Trisaminocyclopropenium Cations as Small-Molecule Organic Fluorophores: Design Guidelines and Bioimaging Applications

Article abstract of DOI:10.1021/acs.joc.0c02026

The discovery of fluorescence two centuries ago ushered in, what is today, an illuminating field of science rooted in the rational design of photochromic molecules for task-specific bio-, material-, and medical-driven applications. Today, this includes applications in bioimaging and diagnosis, photodynamic therapy regimes, in addition to photovoltaic devices and solar cells, among a vast multitude of other usages. In furthering this indispensable area of daily life and modern-day scientific research, we report herein the synthesis of a class of trisaminocyclopropenium fluorophores along with a systematic investigation of their unique molecular and electronic dependent photophysical properties. Among these fluorophores, tris[N(naphthalen-2-ylmethyl)phenylamino] cyclopropenium chloride (TNTPC) displayed a strong photophysical profile including a 0.92 quantum yield ascribed to intramolecular charge transfer and intramolecular through-space conjugation. Moreover, this cyclopropenium-based fluorophore functions as a competent imaging agent for DNA visualization and nuclear counterstaining in cell culture. To facilitate the broader use of these compounds, design principles supported by density functional theory calculations for engineering analogs of this class of fluorophores are offered. Collectively, this study adds to the burgeoning interest in cyclopropenium compounds and their unique properties as fluorophores with uses in bioimaging applications.

Full text of DOI:10.1021/acs.joc.0c02026

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Article
Trisaminocyclopropenium Cations as Small Molecule Organic
Fluorophores: Design Guidelines and Bioimaging Applications
Matt Guest, Roya Mir, Gregory Foran, Brianne Hickson, Aleksandar Necakov, and Travis Dudding
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c02026 • Publication Date (Web): 15 Sep 2020
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Trisaminocyclopropenium Cations as Small Molecule Organic Fluorophores: Design
Guidelines and Bioimaging Applications
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Matt Guest, Roya Mir, Gregory Foran, Brianne Hickson, Aleksandar Necakov
and Travis Dudding*
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Brock University, 1812 Sir Isaac Brock Way, St. Catharines, Ontario, L2S 3A1, Canada
tdudding@brocku.ca
 Design Guidelines
 Intramolecular Charge
Transfer (ICT)
N
 Intramolecular Through
N
N
Space Conjugation (ITSC)
 Coupled ICT-ITSC
Mechanisms for
Enhanced Photophysical
Properties
ICT
ITSC
 Bioimaging Applications
Abstract
The discovery of fluorescence two centuries ago ushered in, what is today, an illuminating
field of science rooted in the rational design of photochromic molecules for task-specific
bio-, material- and medical-driven applications. Today, this includes applications in
bioimaging and diagnosis, photodynamic therapy regimes, in addition to photovoltaic
devices and solar cells, among a vast multitude of other usages. In furthering this
indispensable area of daily life and modern-day scientific research, we report herein the
synthesis of a class of trisaminocyclopropenium fluorophores along with a systematic
investigation of their unique molecular and electronic dependent photophysical properties.
Among these fluorophores, tris[N(naphthalen-2-ylmethyl)phenylamino] cyclopropenium
chloride (TNTPC) displayed a strong photophysical profile including a 0.92 quantum yield
ascribed to intramolecular charge transfer and intramolecular through-space conjugation.
Moreover, this cyclopropenium-based fluorophore functions as a competent imaging agent
for DNA visualization and nuclear counterstaining in cell culture. To facilitate the broader
use of these compounds, design principles supported by density functional theory
calculations for engineering analogs of this class of fluorophores are offered. Collectively,
this study adds to the burgeoning interest in cyclopropenium compounds and their unique
properties as fluorophores with uses in bioimaging applications.
1.0 Introduction
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The phenomena of fluorescence, first described by G. Stokes in 1852,1 has a rich and
fascinating history with branches in physics, biology and chemistry. Today, this illuminating
property finds widespread use in optoelectronics,2 light microscopy,3 light harvesting complexes,4
chemical sensors,5 biological probes6 disease diagnostics7 and cancer therapeutics.8 Paramount to
the ongoing advancement of many of these applications, however, are small organic fluorophores.
This is especially true for fluorescent-based applications targeting responsive materials9 and
molecular machines,10 in addition to single-molecule research11 and bioimaging. 12,13,14 Of course,
this rife utility has inspired the advancement of various structural families of small organic
fluorophores, each with inherent strengths and limitations, including prominent examples, such as
diphenylethene,15 azobenzene,16 stillbene,17 naphthalene,18 spiropyran19 and auxochrome.20
Nonetheless, there remains a need for small organic fluorophores21 that are free from drawbacks
and associated limitations arising from extended -conjugation, (photo/oxidative)-degradation,
complicated chemical syntheses, poor aqueous solubility, inadequate cellular uptake, cytotoxicity,
low fluorescence brightness, small Stokes shifts and low signal-to-noise ratios.22
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Recently, electron-rich π-aromatic cyclopropenium ions emerged as a promising class of
small-molecule fluorophores. In point of fact, Breslow in 1957 synthesized the first
cyclopropenium ion, namely, triphenylcyclopropenium, while shortly thereafter Yoshida reported
an amino-substituted derivative, thus giving rise to tris(dialkylamino)cyclopropenium (TDAC)
ions or more generally trisaminocyclopropeniums (Figure 1A). Since, research into the synthesis
and properties of amino-substituted cyclopropenium ions has been ongoing and they now find use
as catalysts,23 superbases,24 ionic liquids,25 polyelectrolytes26 and nitrogen-based ligands,27
platforms for photoelectronic redox-active chemistries,28 and fluorophores as attested for by our
fluorescent cyclopropenium-based proton sponge derivative Janus (1) 29,30 and a boron-containing
analog coined DAGBO (2), among other derivatives (Figure 1A).31 In reflecting upon these
various developments, we posited the coupling of excitation processes, viz, (1) intramolecular
charge transfer (ICT, black dotted arrows) from a cyclopropenium core (electron-donor, red
sphere) to an appended group (electron-acceptor, blue sphere), such as an arene ring system and
(2) intramolecular through-space conjugation (ITSC, green arrows) — a phenomenon observed in
organic fluorophores — would be feasible in trisaminocyclopropenium systems (Figure 1B).
Notably, trisaminocyclopropenium fluorophores displaying ITSC in solution would be a
particularly significant development, especially if it allowed for large Stoke shifts and high
fluorescence efficiencies, which are of utmost importance in fluorescent imaging. This is
particularly relevant as 1,1,2,2-tetraphenylethane (s-TPE, 3)32 and related compounds display
ITSC contributing to high fluorescence efficiency, albeit only in the solid-state, owing to
aggregation-induced emission (AIE) limiting in vivo applications.
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A) Previous Work
Me
N
N(iPr)2
N(iPr)2
N(iPr)2
F
F
B
Me
Me N HN
N
Me
N(iPr)2
N
N
NMe2
NMe2
Ph
(Janus, 1)
(DAGBO, 2)
Me2N
Ph
Ph
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tris(dialkylamino)cyclopropenium
(TDAC)
1957
1971
2016
2020
Catalysts, Superbases, Ionic liquids,
Polyelectrolytes, Nitrogen-based ligands,
Photoelectronic redox-active chemistries, and Fluorescence
B) Coupling ICT and ITSC in Trisaminocyclopropenium Fluorophores.
electron-donor
NR2
R
Intramolecular
Through-Space
Conjugation
(ITSC)
N
NR2
Intramolecular
Charge Transfer
(ICT)
electron-acceptor
(s-TPE, 3)
This Work
N
N
N
N
N
N
ICT
ITSC
Coupled ICT-ITSC
ICT
ITSC
Trisaminocyclopropenium
Fluorophore
Figure 1. A) Previous development of electron-rich aromatic cyclopropenium ion containing
molecules including fluorescent Janus (1) and DAGBO (2). B) Schematic representation of
Intramolecular Charge Transfer (ICT) and Intramolecular Through Space Conjugation (ITSC) and
the coupling of these processes in trisaminocyclopropenium fluorophores.
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Accordingly, we report vide infra a family of easily synthesized trisaminocyclopropenium
fluorophores varying in size, conjugation, and flexibility (Section 2.1 – 2.2). This includes the
development of tris[N(naphthalen-2-ylmethyl)phenylamino] cyclopropenium chloride (TNTPC)
with superior photophysical properties such as a large Stokes shift of 180 nm, quantum yield (ϕ)
of 0.92, and molar extinction coefficient (ε) of 6.34 x 104 M-1 cm-1 (Section 2.3). Further, insightful
guidelines for designing small trisaminocyclopropenium fluorophores are described (Section 2.4).
Lastly, we disclose trisaminocyclopropenium fluorophore TNTPC functions as a dye for cellular
and DNA visualization (Section 2.5), thus, providing a new small-molecule fluorophore for
fluorescent microscopy and molecular genetics applications. This is a particularly significant
development in light of the pressing need for bright, efficient, and cost-effective counterstains for
different subcellular compartments. Further to this point is the need for compounds that allow for
the visualization of DNA which has grown immensely over the last several years.33,34,35
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2.0 Results and Discussion
2.1 Synthetic Route
At the outset of this work, tetrachlorocyclopropene (4) was reacted with commercially
available secondary amines (5a-5c) or easily prepared amines (5d-5i) to produce the requisite
trisaminocyclopropeniums (6a-6i) in moderate-to-high yields (Scheme 1) (see Experimental
Section for synthetic details).36 All of these new compounds were fully characterized by 1H NMR,
13C NMR, 11B NMR and 19F NMR where required, high-resolution mass spectrometry, ultraviolet-
visible-near infrared (UV-Vis-NIR) and fluorescence spectroscopy (see Supporting Information
Sections S1, S2 and S3).
Scheme 1. Synthesis of trisaminocyclopropenium fluorophores (6a-6i).
2.2 Structural Derivatization and Impact on Photophysical Properties
2.2.1 Heterocyclic or Non-Aromatic Effects (6a–6b)
With synthesized trisaminocyclopropeniums in hand, our efforts initially turned to
investigating the photophysical properties of tris-morpholino functionalized cyclopropenium 6a in
dichloromethane (photophysical properties of fluorophores 6a-6i shown in Figure 2). This
compound displayed an absorption maxima (λabs) of 339 nm with a molar absorption coefficient
of 2.45 x 103 M-1 cm-1 and emission maxima (λem) at 422 nm associated with a 0.05 quantum yield
(ϕ) that collectively provided a respectable Stokes shift of 83 nm. Similarly, tris-substituted N,N-
dicyclohexyl cyclopropenium 6b exhibited an absorbance of 242 nm and molar absorptivity of
4.93 x 103 M-1 cm-1 along with a Stokes shift of 65 nm and 0.06 quantum yield. The modest size
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Stokes shifts and quantum yields of these two somewhat flexible fluorophores is notable given
their lack of π-π conjugation.
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2.2.2 Benzyl, Phenyl and Steric Effects (6c–6g)
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Next, the influence of additional arene groups was explored using tris-substituted N,N-
dibenzyl cyclopropenium 6c, which exhibited a bathochromically shifted absorption band at 274
nm along with an increased molar absorptivity (ε = 2.11 x 104 M-1 cm-1) relative to fluorophore
6b, in addition to an emission band at 304 nm and low quantum yield of 0.07. Likewise, tris-
substituted N,N-(benzyl)phenyl cyclopropenium 6d had a comparable quantum yield (ϕ = 0.06),
while displaying a bathochromically shifted emission (λem= 335 nm) and hypsochromically shifted
absorption (λabs = 250 nm) resulting in a greatly enhanced Stokes shift of 85 nm. Tris-substituted
N,N-(phenyl)-1-phenylethyl cyclopropenium 6e was examined next as a potential case for probing
twisted intramolecular charge transfer (TICT), a phenomenon resulting in decreased quantum
efficiencies and diminished brightness through non-radiative de-excitation pathways.37 Overall,
trisaminocyclopropenium 6e displayed a slightly increased quantum yield (ϕ = 0.07), whereas the
remaining photophysical profile was similar to fluorophore 6d (Figure 2). In contrast, tris-
substituted N,N-(isopropyl)phenyl cyclopropenium 6f•Cl- displayed a bathochromically shifted
absorbance (Δλ = 29 nm; λabs = 278 nm) and hypsochromically shifted emission band (Δλ = 32 nm;
λem = 306 nm) with a decreased molar absorptivity of 1.32 x 104 M-1 cm-1 and dramatically reduced
Stokes shift (Δλ = 28 nm) owing to reduced conjugation originating from exchanging the N-benzyl
groups for N-isopropyl groups.
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Based on our previous reports demonstrating counterion effects on cyclopropeniums, e.g.,
quantum yield and (intra- vs. intermolecular) hydrogen bonding38 and/or reactivity,28e we next
-
examined trisaminocyclopropenium 6f•BF4 in order to assess if switching the counterion had an
influence on fluorescence. Notably, the quantum yield (ϕ = 0.12) of trisaminocyclopropenium
-
6f•BF4 was nearly double that of fluorophore 6f•Cl-, clearly showing the impact of a
tetrafluoroborate counterion versus a chloride counterion. This increased quantum yield is thought
to arises from more effective hydrogen bonding, which is consistent with the greater number of
hydrogen bond acceptor sites of a tetrafluoroborate counteranion relative to a chloride counterion
(see Section 2.5 for models supporting this premise).
Further, in probing the effect of long aliphatic carbon chain substitution, tris-substituted
N,N-(dodecyl)phenyl functionalized cyclopropenium 6g was found to have a large Stokes shift of
90 nm and respectable quantum yield (ϕ = 0.10). Interestingly, the long n-alkyl hydrocarbon chains
of this fluorophore versus the N-isopropyl groups of trisaminocyclopropenium 6f•Cl- resulted in
an increased Stokes shift. Collectively, trisaminocyclopropenium fluorophores 6c–6g highlight the
influence of additional aromatic substitution, restricted rotation and anions on the photophysical
properties of trisaminocyclopropenium fluorophores. To be more specific, with increasing
conjugation brought about by arene groups was greater molar absorptivity’s and bathochromic
shifts in absorption wavelengths. Further, steric imposed constraint of molecular motion reduced
non-radiative decay processes arising from rotational and vibrational degrees of freedom,
ultimately leading to increased quantum yields susceptible to counteranion effects.
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-
BF4
N
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N
O
N
-
6iBF4
  0.74
  66,963 M-1 cm-1
abs  248 nm
em  431 nm
  183 nm
6a
6iCl-
  0.05
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  2,450 M-1 cm-1
abs  339 nm
em  422 nm
  83 nm
  0.92
  63,441 M-1 cm-1
abs  248 nm
em  428 nm
  180 nm
N
N
6b
6h
  0.06
  0.30
  21,128 M-1 cm-1
abs  337 nm
em  421 nm
  85 nm
  4,926 M-1 cm-1
abs  242 nm
em  307 nm
  65 nm
Heterocyclic and
Non-Aromatic
Effects
Naphthyl
Effects
R
R
R
Cyclopropenium
Core
N
N
10
6c
6g
Benzyl, Phenyl
and Steric Effects
  0.07
  0.10
  21,088 M-1 cm-1
abs  274 nm
em  304 nm
  30 nm
  16,457 M-1 cm-1
abs  250 nm
em  340 nm
  90 nm
-
BF4
N
N
-
6d
6fBF4
  0.12
  14,631 M-1 cm-1
abs  274 nm
em  306 nm
  32 nm
N
N
  0.06
  24,683 M-1 cm-1
abs  250 nm
em  335 nm
  85 nm
6fCl-
  0.07
6e
  0.07
  24,738 M-1 cm-1
abs
em  338 nm
  89 nm
  13,216 M-1 cm-1
abs  278 nm
em  306 nm
  28 nm

249 nm
Figure 2. Summary of Fluorescent Data for fluorophores 6a–6i.*
*Experimental fluorescent data for 6a–6i were performed in dichloromethane. -R denotes amino
group substituent bound to the central cyclopropenium core bearing a chloride counterion unless
otherwise stated; ϕ: quantum yield; ε: molar extinction coefficient; λabs: peak UV-Vis absorption
wavelength; λem: peak emission wavelength; Δλ: Stokes shift.
2.2.3 Naphthyl Effects (6h–6i)
Given the proven utility of naphthyl groups in fluorescent molecules,39 our attention turned
to examining tris-substituted N,N-(isopropyl)naphthalen-1-yl cyclopropenium 6h, which
displayed bathochromically shifted absorbance and emission bands (λabs = 337 nm, λem = 421 nm)
compared to all previous fluorophores other than trisaminocyclopropenium 6a. Moreover, in
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demonstrating the beneficial role of naphthyl groups, this fluorophore displayed a quantum yield
of 0.30 and molar absorptivity of 2.11 x 104 M-1 cm-1. Based on this promising lead, we surmised
that increased conjugation brought about by the presence of naphthyl groups would offer
advantages in terms of fluorescence efficiency, so we turned to investigating tris-substituted N,N-
(napthalen-2-ylmethyl)phenyl cyclopropenium 6i•Cl- (TNTPC). Impressively, this small-
molecule trisaminocyclopropenium fluorophore was highly fluorescent, surpassing expectations
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in all key characteristics, including having a 0.92 quantum yield and 180 nm Stokes shift (λabs
=
248 nm, λem = 428 nm) in combination with a molar absorptivity of 6.34 x 104 M-1 cm-1 (see Section
-
2.3). Encouraged by this result, we investigated fluorophore 6i•BF4 with a tetrafluoroborate
counterion to see if the quantum yield would increase relative to chloride counterion containing
-
trisaminocyclopropenium 6i•Cl-, much like that seen for fluorophore 6f•BF4 versus 6f•Cl-. The
fundamental transitions of this fluorophore (λabs = 248 nm, λem = 431 nm, 6.70 x 104 M-1 cm-1)
remained constant; however, the quantum yield decreased to 0.74 and, in doing so, corroborated
the affect anions may have on the quantum yields of cyclopropenium fluorophores.
2.3 Investigation of the Superior Photophysical Profile of TNTPC (6i•Cl-)
Inspired by the impressive and otherwise unprecedented luminescence of
trisaminocyclopropenium TNTPC (Figure 3A), a more in-depth analysis of this fluorophore was
undertaken to better understand the underlying structural and electronic factors contributing to its
photophysical profile. Along with the main absorption and emission maxima mentioned above for
trisaminocyclopropenium TNTPC, we observed a 280 nm shoulder peak with an emission
maximum at 430 nm resulting in a large 150 nm Stokes shifts (Figure 3B/C). In terms of the
fluorescence efficiency of this shoulder absorption it provided a smaller — yet respectable —
quantum yield of 0.30, compared to that of the 248 nm maximum absorbance. Subsequently, in an
attempt to gauge the influence of the cyclopropenium ring on the fluorescence of TNTPC,
structurally simple N-(naphthalen-2-ylmethyl)aniline (5i) was investigated (Figure 5B). Overall,
the photo-spectral profile of this subcomponent (see Supporting Information Section S3) of
trisaminocyclopropenium TNTPC was similar in absorbance and emission wavelengths; however,
it featured a 63% smaller quantum yield of 0.29. Further, in attempting to approximate the effect
of the local environment of the tris-substituents of TNTPC and, with it, gain a better picture of
their influence upon quantum yield, we studied a 3x10-6 M concentration of secondary amine 5i
and observed a moderately increased quantum yield of 0.43, while the absorption and emission
wavelengths remained constant. Based upon these findings it is proposed the cyclopropenium
enhances quantum yield by serving as an electron donor for ICT processes and acting as a platform
for arranging the N-alkyl/aryl substituents in proximity, thereby enabling π,π-stacking interactions
coupled to ITSC.
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TNTPC 6i Cl-
,

B)
CHCl3
C)
CHCl3
CHCl3
Figure 3. A) Molecular structure of trisaminocyclopropenium TNTPC (6i•Cl-). B) Absorption
spectra for trisaminocyclopropenium TNTPC in various solvents at 1x10-6 M . C) Emission
spectra for trisaminocyclopropenium TNTPC in various solvents at 1x10-6 M.
Solvatochromism experiments exploring microenvironmental effects and the ICT character
of trisaminocyclopropenium TNTPC were then conducted (see Supporting Information Section
S4) using solvents chloroform, dichloromethane, ethanol (EtOH) and tetrahydrofuran (THF). The
rationale for performing these experiments being that the Stokes Shift of a fluorophore involving
ICT should be altered by a solvents polarity.40 Across this panel of solvents, the absorption profiles
were qualitatively similar, although noticeably shifted. For instance, in chloroform we observed a
hypsochromically shifted absorption maxima at 240 nm versus ~248 nm in dichloromethane and
EtOH. Meanwhile, in THF, both absorption peaks were bathochromically shifted to 250 nm and
295 nm. Moving to the emission spectra of this fluorophore, we observed remarkable
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solvatochromic effects (Figure 3C). Beginning with chloroform, dominating the emission profile
in this solvent was a single peak at 336 nm for both excitations that was barley visible in
dichloromethane. In THF, a single bathochromically shifted emission band at 440 nm was
observed for both excitations. Lastly, the greatest red shift in emission was in EtOH as seen by
emission peaks at 488 and 484 nm resulting in Stokes shift of 240 nm and 208 nm. We attribute
this observed large bathochromic shift to a significant degree of solvent reorganization allowing
for excited state dipole stabilization21 resulting in energy loss before fluorescent emission and a
reduced highest occupied molecular orbital (HOMO)/lowest unoccupied molecular orbital
(LUMO) energy gap. Clear from these results is positive solvatochromism consistent with π,π-
transitions as the major excitation pathway.
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Figure 4. A) CYL View generated structure of the optimized molecular geometry of
trisaminocyclopropenium TNTPC with side view (top) and top view (bottom) with N-naphthyl
groups A, B and C highlighted. B) GaussView generated molecular orbitals of
trisaminocyclopropenium TNTPC with side view (top) and top view (bottom).
To shed additional light on the fluorescence and ICT character of trisaminocyclopropenium
TNTPC, we turned to computational studies. To this end, using the program MacroModel,41
Monte Carlo conformational searches (MCCS) with the OPLS3 force field42 provided a lowest
energy conformer that was exported to the program Gaussian 09 for further optimization.43 A
chloride counterion was then positioned in different quadrants of this conformer (Figure S63) and
each resulting structure optimized at the M06-2X-D3/6-31G(d,p) level of theory (see Supporting
Information Section S5).44,45 To account for solvent (dichloromethane), the self-consistent reaction
field (SCRF) method using the solvation method based on density (SMD) model was employed,
which accurately treats medium/long ranged attractive noncovalent interactions.46
The optimized lowest energy structure of trisaminocyclopropenium TNTPC revealed a
propeller-like geometry with each N-naphthyl group (rings A, B and C) geared upwards relative
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to the cyclopropenium moiety, whereas the N-phenyl rings were geared downwards (Figure 4A).
Visible also was π,π-stacking between the N-naphthyl and N-phenyl groups as evidenced by short
interplane distances (≤ 3.44 Å) and non-covalent interaction (NCI) plots showing intramolecular
interactions (green isosurfaces) between N-naphthyl rings A and C with N-phenyl rings visualized
by zone 2 in Figure 5A, supporting possible ITSC processes. Further gearing of a single N-naphthyl
group (ring B) away from the N-phenyl ring plane allowed for non-covalent interactions with the
neighboring N-naphthyl group (ring A) in zone 1 (Figure 5A). Meanwhile, MO analysis revealed
a HOMO localized on the cyclopropenium and phenyl rings and a LUMO located principally on
the two electronically conjugated N-naphthyl rings A and B (Figure 4B). Conversely, the electron
density of the LUMO + 1 was located on the third N-naphthyl group (ring C). Based on these
findings, it is inferred the radiative light absorbed by the N-phenyl rings and the cyclopropenium
of TNTPC results in intramolecular energy transfer to the naphthyl groups, where emissive
fluorescence occurs. What is more, evident from these molecular orbital findings and NCI plots
is the likelihood of ITSC as a contributor to fluorescence in TNTPC. While this is a dynamic
system and in actuality, many states contribute to the spectral photophysical profile of TNTPC,
the spatiotemporal static model discussed above offers insights into designing
trisaminocyclopropenium fluorophores.
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Figure 5. A) NCI plots with zone 1 showing interactions between N-naphthyl rings A and B and
zone 2 showing interactions between N-naphthyl rings A/C with N-phenyl rings for
trisaminocyclopropenium TNTPC. B) CYL View generated structure of the optimized molecular
geometry of 5i. C) GaussView generated molecular orbitals of 5i.
2.4 Guidelines for Designing Trisaminocyclopropenium Small Organic Fluorophores
With trisaminocyclopropenium TNTPC established as a small-molecule fluorophore, we
aimed our sights towards finding design principles for engineering trisaminocyclopropenium
fluorescent molecules. Key in this undertaking was identifying factors for modulating desirable
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qualities, including: (1) bathochromically shifted absorption wavelengths; (2) larger Stokes shifts
and (3) higher quantum yield. To this end, we turned to DFT computations to explore the structure
and electronic elements of trisaminocyclopropeniums 6a-6i contributing to ICT and ITSC
processes, HOMO/LUMO gaps and anionic effects (see Supporting Information Section S5
(Figure S75)).
2.4.1 Bathochromically Shifted Absorption Bands
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Given red-shifted excitation of fluorescent molecules is desirable for decreasing photo-
damage of living organisms, we sought to determine what relationship existed between the N-
(hetero)alkyl/aryl substituents and bathochromic shifting of absorbance. Accordingly, in
examining the primary excitation pathway (HOMO  LUMO computed electron transitions), a
smaller energy gap (∆eV) and more bathochromically shifted absorption bands were observed in
moving from fluorophore 6a to 6i as a result of increasing π-conjugation (Figure S75). In
comparing trisaminocyclopropenium fluorophores 6b and 6h, the greater degree of conjugation in
the latter resulted in a 53% decrease of the ∆eV gap, contributing to a 95 nm bathochromic shift
in the absorption band (Figure 6A). A second effect contributing to greater bathochromically
shifted absorbances was the enhancement of push-pull or donor-acceptor character and, with it,
ICT. This trend in push-pull character is observed across the set of fluorophores (Figure S75) and
as an exemplary case is clearly seen from the HOMO and LUMO of trisaminocyclopropenium
fluorophore 6b, wherein the electron-rich cyclopropenium ring donates electron density to the
acceptor N-naphthyl groups (Figure 6A).
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A)
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LUMO
LUMO
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-0.12596 eV
-0.19761 eV
eV = 0.07999 eV
eV = 0.1712 eV
-0.27760 eV
-0.29713 eV
6b
6h
HOMO
HOMO
B)
ITSC
6fCl-
-
6fBF4
Figure 6. A) CYL view generated optimized structures of trisaminocyclopropenium
fluorophores 6b and 6h alongside Gaussian 09 generated molecular orbitals of the HOMO and
LUMO with respective energies in electron volts (eV) and energy differences (∆eV). B) NCI
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plots for trisaminocyclopropeniums 6f•Cl- and 6f•BF4 displaying the bonding contacts between
the Cl– and BF4 counterions and representation of ITSC between the phenyl rings of fluorophore
–
-
6f•BF4 .
2.4.2 Larger Stokes Shifts
The requirement for fluorophores with large Stokes shifts cannot be understated as it is
essential for ensuring high signal-to-noise ratios minimizing interference between excitation
sources and emissions in fluorescent applications.47 That said, many common fluorophores such
as rhodamine48 and BODIPY49 dyes display small stokes shifts (∆λ ≤ 70 nm) resulting in
reabsorption of emitted photons, contributing to background noise. Despite this frequently
encountered deficiency, several of our synthesized trisaminocyclopropeniums displayed large
Stokes shifts, e.g., 6a, 6d, 6e, 6g-6i. Contributing to the large Stokes shifts of these
trisaminocyclopropeniums is flexibility allowing for structural changes in the fluorophores
themselves and reorientation of the surrounding solvent molecules associated with the lowering of
excited state energies materializing in red shifted emission and larger Stokes shifts.22 Alternatively,
trisaminocyclopropenium fluorophores 6c (∆λ = 30 nm) and 6f•Cl- (∆λ = 28 nm), with sterically
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hindered aromatic groups, had small Stokes shifts, whereas fluorophores 6d (∆λ = 85 nm) and 6e
(∆λ = 89 nm), with more flexible N-aryl substitutes, had larger Stokes shift.
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2.4.3 Higher Quantum Yields
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In terms of quantum yields, several factors contributed to high fluorescence efficiency of
these trisaminocyclopropenium fluorophores. First were presumably TICT processes involving the
twisting of groups away from the fluorophore scaffold to form non-emissive and reactive
substituents, which is a common nonradiative de-excitation pathways in donor-acceptor
fluorophores.50 For instance, TICT was likely a major pathway for de-excitation and the low
quantum yields of trisaminocyclopropenium fluorophores 6a-6f•Cl- (ϕ ≤ 0.07), owing to the
presence of rotatable alkylamino groups, including heterocycles and other six-membered ring
systems. In contrast, the presence of N-dodecyl groups, as found in trisaminocyclopropenium
fluorophore 6g, resulted in increased quantum yield (ϕ = 0.10). The rationale for this difference
being van der Waals packing of the N-dodecyl groups disfavoring TICT, while at the same time
placing the aromatic N-phenyl groups in close proximity, allowing for more effective ITSC.
Further, by enlarging the steric bulk of the amino groups – thereby disfavoring TICT in
combination with extending π-conjugation through addition of N-naphthyl substituents – even
larger quantum yields were observed in trisaminocyclopropenium fluorophores 6h (ϕ = 0.30) and
TNTPC (ϕ = 0.92).
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Next, vital for improving quantum yield was the push-pull element of ICT. This is clearly
demonstrated by trisaminocyclopropenium fluorophores 6a–6f containing poorly electron-
donating or withdrawing substituents such as N-cyclohexyl, N-phenyl, or N-benzyl groups,
resulting in low ICT as corroborated by HOMO and LUMO overlap (Figure S75). Alternatively,
trisaminocyclopropenium TNTPC with N-phenyl donor groups and strongly electron-
withdrawing N-naphthyl acceptor groups possessed a large potential ICT gradient leading to
improved charge transfer and enhanced fluorescence efficiency. Moreover, computational models
suggest that the ITSC between the aromatic groups of trisaminocyclopropenium TNTPC allowed
for electron cloud delocalization, supporting increased charge transport. At the same time, the
cyclopropenium moiety, in the present case, though exchangeable for other donor systems, works
to further donate electron density and improve ICT systems.
Lastly, a relatively unexplored avenue, namely the influence of counterions, had a
significant impact on the quantum yields of these fluorophores. Specifically, exchanging the
chloride counterion of trisaminocyclopropenium 6f•Cl- for a tetrafluoroborate counterion as found
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in trisaminocyclopropenium 6f•BF4 nearly doubled the quantum yield. The origin of this effect is
ascribed to differences in hydrogen bonding, as supported by computational optimizations and
NCI plots (Figure 6B), clearly revealing less hydrogen bond contacts with the counterion in
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trisaminocyclopropenium fluorophore 6f•Cl- relative to 6f•BF4 . Notably, this difference resulted
in a more planar geometry and minimal interactions between the N-phenyl rings in the former case.
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Conversely, in trisaminocyclopropenium fluorophore 6f•BF4 , numerous hydrogen bonding
interactions led to a downward gearing of the N-isopropyl groups relative to the cyclopropenium
core which, in turn, forced the N-phenyl groups upwards into closer proximity, allowing for
increased π-π-overlap, materializing in ITSC (Figure 6B). The role of ITSC in
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trisaminocyclopropenium fluorophore 6f•BF4 provides a compelling case for increasing quantum
yield that demonstrates how counterions may be employed to manipulate molecular geometry and
provide enhanced fluorescent properties. Nevertheless, as observed with the decrease in quantum
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yield in exchanging the chloride counterion of fluorophore 6i•Cl- for the BF4 counterion of
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fluorophore 6i•BF4 , the influence of counterions can have both a positive or negative impact.
Taken together, by keeping in mind the above-mentioned observations or, in the broadest sense,
“design principles,” it is feasible to obtain large quantum yields. What is more, if combined with
high molar absorptivity knowingly attained by extending π-conjugation, highly bright small
fluorescent molecules may be developed.
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2.5 Elucidating Biological and Practical Uses of TNTPC
With the exceptional fluorescence of TNTPC determined and design principles in hand,
we hypothesized this hydrophobic trisaminocyclopropenium small-molecule fluorophore might
function as a membrane counterstain for mammalian cells. To test this hypothesis, we added
trisaminocyclopropenium TNTPC solubilized in dimethylsulfoxide (DMSO) to HEK293 (human
embryonic kidney) cells at varying concentrations of 10 nM, 100 nM and 10 μM. Remarkably,
TNTPC formed membrane-associated spherules on the exterior of cells under all concentrations
tested (Figure 7I-A). Although we did not investigate the nature of these spherules in detail, we
hypothesize that they contain TNTPC as a result of their fluorescence emission profile (Figure 7I-
AII,BII,CII), which corresponds with that of TNTPC in solution. These TNTPC-containing
spherules most likely form as a result of phase separation between hydrophobic TNTPC and the
aqueous culture media, along with a concomitant association with the hydrophobic cellular
membrane lipid bilayer through hydrophobic interactions.
Apart from fluorescence emitting from some of the membrane associated spherules, we
observed minimal fluorescence emission from cell membranes treated with either 10 or 100 nM
TNTPC solutions following excitation at 380 nm (Figure 7I-B/C). However, we found that cells
treated with 10 μM TNTPC displayed bright fluorescence emission at 450 nm coming from what
appeared to be the nucleus(Figure 7I-B/C) and showed no morphological difference between
controls prior to the removal of cell media (Figure 7I). To validate this finding we transfected
HeLa cells (Henrietta Lacks, cervical cancer cells) with a DNA plasmid containing a genetically-
encoded expression construct capable of inducing cellular production of the Red fluorescent
protein mScarlet, carrying a nuclear localization sequence (mScarlet-NLS), which served as an
orthogonal fluorescence counterstain. When expressed, mScarlet-NLS localizes specifically to the
nuclei of cells and has a distinct fluorescent excitation/emission profile as compared to TNTPC,
allowing us to spectrally resolve the two. Following treatment of mScarlet-NLS transfected HeLa
cells with TNTPC, 450nm fluorescence can clearly be visualized, and colocalizes with mScarlet
fluorescence in the cellular nucleus (Figure 7II). In contrast, cells treated with DMSO alone exhibit
mScarlet fluorescence, but no fluorescence at 450nm.
Interestingly, in contrast to our hypothesis that TNTPC would serve as a fluorescent
membrane counterstain, we instead observed specific labelling and localization of TNTPC to
cellular nuclei. Considering the specific localization of TNTPC to the nucleus we then looked to
provide a rationale for this intriguing result.
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Figure 7. I – TNTPC fluorescently labels the nuclei of cells. HEK293 cells were imaged 5 minutes
post-treatment in (A, B, C) 1% DMSO cell media, (AI, BI, CI) 1 % DMSO and 10 nM TNTPC
cell media, (AII, BII, CII) 1% DMSO and 100 nM TNTPC cell media, or (AIII, BIII, CIII) 1%
DMSO and 10 μM TNTPC cell media. A, AI, AII, AIII) Bright-field images. B, BI, BII, BIII) 380
excitation and 450 nm emission fluorescence channel to image TNTPC. C, CI, CII, CIII) Merged
images of brightfield and fluorescent images. Spherules (white arrows) are seen to form on cell
membranes at all concentrations tested of TNTPC (10 nM,100 nM and 10 μM ) but fail to be
observed when only DMSO is present. Several of these spherules produce significant amounts of
blue fluorescence (yellow arrows) indicating the presence of TNTPC. Nuclei (green arrows) can
be visualized following 10 μM treatment of TNTPC. II – Validation of TNTPC nuclear staining.
HeLA cells were transfected with a mScarlet-NLS expression construct and imaged 5 minutes
post-treatment in (A, B, C, D) 1% DMSO cell media or (AI, BI, CI, DI) 1% DMSO and 10 μM
TNTPC media A, AI) Bright-field image. B, BI) 380 excitation and 450 nm emission fluorescence
channel to image TNTPC. C, CI) 570 nm excitation and 600 nm emission fluorescence channel
to image mScarlet-NLS. D, DI) Merged images of all channels in the treatment condition. Nuclei
(green arrows) can be visualized following 10 μM treatment, which overlaps with fluorescence
being emitted from nuclei indicated by mScarlet-NLS (magenta arrows). III - Functionality of
TNTPC as a DNA dye and binding agent on non-denaturing agarose gels. A) Wells 1–5 contain
7.5 μg of DNA of varying lengths in 10 μM TNTPC in 25% DMSO B) Wells 6, 7 contain 25%
DMSO and 7.5 μg DNA without TNTPC present. C) Wells 8, 9 contain 10 μM TNTPC in 25%
DMSO without DNA present. Black arrows indicate band visualized at expected length for a given
DNA species. Red arrows represent where a band should have been seen if DNA was visualized.
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In order to determine whether TNTPC is capable of binding to DNA, we next used non-
denaturing agarose gel electrophoresis in order to separate DNA fragments associated with
TNTPC according to their molecular weight. TNTPC strongly associates and migrates with DNA
following gel electrophoresis and allows for the visualization of DNA. We hypothesize this is due
to the positive charges along TNTPC interacting with the negatively charged phosphate backbone
of DNA allowing it to have a stable interaction under an electrical field (Figure 7III-
A).Furthermore, we saw no bands at the expected lengths of DNA when DNA is added to the gel
with DMSO alone and when only TNTPC was added to the gel, no band formation was observed
either (Figure 7III-B/C).
To further investigate whether DNA-binding influences TNTPC fluorescence, we
measured the excitation and emission spectra of this small organic fluorophore in DMSO and
EtOH, both in the presence and absence of DNA (Figure 8). From these measurements, we
observed that in DMSO TNTPC excitation occurred at 385 nm, whereas in EtOH the excitation
peak was greatly blue-shifted to 330 nm. Moreover, the excitation spectra of TNTPC in EtOH
exhibit no difference in response to DNA addition, whereas the excitation spectra in DMSO was
slightly broader in the absence of DNA than when it was present (Figure 8A). In terms of
fluorescence emission, we observed that the emission spectra of TNTPC in EtOH was unchanged
by the addition of DNA, whereas in DMSO a broad emission peak between 480 and 540 nm was
observed in the absence of DNA, and this broad peak became slightly blue-shifted to 440-520 nm
upon DNA addition (Figure 8B).
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Figure 8. Determing the fluorescent profile of 10 μM TNTPC in EtOH or DMSO with or without
10μg of DNA. A) Excitation spectra of DMSO (red), 10 μM TNTPC in DMSO (light blue), 10
μM TNTPC in DMSO with 10μg of DNA (purple), EtOH (teal), 10 μM TNTPC in EtOH
(yellow), and 10 μM TNTPC in EtOH with 10μg of DNA (dark blue). B) Emission spectra of
DMSO (red), 10 μM TNTPC in DMSO (light blue), 10 μM TNTPC in DMSO with 10μg of DNA
(purple), EtOH (teal), 10 μM TNTPC in EtOH (yellow), and 10 μM TNTPC in EtOH with 10μg
of DNA (dark blue). Emission spectra’s of DMSO solutions were excited at 385 nm while emission
spectra’s of EtOH solutions were excited at 330 nm.
Taken together, these findings highlight the use of trisaminocyclopropeniums, specifically,
the small organic fluorophore TNTPC as a viable addition to current counterstain technology for
specific labeling of cellular nuclei and DNA on non-denaturing gels. Moreover, this work provides
the foundation and design guidelines for the broader biologically-oriented application of
trisaminocyclopropeniums as cost-effective alternative staining agents for DNA visualization.
Current work in our group is expanding the repertoire of trisaminocyclopropenium small organic
fluorophore uses in bioimaging applications among other areas, and related findings will be
reported in due course.
3.0 Conclusion
In summary, the utility of cyclopropeniums as small organic fluorophores has been
expanded, including to applications in bioimaging. Accompanying these advancements, guidelines
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for the rational design of trisaminocyclopropenium fluorophores with desirable photophysical
properties and capacity to operate by simultaneous fluorescence-related mechanisms (ICT and
ITSC) were disclosed. Attesting to this fact, was the development of highly fluorescent
trisaminocyclopropenium small organic fluorophore TNTPC with an impressive quantum yield
of 0.92, Stokes shift of 180 nm and molar extinction coefficient of 6.34 x 104 M-1 cm-1; thus,
boding well for future applications involving high-resolution fluorescent techniques where large
Stokes shifts and high fluorescence efficiency are a must. Furthermore, trisaminocyclopropenium
fluorophore TNTPC functioned as a potent counterstain for cellular nuclei as evidenced by the
decoration of cellular membranes and use as a dye for DNA visualization. Collectively, this study
adds to the burgeoning interest in cyclopropenium compounds and their use as fluorophores, now
to bioimaging applications.
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Experimental Section
General Information
Materials were obtained from commercial suppliers and were used without further purification.
Dichloromethane was distilled over CaH, tetrahydrofuran (THF) was distilled over Na and a
benzophenone indicator, and acetonitrile (MeCN) was distilled over Na2CO3. Reactions were
monitored by thin layer chromatography (TLC) using TLC silica gel 60 F254. NMR spectra were
obtained with 1H 300 MHz, 1H 400 MHz or 1H 600 MHz, 13C 75.5 MHz or 13C 150.9 MHz, 19F
292.4 MHz, and 11B 96.3 MHz spectrometers. The chemical shifts are reported as δ values (ppm)
relative to tetramethylsilane (s = singlet, d = doublet, t = triplet, q = quartet, sep. = septet, m =
multiplet). All reactions were performed under an inert nitrogen atmosphere. HRMS (high-
resolution mass spectrometry) spectra were measured using electron ionization (EI), electrospray
ionization (ESI) or fast atom bombardment (FAB) and a time-of-flight (TOF) mass analyzer in
positive ionization mode. Secondary amines 5a-5c were obtained from commercial suppliers and
used without further purification.
Experimental Methods
Cell Culture Conditions
HEK293 cells at passage 15 were cultured in DMEM containing 10% FBS, 2% non-essential
amino acids (NEAA), and 1% penicillin/streptomycin. To enable fluorescent image analysis, cells
were seeded on 35 mm Matek glass bottom collagen coated dishes at 70 percent confluency.
Fluorescent Microscopy
Imaging was completed with a Carl Zeiss Axio Observer. Z1 inverted epifluorescence microscope
equipped with ApoTome.2 optical sectioning and a Hamamatsu ORCA-Flash 4.0 V2 digital
camera. Cells were viewed with a Plan-Apochromat 63x/1.40 Oil DIC M27 microscope objective.
Throughout the experiments, cells were kept in a humidified incubator with 5% CO2 at 37 °C.
Blue fluorescence was detected using a fluorescence channel possessing excitation of 350–385 nm
and emission wavelength filter at 410-460 nm. Both the intensity of fluorescence illumination and
the camera exposure time was held constant throughout all experiments. Cells were imaged before,
immediately after and 5 minutes after treatment with either 20 μL of DMSO, 1 μM, 10 μM, or 1
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mM TNTPC in 100% DMSO into 2 mL of high glucose DMEM media. Images were analyzed and
compiled using FIJI image analysis software.51
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Gel Electrophoresis
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1% by mass agarose gels were run through gel electrophoresis at 80 volts for 30 minutes. Wells
either contained 15 μL of 500 ng/μL DNA treated with either 5 μL of 100% DMSO or 5 μL of 40
μM TNTPC in DMSO to create a 10 μM final concentration. A tertiary control of 20 μL of TNTPC
at 10 μM without DNA was also run. Visualization of gels was completed using a Gel Doc XR
system (BIO RAD).
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General Procedures for 6a-6i
The general procedure for all trisaminocyclopropenium ion derivatives (6a-6i) follows the
example procedure outlined for derivative 6a.
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*For derivatives 6f•BF4 and 6i•BF4 , counter-ion exchanges were performed to the precursor
(6f•Cl- and 6i•Cl- respectively) following the example counterion exchange procedure for
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derivative 6f•BF4 .
Tris[N(morpholino)] cyclopropenium chloride (6a)
To a solution of tetrachlorocyclopropene (140 mg, 0.79 mmol, 1 equiv.), in dichloromethane (5
mL) was added secondary amine 5a (413 mg, 4.74 mmol, 6 equiv.) dropwise under an inert
nitrogen atmosphere at 0 °C. The resulting solution was allowed to warm to room temperature and
stirred for four hours, after which water (5 mL) was added to the reaction mixture. The organic
phase was separated and washed with 1 M HCl (8 mL) and extracted with dichloromethane (3 x
10 mL). The combined organic extracts were dried over MgSO4 and the solvent removed under
reduced pressure. The crude product was purified by recrystallization from dichloromethane/ether
(2:1, 20 mL:10 mL) to afford 6a (186 mg, 0.57 mmol) as an off-white crystalline solid in 72%
yield. m.p. = 94 - 98 °C. 1H NMR (300 MHz, CDCl3): δ = 3.91-3.88 (t, J = 4.7 Hz, 4H), 3.74-3.71
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(t, J = 4.3 Hz, 8H), 3.47-3.43 (t, J = 5.0 Hz, 8H), 3.10-3.07 (t, J = 4.9 Hz, 4H). C {1H} NMR
(75.5 MHz, CDCl3): δ = 117.3, 65.8, 63.5, 50.2, 43.4. HRMS (FAB) m/z: [M+] Calcd for
C15H24N3O3 294.1812; Found 294.1818
Tris[N,N(dicyclohexyl)] cyclopropenium chloride (6b)
Off-white solid, 69% yield (295 mg, 0.48 mmol). Secondary amine: 5b. m.p. = 213 - 215 °C. 1H
NMR (600 MHz, CDCl3): δ = 3.42-3.38 (t, J = 12.0 Hz, 6H), 1.96-1.91 (t, J = 14.2 Hz, 20H), 1.74-
1.72 (d, J = 13.1 Hz, 6H), 1.66-1.60 (q, J = 12.7 Hz, 16H), 1.39-1.33 (q, J = 12.8 Hz, 12H), 1.20-
1.13 (q, J = 13.1 Hz, 6H). 13C {1H} NMR (150.9 MHz, CDCl3): δ = 123.9, 61.1, 32.9, 25.87, 25.0.
HRMS (FAB) m/z: [M+] Calcd for C39H66N3 576.5241; Found 576.5237.
Tris[N,N(dibenzyl)] cyclopropenium chloride (6c)
White solid, 79% yield (350 mg, 0.53 mmol). Secondary amine: 5c. m.p. = 169 - 173 °C. 1H NMR
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(300 MHz, CDCl3): δ = 7.55-7.52 (m, 12H), 7.42-7.32 (m, 18H), 3.88 (s, 12H). C {1H} NMR
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(75.5 MHz, CDCl3): δ = 130.4, 130.1, 129.4, 129.1, 48.7. HRMS (FAB) m/z: [M+] Calcd for
C45H42N3 624.3373; Found 624.3369.
Tris[N,N(benzyl)phenyl] cyclopropenium chloride (6d)
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White solid, 70% yield (389 mg, 0.63 mmol). Secondary amine: 5d. m.p. = 213 - 215 °C. 1H NMR
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(300 MHz, CDCl3): δ = 7.41-7.31 (m, 20H), 7.28-7.25 (m, 10H), 4.37 (s, 6H). C {1H} NMR
(75.5 MHz, CDCl3): δ = 134.3, 131.1, 129.7, 129.5, 129.4, 129.2, 128.7, 124.0, 55.9. HRMS (FAB)
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m/z: [M+] Calcd for C42H36N3 582.2904; Found 582.2881.
Tris[N,N(phenyl)-1-phenylethyl)] cyclopropenium chloride (6e)
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White solid, 68% (208 mg, 0.32 mmol). Secondary amine: 5e. m.p 186 - 188 °C. H NMR (300
MHz, CDCl3): δ 7.42-7.39 (m, 5H), 7.31-7.29 (m, 5H), 7.27-7.21 (m, 20H), 4.55-4.47 (q, J = 6.7
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Hz, 3H), 1.94-1.92 (d, J = 6.9 Hz, 9H). C {1H} NMR (75.5 MHz, CDCl3): δ = 135.1, 134.0,
129.3, 128.8, 128.7, 128.6, 124.5, 64.5, 19.1. HRMS (FAB) m/z: [M+] Calcd for C45H42N3
624.3373; Found 624.3379.
Tris[N,N(isopropyl)phenyl] cyclopropenium chloride (6f•Cl-)
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White solid,78% (475 mg, 1.00 mmol). Secondary amine: 5f. m.p 185 - 186 °C. H NMR (300
MHz, CDCl3): δ 7.42-7.36 (m, 15H), 2.81-2.67 (sep., J = 7.0 Hz, 3H), 0.86-0.83 (d, J = 6.6 Hz,
18H). 13C {1H} NMR (75.5 MHz, CDCl3): δ = 138.3, 130.2, 129.7, 129.1, 115.9, 56.2, 21.4. HRMS
(FAB) m/z: [M+] Calcd for C30H36N3 438.2904; Found 438.2901.
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Tris[N,N(isopropyl)phenyl] cyclopropenium tetrafluoroborate (6f•BF4 )*
To a solution of 6f•Cl- (880 mg, 1.86 mmol, 1 equiv.) in dichloromethane (10 mL) was added
sodium tetrafluoroborate (817 mg, 7.44 mmol, 4 equiv.) in one portion. The resulting solution was
allowed to stir for 24 hours, after which water (5 mL) was added to the reaction mixture. The
organic phase was separated and washed with 1 M HCl (8 mL) and extracted with dichloromethane
(3 x 10 mL). The combined organic extracts were dried over MgSO4 and the solvent was removed
under reduced pressure. The crude product was purified by recrystallization from
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dichloromethane/ether (2:1, 20 mL:10 mL) to afford 6f•BF4 (750 mg, 1.43 mmol) as an off-white
1
crystalline solid in 77% yield. m.p 171 - 174°C. H NMR (300 MHz, CDCl3): δ 7.35-7.31 (m,
15H), 2.74-2.61 (sep., J = 6.9 Hz, 3H), 0.79-0.76 (d, J = 6.9 Hz, 9H). 13C {1H} NMR (75.5 MHz,
CDCl3): δ = 138.2, 130.1, 129.6, 129.1, 115.8, 56.1, 21.3. 11B {1H} NMR (96.3 MHz, CDCl3): δ
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= -0.76. F {1H} NMR (292.4 MHz, CDCl3): δ = -152.96. HRMS (FAB) m/z: [M+] Calcd for
C30H36N3 438.2904; Found 438.2899.
Tris[N,N(dodecyl)phenyl] cyclopropenium chloride (6g)
White solid, 72% yield (591 mg, 0.69 mmol). Secondary amine: 5g. m.p. = 100 - 102 °C. 1H NMR
(300 MHz, CDCl3): δ = 7.45-7.36 (m, 12H), 7.30-7.25 (m, 3H), 3.25-3.19 (t, J = 7.9 Hz, 6H), 1.85-
1.75 (quin., J = 7.2 Hz, 6H), 1.26-1.22 (d, J = 11.4 Hz, 51H), 0.92-0.87 (t, J = 6.4 Hz, 9H). 13C
{1H} NMR (75.5 MHz, CDCl3): δ = 135.6, 130.0, 129.3, 123.5, 53.1, 31.9, 29.6, 29.5, 29.5, 29.4,
29.3, 28.9, 26.6, 25.5, 22.7, 14.1. HRMS (FAB) m/z: [M+] Calcd for C57H90N3 816.7129; Found
816.7133.
Tris[N,N(isopropyl)naphthalen-1-yl] cyclopropenium chloride (6h)
White solid, 73% yield (453 mg, 0.73 mmol). Secondary amine: 5h. m.p. = 223 - 227 °C. 1H NMR
(300 MHz, CDCl3): δ = 8.38 (s, 2H), 7.90-7.87 (d, J = 8.1 Hz, 5H), 7.80 (s, 2H), 7.68-7.63 (t, J =
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7.2 Hz, 4H), 7.59-7.54 (t, J = 7.6 Hz, 4H), 7.42-7.37 (t, J = 7.8 Hz, 4H), 3.95-3.83 (sep., J = 6.2
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Hz, 3H), 1.52-1.50 (d, J = 6.4 Hz, 18H). C {1H} NMR (75.5 MHz, CDCl3): δ = 134.5, 130.4,
129.9, 128.7, 127.6, 127.5, 126.9, 126.9, 124.9, 123.1, 122.1, 29.3, 19.7. HRMS (FAB) m/z: [M+]
Calcd for C52H42N3 588.3373; Found 588.3364.
Tris[N,N(naphthalen-2-ylmethyl)phenyl] cyclopropenium chloride (6i•Cl-)
White solid, 83% yield (138 mg, 0.18 mmol). Secondary amine: 5i. m.p. = 198 - 200 °C. 1H NMR
(300 MHz, CDCl3): δ = 7.87-7.71 (t, J = 6.6 Hz, 12H), 7.58-7.55 (dd, J = 13.4 Hz, J = 7.1 Hz, 3H),
7.46-7.43 (t, J = 11.7 Hz, 6H), 7.40-7.36 (m, 6H), 7.28-7.24 (m, 9H), 4.50 (s, 6H). 13C {1H} NMR
(75.5 MHz, CDCl3): δ = 134.3, 133.4, 132.8, 131.0, 129.7, 129.2, 128.6, 128.2, 127.7, 127.6,
126.9, 126.4, 123.8, 56.1. HRMS (FAB) m/z: [M+] Calcd for C54H42N3 732.3373; Found
732.3368.
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Tris[N(naphthalen-2-ylmethyl)phenyl] cyclopropenium tetrafluoroborate (6i•BF4 )*
Yellow solid, 78% yield (98 mg, 0.12 mmol). Counterion Exchange with: 6i•Cl-. m.p. = 168 °C -
170 °C. 1H NMR (300 MHz, CDCl3): δ = 7.88-7.82 (m, 12H), 7.54-7.48 (m, 9H), 7.25-7.19 (m,
6H), 6.78-7.70 (m, 9H), 4.53 (s, 6H). 13C {1H} NMR (75.5 MHz, CDCl3): δ = 148.2, 137.0, 133.5,
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132.8, 129.3, 128.4, 127.8, 127.7, 126.2, 125.9, 125.7, 117.7, 112.9, 48.5. B {1H} NMR (96.3
MHz, CDCl3): δ = -0.42. 19F {1H} NMR (292.4 MHz, CDCl3): δ = -151.55. HRMS (FAB) m/z:
Calcd for C54H42N3 732.3373; Found 732.3368.
General Procedure for Secondary Amine Derivatives 5a-5i
The general procedure for all secondary amines (5a-5i) follows the example procedure outlined
for derivative 5i (See Supporting Information Scheme S1). Microwave reactions were carried out
on a CEM Discover Microwave reactor with an Exlporer 24 PLS (Model # SP_1303A; Serial #
52289). All microwave reactions were performed in 10 mL vessels with a sealed snap cap at 150
oC for 30 minutes with medium stirring. Reaction mixture temperature and temperature-time
profiles were monitered by Synergy Software (See Supporting Information Graph S1).
N-(naphthalen-2-ylmethyl)aniline (5i)
To a 10 mL microwave vessel containing a stir bar was added aniline (438 mg, 4.71 mmol, 3
equiv.), 2-(bromomethyl)naphthalene (347 mg, 1.57, 1 equiv.) followed by potassium iodide (50
mg, 0.3 mmol, 0.2 equiv.), potassium carbonate (217 mg, 1.57 mmol, 1 equiv.) and acetonitrile
(2 mL). The reaction mixture was microwaved at 150 oC for 30 minutes. The reaction mixture was
gravity filtered and the filtrate concentrated under reduced pressure. The crude product was
purified by column chromatography with hexanes in ethyl acetate (10:1) to afford 5i (301 mg, 1.30
1
mmol) as a light-yellow oil in 83% yield. H NMR (300 MHz, CDCl3): δ = 7.90-7.84 (m, 4H),
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7.56-7.49 (m, 3H), 7.28-7.21 (m, 2H), 6.81-6.72 (m, 3H), 4.54 (s, 2H), 4.17 (s, 1H). C {1H}
NMR (75.5 MHz, CDCl3): δ = 148.2, 137.0, 133.5, 132.8, 129.3, 128.4, 127.8, 127.7, 126.2, 125.9,
125.8, 117.7, 113.0, 48.5. HRMS (EI) m/z: [M+] Calcd for C17H15N 233.1199; Found 233.1197.
N-isopropylnaphthalen-1-amine (5h)
Light red oil, 71% yield (230 mg, 1.24 mmol) Primary amine: 1-naphthylamine; Halogenated
1
compound: 2-bromopropane. H NMR (300 MHz, CDCl3): δ 7.88-7.85 (m, 2H), 7.55-7.49 (m,
2H), 7.47-7.41 (t, J = 7.9 Hz, 1H), δ 7.31-7.28 (d, J = 8.2 Hz, 1H), δ 6.73-6.70 (d, J = 7.5 Hz, 1H),
δ 4.27 (s, 1H), δ 3.96-3.84 (sep, J = 6.2 Hz, 1H), δ 1.43-1.41 (d, J = 6.2 Hz, 6H). 13C {1H} NMR
(75.5 MHz, CDCl3): δ = 142.6, 134.6, 128.8, 126.7, 125.7, 124.6, 123.5, 119.9, 116.8, 104.9, 44.3,
23.0. HRMS (EI) m/z: Calcd for C13H15N 185.1199; Found 185.1199.
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N-dodecylaniline (5g)
Light yellow oil. 79% yield (421 mg, 1.61 mmol). Primary amine: Aniline; Halogenated
compound: iodododecane. 1H NMR (300 MHz, CDCl3): δ 7.27-7.22 (m, 2H), 6.79-6.74 (t, J = 7.3
Hz, 1H), 6.69-6.66 (dd, J = 7.6 Hz, 2H), δ 3.66 (s, 1H), δ 3.19-3.15 (t, J = 7.1 Hz, 2H), δ 1.73-1.64
(q, J = 6.6 Hz, 2H), δ 1.36 (s, 18H), δ 1.00-0.96 (t, J = 6.3 Hz, 3H), 13C {1H} NMR (75.5 MHz,
CDCl3): δ = 148.6, 129.3, 117.1, 112.7, 44.1, 32.0, 29.8, 29.7, 29.7, 29.7, 29.6, 29.4, 27.3, 22.8,
14.2. HRMS (EI) m/z: Calcd for C18H31N 261.2451; Found 261.2449.
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N-isopropylaniline (5f)
Clear oil. 85% yield (335 mg, 2.48 mmol) Primary amine: Aniline; Halogenated compound: 2-
bromopropane. 1H NMR (300 MHz, CDCl3): δ 7.54-7.49 (t, J = 7.3 Hz, 2H), 7.06-7.04 (t, J = 7.2
Hz, 1H), 6.91-6.88 (dd, J = 7.6 Hz, 2H), δ 3.98-3.85 (sep, J = 6.2 Hz, 1H), δ 3.69 (s, 1H), δ 1.51-
1.49 (d, J = 6.2 Hz, 6H). 13C {1H} NMR (75.5 MHz, CDCl3): δ = 147.9, 129.6, 117.2, 113.6, 44.4,
23.2. HRMS (EI) m/z: [M+] Calcd for C9H13N 135.1048; Found 135.1041.
N-(1-phenylethyl)aniline (5e)
Light yellow oil. 76% yield (272 mg, 1.28 mmol) Primary amine: Aniline; Halogenated
compound: 1-(bromoethyl)benzene. 1H NMR (300 MHz, CDCl3): δ 7.47-7.37 (m, 4H), 7.33-7.28
(m, 1H), 7.20-7.15 (m, 2H), δ 6.75-6.70 (t, J = 7.2 Hz, 1H), δ 6.61-6.58 (dd, J = 8.4 Hz, 2H), δ
4.60-4.53 (q, J = 6.7 Hz, 1H), δ 4.09 (s, 1H), δ 1.60-1.58 (d, J = 6.7 Hz, 3H). 13C {1H} NMR (75.5
MHz, CDCl3): δ = 147.4, 145.3, 129.2, 128.7, 126.9, 125.9, 117.3, 113.4, 53.5, 25.1. HRMS (EI)
m/z: [M+] Calcd for C14H15N 197.1199; Found 197.1197.
N-benzylaniline (5d)
Light yellow oil. 88% yield (446 mg, 2.44 mmol) Primary amine: Aniline; Halogenated
compound: 1-(bromoethyl)benzene. 1H NMR (300 MHz, CDCl3): δ 7.43-7.34 (m, 5H), 7.27-7.22
(m, 2H), 6.81-6.76 (t, J = 7.2 Hz, 1H), δ 6.72-6.69 (dd, J = 8.5 Hz, 2H), δ 4.39 (s, 2H), δ 4.07 (s,
13
1H). C {1H} NMR (75.5 MHz, CDCl3): δ = 148.2, 139.5, 129.3, 128.7, 127.6, 127.3, 117.6,
112.9, 48.4. HRMS (EI) m/z: [M+] Calcd for C13H13N 183.1043; Found: 183.1043.
Dibenzylamine (5c)
Obtained from commercial supplier and used without further purification.
Dicyclohexylamine (5b)
Obtained from commercial supplier and used without further purification.
Morpholine (5a)
Obtained from commercial supplier and used without further purification.
Associated Content
Supporting Information
The supporting information is available free of charge at http://pubs.acs.org.
Complete experimental, photophysical and computational methods and data.
Author Information
Corresponding Author
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Travis Dudding – Department of Chemistry, Brock University, St. Catharines, Ontario,
L2S 3A1, Canada; Email: tdudding@brocku.ca
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Author Contributions
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All authors have given approval to the final version of the manuscript.
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Notes
The authors declare no competing financial interest.
Acknowledgments
M.G. acknowledges OGS for funding. G.F. acknowledges OGS for funding. T.D.
acknowledges financial support from the Natural Science and Engineering Research Council
(NSERC) Discovery Grant (2019-04205). Computations were carried out using facilities at
SHARCNET (Shared Hierarchical Academic Research Computing Network: www.sharcnet.ca)
and Compute/Calcul Canada. A.N. acknowledges financial support from the Natural Science and
Engineering Research Council (NSERC) Discovery Grant. The authors acknowledge Prof. Jeff
Stuart for the use of his fluorescence spectrophotometer and Zeiss microscope.
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