Cellular localisation of structurally diverse diphenylacetylene fluorophores

Article abstract of DOI:10.1039/d0ob01153c

Fluorescent probes are increasingly used as reporter molecules in a wide variety of biophysical experiments, but when designing new compounds it can often be difficult to anticipate the effect that changing chemical structure can have on cellular localisa

Full text of DOI:10.1039/d0ob01153c

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Ambler and A. Whiting, Org. Biomol. Chem., 2020, DOI: 10.1039/D0OB01153C.
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DOI: 10.1039/D0OB01153C
Organic & Biomolecular Chemistry
ARTICLE
Cellular localisation of structurally diverse diphenylacetylene
fluorophores
a,d*
a,b,f
David R. Chisholm,†
Joshua G. Hughes,†
Thomas S. Blacker,c Rachel Humann,d,e Candace
Received 00th January 20xx,
Accepted 00th January 20xx
Adams,a Daniel Callaghan,a Alba Pujol,a Nicola K. Lembicz,e Angus J. Bain,c John M. Girkin,f* Carrie A.
Amblera,b* and Andrew Whitinga,d*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Fluorescent probes are increasingly used as reporter molecules in a wide variety of biophysical experiments, but when
designing new compounds it can often be difficult to anticipate the effect that changing chemical structure can have on
cellular localisation and fluorescence behaviour. To provide further chemical rationale for probe design, a series of donor-
acceptor diphenylacetylene fluorophores with varying lipophilicities and structures were synthesised and analysed in
human epidermal cells using a range of cellular imaging techniques. These experiments showed that, within this family, the
greatest determinants of cellular localisation were overall lipophilicity and the presence of ionisable groups. Indeed,
compounds with high LogD values (>5) were found to localise in lipid droplets, but conversion of their ester acceptor groups
to the corresponding carboxylic acids caused a pronounced shift to localisation in the endoplasmic reticulum. Mildly
lipophilic compounds (LogD = 2-3) with strongly basic amine groups were shown to be confined to lysosomes i.e. an acidic
cellular compartment, but sequestering this positively charged motif as an amide resulted in a significant change to
cytoplasmic and membrane localisation. Finally, specific organelles including the mitochondria could be targeted by
incorporating groups such as a triphenylphosphonium moiety. Taken together, this account illustrates a range of guiding
principles that can inform the design of other fluorescent molecules but, moreover, has demonstrated that many of these
diphenylacetylenes have significant utility as probes in a range of cellular imaging studies.
design choices, it is often the case that unexpected behaviours
arise.5 It is, therefore, important to consider these results with
respect not only to the intended application but also to the
Introduction
physical and molecular properties of the compound.
Physicochemical descriptors such as logP, pKa, polar surface
area and membrane permeability will all affect cellular
localisation since each cellular compartment exhibits varying
The design and synthesis of new fluorescent molecules for
cellular imaging purposes has become a rich and burgeoning
field in the past twenty-five years. From probes that detect
particular molecules, to compounds that localise to specific
organelles; this area of chemical biology is now host to an
abundance of elegant molecular design.1–4 When designing new
fluorophores, minor changes in chemical structure can result in
marked differences in cellular behaviour and localisation, and
while many of these changes can be attributed to fairly rational
environmental properties (Fig. 1A), and having
a firm
understanding of how the modulation of these physicochemical
parameters influences the observed behaviour will assist in the
design of more selective compounds.6–9
The incorporation of cellular recognition motifs also adds
complexity when analysing new fluorophores and poses a
number of questions.10 For example, does the targeting motif
exhibit passive behaviour e.g. utilising an intrinsic intracellular
electrochemical gradient? Or is it “active” i.e. requiring
metabolism, or recognition by a fixed concentration of a cellular
species? It is important to bear these questions in mind when
designing imaging experiments since variables such as
fluorophore concentration, treatment time, culture conditions
and cell type will all impact the observed imaging results and
level of perturbation to the biological process under
observation.
a. LightOx Limited, 65 Westgate Road, Newcastle upon Tyne, NE1 1SG, UK
b. Department of Biosciences, Durham University, South Road, Durham, DH1 3LE,
UK
c. Department of Physics & Astronomy, University College London, Gower Street,
London, WC1E 6BT, UK
d. Department of Chemistry, Durham University, Science Laboratories, South
Road, Durham, DH1 3LE, UK
e. High Force Research Limited, Bowburn North Industrial Estate, Bowburn,
Durham, DH6 5PF, UK
f. Centre for Advanced Instrumentation, Department of Physics, Durham
University, South Road, Durham, DH1 3LE, UK
† Joint first authors
* Corresponding authors
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
To understand cellular localisation, an imaging approach is
most often employed.11 Automated computational methods for
analysing fluorescence images are also extremely beneficial for
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high-throughput work, for example in proteomics12 or for corresponding two-photon near-IR irradiation.16,17 When
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analysis of combinatorial libraries of fluorescent compounds.13 designing new chemical derivatives of this structural class, small
DOI: 10.1039/D0OB01153C
Predictive models for subcellular localisation are becoming modifications in chemical structure often resulted in distinct
14
increasingly beneficial for the design of new fluorophores,
localisation behaviour and it became apparent that a more
and there is a great need for informative accounts of subcellular holistic approach to the understanding of these changes was
behaviour; the more structure-based imaging case-studies necessary in order to inform more effective design choices.
there are, the better informed we can be in making molecular Therefore, an investigation into the effect of changing
design choices.15
molecular structure upon subcellular localisation within a family
We recently reported a new class of diphenylacetylene that of related diphenylacetylenes (Fig. 1) was conducted.
elicits cytotoxic activity when activated by UV, violet and
Fig. 1 A) General characteristics of cellular compartments in human cells.18 B) Series of fluorescent diphenylacetylenes with differing substituents, targeting moieties
and physicochemical properties
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Organic & Biomolecular Chemistry
ARTICLE
Phenylpiperazine diphenyl acetylenes 3a and 3b were
synthesised according to Scheme 3, in which commercially
available 1-phenylpiperazine was iodinated using ICl in
AcOH/H2O in a 63% yield on a multi-gram scale. Sonogashira
coupling with alkynes 7a16 and 7c (Scheme 2) was conducted
Results and Discussion
Synthesis
Lipophilic acids 1a and 1b (syntheses reported previously16,19
)
with
5 mol% Pd(PPh3)2Cl2/CuI in triethylamine. Iodo-
and esters 2a and 2b were synthesised by the Sonogashira
coupling of 6-iodo-tetrahydroquinoline 6 (synthesis reported
previously20) with acetylenes 7a16 and 7b19 (Scheme 1).
phenylpiperazine 10 is poorly soluble in triethylamine at RT,
hence, the solution was heated to 60 oC in order to achieve an
acceptable conversion of the starting iodide. SiO2
chromatography, followed by recrystallisation from MeOH or
MeCN provided the desired 3a and 3b in acceptable to good
yields.
Scheme 1 Synthesis of lipophilic esters 2a and 2b.
The tetrahydroquinoline moiety employed in these compounds
is an excellent π-donor for a donor-acceptor fluorophore due to
its rigid structure, but the gem-dimethyl and isopropyl groups
also provide a significant degree of lipophilicity. We anticipated
that it would be difficult to direct such hydrophobic compounds
towards more polar cellular environments and, hence, we
designed an alternative, less lipophilic, phenylpiperazine-based
π-donor scaffold. Phenylpiperazines are widely used in
therapeutics due to their often-favourable effects on
pharmacokinetic properties, but we also envisaged the free
secondary amine would be a useful attachment point for
moieties known to direct cellular localisation.
Scheme 3 Iodination of 1-phenylpiperazine to provide key π-donor building block 10,
followed by Sonogashira coupling to give corresponding diphenylacetylenes 3a and 3b.
We also anticipated that the corresponding primary amine 3c
would be significantly more polar than the tetrahydroquinoline-
based compounds and, accordingly, devised an analogous
synthetic route (Scheme 4) employing a Sonogashira coupling
of iodoaniline derivative 14. Acylation of 4-iodo-N-
methylaniline with chloroacetyl chloride, followed by insertion
of the primary amine group using Gabriel chemistry provided 2-
amino-N-phenylacetamide 13. Reduction using BH3.Me2S
yielded the target aniline 14 which was coupled to 7a to provide
primary amine diphenylacetylene 3c in a 58% yield.
Scheme 2 Synthesis of tert-butyl alkyne acceptor moiety 8a.
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Scheme 4 Synthesis of amine-substituted diphenylacetylene 3c.
The reactive amine groups of 3a-3c enable further modulation
of the properties of the compounds through a single step
conversion, i.e. acylation, alkylation or sulfonylation. To this
end, we exemplified acylation via reaction of 3b with acetyl
chloride to provide the N-Ac diphenylacetylene 4a in an
excellent yield (Scheme 5). Alkylation with commercially
available (3-bromopropyl)triphenylphosphonium bromide was
achieved by heating a solution of 3b in DMF with K2CO3 to
provide triphenylphosphonium-linked 5a in a 66% yield.
Triphenyphosphonium cations are widely used to encourage
accumulation in the mitochondrial matrix due to the significant
negative mitochondrial membrane potential.21,22 We also
sulfonylated 3c using tosyl chloride (Equation 1) to provide the
N-tosyl-piperazine diphenylacetylene, 5b; tosyl sulphonamide
groups have been employed as ER targeting moieties in a
variety of fluorophores.23,24
Equation 1 Sulfonylation of 3c to provide an ER-targeting compound 5b.
Photophysical characterisation
All of the compounds exhibited photophysical properties (Table
1) that were characteristic of other donor-acceptor
diphenylacetylenes.16,17,19 The compounds exhibited absorption
(Fig. 2, Table 1 and ESI Section 2) bands at around 300 nm (S0 →
S2) and 360-400 nm (S0 → S1) due to the formation of
intramolecular charge transfer excited states as a result of the
presence of strong π-donor/acceptor moieties (Figure 2A).
Generally, those compounds with a THQ donor moiety (1a-2b)
exhibited absorptions at longer wavelength and with larger
extinction coefficients compared to those with the more
electron deficient donor structures (3a-5d). Excitation led to
highly solvatochromatic fluorescence emission (Figure 2B) in all
compounds, with a large bathochromic shift evident in EtOH,
indicating that polar solvents stabilise the emissive S1 state. In
toluene, the compounds exhibited quantum yields of up to 0.89
and mean lifetimes of around 1.5 ns, but a significant reduction
in both was observed in EtOH, perhaps due to aggregation in
this much more polar environment. The alkyl amines, 3a-3c,
also exhibited a significantly reduced quantum yield in toluene,
possibly also due to aggregation effects of these more polar
compounds. All compounds exhibited multiexponential
fluorescence decay dynamics in both solvents, indicating the
presence of multiple emissive conformations or environments,
each with a distinct lifetime (see ESI, Table 1).16
Scheme 5 Acetylation of 3b to give N-Ac diphenylacetylene 4a and addition of a
mitochondrial targeting group 5a.
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Figure 2 (A) Absorption spectra of compounds 1a, 2a, 3a, 4a and 5a in toluene (100 µM). (B) Normalised emission spectra of compounds 1a, 2a, 3a, 4a and 5a in toluene
(10 µM).
Table 1: Photophysical properties of compounds 1a-5b in toluene and EtOH.
Compound
Solvent
λabs(max)/nm
ε/M-1cm-1
λem(max)/nm
φ
<τ>/ns
1a
Toluene
EtOH
Toluene
EtOH
Toluene
EtOH
Toluene
EtOH
Toluene
EtOH
Toluene
EtOH
Toluene
EtOH
Toluene
EtOH
390
387
392
387
395
394
392
387
362
357
362
352
377
367
361
357
367
358
382
377
29287
25120
43273
24125
39014
31017
56722
30825
35500
37885
23873
15713
25681
32355
42421
27933
36816
29209
34973
31767
500
525
466
548
498
555
466
553
477
521
482
503
479
527
468
535
498
503
478
535
0.67
0.13
0.84
0.13
0.80
0.06
0.78
0.15
0.19
0.12
0.23
0.14
0.21
0.06
0.97
0.10
0.72
0.13
0.89
0.13
1.62
0.20
1.62
0.15
1.68
0.11
1.67
0.13
1.58
0.08
1.61
0.11
1.45
0.12
1.45
0.06
1.64
0.09
1.58
0.10
1b
2a
2b
3a
3b
3c
4a
5a
5b
Toluene
EtOH
Toluene
EtOH
conducted on groups of whole cells, and to minimise
photobleaching and the effect of z-drift whilst imaging, a large
pinhole size of 5 Airy Units was used to allow low laser power,
maximal depth coverage and fast scan speeds.
Imaging & cellular characterisation
We next conducted a series of imaging experiments to
characterise and understand the cellular localisation of the
compounds and attempted to relate their respective
behaviours to the differences in molecular structure. HaCaT
keratinocytes were treated with varying concentrations of each
compound and incubated for 1 hour, whereupon they were
imaged using a Zeiss 880 confocal microscope.
Whole cell fluorescence emission scan (lambda scan)
experiments with each compound were carried out in order to
understand the differences in in cellulo fluorescence emission
behaviour. The lambda scans (see ESI Section 3) were
Each compound displayed the broad emission that is
characteristic of donor-acceptor diphenylacetylenes due to
emission taking place from excited states that exhibit a variety
of angles about the freely rotatable central acetylene,16,25 and
also showed a similar λmax to those obtained in toluene. The
most obvious differences between compounds are shown in
Fig. 3; foremost of which is the blue-shift in the emission of the
acids 1a/1b compared to the corresponding esters 2a/2b. This
is likely due to deprotonation of the carboxylic acid of 1a/1b
that causes a reduction in the dipolar nature of the excited
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state. Local polarity also appears to play a role; the basic amine
Journal Name
Localisation to the endoplasmic reticulum was VfiierwstAartsicsleeOsnsleinde
26
DOI: 10.1039/D0OB01153C
compounds 3a-c showed more red-shifted emission spectra, in using BODIPY ER-Tracker™ Red (BODIPY TR Glibenclamide)
line with the prediction that their amine groups - protonated at which can be excited at 587 nm and emits with a peak emission
physiological pH - cause a shift in localisation to a more polar wavelength at 615 nm. As shown in Fig. 4, both of the acids, 1a
environment compared to the tetrahydroquinoline-based and 1b, exhibit significant co-localisation with the ER-Tracker
compounds. A further red shift was observed in cells treated (PCC = 0.71 and 0.80, respectively) along with general lipophilic
with 4a, indicating that this compound localises to even more staining. Both compounds also appeared to localise to the
hydrophilic compartments (vide infra). Targeted derivatives, nuclear envelope, highlighted by arrows in Fig. 4, as is typical for
5a/5b, showed similar lambda scans to their parent compounds compounds that interact with the ER due to the continuity
(see ESI Section 3).
between the structures.27 We also used Nile Red as a general
lipophilic stain,28 which similarly showed significant co-
localisation with 1a and 1b (PCC = 0.80 and 0.81, respectively)
and, hence, we can characterise these compounds as non-
specific lipophilic and ER dyes (see ESI Section 4 & 7).
1a
1b
2a
2b
3b
4a
1.0
0.5
0.0
400
500
600
700
Wavelength/nm
Figure 3 Whole cell fluorescence emission scans of HaCaT keratinocytes treated
with 1a, 1b, 2a, 2b, 3b and 4a (1 μM). Emission was recorded on a confocal
microscope via a lambda scan with laser excitation of 405 nm.
With the emission behaviour on a whole cell basis understood,
we next sought to conduct a series of imaging experiments to
gain insights into the differences in localisation behaviour as a
function of compound structure. A co-localisation approach
(see ESI Section 4) was employed in which HaCaT keratinocytes
treated with 1a-5b were co-treated with
a range of
commercially available organelle-specific dyes with different
absorption/emission properties. By spectral analysis, it is
possible to extract the components of the overall fluorescence
signal from each compound and hence overlay the two to
understand the extent of overlap of the signals. For the analysis,
we used a combination of visual inspection along with a
Pearson’s Correlation Coefficient (PCC) statistical analysis (see
ESI Section 6). PCC measures the covariance of each pixel, and
ranges from +1 to -1, indicating a perfect linear relationship and
a perfect inverse relationship, respectively.15
Figure 4 Co-localisation images of lipophilic acids 1a and 1b compared to BODIPY
ER-Tracker™ Red and Nile Red.
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In contrast to the diffuse, non-specific localisation exhibited by significant co-localisation (PCC
ARTICLE
=
0.69, 0.63ViewanArdticle0O.n7lin3e,
DOI: 10.1039/D0OB01153C
lipophilic acids 1a and 1b, the behaviour of the corresponding respectively) with the lysosomal fluorophore, LysoTracker™
methyl esters 2a and 2b was characterised by the presence of Deep Red (Fig. 6) although some subtle differences were
bright punctate spots distributed throughout the cells (Figure evident between the three compounds. Comparing 3a (tert-
5).17 These spots were shown to colocalise with a BODIPY™ butyl ester) with 3b (methyl ester), it was apparent that 3a
493/503 Lipid Droplet probe (PCC = 0.65 and 0.66), and given showed some evidence of localisation in the endoplasmic
the high degree of lipophilicity of 2a and 2b (simulated logP = reticulum (PCC compared to ER-Tracker = 0.48), especially as
5.95 and 6.89, respectively) it is reasonable to suggest that the much of the fluorescence signal was polarised towards one side
compounds are trafficked to lipid rich areas, and are perhaps of the nucleus in some cells. 3a also exhibited some overlap
actively packaged into vesicles for transport out of the cell. The with the lipophilic fluorophore (PCC = 0.42), Nile Red,
subtle nuclear envelope staining evidenced in the case of 1a and suggesting that the presence of the hydrophobic tert-butyl ester
1b was not evident with the corresponding esters, but some group causes enough of an increase in logP (estimated30 logP =
mitochondrial staining according to MitoTracker™ Deep Red FM 4.49 for 3a versus 3.41 for 3b) to bring about some non-specific
co-localisation was evident, although a low PCC value (PCC = hydrophobic interactions with more lipophilic cellular
0.33 and 0.32, respectively) indicated that this was not a very compartments (see ESI). The less hydrophobic compounds, 3b
specific effect, with the majority of the fluorescence signal and 3c showed more specific lysosomal localisation according
evident in the bright punctate structures.
to the statistical analysis (see ESI), although with some subtle
indicators of Golgi, ER and mitochondrial staining.
Figure
6 Co-localisation images of basic alkyl amines 3a-c compared to
LysoTracker™ Deep Red.
When the localisation behaviour of 3b was compared to the
corresponding N-acetyl phenylpiperazine 4a,
a striking
difference was observed. Once the basic phenylpiperazine free
amine is blocked, 4a is likely to remain neutrally charged in cells
since the pKaH of the conjugated amine group is unlikely to be
high enough to be protonated at physiological pH due to
significant π-donation into the extended diphenylacetylene
structure. 4a exhibited much more membrane-based
localisation, where it was brightest towards the exterior of the
cell and around the nuclear envelope (Fig. 7). The compound
exhibited very weak correlation with the ER and mitochondria
(PCC = 0.27 and 0.09, respectively), in contrast with 3a-3c,
although some lysosomal co-localisation was evident (PCC =
0.22), perhaps suggesting that 4a is a substrate for enzymatic
Figure 5 Co-localisation images of lipophilic esters 2a and 2b compared to Nile Red
and MitoTracker Red™.
Upon moving from the highly lipophilic tetrahydroquinoline
diphenylacetylenes 1a-2b to the more polar 3a-3c, a significant
shift in the localisation behaviour was observed. The presence
of the strongly basic unfunctionalised amine groups in 3a-3c
would suggest that these compounds localise to cellular
compartments in which the pH was lower.29 Indeed, 3a-3c were
shown to exhibit distinct lysosomal localisation according to
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degradation through deacetylation, releasing the secondary fluorophores than the sulphonamide 5b within this series of
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DOI: 10.1039/D0OB01153C
amine 3b. The cellular behaviour of 4a was distinct from all the diphenylacetylene fluorophores. Perhaps by reducing the
compounds studied, and underlined the effect of the removal lipophilicity of derivatives of 5b and/or increasing the linker
of ionisable groups along with a structure of relatively higher length for the sulphonamide moiety, more specific ER
polarity (estimated30 logP of 4a/2a = 4.03/5.95) in guiding the localisation could be observed. The subtleties in the
compound to more polar cellular environments.
fluorescence localisation behaviour of 5b does, however,
Where the other compounds had displayed somewhat underline the need to conduct co-localisation studies that
heterogenous behaviour, triphenylphosphonium-substituted employ several fluorophores and techniques along with careful
5a was found to exhibit much more specific localisation. As analysis of each image.
shown in Fig. 8, co-localisation studies involving the
Mitotracker™ mitochondrial dye and 5a highlighted a strong
correlation (PCC = 0.71) between the two signals, indicating that
the compound was likely to be localised in the mitochondria, as
anticipated.22 Statistical analysis also indicated strong
correlation with the ER (PCC = 0.75) and lipophilic (PCC = 0.63)
dyes, however, it was adjudged that the physiology of those
structures associated with 5a were distinct from that of the ER
and lipid environments (particularly in comparison with 2a/2b
for the latter), and hence underlined the need to carefully
examine the cellular images rather than rely solely on the
statistical analysis.
Figure 8 Co-localisation images of substituted triphenylphosphonium compound
5a compared to MitoTracker™ Red and ER tracker Red.
Figure 7 Colocalisation images of acyl amine 4a compared to LysoTracker™ Deep
Red and Nile Red.
Imaging of the tosyl sulphonamide substituted compound
5b (Fig. 9) indicated co-localisation with the ER dye as initially
anticipated (PCC = 0.62),24,31–33 however, analysis of the images
suggested that this was not an entirely specific effect. Indeed,
while weaker correlations were evident with Mitotracker™ (PCC
Figure 9 Colocalisation images of sulphonamide 5b compared to ER tracker Deep
Red and Nile Red.
= 0.41) or Lysotracker™ (PCC = 0.46), the lipophilic dye Nile Red
Next, the relative intensities of the fluorescence in a cellular
was shown to exhibit significant co-localisation with 5b as
environment was assessed. Individual wells of cultured HaCaT
indicated by a high PCC value (0.86). Given the relatively high
epithelial cells were pre-treated with the same concentration of
lipophilicity of 5b (simulated logP = 5.2), as a result of the
each compound (1 μM) and then imaged. Using ImageJ for the
presence of the tosyl group, this behaviour is not entirely
analysis, thresholding was applied to select the cells within the
unexpected. In fact, comparison with 1a and 1b shows that 5b
images and omit any background devoid of cells, and the
behaves similarly to the lipophilic carboxylate compounds i.e.
average intensity per pixel in each image was then calculated
general localisation in non-polar cellular environments such as
and used as the comparison metric. The entirety of each cell
the ER, although without the very bright punctate spots
was included in this analysis rather than just the labelled regions
displayed by the even more lipophilic 2a/2b. Indeed, the
in order to ensure that, whether a compound localised in highly
correlation statistics actually showed that lipophilic
punctate spots or exhibited more broad, diffuse localisation,
carboxylates 1a/1b displayed more specific ER localisation than
the variation was properly accounted for to give a valid
5b (see ESI) and are, therefore, more appropriate ER
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comparison between compounds, rather than just focusing on the fluorescence behaviour of compounds possessing ionisable
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DOI: 10.1039/D0OB01153C
different organelles.
groups (1a/1b, 3a-c, 5a/5b) was heavily influenced by pH
changes, while those compounds lacking ionisable groups
(2a/2b and 4a) exhibited only minor differences as a function of
pH. The carboxylic acids, 1a/1b, exhibited a blue-shift and a
significant increase in intensity at pH 7.5 and 9 compared to the
emission spectrum at pH 5, indicating that deprotonation of the
carboxylic acid causes a major change in the excited state
properties of these compounds. The basic amines, 3a-3c,
displayed a linear increase in emission intensity as the pH was
increased from 5, to 7.5, to 9, along with a mild red shift (ca. 15
nm from pH 5 to 9 for 3a), thus demonstrating that protonation
of the basic amine group elicits an electron-withdrawing
inductive influence upon the conjugated nitrogen atom that
diminishes the strength of charge transfer and, hence, reduces
fluorescence intensity. A very similar effect was also observed
with 5a due to the compound’s basic tertiary amine group. The
sulfonamide 5b exhibited very similar emission spectra at pH 5
and 7.5, but a 20 nm blue-shift and minor increase in intensity
was observed at pH 9, indicating that the emission behaviour of
this compound is also modulated as the pH of the solution nears
the pKa of the sulfonamide proton.
The results of the whole-cell relative intensity experiments
and the pH study demonstrate that, for fluorophores that are
highly sensitive towards local environment, the observed
fluorescence intensity within a cell is not simply a function of
the quantum yield. This is foremost evidenced by compounds
3a-c, which displayed the lowest quantum yields in the
photophysical characterisation (Table 1) yet exhibited some of
the highest intensities in the whole cell intensity study (Fig. 10)
even whilst localising in an acidic environment that attenuates
charge transfer from the key donor nitrogen atom in the
conjugated diphenylacetylene structure according to the pH
experiments. It is, therefore, clear that cellular compartments
exhibit unique environmental and solvation characteristics
whose effects on photophysical properties such as fluorescence
intensity are difficult to predict a priori using measurements
determined in a single-component system. This underlines the
need to undertake careful comparative studies involving both
cellular and solvent-based measurements to inform meaningful
predictions of cellular fluorescence behaviour.
1.0
0.5
0.0
1a 1b 2a 2b 3a 3b 3c 4a 5a 5b
Figure 10 Normalised whole-cell relative fluorescence intensity of each compound (1
M) in HaCaT keratinocytes. A 405 nm laser was used to excite the compounds.
In these experiments (Fig. 10 and ESI Section 5), it was clear that
primary amine 3c and the corresponding N-tosyl sulphonamide
5b provided the most intense fluorescence signal under these
experimental conditions. The greater intensity may be a
function of the 405 nm excitation being closer to the λmax of 3c
and 5b (377 & 382 nm, respectively, in toluene) as a result of
the stronger donor capability of the N,N-disubstituted aniline
structure in comparison to the more strained phenylpiperazine
systems. Conversely, however, those compounds with the even
stronger donor, the rigid tetrahydroquinoline of 1a-2b,
exhibited relatively weaker fluorescence signal, suggesting that
cellular localisation plays a greater role in the relative intensity
within this family of compounds. Furthermore, the other free
amines, particularly 3b, exhibited similarly strong fluorescence
to 3c, and it may be that the lysosomal environment is
particularly insulating for these compounds, limiting non-
radiative processes that are presumably more prevalent in a
more aqueous environment. This change in fluorescence
intensity as a function of environment is further echoed when
comparing the acid 1a (ER and cytoplasmic localisation – more
polar) to the corresponding methyl ester 2a (lipid droplets – less
polar), although those compounds with the naphthalene
acceptor (2a/2b) appeared to be outliers from this trend. The
substituted amine 4a also showed weaker fluorescence
intensity compared to the related 3b and 5a, which correlates
to the observed, more polar, membrane-based localisation and
presumably translates to an increase in non-radiative
quenching of the excited state – a common occurrence with
donor-acceptor fluorophores in more polar, aqueous
enviroments.34
We also performed a series of photostability experiments
comparing the bleaching rate of each compound in live and
fixed (PFA) cells in order to assess their suitability as a general
class of fluorophore for a range of imaging experiments. As
shown in Fig. 11, the compounds typically exhibited similar T1/2
in live and fixed cells although generally with a slight increase in
fixed cells as is often typical. However, while the photophysical
properties of the compounds broadly remained similar upon
fixation, the cellular localisation was found to vary significantly
(see ESI, Section 8).
The major influence of the local environment on the
observed fluorescence was further underlined when we
examined the effect of pH. Solutions of each compound were
prepared in buffers of pH 5, 7.5 and 9 in order to reflect the
typical pH that the compounds may encounter in different
cellular compartments, and the emission spectrum of each
solution was recorded between 400-700 nm with a common,
fixed excitation wavelength of 360 nm. The emission spectra at
each pH were compared (see ESI, Section 6) and showed that
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Journal Name
b
Compound
LogP
5.13
LogDa
2.17
pKa
pKaHc
Localisation
View Article Online
DOI: 10.1039/D0OB01153C
1a
4.5
4.5
-
-
-
-
-
ER
Live
Fixed (PFA)
Lipophilic areas
1.0
0.5
0.0
1b
2a
2b
6.79
5.95
6.89
3.83
5.95
6.89
ER
Lipophilic areas
Lipid droplets
Lipophilic areas
-
Lipid droplets
Lipophilic areas
3a
3b
3c
4a
4.49
3.41
3.45
4.03
3.27
2.19
2.50
4.03
-
-
-
-
8.6
8.6
8.3
3.2
Lysosome
Lysosome
Lysosome
1a 1b 2a 2b 3a 3b 3c 4a 5a 5b
Figure 11 Photostability assessment of each compound in live and fixed HaCaT
keratinocytes. A 405 nm laser was used to excite the compounds.
Significant changes in fluorescent probe localisation are
common when cells are fixed due to changes in subcellular
structure, membrane integrity and permeability, local polarity,
pH and chemical modification. Given that the photophysical
properties of the compounds are very similar when fixed and
live cells are compared, we can be confident that the
compounds remain unmodified on fixation using a variety of
methods (paraformaldehyde, alcohols) but instead are subject
to changes in localisation due to the effect of fixation on the cell,
and hence, we had to exercise great care when analysing
fluorescence data from fixed cell samples. In contrast,
compounds 1a-5b were found to exhibit highly consistent
localisation in all live experiments, and have good utility in this
modality for a variety of imaging applications.
Cell membrane
Cytoplasm
5a
5b
8.10
5.20
5.74
5.20
-
6.2
-
Mitochondria
10.1
ER
Lipophilic areas
Table 2: Calculated physical properties of compounds 1a-5b related to cellular
behaviour. All physical properties were calculated using ACD/I-Lab 2.0.30 a LogD at pH 7.4.
b pKa of the strongest acidic proton. C pKaH of the strongest basic group.
Conclusions
A group of structurally varied diphenylacetylene fluorophores
have been described and characterised as novel imaging agents
for a variety of cellular experiments. We have shown that
through modification of their component structures in a
considered manner the localisation behaviour of this class of
compound can be directly influenced. This was most obviously
evidenced by examining the effect of going from a highly
lipophilic tetrahydroquinoline donor system (2a) to a much
more polar phenylpiperazine (3b) and to the corresponding N-
acetyl compound (4a), where we observe a shift in localisation
from lipid-based to lysosomal to membrane-based that is
commensurate with the changes in logP/logD and pKaH
between structures. The observations and guidelines observed
herein can assist in directing the design and synthesis of not
only the next generation of this class of fluorophore but also of
other donor-acceptor fluorophore systems, which are a
burgeoning class of compound both in the cellular imaging
arena and a wide range of other applications including liquid
crystals, plastics and sensor applications.
With all of these analyses in hand, we can begin to draw
meaningful molecular conclusions (Table 2) on the cellular
behaviour of the compounds. Perhaps the most significant
determinant of cellular localisation within this family is the
presence (or absence) of ionisable groups, which tend to
override the inherent hydrophobicity of the core
diphenylacetylene structure. For those compounds without
ionisable groups, structures with high logP values exhibit
localisation in more lipophilic cellular compartments. Reduction
of lipophilicity, as is the case when comparing the
tetrahydroquinoline 2a and the N-Ac phenylpiperazine 4a,
causes
a
pronounced shift to more membrane-
based/cytoplasmic environments. The addition of cellular
recognition motifs (5a/5b) resulted in selective uptake to those
areas, although the sulphonamide 5b showed relatively
heterogenous localisation behaviour compared to the
triphenylphosphonium compound 5a. Importantly, 5a/5b
showed markedly different behaviour from their respective
parent compounds (3b and 3c, respectively), indicating that this
structural class of fluorophore is suitable for derivatisation with
other recognition motifs, and a wide variety of modifications Experimental
could be easily implemented to further influence cellular
localisation behaviour.10
General chemical information
Reagents were purchased from Sigma-Aldrich, Acros Organics,
Alfa-Aesar and Fluorochem. Reagents were purified, if required,
by recrystallisation or distillation/sublimation under vacuum.
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Solvents were used as supplied from Fisher Scientific or Sigma replaced with Ar. Compound 7b19 (1.41 g, 7.14 mmol),
View Article Online
DOI: 10.1039/D0OB01153C
Aldrich, and dried before use if required with appropriate drying Pd(PPh3)2Cl2 (470 mg, 0.67 mmol) and CuI (132 mg, 0.69 mmol)
agents. Thin-layer chromatography (TLC) was conducted using were added under Ar and the resultant suspension was stirred
Merck Millipore silica gel 60G F254 25 glass plates and/or TLC- at RT for 72 h. The suspension was diluted with heptane/EtOAc
PET foils of aluminium oxide with fluorescent indicator 254 nm (9:1), passed through a short Celite/SiO2 plug and the extracts
(40 × 80 mm) with visualisation by UV lamp or appropriate were evaporated to give a crude solid. This was purified by SiO2
staining agents. Flash column chromatography was performed chromatography (9:1, heptane/EtOAc), followed by
using SiO2 from Sigma-Aldrich (230-400 mesh, 40-63 μM, 60 Å), recrystallisation from EtOH to give compound 2b as a yellow
and monitored using TLC. NMR spectra were recorded using solid (1.76 g, 60%): 1H NMR (700 MHz, CDCl3) δ 1.23 (d, J = 6.6
Varian VNMRS-700, Varian VNMRS-600, Bruker Avance-400 or Hz, 6H), 1.30 (s, 6H), 1.66 – 1.73 (m, 2H), 3.18 – 3.27 (m, 2H),
Varian Mercury-400 spectrometers operating at ambient probe 3.98 (s, 3H), 4.16 (hept, J = 6.6 Hz, 1H), 6.66 (d, J = 8.6 Hz, 1H),
temperature. NMR peaks are reported as singlet (s), doublet (d), 7.28 (dd, J = 8.6, 2.1 Hz, 1H), 7.41 (d, J = 2.1 Hz, 1H), 7.62 (dd, J
triplet (t), quartet (q), broad (br), septet (sept), combinations = 8.5, 1.7 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.88 (d, J = 8.5 Hz, 1H),
thereof, or as a multiplet (m), with reference to the following 8.01 (d, J = 1.5 Hz, 1H), 8.05 (dd, J = 8.6, 1.7 Hz, 1H), 8.56 (s, 1H);
deuterated solvent signals: CDCl3 (1H = 7.26 ppm, 13C = 77.0 13C NMR (176 MHz, CDCl3) δ 18.9, 29.9, 32.0, 36.5, 36.6, 47.2,
ppm), (CD3)2SO (1H = 2.50 ppm, 13C = 39.5 ppm). ESMS was 52.2, 87.1, 93.6, 108.1, 110.4, 124.4, 125.8, 127.4, 127.7, 129.1,
performed using a TQD (Waters Ltd., UK) mass spectrometer 129.4, 129.4, 130.0, 130.8, 130.9, 131.3, 131.5, 135.3, 144.6,
with an Acquity UPLC (Waters Ltd., UK), and accurate mass 167.1; IR (ATR) vmax/cm–1 2969m, 2929m, 2867w, 2191m,
measurements were obtained using a QtoF Premier mass 1717s, 1625m, 1605s, 1594s, 1434s, 1280s, 1188s, 1179s,
spectrometer with an Acquity UPLC (Waters Ltd., UK). ASAP 1096m, 892m; MS(ES): m/z = 412.2 [M+H]+; HRMS (ES) calcd.
measurements were performed using an LCT Premier XE mass for C28H30NO2 [M+H]+: 412.2271, found 412.2273.
spectrometer and an Acquity UPLC (Waters Ltd., UK). IR spectra
were recorded using a Perkin Elmer FTIR spectrometer. Tert-butyl (2E)-3-(4-ethynylphenyl)prop-2-enoate, 7c
Physicochemical values were estimated using ACD/I-Lab 2.0
Et3N (250 mL) was degassed by sparging with Ar for 1 h. 4-
online software (https://www.acdlabs.com/resources/ilab/).30
Bromobenzaldehyde (18.5 g, 100.0 mmol), Pd(PPh3)2Cl2 (1.4 g,
2.00 mmol), CuI (0.38 g, 2.00 mmol) and trimethylsilylacetylene
(15.2 mL, 110.0 mmol) were then added under Ar and the
resultant suspension was stirred at RT for 16 h. The suspension
was diluted with heptane, passed through a short Celite/SiO2
Methyl
(2E)-3-(4-{2-[4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroq
uinolin-6-yl]ethynyl}phenyl)prop-2-enoate, 2a
A solution of compound 620 (1.72 g, 5.22 mmol) in Et3N (34 mL)
was degassed by sonication, before the atmosphere was
replaced with Ar. Compound 7a16 (1.02 g, 5.48 mmol),
plug and the extracts were evaporated to give a crude dark solid
o
(24 g). This was purified by Kugelrohr distillation (130-150 C,
9.0 Torr) to give compound 8 as an off-white solid (21.5 g,
Pd(PPh3)2Cl2 (370 mg, 0.53 mmol) and CuI (99 mg, 0.52 mmol)
>100%), which was carried to the next step without further
were added under Ar and the resultant suspension was stirred
purification. Tert-butyl diethylphosphonoacetate (14.4 mL, 61.5
at RT for 72 h. The suspension was diluted with heptane, passed
mmol) and LiCl (2.54 g, 60.0 mmol) were added to anhydrous
through a short Celite/SiO2 plug and the extracts were
THF (100 mL) at 0 oC and the resultant solution was stirred for
evaporated to give a crude solid. This was purified by SiO2
chromatography (9:1, heptane/EtOAc), followed by
recrystallisation from EtOH to give compound 2a as a yellow
solid (1.16 g, 58%): 1H NMR (700 MHz, CDCl3) δ 1.22 (d, J = 6.6
Hz, 6H), 1.28 (s, 6H), 1.64 – 1.73 (m, 2H), 3.17 – 3.25 (m, 2H),
3.81 (s, 3H), 4.15 (hept, J = 6.6 Hz, 1H), 6.43 (d, J = 16.0 Hz, 1H),
6.64 (d, J = 8.7 Hz, 1H), 7.24 (dd, J = 8.6, 2.1 Hz, 1H), 7.36 (d, J =
2.1 Hz, 1H), 7.45 – 7.50 (m, 4H), 7.67 (d, J = 16.0 Hz, 1H); 13
15 min, whereupon compound 8 (10.1 g, 50.0 mmol) was
added. To this solution was slowly added DBU (8.2 mL, 55.0
mmol), and the resultant slurry was stirred at RT for 16 h. This
was poured into crushed ice, and extracted with EtOAc. The
organics were washed with H2O and brine, dried (MgSO4) and
evaporated to give a crude white solid (18 g). This was purified
by recrystallisation from heptane to give compound 9 as a
C
1
colourless crystalline solid (10.99 g, 73%): H NMR (400 MHz,
NMR (176 MHz, CDCl3) δ 18.9, 29.9, 32.0, 36.5, 36.6, 47.2, 51.7,
86.8, 94.1, 108.0, 110.4, 117.6, 126.6, 127.9, 129.3, 130.8,
131.5, 131.5, 133.0, 144.2, 144.6, 167.4; IR (ATR) vmax/cm–
1 3031w, 2969m, 2930m, 2867w, 2190m, 1716s, 1632m, 1592s,
1433s, 1322s, 1269s, 1203s, 1168s, 1139s, 829m; MS(ES): m/z =
388.2 [M+H]+; HRMS (ES) calcd. for C26H30NO2 [M+H]+:
388.2271, found 388.2271.
CDCl3) δ 0.25 (s, 9H), 1.53 (s, 9H), 6.36 (d, J = 16.0 Hz, 1H), 7.40
– 7.49 (m, 4H), 7.54 (d, J = 16.0 Hz, 1H). Compound 9 (10.95 g,
36.4 mmol) and K2CO3 (7.55 g, 54.6 mmol) were added to
MeOH/DCM (200 mL, 1:3) and the resultant solution was stirred
at RT for 3 h. The solution was diluted with DCM, and the
organics washed with sat. NH4Cl and H2O, dried (MgSO4) and
evaporated to give a crude solid (8 g). This purified by
recrystallisation from heptane to give compound 7c as a
Methyl
1
colourless crystalline solid (5.96 g, 72%): H NMR (600 MHz,
6-{2-[4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin
-6-yl]ethynyl}naphthalene-2-carboxylate, 2b
A solution of compound 620 (2.55 g, 7.75 mmol) in Et3N (60 mL)
was degassed by sonication, before the atmosphere was
CDCl3) δ 1.53 (s, 9H), 3.17 (s, 1H), 6.36 (d, J = 16.0 Hz, 1H), 7.43
– 7.49 (m, 4H), 7.54 (d, J = 16.0 Hz, 1H); 13C NMR (151 MHz,
CDCl3) δ 28.1, 79.0, 80.6, 83.2, 121.2, 123.5, 127.7, 132.5, 135.0,
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142.4, 166.0; IR (ATR) vmax/cm–1 3281m, 3064w, 3000w, 2980w, 6.94 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H),
View Article Online
2936w, 1691s, 1641m, 1370m, 1296s, 1153s, 1002m, 980m, 7.67 (d, J = 16.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 2H); 13
DOI: 10C.10N3M9/RD0(1O5B101M15H3zC,
832s; MS(ASAP): m/z = 228.1 [M+H]+; HRMS (ASAP) calcd. for DMSO-d6) δ 44.9, 47.5, 51.5, 87.6, 92.7, 110.7, 114.5, 118.3, 124.9,
C15H16O2 [M+H]+: 228.1150, found 228.1161.35
128.6, 131.3, 132.5, 133.5, 143.6, 151.2, 166.6; IR (ATR) vmax/cm–
1 3039w, 2952w, 2909w, 2830w, 2204w, 2173w, 1698s, 1630s,
1593m, 1518m, 1312m, 1243s, 1168s, 987m, 831s, 817s;
MS(ASAP): m/z = 347.2 [M+H]+; HRMS (ASAP) calcd. for C22H23N2O2
[M+H]+: 347.1760, found 347.1736.
1-(4-Iodophenyl)piperazine, 10
To a vigorously stirred solution of 1-phenylpiperazine (20.5 mL,
134.0 mmol) in AcOH/H2O (3:1, 84 mL) at 55 oC was added
dropwise a solution of ICl (24.0 g, 148.0 mmol) in AcOH/H2O
(3:1, 124 mL). The resultant slurry was further stirred for 1 h and 2-Chloro-N-(4-iodophenyl)-N-methylacetamide, 11
then cooled to RT and stirred for a further 16 h. The slurry was
4-Iodo-N-methylaniline (13.9 g, 59.7 mmol) was dissolved in DCM
poured into crushed ice, and 20% aq. NaOH added until the
(100 mL), whereupon chloroacetyl chloride (5.2 mL, 65.7 mmol) and
solution was at pH 13. The resultant precipitate was isolated by
Et3N (9.2 mL, 65.7 mmol) were added and the resultant mixture was
filtration, washed with H2O and dried to give a crude yellow
stirred for 16 h at RT. The solution was then diluted with DCM,
solid. This was purified by recrystallisation from MeOH/H2O
washed with sat. NH4Cl and H2O, dried (MgSO4) and evaporated to
give a crude solid. This was purified by SiO2 chromatography (8:2,
(1:1) to give compound 10 as a beige solid (24.2 g, 63%): 1H NMR
(600 MHz, CDCl3) δ 2.97 – 3.03 (m, 4H), 3.07 – 3.14 (m, 4H), 6.65
heptane/EtOAc) to give compound 11 as an off-white solid (8.26 g,
– 6.69 (m, 2H), 7.48 – 7.52 (m, 2H); 13C NMR (151 MHz, CDCl3) δ
45%): 1H NMR (600 MHz, CDCl3) δ 3.28 (s, 3H), 3.83 (s, 2H), 6.95 –
45.9, 49.9, 81.4, 118.0, 137.7, 151.3; IR (ATR) vmax/cm–1 3032w,
7.06 (m, 2H), 7.78 (d, J = 8.1 Hz, 2H); 13C NMR (151 MHz, CDCl3) δ
2955w, 2829m, 1582m, 1489m, 1243s, 914m, 803s;
37.9, 41.2, 93.9, 129.0, 139.3, 142.4, 166.1; IR (ATR) vmax/cm-
MS(ASAP): m/z = 289.0 [M+H]+; HRMS (ASAP) calcd. for C10H13N2I
1 2996w, 2947w, 1664s, 1480m, 1371m, 1260m, 1009m, 824m,
[M]+: 288.0124, found 288.0114.36
552s; MS (ASAP) m/z = 310.0 [M+H]+; HRMS (ASAP) calcd. for
C9H10ONICl [M+H]+: 309.9496, found 309.9494.
Tert-butyl
(2E)-3-(4-{2-[4-(piperazin-1-yl)phenyl]ethynyl}phenyl)prop-2-enoa
te, 3a
2-Amino-N-(4-iodophenyl)-N-methylacetamide, 13
Et3N (80 mL) was degassed by sparging with Ar for 1 h. Compound Compound 11 (8.23 g, 26.6 mmol) and potassium phthalimide (7.39
10 (2.16 g, 7.5 mmol), compound 7c (1.80 g, 7.88 mmol), g, 39.9 mmol) were dissolved in DMF (40 mL) and the resultant
Pd(PPh3)2Cl2 (260 mg, 0.39 mmol) and CuI (71 mg, 0.39 mmol) were mixture was heated to 120 oC and stirred for 5 h. The solution was
then added under Ar and the resultant suspension was stirred at cooled, and diluted with H2O. The resultant precipitate was isolated
60 oC for 24 h. The solvent was then evaporated to give a crude by filtration, washed with H2O and then recrystallised from EtOH to
solid which was purified by SiO2 chromatography (9:1, DCM/MeOH, give compound 12 as a white solid (9.26 g, 83%), which was carried
1% Et3N) and then recrystallisation from MeOH to give compound 3a directly to the next step. Compound 12 (9.2 g, 11.51 mmol) was
as a yellow solid (2.11 g, 72%): 1H NMR (400 MHz, CDCl3) δ 1.53 (s, dissolved in EtOH (50 mL) and the resultant mixture was heated to
9H), 3.22-3.28 (m, 4H), 3.38-3.45 (m, 4H), 6.37 (d, J = 15.9 Hz, 1H), reflux, whereupon hydrazine hydrate (64%, 1.22 mL, 24.09 mmol)
6.77 – 6.95 (m, 2H), 7.33 – 7.53 (m, 6H), 7.56 (d, J = 15.9 Hz, 1H); 13
C
was added and the mixture was stirred at reflux for 3 h. The
NMR (176 MHz, CDCl3) δ 28.2, 80.6, 87.8, 92.2, 113.1, 115.2, 120.5, suspension was then cooled and the resultant precipitate was
125.5, 127.8, 127.8, 131.7, 132.8, 133.9, 142.7, 166.2; IR filtered. The filtrate was evaporated to give a crude oily solid (7 g),
(ATR) vmax/cm–1 2967w, 2916w, 2830w, 2212w, 1687s, 1629m, which was purified by SiO2 chromatography (9:1, DCM/MeOH with
1595m, 1518m, 1326m, 1241m, 1159m, 1128m, 986m, 831s, 819s; 1% Et3N) to give compound 13 as a crystalline white solid (5.97 g,
MS(ASAP): m/z = 389.2 [M+H]+; HRMS (ASAP) calcd. for C25H29N2O2 94%): 1H NMR (600 MHz, CDCl3) δ 3.13 (s, 2H), 3.25 (s, 3H), 6.92 (d, J
[M+H]+: 389.2229, found 389.2231.
= 8.0 Hz, 2H), 7.74 (d, J = 8.0 Hz, 2H); 13C NMR (151 MHz, CDCl3) δ
37.3, 44.1, 93.3, 129.1, 139.1, 142.4, 172.6; IR (ATR) vmax/cm–
1 3365m, 3301w, 3055w, 2947w, 2885w, 1649s, 1570m, 1486m,
1423m, 1345m, 1109m, 1013m, 892s; MS(ES): m/z = 291.1 [M+H]+;
HRMS (ES) calcd. for C9H12N2OI [M+H]+: 290.9994, found 291.0012.
Methyl
(2E)-3-(4-{2-[4-(piperazin-1-yl)phenyl]ethynyl}phenyl)prop-2-enoa
te, 3b
Et3N (150 mL) was degassed by sparging with Ar for 1 h. Compound
10 (4.50 g, 15.6 mmol), compound 7a (3.05 g, 16.4 mmol),
N-(2-Aminoethyl)-4-iodo-N-methylaniline, 14
Pd(PPh3)2Cl2 (550 mg, 0.78 mmol) and CuI (150 mg, 0.78 mmol) were Compound 13 (5.72 g, 19.72 mmol) was dissolved in anhydrous
then added under Ar and the resultant suspension was stirred at toluene (50 mL) under N2, whereupon BH3.Me2S (2.0 M, 10.35 mL,
60 oC for 24 h. The solvent was then evaporated to give a crude 20.70 mmol) was added and the resultant solution was stirred at
solid which was purified by SiO2 chromatography (9:1, DCM/MeOH, reflux for 16 h. The solution was cooled, and 10% Na2CO3 was added,
1% Et3N) and then recrystallisation from MeOH to give compound 3b whereupon the solution was stirred vigorously for 10 mins. The
as a yellow solid (2.74 g, 51%): 1H NMR (600 MHz, DMSO-d6) δ 2.82- solution was then diluted with EtOAc, washed with H2O and brine,
2.94 (m, 4H), 3.14-3.24 (m, 4H), 3.73 (s, 3H), 6.67 (d, J = 16.0 Hz, 1H), dried (MgSO4) and evaporated to give a crude yellow oil (4.4 g). This
12 | J. Name., 2012, 00, 1-3
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was purified by SiO2 chromatography (9:1, DCM:MeOH, 0.5% Et3N) Compound 3b (0.35 g, 1.01 mmol) was dissolved in anhydrous DMF
View Article Online
to give compound 14 as a yellow oil (3.46 g, 64%), which was carried (10 mL) under Ar, whereupon K2CO3 (0.16D7OgI:,110..1203m9/mDo0Ol)Ba0n1d15(33C-
immediately to the next step: 1H NMR (400 MHz, CDCl3) δ 2.90 (t, J = bromopropyl)triphenylphosphonium bromide (0.47 g, 1.01 mmol)
6.6 Hz, 2H), 2.93 (s, 3H), 3.36 (t, J = 6.65 Hz, 2H), 6.47 – 6.57 (m, 2H), were added and the resultant solution was stirred at 80 oC for 16 h.
7.41 – 7.49 (m, 2H).
The solution was cooled, diluted with H2O and extracted with EtOAc.
The organics were washed with H2O and brine, dried (MgSO4) and
evaporated to give a crude yellow solid (0.5 g). This was purified by
Methyl (2E)-3-[4-(2-{4-[(2-
aminoethyl)(methyl)amino]phenyl}ethynyl)phenyl]prop-2-enoate,
3c
SiO2 chromatography
(95:5,
DCM/MeOH)
and
further
recrystallisation from a DCM/heptane solution to give compound 5a
as a yellow solid (0.44 g, 60%): 1H NMR (600 MHz, CDCl3) δ 1.82-1.91
Compound 14 (3.46 g, 12.53 mmol) was dissolved in Et3N (120 mL) (m, 2H), 2.52-2.58 (m, 4H), 2.74 (t, J = 6.3 Hz, 2H), 3.16-3.23 (m, 4H),
and the solution was degassed by sparging with Ar for 1 3.79 (s, 3H), 3.91-3.99 (m, 2H), 6.41 (d, J = 16.0 Hz, 1H), 6.77 – 6.84
h. Compound 7a (2.57 g, 13.8 mmol), Pd(PPh3)2Cl2 (440 mg, 0.63 (m, 2H), 7.32 – 7.42 (m, 2H), 7.39 – 7.52 (m, 4H), 7.64 (d, J = 16.0 Hz,
mmol) and CuI (120 mg, 0.63 mmol) were then added under Ar and 1H), 7.66-7.73 (m, 6H), 7.75-7.81 (m, 3H), 7.81 – 7.90 (m, 6H); 13C
the resultant suspension was stirred at 60 oC for 72 h. The solvent NMR (151 MHz, CDCl3) δ 19.8 (d, J = 3.2 Hz), 20.1 (d, J = 51.8 Hz), 47.9,
was then evaporated to give a crude solid which was purified by 51.7, 52.7, 57.1 (d, J = 16.5 Hz), 87.6, 92.5, 112.7, 114.9, 117.9, 118.2,
SiO2 chromatography (9:1, DCM/MeOH, 0.5% Et3N) to give 118.7, 125.8, 127.9, 130.4 (d, J = 12.5 Hz), 131.7, 132.7, 133.4, 133.6
compound 3c as a yellow solid (2.44 g, 58%): 1H NMR (600 (d, J = 10.0 Hz), 135.0 (d, J = 3.1 Hz), 144.0, 150.8, 167.3; IR
MHz, DMSO-d6) δ 2.94 (t, J = 7.0 Hz, 2H), 2.97 (s, 3H), 3.56 (t, J = 7.0 (ATR) vmax/cm–1 3362br, 2952w, 2876w, 2826w, 2206w, 1703m,
Hz, 2H), 3.73 (s, 3H), 6.67 (d, J = 16.0 Hz, 1H), 6.79 (d, J = 9.0 Hz, 2H), 1630m, 1595s, 1519s, 1437s, 1425m, 1324m, 1240s, 1169s, 1111s,
7.40 (d, J = 8.9 Hz, 2H), 7.47 – 7.54 (m, 2H), 7.67 (d, J = 16.0 Hz, 1H), 996s, 823s; MS(ES): m/z = 649.4 [M]+; HRMS (ES) calcd. for
7.74 (d, J = 8.3 Hz, 2H); 13C NMR (151 MHz, DMSO-d6) δ 36.3, 38.1, C43H42N2O2P [M]+: 649.2984, found 649.2991.
49.6, 51.5, 87.4, 93.1, 108.6, 111.9, 118.2, 118.2, 125.1, 128.6, 131.2,
132.7, 133.3, 143.6, 148.9, 166.6; IR (ATR) vmax/cm–1 3403br, 3042w,
Methyl (2E)-3-{4-[2-(4-{methyl[2-(4-
methylbenzenesulfonamido)ethyl]amino}phenyl)ethynyl]phenyl}p
2952w, 2888w, 2208m, 1698s, 1632m, 1608m, 1594s, 1522s, 1313s,
1169s, 1134s, 817s; MS(ASAP): m/z = 335.2 [M+H]+; HRMS (ASAP)
rop-2-enoate, 5b
calcd. for C21H23N2O2[M+H]+: 335.1760, found 335.1743.
Compound 3c (0.35 g, 1.05 mmol) was dissolved in DCM (30 mL),
whereupon p-toluenesulfonyl chloride (0.24 g, 1.26 mmol) and Et3N
Methyl
(0.18 mL, 1.26 mmol) were added and the resultant solution was
(2E)-3-(4-{2-[4-(4-acetylpiperazin-1-yl)phenyl]ethynyl}phenyl)prop stirred at RT for 16 h. The solution was diluted with DCM, washed
-2-enoate, 4a
with H2O, dried (MgSO4) and evaporated to give a crude yellow solid
(0.5 g). This was purified by SiO2 chromatography (99:1, DCM/MeOH)
to give compound 5b as a yellow solid (0.47 g, 92%): 1H NMR (600
MHz, CDCl3) δ 2.42 (s, 3H), 2.92 (s, 3H), 3.15 (q, J = 6.4 Hz, 2H), 3.48
(t, J = 6.4 Hz, 2H), 3.81 (s, 3H), 4.78 (t, J = 6.4 Hz, 1H), 6.43 (d, J = 16.0
Hz, 1H), 6.57 – 6.62 (m, 2H), 7.29 (d, J = 8.1 Hz, 2H), 7.34 – 7.39 (m,
2H), 7.45 – 7.52 (m, 4H), 7.66 (d, J = 16.0 Hz, 1H), 7.70 – 7.74 (m,
2H); 13C NMR (151 MHz, CDCl3) δ 21.5, 38.6, 40.3, 51.7, 52.2, 87.5,
92.8, 110.5, 112.0, 117.9, 126.0, 127.0, 128.0, 129.8, 131.6, 133.0,
133.3, 136.7, 143.6, 144.1, 148.8, 167.4; IR (ATR) vmax/cm–1 3241br,
2949w, 2921w, 2857w, 2210m, 1711m, 1632w, 1595s, 1524s,
1320m, 1156s, 1145s, 819s; MS(ASAP): m/z = 489.2 [M+H]+; HRMS
(ASAP) calcd. for C28H29N2O4S [M+H]+: 489.1848, found 489.1866.
Compound 3b (0.35 g, 1.01 mmol) was dissolved in DCM (10
mL), whereupon acetyl chloride (86 μL, 1.21 mmol) and pyridine
(98 μL, 1.21 mmol) were added and the resultant solution was
stirred at RT for 16 h. The solution was diluted with DCM,
washed with sat. NH4Cl and H2O, dried (MgSO4) and evaporated
to give a crude yellow solid (0.4 g). This was purified by
SiO2 chromatography (97.5:2.5, DCM/MeOH) to give compound
1
4a as a yellow solid (0.38 g, 97%): H NMR (600 MHz, CDCl3) δ
2.15 (s, 3H), 3.24 (t, J = 5.3 Hz, 2H), 3.27 (t, J = 5.3 Hz, 2H), 3.63
(t, J = 5.2 Hz, 2H), 3.78 (t, J = 5.3 Hz, 2H), 3.81 (s, 3H), 6.44 (d, J
= 16.0 Hz, 1H), 6.88 (d, J = 8.4 Hz, 2H), 7.41 – 7.47 (m, 2H), 7.46
– 7.54 (m, 4H), 7.67 (d, J = 16.0 Hz, 1H); 13C NMR (151 MHz,
CDCl3) δ 21.3, 41.1, 45.9, 48.3, 48.6, 51.7, 88.0, 92.1, 113.8, General photophysical information
115.6, 118.1, 125.7, 128.0, 131.8, 132.9, 133.7, 144.0, 150.5, Absorption and Emission Spectra
167.3, 169.0; IR (ATR) vmax/cm–1 3039w, 2947w, 2836w, 2205w,
Absorption and emission spectra were obtained using a CLARIOstar
2173w, 1699m, 1627s, 1594m, 1521m, 1446m, 1425m, 1311m,
1236s, 1164s, 994s, 835s, 822s; MS(ASAP): m/z = 388.2 [M+H]+;
HRMS (ASAP) calcd. for C24H24N2O3 [M+H]+: 388.1787, found
388.1793.
(BMG Labtech) plate reader. For absorption spectra, 100 µM
solutions were added to the wells of a UV-transparent 96-well
microplate (Corning) and absorbances were recorded at 1 nm
intervals. These curves were scaled using extinction coefficient
measurements at 400 nm made using a USB4000 spectrometer
(OceanOptics) coupled to a xenon white light source, with the dyes
held in a 3 mm path length optical cuvette (Hellma). Emission spectra
were obtained at 1 nm intervals from 10 µM solutions in microplates
using 355 (±5) nm excitation and an 8 nm emission bandwidth.
(3-{4-[4-(2-{4-[(1E)-3-methoxy-3-oxoprop-1-en-1-
yl]phenyl}ethynyl)phenyl]piperazin-1-yl}propyl)triphenylphospho
nium bromide, 5a
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 13
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Organic & Biomolecular Chemistry
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Page 14 of 17
ARTICLE
Journal Name
Quantum Yield Measurement
media. For all experiments a minimum of three repeats were
taken.
View Article Online
DOI: 10.1039/D0OB01153C
Quantum yields were measured by applying the reference sample
method37 in a 96-well plate format using the CLARIOstar plate
reader. Acridine yellow in ethanol (quantum yield = 0.4738) was used
as the reference. Seven wells of both reference and sample solutions
were produced by varying the concentration of the dye, aiming for
absorbances between 0.01 and 0.07. Across all compounds, these
corresponded to concentrations in the 10-100 µM range. The
absorbances of each well were recorded between 350-360 nm and
the corresponding fluorescence intensity was measured between
380-720 nm. Linear fits of the fluorescence against absorbance
datasets were performed for each sample using Origin (Originlab).
Quantum yields were obtained by dividing the gradient of the sample
data by the gradient of the reference data, correcting for solvent
refractive index using literature values.39
Imaging
Cells were imaged using either a Zeiss 880 Laser Scanning
Confocal Microscope (LSCM) with Airyscan detection or a Leica
SP5 LSCM. Live cells were imaged inside an environmental
chamber at 37 oC and 5% CO2. Samples were fixed by treatment
with paraformaldehyde/PBS (PFA, 4%) for 20 min at RT. Samples
imaged on the Zeiss 880 microscope were excited with a 405
nm, 488 nm, 594 nm or 633 nm laser and imaged with a Plan-
Apochromat Leica 63x 1.2NA HCX PL APO Oil UV long working
distance objective lens.
Colocalisation
Cells plated on 8-well chambered plates were co-stained with
compounds 1a-5b and live cell markers for various organelles
Fluorescence Lifetimes
Fluorescence decay measurements were performed as previously40 for 30 min and then washed twice with PBS. PBS was added to
using an Ortec modular time-correlated single photon counting each well for imaging purposes. Co-localisation analysis was
(TCSPC) system. 250 kHz pulsed excitation at 350 nm was obtained conducted using the ImageJ tool Coloc2, in which the
by using a barium borate (BBO) crystal to frequency double the 700 background was subtracted, and an ROI was selected in each
nm output of an optical parametric amplifier pumped by a case. The Zeiss 880 microscope was used for this work.
Ti:sapphire regenerative amplifier system (all Coherent). Emission
events were collected for 10 minutes through 435-650 nm emission MitoTracker® Red
filtering. Multiexponential decay fitting was performed in Origin Cells were co-stained with compounds 1a-5b and 200 nM
흌푹ퟐ
(Originlab), with
measurements.
values averaging 1.6 (±0.4) across all MitoTracker® Red (ThermoFisher, cat. no. M22425) in media for
30 min, then rinsed twice with PBS (pH 7.2). MitoTracker was
excited using a 633 nm laser.
pH versus fluorescence
Buffer solutions at pH 5 (0.1 M NaOAc), pH 7.5 (0.01 M
phosphate buffer, 0.027 M KCl, 0.137 M NaCl) and pH 9 (0.1 M
sodium borate) were freshly prepared and the pH was
measured immediately before the experiment (final pH values:
5.01, 7.50 and 9.01, respectively). Compound solutions in each
buffer were prepared by dilution from a freshly prepared 10 µM
stock solution in DMSO, to a concentration of 326 nM (3.3% v/v
DMSO). The solutions of each compound were analysed in
quartz cuvettes using a Varian Cary Eclipse Fluorescence
Spectrophotometer by recording the fluorescence spectrum
between 400-700 nm with excitation at 360 nm, with an
excitation slit width of 5 nm and an emission slit width of 10 nm.
The background fluorescence from the buffer solutions was
subtracted to give the fluorescence spectra of each compound
at pH 5, 7.5 and 9.
Nile Red
Cells were co-stained with compounds 1a-5b and 10 μg/mL Nile
Red (Fisher Scientific, cat. no. 11549116) in media for 30 min,
then rinsed twice with PBS (pH 7.2). Nile Red was excited using
a 594 nm laser.
BODIPY™ 493/503
Cells were co-stained with compounds 1a-5b and 2 μM
BODIPY™ 493/503 (Fisher Scientific, cat. no. D3922) in media for
30 min, then rinsed twice with PBS (pH 7.2). BODIPY™ 493/503
was excited using a 488 nm laser.
LysoTracker™ Red DND-99
Cells were co-stained with compounds 1a-5b and 50 nM
LysoTracker™ Red DND-99 (Fisher Scientific, cat. no. 12090146)
in media for 30 min, then rinsed twice with PBS (pH 7.2).
LysoTracker™ Red DND-99 was excited using a 594 nm laser.
General biological information
Cells
HaCaT human epidermal keratinocytes were purchased from a
commercial supplier (Thermo Fisher) and the cell line was
cultured at 37 oC/5% CO2 in DMEM (Gibco cat. no. 10566, high
glucose, GlutaMAX supplement) with 10% foetal calf serum and
1% penicillin/streptomycin. For experiments, cells were plated
in 8-well chambered coverslips at concentrations between 2.5
× 104 to 10.0 × 104 cells per mL. Cells were treated for 30 min
with 1 μM concentrations (unless otherwise stated) of
compounds 1a-5b (from 10 mM stock solutions in DMSO) in
BODIPY™ ER-Tracker™ Red
Cells were co-stained with compounds 1a-5b and 1 μM
BODIPY™ ER-Tracker™ Red (Fisher Scientific, cat. no. 11584746)
in media for 30 min, then rinsed twice with PBS (pH 7.2).
BODIPY™ ER-Tracker™ Red was excited using a 594 nm laser.
Relative Intensity
Cells plated in 8-well plates were treated with compounds 1a-
5b (1 μM) and fixed with PFA. The samples were imaged using a
405 nm laser at 0.2% and the detector was set to the range of
14 | J. Name., 2012, 00, 1-3
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Page 15 of 17
Organic & Biomolecular Chemistry
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Journal Name
ARTICLE
415-735 nm and a gain of 500. The pinhole was set to 4.88 Airy
units to maximise the amount of light collected. Using ImageJ
for the analysis, thresholding at a low tolerance level was used
to select the cells in the images, removing any background areas
devoid of cells. The normalised average intensity per pixel was
used as the comparison metric between the compounds. The
Zeiss 880 microscope was used for this work.
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View Article Online
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and M. Martin-Fernandez, PLoS One, ,
Lambda Scans
Three lambda scans were performed for each compound - each
on different cell samples. To reduce noise, each sample had the
line averaging set to 2, however there was no frame averaging.
The three scans were processed in ImageJ by taking the
intensity at each frame (wavelength step) and then averaging.
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S. H. Alamudi, R. Satapathy, J. Kim, D. Su, H. Ren, R. Das, L.
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To further account for photobleaching, snapshots were taken 10
before and after each lambda scan with a detector range of 415-
735 nm. The intensity of the snapshots was measured using 11
ImageJ and fit to an inverse exponential model by using the
solver function in Microsoft Excel to perform least squares 12
analysis. The lambda scan data was adjusted accordingly. The
P. Gao, W. Pan, N. Li and B. Tang, Chem. Sci., 2019, 10,
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compounds 1a, 1b & 4a and 1 μM for the rest of the compounds 15
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The samples were imaged at 10% power and the detector was 16
set to a gain of 700. 100 data points were taken, and ImageJ was
used to calculate the average pixel intensity of each frame. The
resultant data was fit to an inverse mono-exponential model
using the solver function in Microsoft Excel to perform least
squares analysis. The half-life (number of frames taken to reach 17
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18
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Conflicts of interest
19
D. R. Chisholm, C. W. E. Tomlinson, G.-L. Zhou, C. Holden,
V. Affleck, R. Lamb, K. Newling, P. Ashton, R. Valentine, C.
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A. W. and C. A. A. are LightOx Limited shareholders, the company
licensed to pursue commercial applications of the novel compounds
described in this manuscript.
20
Acknowledgements
D. R. C. thanks the EPSRC, BBSRC and High Force Research
Limited for DTA funding.
21
22
Notes and references
23
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Organic & Biomolecular Chemistry
lipid droplets
very hydrophobic
highly lipophilic
tetrahydroquinoline
moiety
lysosomes
pH = 4.7
basic piperazine moiety
increasing hydrophilicity
mitochondria
negative membrane
potential
positively charged
targeting moiety