Abs PBS+BSA
Ems PBS
Ems PBS+BSA
S16
Figure S11. Normalized absorption and emission spectra for 5 (5 µM) in PBS and with the addition of 600
µM BSA. Emission spectra were recorded with excitation at 520 nm. The normalization of the emission
spectrum with the addition of BSA (red dash) is corrected for intensity to show the effect of BSA.
Figure S12. Normalized absorption and emission spectra for 6 (5 µM) in PBS and with the addition of 600
µM BSA. Emission spectra were recorded with excitation at 520 nm. The normalization of the emission
spectrum with the addition of BSA (red dash) is corrected for intensity to show the effect of BSA.
400 500 600 700 800
0,0
0,2
0,4
0,6
0,8
1,0
No
rma
lize
d a
bso
rban
ce,
em
issio
n
Wavelength (nm)
Abs PBS
Abs PBS+BSA
Ems PBS
Ems PBS+BSA
400 500 600 700 800
0,0
0,2
0,4
0,6
0,8
1,0
No
rma
lize
d a
bso
rban
ce,
em
issio
n
Wavelength (nm)
Abs PBS
Abs PBS+BSA
Ems PBS
Ems PBS+BSA
S17
Figure S13. Normalized absorption spectra of 1 and 5 in water and Pr2DAOTA+ BF4- in MeCN. The two latter
spectra appear similar in broadness, whereas 1 appears to be broader, which is also indicated in the less
pronounced shoulder of 1 around 520 nm.
400 450 500 550 600
0,0
0,2
0,4
0,6
0,8
1,0
No
rma
lize
d a
bso
rban
ce
Wavelength (nm)
1 (H2O)
5 (H2O)
Pr2DAOTA+ (MeCN)
S18
Time resolved fluorescence data for 1 to 6
Figure S14. Fluorescence decay profiles of 1 in water (top left), PBS (top right), PBS with 600 µM BSA
(bottom left) and D2O (bottom right).
S19
Figure S15. Fluorescence decay profiles of 2 in water (top left), PBS (top right), PBS with 600 µM BSA
(bottom left) and D2O (bottom right).
S20
Figure S16. Fluorescence decay profiles of 3 in water (top left), PBS (top right), PBS with 600 µM BSA
(bottom left) and D2O (bottom right).
S21
Figure S17. Fluorescence decay profiles of 4 in water (top left), PBS (top right), PBS with 600 µM BSA
(bottom left) and D2O (bottom right).
S22
Figure S18. Fluorescence decay profiles of 5 in water (top left), PBS (top right), PBS with 600 µM BSA
(bottom left) and D2O (bottom right).
S23
Figure S19. Fluorescence decay profiles of 6 in water (top left), PBS (top right), PBS with 600 µM BSA
(bottom left) and D2O (bottom right).
S24
Determination of quantum yields for 1 to 6
Fluorescence quantum yields were measured in two separate sessions, with each their determination of
the reference. Results are summarized in the tables below. Full spectra can be found on the following
pages.
Table S1. Summarized results of fluorescence quantum yield determination of 1-3. Figures S20-S31.
Dye Solvent Slope R2 Relative slope Φf
Rhodamine 6G Ethanol 2,89E+08 0,99627 1 0,950
1 Water 9,82E+07 0,9996 0,340 0,311
2 Water 8,53E+07 0,99865 0,296 0,270
3 Water 8,78E+07 0,99986 0,304 0,278
Table S2. Summarized results of fluorescence quantum yield determination of 4-6. Figures S32-S43.
Dye Solvent Slope R2 Relative slope Φf
Rhodamine 6G Ethanol 2,85E+08 0,99996 1 0,950
4 Water 1,74E+08 0,99992 0,611 0,558
5 Water 1,75E+08 0,99401 0,615 0,561
6 Water 1,14E+08 0,99703 0,398 0,364
S25
Quantum yield titration of Rhodamine 6G in absolute ethanol (as reference for 1-3)
Figure S20. Absolute (left) and normalized (right) absorption spectra for the quantum yield titration of
Rhodamine 6G in absolute ethanol as a reference for determining quantum yields of 1-3. The vertical
dashed line denotes the excitation wavelength used for measuring the emission spectra presented in figure
S14.
Figure S21. Absolute (left) and normalized (right) emission spectra for the quantum yield titration of
Rhodamine 6G in absolute ethanol as a reference for determining quantum yields of 1-3. Excitation at 500
nm.
400 450 500 550 600
0,00
0,02
0,04
0,06
0,08
Absorb
ance
Wavelength (nm)
R6G_06µL
R6G_12µL
R6G_18µL
R6G_24µL
R6G_30µL
500
400 450 500 550 600
0,0
0,2
0,4
0,6
0,8
1,0
Absorb
ance, norm
aliz
ed
Wavelength (nm)
R6G_06µL
R6G_12µL
R6G_18µL
R6G_24µL
R6G_30µL
550 600 650 700 750 800
0
40000
80000
120000
160000
200000
240000
Em
issio
n (
counts
)
Wavelength (nm)
R6G_06µL
R6G_12µL
R6G_18µL
R6G_24µL
R6G_30µL
550 600 650 700 750 800
0,0
0,2
0,4
0,6
0,8
1,0
Em
issio
n, norm
aliz
ed
Wavelength (nm)
R6G_06µL
R6G_12µL
R6G_18µL
R6G_24µL
R6G_30µL
S26
Figure S22. Linear fit of the integrated emission spectra as a function of sample absorbance for the spectra
of Rhodamine 6G in figures S13 and S14.
0,01 0,02 0,03 0,04 0,05 0,06
2000000
4000000
6000000
8000000
10000000
12000000
14000000
16000000 Power_corr
AU
C
f(A)
R-Square = 0,99627
Intercept = -594310,36719, Slope = 2,88622E8
X Intercept = 0,00206
S27
Quantum yield titration of 1 in water
Figure S23. Absolute (left) and normalized (right) absorption spectra for the quantum yield titration of 1 in
water. The vertical dashed line denotes the excitation wavelength used for measuring the emission spectra
presented in figure S17.
Figure S24. Absolute (left) and normalized (right) emission spectra for the quantum yield titration of 1 in
water. Excitation at 500 nm.
400 450 500 550 600
0,00
0,02
0,04
0,06
Absorb
ance
Wavelength (nm)
NBI-514_06µL
NBI-514_12µL
NBI-514_18µL
NBI-514_24µL
NBI-514_30µL
500
400 450 500 550 600
0,0
0,2
0,4
0,6
0,8
1,0
Absorb
ance, norm
aliz
ed
Wavelength (nm)
NBI-514_06µL
NBI-514_12µL
NBI-514_18µL
NBI-514_24µL
NBI-514_30µL
550 600 650 700 750 800
0
5000
10000
15000
20000
25000
30000
35000
Em
issio
n (
counts
)
Wavelength (nm)
NBI-514_06µL
NBI-514_12µL
NBI-514_18µL
NBI-514_24µL
NBI-514_30µL
550 600 650 700 750 800
0,0
0,2
0,4
0,6
0,8
1,0
Em
issio
n, norm
aliz
ed
Wavelength (nm)
NBI-514_06µL
NBI-514_12µL
NBI-514_18µL
NBI-514_24µL
NBI-514_30µL
S28
Figure S25. Linear fit of the integrated emission spectra as a function of sample absorbance for the spectra
of 1 in figures S16 and S17.
0,005 0,010 0,015 0,020 0,025 0,030 0,035
500000
1000000
1500000
2000000
2500000
3000000
3500000 Power_corr
AU
C
f(A)
R-Square = 0,9996
Intercept = 162795,92029, Slope = 9,815E7
X Intercept = -0,00166
S29
Quantum yield titration of 2 in water
Figure S26. Absolute (left) and normalized (right) absorption spectra for the quantum yield titration of 2 in
water. The vertical dashed line denotes the excitation wavelength used for measuring the emission spectra
presented in figure S20.
Figure S27. Absolute (left) and normalized (right) emission spectra for the quantum yield titration of 2 in
water. Excitation at 500 nm.
400 500 600
0,00
0,02
0,04
0,06
0,08
Ab
so
rban
ce
Wavelength (nm)
NBI-504_20µL
NBI-504_40µL
NBI-504_60µL
NBI-504_80µL
NBI-504_100µL
500
400 500 600
0,0
0,2
0,4
0,6
0,8
1,0
Ab
so
rban
ce,
no
rmaliz
ed
Wavelength (nm)
NBI-504_20µL
NBI-504_40µL
NBI-504_60µL
NBI-504_80µL
NBI-504_100µL
550 600 650 700 750 800
0
10000
20000
30000
40000
50000
Em
issio
n (
counts
)
Wavelength (nm)
NBI-504_20µL
NBI-504_40µL
NBI-504_60µL
NBI-504_80µL
NBI-504_100µL
550 600 650 700 750 800
0,0
0,2
0,4
0,6
0,8
1,0
Em
issio
n, norm
aliz
ed
Wavelength (nm)
NBI-504_20µL
NBI-504_40µL
NBI-504_60µL
NBI-504_80µL
NBI-504_100µL
S30
Figure S28. Linear fit of the integrated emission spectra as a function of sample absorbance for the spectra
of 2 in figures S19 and S20.
0,005 0,010 0,015 0,020 0,025 0,030 0,035 0,040
1000000
1500000
2000000
2500000
3000000
3500000
4000000 AUC
AU
C
f(A)
R-Square = 0,99865
Intercept = 271633,6395, Slope = 8,53156E7
X Intercept = -0,00318
S31
Quantum yield titration of 3 in water
Figure S29. Absolute (left) and normalized (right) absorption spectra for the quantum yield titration of 3 in
water. The vertical dashed line denotes the excitation wavelength used for measuring the emission spectra
presented in figure S23.
Figure S30. Absolute (left) and normalized (right) emission spectra for the quantum yield titration of 3 in
water. Excitation at 500 nm.
400 450 500 550 600
0,00
0,02
0,04
0,06
0,08
Absorb
ance
Wavelength (nm)
NBI-503_06µL
NBI-503_12µL
NBI-503_18µL
NBI-503_24µL
NBI-503_30µL
500
400 450 500 550 600
0,0
0,2
0,4
0,6
0,8
1,0
Absorb
ance, norm
aliz
ed
Wavelength (nm)
NBI-503_06µL
NBI-503_12µL
NBI-503_18µL
NBI-503_24µL
NBI-503_30µL
550 600 650 700 750 800
0
8000
16000
24000
32000
40000
Em
issio
n (
counts
)
Wavelength (nm)
NBI-503_06µL
NBI-503_12µL
NBI-503_18µL
NBI-503_24µL
NBI-503_30µL
550 600 650 700 750 800
0,0
0,2
0,4
0,6
0,8
1,0
Em
issio
n n
orm
aliz
ed
Wavelength (nm)
NBI-503_06µL
NBI-503_12µL
NBI-503_18µL
NBI-503_24µL
NBI-503_30µL
S32
Figure S31. Linear fit of the integrated emission spectra as a function of sample absorbance for the spectra
of 3 in figures S22 and S23.
0,010 0,015 0,020 0,025 0,030 0,035 0,040 0,045 0,050
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000 AUC
AU
C
f(A)
R-Square = 0,99986
Intercept = -16912,24296, Slope = 8,77812E7
X Intercept = 1,92664E-4
S33
Quantum yield titration of Rhodamine 6G in absolute ethanol (as reference for 4-6)
Figure S32. Absolute (left) and normalized (right) absorption spectra for the quantum yield titration of
Rhodamine 6G in absolute ethanol as a reference for determining quantum yields of 4-6. The vertical
dashed line denotes the excitation wavelength used for measuring the emission spectra presented in figure
S26.
Figure S33. Absolute (left) and normalized (right) emission spectra for the quantum yield titration of
Rhodamine 6G in absolute ethanol as a reference for determining quantum yields of 4-6. Excitation at 500
nm.
400 450 500 550 600
0,00
0,02
0,04
0,06
0,08
0,10
Absorb
ance
Wavelength [nm]
R6G_06µL
R6G_12µL
R6G_18µL
R6G_24µL
R6G_30µL
500
400 450 500 550 600
0,0
0,2
0,4
0,6
0,8
1,0
Ab
so
rban
ce,
no
rmaliz
ed
Wavelength [nm]
R6G_06µL
R6G_12µL
R6G_18µL
R6G_24µL
R6G_30µL
550 600 650 700 750 800
0
80000
160000
240000
320000
400000
Em
issio
n (
co
un
ts)
Wavelength [nm]
R6G_06µL
R6G_12µL
R6G_18µL
R6G_24µL
R6G_30µL
550 600 650 700 750 800
0,0
0,2
0,4
0,6
0,8
1,0
Em
issio
n, n
orm
aliz
ed
Wavelength [nm]
R6G_06µL
R6G_12µL
R6G_18µL
R6G_24µL
R6G_30µL
S34
Figure S34. Linear fit of the integrated emission spectra as a function of sample absorbance for the spectra
of Rhodamine 6G in figures S25 and S26.
0,01 0,02 0,03 0,04 0,05 0,06 0,07
2000000
4000000
6000000
8000000
10000000
12000000
14000000
16000000
18000000
20000000 AUC
AU
C
f(A)
R-Square = 0,99996
Intercept = -12194,88716, Slope = 2,85089E8
X Intercept = 4,27758E-5
S35
Quantum yield titration of 4 in water
Figure S35. Absolute (left) and normalized (right) absorption spectra for the quantum yield titration of 4 in
water. The vertical dashed line denotes the excitation wavelength used for measuring the emission spectra
presented in figure S29.
Figure S36. Absolute (left) and normalized (right) emission spectra for the quantum yield titration of 4 in
water. Excitation at 500 nm.
400 450 500 550 600
0,00
0,02
0,04
0,06
0,08
Ab
so
rban
ce
Wavelength [nm]
NBI-327_06µL
NBI-327_12µL
NBI-327_18µL
NBI-327_24µL
NBI-327_30µL
500
400 450 500 550 600
0,0
0,2
0,4
0,6
0,8
1,0
Ab
so
rban
ce,
no
rmaliz
ed
Wavelength [nm]
NBI-327_06µL
NBI-327_12µL
NBI-327_18µL
NBI-327_24µL
NBI-327_30µL
550 600 650 700 750 800
0
20000
40000
60000
80000
100000
120000
140000
160000
Em
issio
n, counts
Wavelength [nm]
NBI-327_06µL
NBI-327_12µL
NBI-327_18µL
NBI-327_24µL
NBI-327_30µL
550 600 650 700 750 800
0,0
0,2
0,4
0,6
0,8
1,0
Em
issio
n, norm
aliz
ed
Wavelength [nm]
NBI-327_06µL
NBI-327_12µL
NBI-327_18µL
NBI-327_24µL
NBI-327_30µL
S36
Figure S37. Linear fit of the integrated emission spectra as a function of sample absorbance for the spectra
of 4 in figures S28 and S29.
0,01 0,02 0,03 0,04 0,05 0,06
2000000
4000000
6000000
8000000
10000000
AUC
AU
C
f(A)
R-Square = 0,99992
Intercept = 84583,54829, Slope = 1,74268E8
X Intercept = -4,85366E-4
S37
Quantum yield titration of 5 in water
Figure S38. Absolute (left) and normalized (right) absorption spectra for the quantum yield titration of 5 in
water. The vertical dashed line denotes the excitation wavelength used for measuring the emission spectra
presented in figure S32.
Figure S39. Absolute (left) and normalized (right) emission spectra for the quantum yield titration of 5 in
water. Excitation at 500 nm.
400 450 500 550 600
0,00
0,02
0,04
0,06
Ab
so
rban
ce
Wavelength [nm]
NBI-507_06µL
NBI-507_12µL
NBI-507_18µL
NBI-507_24µL
NBI-507_30µL
500
400 450 500 550 600
0,0
0,2
0,4
0,6
0,8
1,0
Ab
so
rban
ce,
no
rmaliz
ed
Wavelength [nm]
NBI-507_06µL
NBI-507_12µL
NBI-507_18µL
NBI-507_24µL
NBI-507_30µL
550 600 650 700 750 800
0
20000
40000
60000
80000
100000
Em
issio
n (
counts
)
Wavelength [nm]
NBI-507_06µL
NBI-507_12µL
NBI-507_18µL
NBI-507_24µL
NBI-507_30µL
550 600 650 700 750 800
0,0
0,2
0,4
0,6
0,8
1,0
Em
issio
n, norm
aliz
ed
Wavelength [nm]
NBI-507_06µL
NBI-507_12µL
NBI-507_18µL
NBI-507_24µL
NBI-507_30µL
S38
Figure S40. Linear fit of the integrated emission spectra as a function of sample absorbance for the spectra
of 5 in figures S31 and S32.
0,005 0,010 0,015 0,020 0,025 0,030 0,035 0,040
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000 AUC
AU
C
f(A)
R-Square = 0,99401
Intercept = 172650,99433, Slope = 1,75343E8
X Intercept = -9,84649E-4
S39
Quantum yield titration of 6 in water
Figure S41. Absolute (left) and normalized (right) absorption spectra for the quantum yield titration of 6 in
water. The vertical dashed line denotes the excitation wavelength used for measuring the emission spectra
presented in figure S35.
Figure S42. Absolute (left) and normalized (right) emission spectra for the quantum yield titration of 6 in
water. Excitation at 500 nm.
400 450 500 550 600
0,00
0,02
0,04
0,06
0,08
Ab
so
rban
ce
Wavelenght [nm]
NBI-506_08µL
NBI-506_16µL
NBI-506_24µL
NBI-506_32µL
NBI-506_40µL
500
400 450 500 550 600
0,0
0,2
0,4
0,6
0,8
1,0
Ab
so
rban
ce,
no
rmaliz
ed
Wavelength [nm]
NBI-506_08µL
NBI-506_16µL
NBI-506_24µL
NBI-506_32µL
NBI-506_40µL
550 600 650 700 750 800
0
20000
40000
60000
80000
100000
Em
issio
n (
counts
)
Wavelength (nm)
NBI-506_08µL
NBI-506_16µL
NBI-506_24µL
NBI-506_32µL
NBI-506_40µL
550 600 650 700 750 800
0,0
0,2
0,4
0,6
0,8
1,0
Em
issio
n, norm
aliz
ed
Wavelength [nm]
NBI-506_08µL
NBI-506_16µL
NBI-506_24µL
NBI-506_32µL
NBI-506_40µL
S40
Figure S43. Linear fit of the integrated emission spectra as a function of sample absorbance for the spectra
of 6 in figures S34 and S35.
0,01 0,02 0,03 0,04 0,05 0,06
1000000
2000000
3000000
4000000
5000000
6000000
7000000 AUC
AU
C
f(A)
R-Square = 0,99703
Intercept = 227038,22384, Slope = 1,13557E8
X Intercept = -0,002
S41
NMR spectra of 1 to 6 and 8 to 12
Figure S44.1H-NMR of 1 in CD3OD at 500 MHz.
S42
Figure S45.13C-NMR-APT of 1 in CD3OD at 126 MHz. Spectrum is phased to show nuclei with an uneven
number of protons as positive.
Figure S46.19F-NMR of 1 in CD3OD at 470 MHz. The peak at -77.87 is the external reference of neat TFA.
S43
Figure S47. 1H-NMR of 2 in D2O at 500 MHz.
S44
Figure S48.1H-NMR of 3 in D2O at 500 MHz.
Figure S49.13C-NMR-APT of 3 in D2O at 126 MHz. Spectrum is phased to show nuclei with an uneven
number of protons as positive. Peaks at 57.1 (-), 21.8 (-), 17.7 (-) and 0.0 (+) are the internal DSS reference.
S45
Figure S50.1H-NMR of 4 in CD3OD at 500 MHz.
Figure S51.13C-NMR-APT of 4 in CD3OD at 126 MHz. Spectrum is phased to show nuclei with an uneven
number of protons as positive.
S46
Figure S52.19F-NMR of 4 in CD3OD at 470 MHz. The peak at -77.87 is the external reference of neat TFA.
S47
Figure S53.1H-NMR of 5 in D2O at 500 MHz.
Figure S54.13C-NMR-APT of 5 in D2O at 126 MHz. Spectrum is phased to show nuclei with an uneven
number of protons as positive. Peaks at 57.1 (-), 21.8 (-), 17.7 (-) and 0.0 (+) are the internal DSS reference.
S48
Figure S55.19F-NMR of 5 in D2O at 470 MHz. The peak at -77.87 is the external reference of neat TFA.
S49
Figure S56.1H-NMR of 6 in D2O at 500 MHz.
Figure S57.19F-NMR of 6 in D2O at 470 MHz. The peak at -77.87 is the external reference of neat TFA.
S50
Figure S58.1H-NMR of 8 in CD3CN at 500 MHz.
Figure S59.13C-NMR-APT of 8 in CD3CN at 126 MHz. Spectrum is phased to show nuclei with an uneven
number of protons as positive.
S51
Figure S60.19F-NMR of 8 in CD3CN at 470 MHz. The peak at -77.87 is the external reference of neat TFA.
S52
Figure S61.1H-NMR of 9 in CD3CN at 500 MHz.
Figure S62.13C-NMR-APT of 9 in CD3CN at 126 MHz. Spectrum is phased to show nuclei with an uneven
number of protons as positive.
S53
Figure S63.19F-NMR of 9 in CD3CN at 470 MHz. The peak at -77.87 is the external reference of neat TFA.
S54
Figure S64.1H-NMR of 10 in D2O at 500 MHz.
Figure S65.13C-NMR-APT of 10 in D2O at 126 MHz. Spectrum is phased to show nuclei with an uneven
number of protons as positive. Peaks at 57.1 (-), 21.8 (-), 17.7 (-) and 0.0 (+) are the internal DSS reference.
S55
Figure S66.19F-NMR of 10 in D2O at 470 MHz. The peak at -77.87 is the external reference of neat TFA.
S56
Figure S67.1H-NMR of 11 in CD3CN at 500 MHz.
Figure S68.13C-NMR-APT of 11 in CD3CN at 126 MHz. Spectrum is phased to show nuclei with an uneven
number of protons as positive.
S57
Figure S69.19F-NMR of 11 in CD3CN at 470 MHz. The peak at -77.87 is the external reference of neat TFA.
S58
Figure S70.1H-NMR of 12 in D2O at 500 MHz.
Figure S71.19F-NMR of 12 in D2O at 470 MHz. The peak at -77.87 is the external reference of neat TFA.
S59
References 1. A. M. Brouwer, Pure and Applied Chemistry, 2011, 83, 2213-2228. 2. C. Wurth, M. Grabolle, J. Pauli, M. Spieles and U. Resch-Genger, Nat Protoc, 2013, 8, 1535-1550. 3. U. Resch-Genger and K. Rurack, Pure and Applied Chemistry, 2013, 85, 2005-2026. 4. J. V. Herraez and R. Belda, J Solution Chem, 2006, 35, 1315-1328. 5. J. C. Martin and R. G. Smith, Journal of the American Chemical Society, 1964, 86, 2252-&. 6. B. W. Laursen and F. C. Krebs, Chem-Eur J, 2001, 7, 1773-1783. 7. M. G. Barlow, M. Green, R. N. Haszeldine and H. G. Higson, Journal of the Chemical Society B:
Physical Organic, 1966, DOI: 10.1039/j29660001025.
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